Test Methods for Evaluating Solid Waste
Physical/Chemical Methods
Final (Promulgated) Updates II and MA 3
Volume 3
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METHOD 8275
THERMAL CHROMATOGRAPHY/MASS SPECTROMETRY (TC/MS) FOR
SCREENING SEMIVOLATILE ORGANIC COMPOUNDS
1.0 SCOPE AND APPLICATION
1.1 Method 8275 is a screening technique that may be used for the
qualitative identification of semivolatile organic compounds in extracts prepared
from nonaqueous solid wastes and soils. It is not intended for use as a rigorous
quantitative method. Direct injection of a sample may be used in limited
applications. The following analytes can be qualitatively determined by this
method:
Compound Name CAS No.8
2-Chlorophenol 95-57-8
4-Methylphenol 106-44-5
2,4-Dichlorophenol 120-83-2
Naphthalene 91-20-3
4-Chloro-3-methylphenol 59-50-7
1-Chloronaphthalene 90-13-1
2,4-Dinitrotoluene 121-14-2
Fluorene 86-73-7
Diphenylamine 122-39-4
Hexachlorobenzene 118-74-1
Dibenzothiophene 132-65-0
Phenanthrene 85-01-8
Carbazole 86-74-8
Aldrin 309-00-2
Pyrene 129-00-0
Benzo(k)fluoranthene 207-08-9
Benzo(a)pyrene 50-32-8
a Chemical Abstract Services Registry Number.
1.2 Method 8275 can be used to qualitatively identify most neutral,
acidic, and basic organic compounds that can be thermally desorbed from a sample,
and are capable of being eluted without derivatization as sharp peaks from a gas
chromatographic fused-silica capillary column coated with a slightly polar
silicone.
1.3 This method is restricted to use by or under the supervision of
analysts experienced in the use of gas chromatograph/mass spectrometers and
skilled in the interpretation of mass spectra. Each analyst must demonstrate the
ability to generate acceptable results with this method.
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2.0 SUMMARY OF METHOD
2.1 A portion of the sample (0.010-0.100 g) is weighed into a sample
crucible. The crucible is placed in a pyrocell and heated. The compounds
desorbed from the sample are detected using a flame ionization detector (FID).
The FID response is used to calculate the optimal amount of sample needed for
mass spectrometry. A second sample is desorbed and the compounds are condensed
on the head of a fused silica capillary column. The column is heated using a
temperature program, and the effluent from the column is introduced into the mass
spectrometer.
3.0 INTERFERENCES
3.1 Contamination by carryover can occur whenever low-level samples are
analyzed after high-level samples. Whenever an unusually concentrated sample is
encountered, it should be followed by the analysis of an empty (clean) crucible
to check for cross contamination.
4.0 APPARATUS AND MATERIALS
4.1 Thermal Chromatograph (TC) System
4.1.1 Thermal chromatograph™, Ruska Laboratories, or equivalent.
4.1.2 Column - 30 m x 0.25 mm ID (or 0.32 mm ID), 1 ^m film
thickness, silicone-coated, fused-silica capillary column (J&W Scientific
DB-5 or equivalent).
4.1.3 Flame Ionization detector (FID).
4.2 Mass Spectrometer (MS) system
4.2.1 Mass Spectrometer - Capable of scanning from 35 to 500 amu
every one second or less, using 70 volts (nominal) electron energy in the
electron impact ionization mode.
4.2.2 TC/MS interface - Any GC-to-MS interface producing acceptable
calibration data in the concentration range of interest may be used.
4.2.3 Data System - A computer must be interfaced to the mass
spectrometer. The data system must allow the continuous acquisition and
storage on machine-readable media of all mass spectra obtained throughout
the duration of the chromatographic program. The computer must have
software that can search any GC/MS data file for ions of a specific mass
(or group of masses) and that can plot such ion abundances versus time or
scan number. This type of plot is defined as an extracted ion
chromatogram (EIC). Software must also be available that allows for
integration of the abundances in, and EIC between, specified time or scan-
number limits.
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4.3 Tools and equipment
4.3.1 Fused quartz spatula.
4.3.2 Fused quartz incinerator ladle.
4.3.3 Metal forceps for sample crucible.
4.3.4 Sample crucible storage dishes.
4.3.5 Porous fused quartz sample crucibles with lids.
4.3.6 Sample crucible cleaning incinerator.
4.3.7 Cool ing rack.
4.3.8 Microbalance, 1 g capacity, 0.000001 g sensitivity, Mettler
Model M-3 or equivalent.
4.4 Vials - 10 mL, glass with Teflon lined screw-caps or crimp tops.
4.5 Volumetric flasks, Class A - 10 ml to 1000 ml.
5.0 REAGENTS
5.1 Reagent grade inorganic chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available.
5.2 Solvents
5.2.1 Methanol, CH3OH - Pesticide grade or equivalent.
5.2.2 Acetone, CH3COCH3 - Pesticide grade or equivalent.
5.2.3 Toluene, C6H5CH3 - Pesticide grade or equivalent.
5.2.4 Methylene chloride, CH2C12 - Pesticide grade or equivalent.
5.2.5 Carbon disulfide, CS2 - Pesticide grade or equivalent.
5.2.6 Hexane, C6H14 - Pesticide grade or equivalent.
5.2.7 Other suitable solvents - Pesticide grade or equivalent.
5.3 Stock Standard solutions - Standard solutions may be prepared from
pure standard materials or purchased as certified solutions.
5.3.1 Prepare stock standard solutions by weighing about 0.01 g of
pure material. Dissolve the material in pesticide quality acetone, or
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other suitable solvent, and dilute to 10 ml in a volumetric flask. Larger
volumes may be used at the convenience of the analyst.
5.3.2 Transfer the stock standard solutions into glass vials with
Teflon lined screw-caps or crimp tops. Store at -10°C to -20°C or less
and protect from light. Stock standard solutions should be checked
frequently for signs of degradation or evaporation, especially prior to
use in preparation of calibration standards.
5.3.3 Stock standard solutions must be replaced after 1 year, or
sooner if comparison with quality control check samples indicates a
problem.
5.4 Internal Standard solutions - The internal standards recommended are
l,4-dichlorobenzene-d4, naphthalene-ds, acenaphthene-d10, phenanthrene-d10,
cnrysene-d12, and perylene-d12. Other compounds may be used as internal standards
as long as the requirements given in Sec. 7 are met. Dissolve about 0.200 g of
each compound with a small volume of carbon disulfide. Transfer to a 50 ml
volumetric flask and dilute to volume with methylene chloride, so that the final
solvent is approximately 20/80 (V/V) carbon disulfide/methylene chloride. Most
of the compounds are also soluble in small volumes of methanol, acetone, or
toluene, except for perylene-d12. Prior to each analysis, deposit about 10 ^L
of the internal standard onto the sample in the crucible. Store internal
standard solutions at 4°C or less before, and between, use.
5.5 Calibration standards - Prepare calibration standards within the
working range of the TC/MS system. Each standard should contain each analyte or
interest (e.g. some or all of the compounds listed in Sec. 1.1 may be included).
Each aliquot of calibration standard should be spiked with internal standards
prior to analysis. Stock solutions should be stored at -10°C to -20°C and should
be freshly prepared once a year, or sooner if check standards indicate a problem.
The daily calibration standard should be prepared weekly, and stored at 4°C.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this Chapter, Organic Analytes,
Sec. 4.1.
7.0 PROCEDURE
7.1 Crucible Preparation
7.1.1 Turn on the incinerator and let it heat for at least 10
minutes. The bore of the incinerator should be glowing red.
7.1.2 Load the sample crucible and lid into the incinerator ladle
and insert into the incinerator bore. Leave in the incinerator for 5
minutes, then remove and place on the cooling rack.
7.1.3 Allow the crucibles and lids to cool for five minutes before
placing them in the storage dishes.
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CAUTION: Do not touch the crucibles with your fingers. This can
result in a serious burn, as well as contamination of
the crucible. Always handle the sample crucibles and
lids with forceps and tools specified.
7.1.4 All sample crucibles and lids required for the number of
analyses planned should be cleaned and placed in the storage dishes ready
for use.
7.2 Sample Preparation and Loading
7.2.1 The analyst should take care in selecting a sample for
analysis, since the sample size is generally limited to 0.100 g or less.
This implies that the sample should be mixed as thoroughly as possible
before taking an aliquot. Because the sample size is limited, the analyst
may wish to analyze several aliquots for determination.
7.2.2 The sample should be mixed or ground such that a 0.010 to
0.100 g aliquot can be removed. Remove one sample crucible from the
storage dish and place it on the microbalance. Establish the tare weight.
Remove the sample crucible from the balance with the forceps and place it
on a clean surface.
7.2.3 Load an amount of sample into the sample crucible using the
fused quartz spatula. Place the assembly on the microbalance and
determine the weight of the sample. For severely contaminated samples,
less than 0.010 g will suffice, while 0.050-0.100 g is needed for low
concentrations of contaminants. Place the crucible lid on the crucible;
the sample is now ready for analysis.
7.3 FID Analysis
7.3.1 Load the sample into the TC. Hold the sample at 30°C for 2
minutes followed by linear temperature programmed heating to 260°C at
30°C/minute. Follow the temperature program with an isothermal heating
period of 10 minutes at 260°C, followed by cooling back to 30°C. The total
analysis cycle time is 24.2 minutes
7.3.2 Monitor the FID response in real time during analysis, and
note the highest response in millivolts (mV). Use this information to
determine the proper weight of sample needed for combined thermal
extraction/gas chromatography/mass spectrometry.
7.4 Thermal Extraction/GC/MS
7.4.1 Prepare a calibration curve using a clean crucible and lid by
spiking the compounds of interest at five concentrations into the crucible
and applying the internal standards to the crucible lid. Analyze these
standards and establish response factors at different concentrations.
7.4.2 Weigh out the amount of fresh sample that will provide
approximately 1000 to 3000 mv response. For example, if 0.010 g of sample
gives an FID response of 500 mv, then 0.020 to 0.060 g (0.040 g ± 50 %)
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should be used. If 0.100 g gives 8000 mv, then 0.025 g ± 50 % should be
used.
7.4.3 After weighing out the sample into the crucible, deposit the
internal -standards (10 juL) onto the sample. Load the crucible into the
pyrocell, using the same temperature program in Sec. 7.3.1. Hold the
capillary at 5°C during this time to focus the released semivolatiles (the
intermediate trap is held at 330°C to pass all compounds onto the column).
Maintain the splitter zone at 310°C, and the GC/MS transfer line at 285°C.
After the isothermal heating period is complete, temperature program the
column from 5°C to 285°C at 10°C/nrinute and hold at 285°C for 5 minutes.
Acquire data during the entire run time.
7.4.4 If the response for any quantitation ion exceeds the initial
calibration curve range of the TC/MS system, a smaller sample should be
analyzed.
7.5 Data Interpretation
7.5.1 Qualitative Analysis
7.5.1.1 The qualitative identification of compounds
determined by this method is based on retention time, and on
comparison of the sample mass spectrum, after background correction,
with characteristic ions in a reference mass spectrum. The
reference mass spectrum must be generated by the laboratory using
the conditions of this method. The characteristic ions from the
reference mass spectrum are defined to be the three ions of greatest
relative intensity, or any ions over 30% relative intensity if less
than three such ions occur in the reference spectrum. Compounds
should be identified as present when the criteria below are met.
7.5.1.1.1 The intensities of the characteristic ions
of a compound maximize in the same scan or within one scan of
each other. Selection of a peak by a data system target
compound search routine where the search is based on the
presence of a target chromatographic peak containing ions
specific for the target compound at a compound-specific
retention time will be accepted as meeting this criterion.
7.5.1.1.2 The RRT of the sample component is within
± 0.06 RRT units of the RRT of the standard component.
7.5.1.1.3 The relative intensities of the
characteristic ions agree within 30% of the relative
intensities of these ions in the reference spectrum.
(Example: For an ion with an abundance of 50% in the
reference spectrum, the corresponding abundance in a sample
spectrum can range between 20% and 80%.)
7.5.1.1.4 Structural isomers that produce very similar
mass spectra should be identified as individual isomers if
they have sufficiently different GC retention times.
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Sufficient GC resolution is achieved if the height of the
valley between two isomer peaks is less than 25% of the sum of
the two peak heights. Otherwise, structural isomers are
identified as isomeric pairs.
7.5.1.1.5 Identification is hampered when sample
components are not resolved chromatographically and produce
mass spectra containing ions contributing by more than one
analyte. When gas chromatographic peaks obviously represent
more than one sample component (i.e., a broadened peak with
shoulder(s) or a valley between two or more maxima),
appropriate selection of analyte spectra and background
spectra is important. Examination of extracted ion current
profiles of appropriate ions can aid in the selection of
spectra, and in qualitative identification of compounds. When
analytes coelute (i.e., only one chromatographic peak is
apparent), the identification criteria can be met, but each
analyte spectrum will contain extraneous ions contributed by
the coeluting compound.
7.5.1.2 For samples containing components not associated
with the calibration standards, a library search may be made for the
purpose of tentative identification. The necessity to perform this
type of identification will be determined by the purpose of the
analyses being conducted. Computer generated library search
routines should not use normalization routines that would
misrepresent the library or unknown spectra when compared to each
other. For example, the RCRA permit or waste delisting requirements
may require the reporting of non-target analytes. Only after visual
comparison of sample spectra with the nearest library searches will
the mass spectral interpretation specialist assign a tentative
identification. Guidelines for making tentative identification are:
(1) Relative intensities of major ions in the reference
spectrum (ions > 10% of the most abundant ion) should be present in
the sample spectrum.
(2) The relative intensities of the major ions should agree
within + 20%. (Example: For an ion with an abundance of 50% in the
standard spectrum, the corresponding sample ion abundance must be
within 30 and 70%).
(3) Molecular ions present in the reference spectrum should
be present in the sample spectrum.
(4) Ions present in the sample spectrum but not in the
reference spectrum should be reviewed for possible background
contamination or presence of coeluting compounds.
(5) Ions present in the reference spectrum but not in the
sample spectrum should be reviewed for possible subtraction from the
sample spectrum because of background contamination or coeluting.
Data system library reduction programs can sometimes create these
discrepancies.
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8.0 QUALITY CONTROL
8.1 Refer to Chapter One and Method 8000 for specific quality control
procedures.
9.0 METHOD PERFORMANCE
9.1 Table 1 presents method performance data, generated using spiked soil
samples. Method performance data in an aqueous matrix are not available.
10.0 REFERENCES
1. Zumberge, J.E., C. Sutton, R.D. Worden, T. Junk, T.R. Irvin, C.B. Henry,
V. Shirley, and E.B. Overton, "Determination of Semi-Volatile Organic
Pollutants in Soils by Thermal Chromatography-Mass Spectrometry (TC/MS):
an Assessment for Field Analysis," in preparation.
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TABLE 1
METHOD PERFORMANCE, SOIL MATRIX
Analyte
2-Chlorophenol
4-Methyl phenol
2,4-Dichlorophenol
Naphthalene
4-Chloro-3-methyl -phenol
1-Chloronaphthalene
2,4-Dinitrotoluene
Fluorene
Diphenylamine
Hexachlorobenzene
Dibenzothiophene
Phenanthrene
Carbazole
Aldrin
Pyrene
Benzo( k) fl uoranthene
Benzo(a)pyrene
Average
Clay
30
10
23
77
9
96
7
9
5
68
20
11
4
3
7
4
4
% Recovery"
Silt
22
77
20
120
12
103
10
25
6
64
35
31
8
19
19
9
8
Subsoil
2
7
26
63
9
70
10
19
6
80
50
40
9
15
20
11
11
Mean
Recovery
18
31
23
87
10
90
9
18
6
71
35
24
7
12
15
8
8
Percent theoretical recovery based upon linearity of injections deposited on
the crucible lid (slope and y-intercept). Average of 9 replicates (-10 mg
soil spiked with 50 ppm of analyte); 3 different instruments at 3 different
laboratories.
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TABLE 2
CHARACTERISTIC IONS FOR SEMIVOLATILE COMPOUNDS
Compound
Primary
Ion
Secondary
Ion(s)
2-Chlorophenol
4-Methylphenol
2,4-Dichlorophenol
Naphthalene
4-Chloro-3-methyl-phenol
1-Chloronaphthalene
2,4-Dinitrotoluene
Fluorene
Diphenylamine
Hexachlorobenzene
Phenanthrene
Aldrin
Pyrene
Benzo(k)fluoranthene
Benzo(a)pyrene
128
107
162
128
107
162
165
166
169
284
178
66
202
252
252
64,130
107,108,77,79,90
164,98
129,127
144,142
127,164
63,89
165,167
168,167
142,249
179,176
263,220
200,203
253,125
253,125
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METHOD 8275
THERMAL CHROMATOGRAPHY/MASS SPECTROMETRY (TC/MS) FOR
SCREENING SEMIVOLATILE ORGANIC COMPOUNDS
Start
7.1 Prepare
crucible
7.2.2
Establish
tare weight
of crucible.
7.2.3 Place
sample in
crucible; establish
weight.
7.3.1 FID
Analysis using
linear temp.
programmed
heating.
7.3.2 Using
FID response,
determine
sample weight
for TE/GC/MS.
7.4.1 Prepare
calibration
curve.
7.4.2 Prepare
amount of
sample for
appropriate
FID response.
7.4.3 Weigh
sample into
crucible; use
temp, program
m Sec. 7.3.1 .
7.4.4 Use
smaller
sample.
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00
s>
O
o
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METHOD 8290
POLYCHLORINATED DIBENZODIOXINS (PCDDs) AND POLYCHLORINATED DIBENZOFURANS
(PCDFs)BY HIGH-RESOLUTION GAS CHROMATOGRAPHY/HIGH-RESOLUTION
MASS SPECTROMETRY (HRGC/HRMS)
1.0 SCOPE AND APPLICATION
1.1 This method provides procedures for the detection and quantitative
measurement of polychlorinated dibenzo-p-dioxins (tetra- through octachlorinated
homologues; PCDDs}, and polychlorinated dibenzofurans (tetra- through
octachlorinated homologues; PCDFs) in a variety of environmental matrices and at
part-per-trillion (ppt) to part-per-quadrillion (ppq) concentrations. The
following compounds can be determined by this method:
Compound Name
CAS No8
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
1,2,3,7,8-Pentachlorodibenzo-p-dioxin (PeCDD)
1,2,3,6,7,8-Hexachlorodibenzo-p-dioxin (HxCDD)
1,2,3,4,7,8-Hexachlorodibenzo-p-dioxin (HxCDD)
1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin (HxCDD)
1,2,3,4,6,7,8-Heptachlorodibenzo-p-dioxin (HpCDD)
1,2,3,4,6,7,8,9-Octachlorodibenzo-p-dioxin (OCDD)
2,3,7,8-Tetrachlorodibenzofuran (TCDF)
1,2,3,7 , 8- Pentachl orodi benzof uran ( PeCDF )
2,3,4,7,8-Pentachlorodibenzofuran (PeCDF)
1,2,3,6,7,8-Hexachlorodibenzofuran (HxCDF)
1,2,3,7 , 8, 9-Hexachl orodi benzof uran (HxCDF)
1,2,3,4,7 , 8-Hexachl orodi benzof uran (HxCDF )
2,3,4,6,7, 8-Hexachl orodi benzof uran (HxCDF)
1,2,3,4,6,7 , 8-Heptachl orodi benzof uran (HpCDF)
1,2,3,4,7,8,9-Heptachlorodibenzofuran (HpCDF)
1,2,3,4,6,7,8,9-Octachlorodibenzofuran (OCDF)
1746-01-6
40321-76-4
57653-85-7
39227-28-6
19408-74-3
35822-39-4
3268-87-9
51207-31-9
57117-41-6
57117-31-4
57117-44-9
72918-21-9
70648-26-9
60851-34-5
67562-39-4
55673-89-7
39001-02-0
8 Chemical Abstract Service Registry Number
1.2 The analytical method calls for the use of high-resolution gas
chromatography and high-resolution mass spectrometry (HRGC/HRMS) on purified
sample extracts. Table 1 lists the various sample types covered by this
analytical protocol, the 2,3,7,8-TCDD-based method cal ibration 1 imits (MCLs), and
other pertinent information. Samples containing concentrations of specific
congeneric analytes (PCDDs and PCDFs) considered within the scope of this method
that are greater than ten times the upper MCLs must be analyzed by a protocol
designed for such concentration levels, e.g., Method 8280. An optional method
for reporting the analytical results using a 2,3,7,8-TCDD toxicity equivalency
factor (TEF) is described.
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1.3 The sensitivity of this method is dependent upon the level of inter-
ferences within a given matrix. The calibration range of the method for all
water sample is 10 to 2000 ppq for TCDD/TCDF and PeCDD/PeCDF, and 1.0 to 200 ppt
for a 10 g soil, sediment, fly ash, or tissue sample for the same analytes
(Table 1). Analysis of a one-tenth aliquot of the sample permits measurement of
concentrations up to 10 times the upper MCL. The actual limits of detection and
quantitation will differ from the lower MCL, depending on the complexity of the
matrix.
1.4 This method is designed for use by analysts who are experienced with
residue analysis and skilled in HR6C/HRMS.
1.5 Because of the extreme toxicity of many of these compounds, the
analyst must take the necessary precautions to prevent exposure to materials
known or believed to contain PCDDs or PCOFs. It is the responsibility of the
laboratory personnel to ensure that safe handling procedures are employed. Sec.
11 of this method discusses safety procedures.
2.0 SUMMARY OF METHOD
2.1 This procedure uses matrix specific extraction, analyte specific
VHRMS analysis techniques.
2.1 This procedure uses matrix sp<
cleanup, and HRGC/HRMS analysis techniques.
2.2 If interferences are encountered, the method provides selected
cleanup procedures to aid the analyst in their elimination. A simplified
analysis flow chart is presented at the end of this method.
2.3 A specified amount (see Table 1) of soil, sediment, fly ash, water,
sludge (including paper pulp), still bottom, fuel oil, chemical reactor residue,
fish tissue, or human adipose tissue is spiked with a solution containing
specified amounts of each of the nine isotopically (13C12) labeled PCDDs/PCDFs
listed in Column 1 of Table 2. The sample is then extracted according to a
matrix specific extraction procedure. Aqueous samples that are judged to contain
1 percent or more solids, and solid samples that show an aqueous phase, are
filtered, the solid phase (including the filter) and the aqueous phase extracted
separately, and the extracts combined before extract cleanup. The extraction
procedures are:
a) Toluene: Soxhlet extraction for soil, sediment, fly ash, and paper
pulp samples;
b) Methylene chloride: liquid-liquid extraction for water samples;
c) Toluene: Dean-Stark extraction for fuel oil, and aqueous sludge
samples;
d) Toluene extraction for still bottom samples;
e) Hexane/methylene chloride: Soxhlet extraction or methylene
chloride: Soxhlet extraction for fish tissue samples; and
f) Methylene chloride extraction for human adipose tissue samples.
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g) As an option, all solid samples (wet or dry) may be extracted with
toluene using a Soxhlet/Dean Stark extraction system.
The decision for the selection of an extraction procedure for chemical
reactor residue samples is based on the appearance (consistency, viscosity) of
the samples. Generally, they can be handled according to the procedure used for
still bottom (or chemical sludge) samples.
2.4 The extracts are submitted to an acid-base washing treatment and
dried. Following a solvent exchange step, the extracts are cleaned up by column
chromatography on alumina, silica gel, and activated carbon.
2.4.1 The extracts from adipose tissue samples are treated with
silica gel impregnated with sulfuric acid before chromatography on acidic
silica gel, neutral alumina, and activated carbon.
2.4.2 Fish tissue and paper pulp extracts are subjected to an acid
wash treatment only, prior to chromatography on alumina and activated
carbon.
2.5 The preparation of the final extract for HRGC/HRMS analysis is
accomplished by adding 10 to 50 pi (depending on the matrix) of a nonane
solution containing 50 pg//uL of the recovery standards 13C12-1,2,3,4-TCDD and
13C12-l,2,3,7,8,9-HxCDD (Table 2). The former is used to determine the percent
recoveries of tetra- and pentachlorinated PCDD/PCDF congeners, while the latter
is used to determine the percent recoveries of the hexa-, hepta- and
octachlorinated PCDD/PCDF congeners.
2.6 Two fj.1 of the concentrated extract are injected into an HRGC/HRMS
system capable of performing selected ion monitoring at resolving powers of at
least 10,000 (10 percent valley definition).
2.7 The identification of OCDD and nine of the fifteen 2,3,7,8-
substituted congeners (Table 3), for which a 13C-labeled standard is available
in the sample fortification and recovery standard solutions (Table 2), is based
on their elution at their exact retention time (within 0.005 retention time units
measured in the routine calibration) and the simultaneous detection of the two
most abundant ions in the molecular ion region. The remaining six 2,3,7,8-
substituted congeners (i.e., 2,3,4,7,8-PeCDF; 1,2,3,4,7,8-HxCDD; 1,2,3,6,7,8-
HxCDF; 1,2,3,7,8,9-HxCDF; 2,3,4,6,7,8-HxCDF, and 1,2,3,4,7,8,9-HpCDF), for which
no carbon-labeled internal standards are available in the sample fortification
solution, and all other PCDD/PCDF congeners are identified when their relative
retention times fall within their, respective PCDD/PCDF retention time windows,
as established from the routine calibration data, and the simultaneous detection
of the two most abundant ions in the molecular ion region. The identification
of OCDF is based on its retention time relative to 13C12-OCDD and the simultaneous
detection of the two most abundant ions in the molecular ion region.
Identification also is based on a comparison of the ratios of the integrated ion
abundance of the molecular ion species to their theoretical abundance ratios.
2.8 Quantitation of the individual congeners, total PCDDs and total PCDFs
is achieved in conjunction with the establishment of a multipoint (five points)
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calibration curve for each homologue, during which each calibration solution is
analyzed once.
3.0 INTERFERENCES
3.1 Solvents, reagents, glassware and other sample processing hardware
may yield discrete artifacts or elevated baselines that may cause misinter-
pretation of the chromatographic data (see references 1 and 2.) All of these
materials must be demonstrated to be free from interferants under the conditions
of analysis by performing laboratory method blanks. Analysts should avoid using
PVC gloves.
3.2 The use of high purity reagents and solvents helps minimize
interference problems. Purification of solvents by distillation in all-glass
systems may be necessary.
3.3 Interferants coextracted from the sample will vary considerably from
matrix to matrix. PCDDs and PCDFs are often associated with other interfering
chlorinated substances such as polychlorinated biphenyls (PCBs), polychlorinated
diphenyl ethers (PCDPEs), polychlorinated naphthalenes, and polychlorinated
alkyldibenzofurans, that may be found at concentrations several orders of
magnitude higher than the analytes of interest. Retention times of target
analytes must be verified using reference standards. These values must
correspond to the retention time windows established in Sec. 8.1.1.3. While
cleanup techniques are provided as part of this method, unique samples may
require additional cleanup steps to achieve lower detection limits.
3.4 A high-resolution capillary column (60 m DB-5, J&W Scientific, or
equivalent) is used in this method. However, no single column is known to
resolve all isomers. The 60 m DB-5 GC column is capable of 2,3,7,8-TCDD isomer
specificity (Sec. 8.1.1). In order to determine the concentration of the
2,3,7,8-TCDF (if detected on the DB-5 column), the sample extract must be
reanalyzed on a column capable of 2,3,7,8-TCDF isomer specificity (e.g., DB-225,
SP-2330, SP-2331, or equivalent).
4.0 APPARATUS AND MATERIALS
4.1 High-Resolution Gas Chromatograph/High-Resolution Mass
Spectrometer/Data System (HRGC/HRMS/DS) - The GC must be equipped for temperature
programming, and all required accessories must be available, such as syringes,
gases, and capillary columns.
4.1.1 GC Injection Port - The GC injection port must be designed for
capillary columns. The use of splitless injection techniques is
recommended. On column 1 /zL injections can be used on the 60 m DB-5
column. The use of a moving needle injection port is also acceptable.
When using the method described in this protocol, a 2 /uL injection volume
is used consistently (i.e., the injection volumes for all extracts,
blanks, calibration solutions and the performance check samples are 2 /iL).
One juL injections are allowed; however, laboratories must remain
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consistent throughout the analyses by using the same injection volume at
all times.
4.1.2 Gas Chromatograph/Mass Spectrometer (GC/MS) Interface - The
GC/MS interface components should withstand 350°C. The interface must be
designed so that the separation of 2,3,7,8-TCDD from the other TCDD
isomers achieved in the gas chromatographic column is not appreciably
degraded. Cold spots or active surfaces (adsorption sites) in the GC/MS
interface can cause peak tailing and peak broadening. It is recommended
that the GC column be fitted directly into the mass spectrometer ion
source without being exposed to the ionizing electron beam. Graphite
ferrules should be avoided in the injection port because they may adsorb
the PCDDs and PCDFs. Vespel™, or equivalent, ferrules are recommended.
4.1.3 Mass Spectrometer - The static resolving power of the
instrument must be maintained at a minimum of 10,000 (10 percent valley).
4.1.4 Data System - A dedicated data system is employed to control
the rapid multiple-ion monitoring process and to acquire the data.
Quantitation data (peak areas or peak heights) and SIM traces (displays of
intensities of each ion signal being monitored including the lock-mass ion
as a function of time) must be acquired during the analyses and stored.
Quantitations may be reported based upon computer generated peak areas or
upon measured peak heights (chart recording). The data system must be
capable of acquiring data at a minimum of 10 ions in a single scan. It is
also recommended to have a data system capable of switching to different
sets of ions (descriptors) at specified times during an HRGC/HRMS
acquisition. The data system should be able to provide hard copies of
individual ion chromatograms for selected gas chromatographic time
intervals. It should also be able to acquire mass spectral peak profiles
(Sec. 8.1.2.3) and provide hard copies of peak profiles to demonstrate the
required resolving power. The data system should permit the measurement
of noise on the base line.
NOTE: The detector ADC zero setting must allow peak-to-peak measure-
ment of the noise on the base line of every monitored channel
and allow for good estimation of the instrument resolving
power. In Figure 2, the effect of different zero settings on
the measured resolving power is shown.
4.2 GC Columns
4.2.1 In order to have an isomer specific determination for 2,3,7,8-
TCDD and to allow the detection of OCDD/OCDF within a reasonable time
interval in one HRGC/HRMS analysis, use of the 60 m DB-5 fused silica
capillary column is recommended. Minimum acceptance criteria must be
demonstrated and documented (Sec. 8.2.2). At the beginning of each 12
hour period (after mass resolution and GC resolution are demonstrated)
during which sample extracts or concentration calibration solutions will
be analyzed, column operating conditions must be attained for the required
separation on the column to be used for samples. Operating conditions
known to produce acceptable results with the recommended column are shown
in Sec. 7.6.
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4.2.2 Isomer specificity for all 2,3,7,8-substituted PCDDs/PCDFs
cannot be achieved on the 60 m DB-5 GC column alone. In order to
determine the proper concentrations of the individual 2,3,7,8-substituted
congeners, the sample extract must be reanalyzed on another GC column that
resolves the isomers.
4.2.3 30 m DB-225 fused silica capillary column, (J&W Scientific) or
equivalent.
4.3 Miscellaneous Equipment and Materials - The following list of items
does not necessarily constitute an exhaustive compendium of the equipment needed
for this analytical method.
4.3.1 Nitrogen evaporation apparatus with variable flow rate.
4.3.2 Balances capable of accurately weighing to 0.01 g and
0.0001 g.
4.3.3 Centrifuge.
4.3.4 Water bath, equipped with concentric ring covers and capable
of being temperature controlled within + 2°C.
4.3.5 Stainless steel or glass container large enough to hold
contents of one pint sample containers.
4.3.6 Glove box.
4.3.7 Drying oven.
4.3.8 Stainless steel spoons and spatulas.
4.3.9 Laboratory hoods.
4.3.10 Pipets, disposable, Pasteur, 150 mm long x 5 mm ID.
4.3.11 Pipets, disposable, serological, 10 ml, for the
preparation of the carbon columns specified in Sec. 7.5.3.
4.3.12 Reaction vial, 2 ml, silanized amber glass (Reacti-vial,
or equivalent).
4.3.13 Stainless steel meat grinder with a 3 to 5 mm hole size
inner plate.
4.3.14 Separatory funnels, 125 mL and 2000 ml.
4.3.15 Kuderna-Danish concentrator, 500 ml, fitted with 10 ml
concentrator tube and three ball Snyder column.
4.3.16 Teflon™ or carborundum (silicon carbide) boiling chips
(or equivalent), washed with hexane before use.
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NOTE: Teflon™ boiling chips may float in methylene chloride, may
not work in the presence of any water phase, and may be
penetrated by nonpolar organic compounds.
4.3.17 Chromatographic columns, glass, 300 mm x 10.5 mm, fitted
with Teflon™ stopcock.
4.3.18 Adapters for concentrator tubes.
4.3.19 Glass fiber filters, 0.70 jum, Whatman GFF, or
equivalent.
4.3.20 Dean-Stark trap, 5 or 10 ml, with T-joints, condenser
and 125 ml flask.
4.3.21 Continuous liquid-liquid extractor.
4.3.22 All glass Soxhlet apparatus, 500 ml flask.
4.3.23 Soxhlet/Dean Stark extractor (optional), all glass, 500
ml flask.
4.3.24 Glass funnels, sized to hold 170 ml of liquid.
4.3.25 Desiccator.
4.3.26 Solvent reservoir (125 ml), Kontes; 12.35 cm diameter
(special order item), compatible with gravity carbon column.
4.3.27 Rotary evaporator with a temperature controlled water
bath.
4.3.28 High speed tissue homogenizer, equipped with an EN-8
probe, or equivalent.
4.3.29 Glass wool, extracted with methylene chloride, dried and
stored in a clean glass jar.
4.3.30 Extraction jars, glass, 250 ml, with teflon lined screw
cap.
4.3.31 Volumetric flasks, Class A - 10 ml to 1000 ml.
4.3.32 Glass vials, 1 dram (or metric equivalent).
NOTE: Reuse of glassware should be minimized to avoid the risk of
contamination. All glassware that is reused must be
scrupulously cleaned as soon as possible after use, according
to the following procedure: Rinse glassware with the last
solvent used in it. Wash with hot detergent water, then rinse
with copious amounts of tap water and several portions of
organic-free reagent water. Rinse with high purity acetone
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and hexane and store it inverted or capped with solvent rinsed
aluminum foil in a clean environment.
5.0 REAGENTS AND STANDARD SOLUTIONS
5.1 Organic-free reagent water - All references to water in this method
r to organic-free reagent water, as defined in Chapter One.
5.2 Column Chromatography Reagents
5.2.1 Alumina, neutral, 80/200 mesh (Super 1, Woelm®, or
equivalent). Store in a sealed container at room temperature, in a
desiccator, over self-indicating silica gel.
5.2.2 Alumina, acidic AG4, (Bio Rad Laboratories catalog #132-1240,
or equivalent). Soxhlet extract with methylene chloride for 24 hours if
blanks show contamination, and activate by heating in a foil covered glass
container for 24 hours at 190°C. Store in a glass bottle sealed with a
Teflon™ lined screw cap.
5.2.3 Silica gel, high purity grade, type 60, 70-230 mesh; Soxhlet
extract with methylene chloride for 24 hours if blanks show contamination,
and activate by heating in a foil covered glass container for 24 hours at
190°C. Store in a glass bottle sealed with a Teflon™ lined screw cap.
5.2.4 Silica gel impregnated with sodium hydroxide. Add one part
(by weight) of 1 M NaOH solution to two parts (by weight) silica gel
(extracted and activated) in a screw cap bottle and mix with a glass rod
until free of lumps. Store in a glass bottle sealed with a Teflon™ lined
screw cap.
5.2.5 Silica gel impregnated with 40 percent (by weight) sulfuric
acid. Add two parts (by weight) concentrated sulfuric acid to three parts
(by weight) silica gel (extracted and activated), mix with a glass rod
until free of lumps, and store in a screw capped glass bottle. Store in
a glass bottle sealed with a Teflon™ lined screw cap.
5.2.6 Celite 545® (Supelco), or equivalent.
5.2.7 Active carbon AX-21 (Anderson Development Co., Adrian, MI), or
equivalent, prewashed with methanol and dried in vacuo at 110°C. Store in
a glass bottle sealed with a Teflon™ lined screw cap.
5.3 Reagents
5.3.1 Sulfuric acid, H2S04, concentrated, ACS grade, specific gravity
1.84.
5.3.2 Potassium hydroxide, KOH, ACS grade, 20 percent (w/v) in
organic-free reagent water.
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5.3.3 Sodium chloride, NaCl, analytical reagent, 5 percent (w/v) in
organic-free reagent water.
5.3.4 Potassium carbonate, K2C03, anhydrous, analytical reagent.
5.4 Desiccating agent
5.4.1 Sodium sulfate (powder, anhydrous), Na2S04. Purify by heating
at 400°C for 4 hours in a shallow tray, or by precleaning the sodium
sulfate with methylene chloride. If the sodium sulfate is precleaned with
methylene chloride, a method blank must be analyzed, demonstrating that
there is no interference from the sodium sulfate.
5.5 Solvents
5.5.1 Methylene chloride, CH2C12. High purity, distilled in glass
or highest available purity.
5.5,2 Hexane,
available purity.
C6H14.
High purity, distilled in glass or highest
5.5.3 Methanol, CH3OH.
available purity.
High purity, distilled in glass or highest
5.5.4 Nonane,
available purity.
C9H20.
High purity, distilled in glass or highest
5.5.5 Toluene, C6H5CH3. High purity, distilled in glass or highest
available purity.
5.5.6 Cyclohexane, C6H12.
available purity.
5.5.7 Acetone, CH3COCH3.
available purity.
High purity, distilled in glass or highest
High purity, distilled in glass or highest
5.6 High-Resolution Concentration Calibration Solutions (Table 5) - Five
nonane solutions containing unlabeled (totaling 17) and carbon-labeled (totaling
11) PCDDs and PCDFs at known concentrations are used to calibrate the instrument.
The concentration ranges are homologue dependent, with the lowest values for the
tetrachlorinated dioxin and furan (1.0 pg/juL) and the highest values for the
octachlorinated congeners (1000 pg/juL).
5.6.1 Depending on the availability of materials, these high-
resolution concentration calibration solutions may be obtained from the
Environmental Monitoring Systems Laboratory, U.S. EPA, Cincinnati, Ohio.
However, additional secondary standards must be obtained from commercial
sources, and solutions should be prepared in the analyst's laboratory. It
is the responsibility of the laboratory to ascertain that the calibration
solutions received (or prepared) are indeed at the appropriate
concentrations before they are used to analyze samples.
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5.6.2 Store the concentration calibration solutions in 1 ml
mini vials at room temperature in the dark.
5.7 GC Column Performance Check Solution - This solution contains the
first and last eluting isomers for each homologous series from tetra- through
heptachlorinated congeners. The solution also contains a series of other TCDD
isomers for the purpose of documenting the chromatographic resolution. The
13C12-2,3,7,8-TCDD is also present. The laboratory is required to use nonane as
the solvent and adjust the volume so that the final concentration does not exceed
100 pg/AtL per congener. Table 7 summarizes the qualitative composition (minimum
requirement) of this performance evaluation solution.
5.8 Sample Fortification Solution - This nonane solution contains the
nine internal standards at the nominal concentrations that are listed in Table 2.
The solution contains at least one carbon-labeled standard for each homologous
series, and it is used to measure the concentrations of the native substances.
(Note that 13C12-OCDF is not present in the solution.)
5.9 Recovery Standard Solution - This nonane solution contains two
recovery standards, 13C12-1,2,3,4-TCDD and 13C12-l>2,3,7,8,9-HxCDD, at a nominal
concentration of 50 pg/>L per compound. 10 to 50 juL of this solution will be
spiked into each sample extract before the final concentration step and HRGC/HRMS
analysis.
5.10 Matrix Spike Fortification Solution - Solution used to prepare the
MS and MSD samples. It contains all unlabeled analytes listed in Table 5 at con-
centrations corresponding to the HRCC 3.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this chapter, Organic Analytes,
Sec. 4.1.
6.2 Sample Collection
6.2.1 Sample collection personnel should, to the extent possible,
homogenize samples in the field before filling the sample containers.
This should minimize or eliminate the necessity for sample homogenization
in the laboratory. The analyst should make a judgment, based on the
appearance of the sample, regarding the necessity for additional mixing.
If the sample is clearly not homogeneous, the entire contents should be
transferred to a glass or stainless steel pan for mixing with a stainless
steel spoon or spatula before removal of a sample portion for analysis.
6.2.2 Grab and composite samples must be collected in glass
containers. Conventional sampling practices must be followed. The bottle
must not be prewashed with sample before collection. Sampling equipment
must be free of potential sources of contamination.
6.3 Grinding or Blending of Fish Samples - If not otherwise specified by
the U.S. EPA, the whole fish (frozen) should be blended or ground to provide a
homogeneous sample. The use of a stainless steel meat grinder with a 3 to 5 mm
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hole size inner plate is recommended. In some circumstances, analysis of fillet
or specific organs of fish may be requested by the U.S. EPA. If so requested,
the above whole fish requirement is superseded.
6.4 Storage and Holding Times - All samples, except fish and adipose
tissue samples, must be stored at 4°C in the dark, extracted within 30 days and
completely analyzed within 45 days of extraction. Fish and adipose tissue
samples must be stored at -20°C in the dark, extracted within 30 days and
completely analyzed within 45 days of collection. Whenever samples are analyzed
after the holding time expiration date, the results should be considered to be
minimum concentrations and should be identified as such.
NOTE: The holding times listed in Sec. 6.4 are recommendations. PCDDs and
PCDFs are very stable in a variety of matrices, and holding times
under the conditions listed in Sec. 6.4 may be as high as a year for
certain matrices. Sample extracts, however, should always be
analyzed within 45 days of extraction.
6.5 Phase Separation - This is a guideline for phase separation for very
wet (>25 percent water) soil, sediment and paper pulp samples. Place a 50 g
portion in a suitable centrifuge bottle and centrifuge for 30 minutes at
2,000 rpm. Remove the bottle and mark the interface level on the bottle.
Estimate the relative volume of each phase. With a disposable pipet, transfer
the liquid layer into a clean bottle. Mix the solid with a stainless steel
spatula and remove a portion to be weighed and analyzed (percent dry weight
determination, extraction). Return the remaining solid portion to the original
sample bottle (empty) or to a clean sample bottle that is properly labeled, and
store it as appropriate. Analyze the solid phase by using only the soil,
sediment and paper pulp method. Take note of, and report, the estimated volume
of liquid before disposing of the liquid as a liquid waste.
6.6 Soil, Sediment, or Paper Sludge (Pulp) Percent Dry Weight
Determination - The percent dry weight of soil, sediment or paper pulp samples
showing detectable levels (see note below) of at least one 2,3,7,8-substituted
PCDD/PCDF congener is determined according to the following procedure. Weigh a
10 g portion of the soil or sediment sample (+ 0.5 g) to three significant
figures. Dry it to constant weight at 110°C in an adequately ventilated oven.
Allow the sample to cool in a desiccator. Weigh the dried solid to three
significant figures. Calculate and report the percent dry weight. Do not use
this solid portion of the sample for extraction, but instead dispose of it as
hazardous waste.
NOTE: Until detection limits have been established (Sec. 1.3), the lower
MCLs (Table 1) may be used to estimate the minimum detectable
levels.
% dry weight = q of dry sample x 100
g of sample
CAUTION: Finely divided soils and sediments contaminated with
PCDDs/PCDFs are hazardous because of the potential for
inhalation or ingestion of particles containing PCDDs/PCDFs
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(including 2,3,7,8-TCDD). Such samples should be handled in
a confined environment (i.e., a closed hood or a glove box).
6.7 Lipid Content Determination
6.7.1 Fish Tissue - To determine the lipid content of fish tissue,
concentrate 125 ml of the fish tissue extract (Sec. 7.2.2), in a tared 200
ml round bottom flask, on a rotary evaporator until a constant weight (W)
is achieved.
100 (W)
Percent lipid =
10
Dispose of the lipid residue as a hazardous waste if the results of
the analysis indicate the presence of PCDDs or PCDFs.
6.7.2 Adipose Tissue - Details for the determination of the adipose
tissue lipid content are provided in Sec. 7.3.3.
7.0 PROCEDURE
7.1 Internal standard addition
7.1.1 Use a portion of 1 g to 1000 g (+ 5 percent) of the sample to
be analyzed. Typical sample size requirements for different matrices are
given in Sec. 7.4 and in Table 1. Transfer the sample portion to a tared
flask and determine its weight.
7.1.2 Except for adipose tissue, add an appropriate quantity of the
sample fortification mixture (Sec. 5.8) to the sample. All samples should
be spiked with 100 /xL of the sample fortification mixture to give internal
standard concentrations as indicated in Table 1. As an example, for 13C12-
2,3,7,8-TCDD, a 10 g soil sample requires the addition of 1000 pg of 13C12-
2,3,7,8-TCDD to give the required 100 ppt fortification level. The fish
tissue sample (20 g) must be spiked with 200 jiL of the internal standard
solution, because half of the extract will be used to determine the lipid
content (Sec. 6.7.1).
7.1.2.1 For the fortification of soil, sediment, fly ash,
water, fish tissue, paper pulp and wet sludge samples, mix the
sample fortification solution with 1.0 mL acetone.
7.1.2.2 Do not dilute the nonane solution for the other
matrices.
7.1.2.3 The fortification of adipose tissue is carried out
at the time of homogenization (Sec. 7.3.2.3).
7.2 Extraction and Purification of Fish and Paper Pulp Samples
7.2.1 Add 60 g anhydrous sodium sulfate to a 20 g portion of a
homogeneous fish sample (Sec. 6.3) and mix thoroughly with a stainless
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steel spatula. After breaking up any lumps, place the fish/sodium sulfate
mixture in the Soxhlet apparatus on top of a glass wool plug. Add 250 ml
methylene chloride or hexane/methylene chloride (1:1) to the Soxhlet
apparatus and reflux for 16 hours. The solvent must cycle completely
through the system five times per hour. Follow the same procedure for the
partially dewatered paper pulp sample (using a 10 g sample, 30 g of
anhydrous sodium sulfate and 200 ml of toluene).
NOTE: As an option, a Soxhlet/Dean Stark extractor system may be
used, with toluene as the solvent. No sodium sulfate is added
when using this option.
7.2.2 Transfer the fish extract from Sec. 7.2.1 to a 250 ml
volumetric flask and fill to the mark with methylene chloride. Mix well,
then remove 125 ml for the determination of the lipid content (Sec.
6.7.1). Transfer the remaining 125 ml of the extract, plus two 15 ml
hexane/methylene chloride rinses of the volumetric flask, to a KD
apparatus equipped with a Snyder column. Quantitatively transfer all of
the paper pulp extract to a KD apparatus equipped with a Snyder column.
NOTE: As an option, a rotary evaporator may be used in place of the
KD apparatus for the concentration of the extracts.
7.2.3 Add a Teflon™, or equivalent, boiling chip. Concentrate the
extract in a water bath to an apparent volume of 10 ml. Remove the
apparatus from the water bath and allow to cool for 5 minutes.
7.2.4 Add 50 ml hexane and a new boiling chip to the KD flask.
Concentrate in a water bath to an apparent volume of 5 ml_. Remove the
apparatus from the water bath and allow to cool for 5 minutes.
NOTE: The methylene chloride must have been completely removed
before proceeding with the next step.
7.2.5 Remove and invert the Snyder column and rinse it into the KD
apparatus with two 1 ml portions of hexane. Decant the contents of the KD
apparatus and concentrator tube into a 125 ml separatory funnel. Rinse
the KD apparatus with two additional 5 ml portions of hexane and add the
rinses to the funnel. Proceed with the cleanup according to the
instructions starting in Sec. 7.5.1.1, but omit the procedures described
in Sees. 7.5.1.2 and 7.5.1.3.
7.3 Extraction and Purification of Human Adipose Tissue
7.3.1 Human adipose tissue samples must be stored at a temperature
of -20°C or lower from the time of collection until the time of analysis.
The use of chlorinated materials during the collection of the samples must
be avoided. Samples are handled with stainless steel forceps, spatulas,
or scissors. All sample bottles (glass) are cleaned as specified in the
note at the end of Sec. 4.3. Teflon™ lined caps should be used.
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NOTE: The specified storage temperature of -20°C is the maximum
storage temperature permissible for adipose tissue samples.
Lower storage temperatures are recommended.
7.3.2 Adipose Tissue Extraction
7.3.2.1 Weigh, to the nearest 0.01 g, a 10 g portion of a
frozen adipose tissue sample into a culture tube (2.2 x 15 cm).
NOTE: The sample size may be smaller, depending on
availability. In such a situation, the analyst is
required to adjust the volume of the internal standard
solution added to the sample to meet the fortification
level stipulated in Table 1.
7.3.2.2 Allow the adipose tissue specimen to reach room
temperature (up to 2 hours).
7.3.2.3 Add 10 ml methylene chloride and 100 jitL of the
sample fortification solution. Homogenize the mixture for
approximately 1 minute with a tissue homogenizer.
7.3.2.4 Allow the mixture to separate, then remove the
methylene chloride extract from the residual solid material with a
disposable pipet. Percolate the methylene chloride through a filter
funnel containing a clean glass wool plug and 10 g anhydrous sodium
sulfate. Collect the dried extract in a graduated 100 ml volumetric
flask.
7.3.2.5 Add a second 10 ml portion of methylene chloride
to the sample and homogenize for 1 minute. Decant the solvent, dry
it, and transfer it to the 100 ml volumetric flask (Sec. 7.3.2.4).
7.3.2.6 Rinse the culture tube with at least two
additional portions of methylene chloride (10 ml each), and transfer
the entire contents to the filter funnel containing the anhydrous
sodium sulfate. Rinse the filter funnel and the anhydrous sodium
sulfate contents with additional methylene chloride (20 to 40 ml)
into the 100 ml flask. Discard the sodium sulfate.
7.3.2.7 Adjust the volume to the 100 ml mark with
methylene chloride.
7.3.3 Adipose Tissue Lipid Content Determination
7.3.3.1 Preweigh a clean 1 dram (or metric equivalent)
glass vial to the nearest 0.0001 g on an analytical balance tared to
zero.
7.3.3.2 Accurately transfer 1.0 ml of the final extract
(100 ml) from Sec. 7.3.2.7 to the vial. Reduce the volume of the
extract on a water bath (50-60°C) by a gentle stream of purified
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nitrogen until an oily residue remains. Nitrogen blowdown is
continued until a constant weight is achieved.
NOTE: When the sample size of the adipose tissue is smaller
than 10 g, then the analyst may use a larger portion (up
to 10 percent) of the extract defined in Sec. 7.3.2.7
for the lipid determination.
7.3.3.3 Accurately weigh the vial with the residue to the
nearest 0.0001 g and calculate the weight of the lipid present in
the vial based on the difference of the weights.
7.3.3.4 Calculate the percent lipid content of the
original sample to the nearest 0.1 percent as shown below:
Lipid content, LC (%) = x 100
where:
Wat X Va|
W,r = weight of the lipid residue to the nearest 0.0001
g calculated from Sec. 7.3.3.3,
Vext = total volume (100 ml) of the extract in ml from
Sec. 7.3.2.7,
Wat = weight of the original adipose tissue sample to
the nearest 0.01 g from Sec. 7.3.2.1, and
Val = volume of the aliquot of the final extract in ml
used for the quantitative measure of the lipid
residue (1.0 ml) from Sec. 7.3.3.2.
7.3.3.5 Record the lipid residue measured in Sec. 7.3.3.3
and the percent lipid content from Sec. 7.3.3.4.
7.3.4 Adipose Tissue Extract Concentration
7.3.4.1 Quantitatively transfer the remaining extract from
Sec. 7.3.3.2 (99.0 ml) to a 500 ml Erlenmeyer flask. Rinse the
volumetric flask with 20 to 30 ml of additional methylene chloride
to ensure quantitative transfer.
7.3.4.2 Concentrate the extract on a rotary evaporator and
a water bath at 40°C until an oily residue remains.
7.3.5 Adipose Tissue Extract Cleanup
7.3.5.1 Add 200 mL hexane to the lipid residue in the 500
mL Erlenmeyer flask and swirl the flask to dissolve the residue.
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7.3.5.2 Slowly add, with stirring, 100 g of 40 percent
(w/w) sulfuric acid-impregnated silica gel. Stir with a magnetic
stirrer for two hours at room temperature.
7.3.5.3 Allow the solid phase to settle, and decant the
liquid through a filter funnel containing 10 g anhydrous sodium
sulfate on a glass wool plug, into another 500 ml Erlenmeyer flask.
7.3.5.4 Rinse the solid phase with two 50 ml portions of
hexane. Stir each rinse for 15 minutes, decant, and dry as
described under Sec. 7.3.5.3. Combine the hexane extracts from Sec.
7.3.5.3 with the rinses.
7.3.5.5 Rinse the sodium sulfate in the filter funnel with
an additional 25 ml hexane and combine this rinse with the hexane
extracts from Sec. 7.3.5.4.
7.3.5.6 Prepare an acidic silica column as follows: Pack
a 2 cm x 10 cm chromatographic column with a glass wool plug, add
approximately 20 ml hexane, add 1 g silica gel and allow to settle,
then add 4 g of 40 percent (w/w) sulfuric acid-impregnated silica
gel and allow to settle. Elute the excess hexane from the column
until the solvent level reaches the top of the chromatographic
packing. Verify that the column does not have any air bubbles and
channels.
7.3.5.7 Quantitatively transfer the hexane extract from
the Erlenmeyer flask (Sees. 7.3.5.3 through 7.3.5.5) to the silica
gel column reservoir. Allow the hexane extract to percolate through
the column and collect the eluate in a 500 mi KD apparatus.
7.3.5.8 Complete the elution by percolating 50 ml hexane
through the column into the KD apparatus. Concentrate the eluate on
a steam bath to approximately 5 ml. Use nitrogen blowdown to bring
the final volume to about 100 /LtL.
NOTE: If the silica gel impregnated with 40 percent sulfuric
acid is highly discolored throughout the length of the
adsorbent bed, the cleaning procedure must be repeated
beginning with Sec. 7.3.5.1.
7.3.5.9 The extract is ready for the column cleanups
described in Sees. 7.5.2 through 7.5.3.6.
7.4 Extraction and Purification of Environmental and Waste Samples
7.4.1 Sludge/Wet Fuel Oil
7.4.1.1 Extract aqueous sludge or wet fuel oil samples by
refluxing a sample (e.g., 2 g) with 50 ml toluene in a 125 ml flask
fitted with a Dean-Stark water separator. Continue refluxing the
sample until all the water is removed.
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NOTE: If the sludge or fuel oil sample dissolves in toluene,
treat it according to the instructions in Sec. 7.4.2
below. If the labeled sludge sample originates from
pulp (paper mills), treat it according to the
instructions starting in Sec. 7.2, but without the
addition of sodium sulfate.
7.4.1.2 Cool the sample, filter the toluene extract
through a glass fiber filter, or equivalent, into a 100 ml round
bottom flask.
7.4.1.3 Rinse the filter with 10 ml toluene and combine
the extract with the rinse.
7.4.1.4 Concentrate the combined solutions to near dryness
on a rotary evaporator at 50°C. Use of an inert gas to concentrate
the extract is also permitted. Proceed with Sec. 7.4.4.
7.4.2 Still Bottom/Oil
7.4.2.1 Extract still bottom or oil samples by mixing a
sample portion (e.g., 1.0 g) with 10 ml toluene in a small beaker
and filtering the solution through a glass fiber filter (or
equivalent) into a 50 ml round bottom flask. Rinse the beaker and
filter with 10 ml toluene.
7.4.2.2 Concentrate the combined toluene solutions to near
dryness on a rotary evaporator at 50°C. Proceed with Sec. 7.4.4.
7.4.3 Fly Ash
NOTE: Because of the tendency of fly ash to "fly", all handling
steps should be performed in a hood in order to minimize
contamination.
7.4.3.1 Weigh about 10 g fly ash to two decimal places and
transfer to an extraction jar. Add 100 fil sample fortification
solution (Sec. 5.8), diluted to 1 ml with acetone, to the sample.
Add 150 ml of 1 M HC1 to the fly ash sample. Seal the jar with the
Teflon™ lined screw cap and shake for 3 hours at room temperature.
7.4.3.2 Rinse a glass fiber filter with toluene, and
filter the sample through the filter paper, placed in a Buchner
funnel, into a 1 L flask. Wash, the fly ash cake with approximately
500 ml organic-free reagent water and dry the filter cake overnight
at room temperature in a desiccator.
7.4.3.3 Add 10 g anhydrous powdered sodium sulfate, mix
thoroughly, let sit in a closed container for one hour, mix again,
let sit for another hour, and mix again.
7.4.3.4 Place the sample and the filter paper into an
extraction thimble, and extract in a Soxhlet extraction apparatus
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charged with 200 ml toluene for 16 hours using a five cycle/hour
schedule.
NOTE: As an option, a Soxhlet/Dean Stark extractor system may
be used, with toluene as the solvent. No sodium sulfate
is added when using this option.
7.4.3.5 Cool and filter the toluene extract through a
glass fiber filter into a 500 ml round bottom flask. Rinse the
filter with 10 ml toluene. Add the rinse to the extract and
concentrate the combined toluene solutions to near dryness on a
rotary evaporator at 50°C. Proceed with Sec. 7.4.4.
7.4.4 Transfer the concentrate to a 125 ml separatory funnel using
15 ml hexane. Rinse the flask with two 5 ml portions of hexane and add
the rinses to the funnel. Shake the combined solutions in the separatory
funnel for two minutes with 50 ml of 5 percent sodium chloride solution,
discard the aqueous layer, and proceed with Sec. 7.5.
7.4.5 Aqueous samples
7.4.5.1 Allow the sample to come to ambient temperature,
then mark the water meniscus on the side of the 1 L sample bottle
for later determination of the exact sample volume. Add the
required acetone diluted sample fortification solution (Sec. 5.8).
7.4.5.2 When the sample is judged to contain 1 percent or
more solids, the sample must be filtered through a glass fiber
filter that has been rinsed with toluene. If the suspended solids
content is too great to filter through the 0.45 jum filter,
centrifuge the sample, decant, and then filter the aqueous phase.
NOTE: Paper mill effluent samples normally contain 0.02%-0.2%
solids, and would not require filtration. However, for
optimum analytical results, all paper mill effluent
samples should be filtered, the isolated solids and
filtrate extracted separately, and the extracts
recombined.
7.4.5.3 Combine the solids from the centrifuge bottle(s)
with the particulates on the filter and with the filter itself and
proceed with the Soxhlet extraction as specified in Sees. 7.4.6.1
through 7.4.6.4. Remove and invert the Snyder column and rinse it
down into the KD apparatus with two 1 ml portions of hexane.
7.4.5.4 Pour the aqueous filtrate into a 2 L separatory
funnel. Add 60 ml methylene chloride to the sample bottle, seal and
shake for 30 seconds to rinse the inner surface. Transfer the
solvent to the separatory funnel and extract the sample by shaking
the funnel for two minutes with periodic venting.
7.4.5.5 Allow the organic layer to separate from the water
phase for a minimum of 10 minutes. If the emulsion interface
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between layers is more than one third the volume of the solvent
layer, the analyst must employ mechanical techniques to complete the
phase separation (e.g., glass stirring rod).
7.4.5.6 Collect the methylene chloride into a KD apparatus
(mounted with a 10 ml concentrator tube) by passing the sample
extracts through a filter funnel packed with a glass wool plug and
5 g anhydrous sodium sulfate.
NOTE: As an option, a rotary evaporator may be used in place
of the KD apparatus for the concentration of the
extracts.
7.4.5.7 Repeat the extraction twice with fresh 60 ml
portions of methylene chloride. After the third extraction, rinse
the sodium sulfate with an additional 30 ml methylene chloride to
ensure quantitative transfer. Combine all extracts and the rinse in
the KD apparatus.
NOTE: A continuous liquid-liquid extractor may be used in
place of a separatory funnel when experience with a
sample from a given source indicates that a serious
emulsion problem will result or an emulsion is
encountered when using a separatory funnel. Add 60 ml
methylene chloride to the sample bottle, seal, and shake
for 30 seconds to rinse the inner surface. Transfer the
solvent to the extractor. Repeat the rinse of the
sample bottle with an additional 50 to 100 ml portion of
methylene chloride and add the rinse to the extractor.
Add 200 to 500 mL methylene chloride to the distilling
flask, add sufficient organic-free reagent water (Sec.
5.1) to ensure proper operation, and extract for
24 hours. Allow to cool, then detach the distilling
flask. Dry and concentrate the extract as described in
Sees. 7.4.5.6 and 7.4.5.8 through 7.4.5.10. Proceed
with Sec. 7.4.5.11.
7.4.5.8 Attach a Snyder column and concentrate the extract
on a water bath until the apparent volume of the liquid is 5 ml.
Remove the KD apparatus and allow it to drain and cool for at least
10 minutes.
7.4.5.9 Remove the Snyder column, add 50 ml hexane, add
the concentrate obtained from the Soxhlet extraction of the
suspended solids (Sec. 7.4.5.3), if applicable, re-attach the Snyder
column, and concentrate to approximately 5 ml. Add a new boiling
chip to the KD apparatus before proceeding with the second
concentration step.
7.4.5.10 Rinse the flask and the lower joint with two 5 ml
portions of hexane and combine the rinses with the extract to give
a final volume of about 15 ml.
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7.4.5.11 Determine the original sample volume by filling
the sample bottle to the mark with water and transferring the water
to a 1000 ml graduated cylinder. Record the sample volume to the
nearest 5 ml. Proceed with Sec. 7.5.
7.4.6 Soil/Sediment
7.4.6.1 Add 10 g anhydrous powdered sodium sulfate to the
sample portion (e.g., 10 g) and mix thoroughly with a stainless
steel spatula. After breaking up any lumps, place the soil/sodium
sulfate mixture in the Soxhlet apparatus on top of a glass wool plug
(the use of an extraction thimble is optional).
NOTE: As an option, a Soxhlet/Dean Stark extractor system may
be used, with toluene as the solvent. No sodium sulfate
is added when using this option.
7.4.6.2 Add 200 to 250 ml toluene to the Soxhlet apparatus
and reflux for 16 hours. The solvent must cycle completely through
the system five times per hour.
NOTE: If the dried sample is not of free flowing consistency,
more sodium sulfate must be added.
7.4.6.3 Cool and filter the extract through a glass fiber
filter into a 500 ml round bottom flask for evaporation of the
toluene. Rinse the filter with 10 ml of toluene, and concentrate
the combined fractions to near dryness on a rotary evaporator at
50°C. Remove the flask from the water bath and allow to cool for
5 minutes.
7.4.6.4 Transfer the residue to a 125 ml separatory
funnel, using 15 ml of hexane. Rinse the flask with two additional
portions of hexane, and add the rinses to the funnel. Proceed with
Sec. 7.5.
7.5 Cleanup
7.5.1 Partition
7.5.1.1 Partition the hexane extract against 40 ml of
concentrated sulfuric acid. Shake for two minutes. Remove and
discard the sulfuric acid layer (bottom). Repeat the acid washing
until no color is visible in the acid layer (perform a maximum of
four acid washings).
7.5.1.2 Omit this step for the fish sample extract.
Partition the extract against 40 ml of 5 percent (w/v) aqueous
sodium chloride. Shake for two minutes. Remove and discard the
aqueous layer (bottom).
7.5.1.3 Omit this step for the fish sample extract.
Partition the extract against 40 mL of 20 percent (w/v) aqueous
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potassium hydroxide (KOH). Shake for two minutes. Remove and
discard the aqueous layer (bottom). Repeat the base washing until
no color is visible in the bottom layer (perform a maximum of four
base washings). Strong base (KOH) is known to degrade certain
PCDDs/PCDFs, so contact time must be minimized.
7.5.1.4 Partition the extract against 40 ml of 5 percent
(w/v) aqueous sodium chloride. Shake for two minutes. Remove and
discard the aqueous layer (bottom). Dry the extract by pouring it
through a filter funnel containing anhydrous sodium sulfate on a
glass wool plug, and collect it in a 50 mi round bottom flask.
Rinse the funnel with the sodium sulfate with two 15 mL portions of
hexane, add the rinses to the 50 ml flask, and concentrate the
hexane solution to near dryness on a rotary evaporator (35°C water
bath), making sure all traces of toluene (when applicable) are
removed. (Use of blowdown with an inert gas to concentrate the
extract is also permitted.)
7.5.2 Silica/Alumina Column Cleanup
7.5.2.1 Pack a gravity column (glass, 30 cm x 10.5 mm),
fitted with a Teflon™ stopcock, with silica gel as follows: Insert
a glass wool plug into the bottom of the column. Place 1 g silica
gel in the column and tap the column gently to settle the silica
gel. Add 2 g sodium hydroxide-impregnated silica gel, 4 g sulfuric
acid-impregnated silica gel, and 2 g silica gel. Tap the column
gently after each addition. A small positive pressure (5 psi) of
clean nitrogen may be used if needed. Elute with 10 ml hexane and
close the stopcock just before exposure of the top layer of silica
gel to air. Discard the eluate. Check the column for channeling.
If channeling is observed, discard the column. Do not tap the
wetted column.
7.5.2.2 Pack a gravity column (glass, 300 mm x 10.5 mm),
fitted with a Teflon™ stopcock, with alumina as follows: Insert a
glass wool plug into the bottom of the column. Add a 4 g layer of
sodium sulfate. Add a 4 g layer of Woelm® Super 1 neutral alumina.
Tap the top of the column gently. Woelm® Super 1 neutral alumina
need not be activated or cleaned before use, but it should be stored
in a sealed desiccator. Add a 4 g layer of anhydrous sodium sulfate
to cover the alumina. Elute with 10 ml hexane and close the
stopcock just before exposure of the sodium sulfate layer to air.
Discard the eluate. Check the column for channeling. If channeling
is observed, discard the column. Do not tap a wetted column.
NOTE: Optionally, acidic alumina (Sec. 5.2.2) can be used in
place of neutral alumina.
7.5.2.3 Dissolve the residue from Sec. 7.5.1.4 in 2 ml
hexane and apply the hexane solution to the top of the silica gel
column. Rinse the flask with enough hexane (3-4 ml) to complete the
quantitative transfer of the sample to the surface of the silica
gel.
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7.5.2.4 Elute the silica gel column with 90 ml of hexane,
concentrate the eluate on a rotary evaporator (35°C water bath) to
approximately 1 mL, and apply the concentrate to the top of the
alumina column (Sec. 7.5.2.2). Rinse the rotary evaporator flask
twice with 2 mL of hexane, and add the rinses to the top of the
alumina column.
7.5.2.5 Add 20 ml hexane to the alumina column and elute
until the hexane level is just below the top of the sodium sulfate.
Do not discard the eluted hexane, but collect it in a separate flask
and store it for later use, as it may be useful in determining where
the labeled analytes are being lost if recoveries are not
satisfactory.
7.5.2.6 Add 15 ml of 60 percent methylene chloride in
hexane (v/v) to the alumina column and collect the eluate in a
conical shaped (15 ml) concentration tube. With a carefully
regulated stream of nitrogen, concentrate the 60 percent methylene
chloride/hexane fraction to about 2 mL.
7.5.3 Carbon Column Cleanup
7.5.3.1 Prepare an AX-21/Celite 545® column as follows:
Thoroughly mix 5.40 g active carbon AX-21 and 62.0 g Celite 545® to
produce an 8 percent (w/w) mixture. Activate the mixture at 130°C
for 6 hours and store it in a desiccator.
7.5.3.2 Cut off both ends of a 10 ml disposable
serological pipet to give a 10 cm long column. Fire polish both
ends and flare, if desired. Insert a glass wool plug at one end,
then pack the column with enough Celite 545® to form a 1 cm plug,
add 1 g of the AX-21/Celite 545® mixture, top with additional Celite
545® (enough for a 1 cm plug), and cap the packing with another
glass wool plug.
NOTE: Each new batch of AX-21/Celite 545® must be checked as
follows: Add 50 ^l of the continuing calibration
solution to 950 ^L hexane. Take this solution through
the carbon column cleanup step, concentrate to 50 /xL
and analyze. If the recovery of any of the analytes is
<80 percent, discard this batch of AX-21/Celite 545®.
7.5.3.3 Rinse the AX-21/Celite 545® column with 5 ml of
toluene, followed by 2 mL of 75:20:5 (v/v) methylene chloride/
methanol/toluene, 1 mL of 1:1 (v/v) cyclohexane/methylene chloride,
and 5 mL hexane. The flow rate should be less than 0.5 mL/min.
Discard the rinses. While the column is still wet with hexane, add
the sample concentrate (Sec. 7.5.2.6) to the top of the column.
Rinse the concentrator tube (which contained the sample concentrate)
twice with 1 mL hexane, and add the rinses to the top of the column.
7.5.3.4 Elute the column sequentially with two 2 mL
portions of hexane, 2 mL cyclohexane/methylene chloride (50:50,
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v/v), and 2 mi methylene chloride/methanol/toluene (75:20:5, v/v).
Combine these eluates; this combined fraction may be used as a check
on column efficiency.
7.5.3.5 Turn the column upside down and elute the
PCDD/PCDF fraction with 20 ml toluene. Verify that no carbon fines
are present in the eluate. If carbon fines are present in the
eluate, filter the eluate through a glass fiber filter (0.45 /xm) and
rinse the filter with 2 ml toluene. Add the rinse to the eluate.
7.5.3.6 Concentrate the toluene fraction to about 1 mL on
a rotary evaporator by using a water bath at 50°C. Carefully
transfer the concentrate into a 1 ml minivial and, again at elevated
temperature (50°C), reduce the volume to about 100 ^l using a stream
of nitrogen and a sand bath. Rinse the rotary evaporator flask
three times with 300 juL of a solution of 1 percent toluene in
methylene chloride, and add the rinses to the concentrate. Add
10 jiiL of the nonane recovery standard solution (Sec. 5.9) for soil,
sediment, water, fish, paper pulp and adipose tissue samples, or 50
;ul_ of the recovery standard solution for sludge, still bottom and
fly ash samples. Store the sample at room temperature in the dark.
7.6 Chromatographic/Mass Spectre-metric Conditions and Data Acquisition
Parameters
7.6.1 Gas Chromatograph
Column coating: DB-5
Film thickness: 0.25 jiiti
Column dimension: 60 m x 0.32 mm
Injector temperature: 270°C
Splitless valve time: 45 s
Interface temperature: Function of the final temperature
Temperature program:
Stage
Init.
Temp.
(°C)
Init.
Hold Time
(min)
Temp.
Ramp
(°C/min)
Final
Temp.
(°C)
Final
Hold
Time (min)
1 200 2 5 220 16
2 5 235 7
3 .5 330 5
Total time: 60 min
7.6.2 Mass Spectrometer
7.6.2.1 The mass spectrometer must be operated in a
selected ion monitoring (SIM) mode with a total cycle time
(including the voltage reset time) of one second or less (Sec.
7.6.3.1). At a minimum, the ions listed in Table 6 for each of the
five SIM descriptors must be monitored. Note that with the
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exception of the last descriptor (OCDD/OCDF), all descriptors
contain 10 ions. The selection (Table 6) of the molecular ions M
and M+2 for 13C-HxCOF and 13C-HpCDF rather than M+2 and M+4 (for
consistency) was made to eliminate, even under high-resolution mass
spectrometric conditions, interferences occurring in these two ion
channels for samples containing high levels of native HxCDDs and
HpCDDs. It is important to maintain the same set of ions for both
calibration and sample extract analyses. The selection of the lock-
mass ion is left to the performing laboratory.
NOTE: At the option of the analyst, the tetra- and
pentachlorinated dioxins and furans can be
combined into a single descriptor.
7.6.2.2 The recommended mass spectrometer tuning
conditions are based on the groups of monitored ions shown in Table
6. By using a PFK molecular leak, tune the instrument to meet the
minimum required resolving power of 10,000 (10 percent valley) at
m/z 304.9824 (PFK) or any other reference signal close to m/z
303.9016 (from TCDF). By using peak matching conditions and the
aforementioned PFK reference peak, verify that the exact mass of m/z
380.9760 (PFK) is within 5 ppm of the required value. Note that the
selection of the low- and high-mass ions must be such that they
provide the largest voltage jump performed in any of the five mass
descriptors (Table 6).
7.6.3 Data Acquisition
7.6.3.1 The total cycle time for data acquisition must be
< 1 second. The total cycle time includes the sum of all the dwell
times and voltage reset times.
7.6.3.2 Acquire SIM data for all the ions listed in the
five descriptors of Table 6.
7.7 Calibration
7.7.1 Initial Calibration - Initial calibration is required before
any samples are analyzed for PCDDs and PCDFs. Initial calibration is also
required if any routine calibration (Sec. 7.7.3) does not meet the
required criteria listed in Sec. 7.7.2.
7.7.1.1 All five high-resolution concentration calibration
solutions listed in Table 5 must be used for the initial
calibration.
7.7.1.2 Tune the instrument with PFK as described in
Sec. 7.6.2.2.
7.7.1.3 Inject 2 /xL of the GC column performance check
solution (Sec. 5.7) and acquire SIM mass spectral data as described
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earlier in Sec. 7.6.2. The total cycle time must be < 1 second. The
laboratory must not perform any further analysis until it is demon-
strated and documented that the criterion listed in Sec. 8.2.1 was
met.
7.7.1.4 By using the same GC (Sec. 7.6.1) and MS
(Sec. 7.6.2) conditions that produced acceptable results with the
column performance check solution, analyze a 2 nL portion of each
of the five concentration calibration solutions once with the
following mass spectrometer operating parameters.
7.7.1.4.1 The ratio of integrated ion current for the
ions appearing in Table 8 (homologous series quantitation
ions) must be within the indicated control limits (set for
each homologous series) for all unlabeled calibration
standards in Table 5.
7.7.1.4.2 The ratio of integrated ion current for the
ions belonging to the carbon-labeled internal and recovery
standards (Table 5) must be within the control limits
stipulated in Table 8.
NOTE: Sees. 7.7.1.4.1 and 7.7.1.4.2 require that 17 ion
ratios from Sec. 7.7.1.4.1 and 11 ion ratios from
Sec. 7.7.1.4.2 be within the specified control
limits simultaneously in one run. It is the
laboratory's responsibility to take corrective
action if the ion abundance ratios are outside
the limits.
7.7.1.4.3 For each selected ion current profile (SICP)
and for each GC signal corresponding to the elution of a
target analyte and of its labeled standards, the signal-to-
noise ratio (S/N) must be better than or equal to 2.5.
Measurement of S/N is required for any GC peak that has an
apparent S/N of less than 5:1. The result of the calculation
must appear on the SICP above the GC peak in question.
7.7.1.4.4 Referring to Table 9, calculate the 17
relative response factors (RF) for unlabeled target analytes
[RF(n); n = 1 to 17] relative to their appropriate internal
standards (Table 5) and the nine RFs for the labeled 13C12
internal standards [RF(m); m = 18 to 26)] relative to the two
recovery standards (Table 5) according to the following
formulae:
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Ax x Qi8 Ai8 x Qre
RFn = RFm =
Qx x Ais Qi8 x Are
where:
Ax = sum of the integrated ion abundances of the
quantitation ions (Tables 6 and 9) for
unlabeled PCDDs/PCDFs,
Ais = sum of the integrated ion abundances of the
quantitation ions (Tables 6 and 9) for the
labeled internal standards,
Ars = sum of the integrated ion abundances of the
quantitation ions (Tables 6 and 9) for the
labeled recovery standards,
Qis = quantity of the internal standard injected
(P9),
Qrs = quantity of the recovery standard injected
(pg), and
Qx = quantity of the unlabeled PCDD/PCDF analyte
injected (pg}.
The RFn and RFm are dimensionless quantities; the units
used to express Qis, Qre and Qx must be the same.
7.7.1.4.5 Calculate the RF and their respective
percent relative standard deviations (%RSD) for the five
calibration solutions:
5
RFn = 1/5 I RFnU)
Where n represents a particular PCDD/PCDF (2,3,7,8-
substituted) congener (n = 1 to 17; Table 9), and j is the
injection number (or calibration solution number; j = 1 to
5).
7.7.1.4.6 The relative response factors to be used for
the determination of the concentration of total isomers in a
homologous series (Table 9) are calculated as follows:
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7.7.1.4.6.1 For congeners that belong to a
homologous series containing only one isomer (e.g., OCDD
and OCDF) or only one 2,3,7,8-substituted isomer
(Table 4; TCDD, PeCDD, HpCDD, and_TCDF), the mean RF
used will be the same as the mean RF determined in Sec.
7.7.1.4.5.
NOTE: The calibration solutions do not contain
13C12-OCDF as an internal standard. This is
because a minimum resolving power of 12,000
is required to resolve the [M+6]+ ion of
13C12-OCDF from the [M+2]+ ion of OCDD (and
[M+4]+ from 13C12-OCDF with [M]+ of OCDD).
Therefore, the RF for OCDF is calculated
relative to 13C12-OCDD.
7.7.1.4.6.2 For congeners that belong to a
homologous series containing more than_ one
2,3,7,8-substituted isomer (Table 4), the mean RF used
for those homologous series will be the mean of the RFs
calculated for all individual 2,3,7,8-substituted
congeners using the equation below:
1 t
RFk = - I RFn
where:
k = 27 to 30 (Table 9), with 27 = PeCDF; 28 =
HxCDF; 29 = HxCDD; and 30 = HpCDF,
t = total number of 2,3,7,8-substituted isomers
present in the calibration solutions (Table
5) for each homologous series (e.g., two
for PeCDF, four for HxCDF, three for HxCDD,
two for HpCDF).
NOTE: Presumably, the HRGC/HRMS response factors
of different isomers within a homologous
series are different. However, this
analytical protocol will make the
assumption that the HRGC/HRMS responses of
all isomers in a homologous series that do
not have the 2,3,7,8-substitution pattern
are the same as the responses of one or
more of the 2,3,7,8-substituted isomer(s)
in that homologous series.
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7.7.1.4.7 Relative response factors [RFm] to be used
for the determination of the percent recoveries for the nine
internal standards are calculated as follows:
RFm
5
RFm = 1/5 I RF
where:
m = 18 to 26 (congener type) and j = 1 to 5
(injection number),
Aism = sum of the integrated ion abundances of the
quantitation ions (Tables 6 and 9) for a
given internal standard (m = 18 to 26),
Ars = sum of the integrated ion abundances of the
quantitation ions (Tables 6 and 9) for the
appropriate recovery standard (see Table 5,
footnotes),
Qre> Q,sm = quantities of, respectively, the recovery
standard (rs) and a particular internal
standard (is = m) injected (pg),
RFm = relative response factor of a particular
internal standard (m) relative to an
appropriate recovery standard, as
determined from one injection, and
RFm = calculated mean relative response factor of
a particular internal standard (m) relative
to an appropriate recovery standard, as
determined from the five initial calibra-
tion injections (j).
7.7.2 Criteria for Acceptable Calibration - The criteria listed
below for acceptable calibration must be met before sample analyses are
performed.
7.7.2.1 The percent relative standard deviations for the
mean response factors [RFn and RFm] from the 17 unlabeled standards
must not exceed ± 20 percent, and those for the nine labeled
reference compounds must not exceed + 30 percent.
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7.7.2.2 The S/N for the GC signals present in every SICP
(including the ones for the labeled standards) must be > 10.
7.7.2.3 The ion abundance ratios (Table 8) must be within
the specified control limits.
NOTE: If the criterion for acceptable calibration
listed in Sec. 7.7.2.1 is met, the analyte-
specific RF can then be considered independent of
the analyte quantity for the calibration concen-
tration range. The mean RFs will be used for all
calculations until the routine calibration
criteria (Sec. 7.7.4) are no longer met. At such
time, new mean RFs will be calculated from a new
set of injections of the calibration solutions.
7.7.3 Routine Calibration (Continuing Calibration Check) - Routine
calibrations must be performed at the beginning of a 12-hour period after
successful mass resolution and GC resolution performance checks. A
routine calibration is also required at the end of a 12-hour shift.
7.7.3.1 Inject 2 pi. of the concentration calibration
solution HRCC-3 standard (Table 5). By using the same HRGC/HRMS
conditions as used in Sees. 7.6.1 and 7.6.2, determine and document
an acceptable calibration as provided in Sec. 7.7.4.
7.7.4 Criteria for Acceptable Routine Calibration - The following
criteria must be met before further analysis is performed.
7.7.4.1 The measured RFs [RFn for the unlabeled standards]
obtained during the routine calibration runs must be within + 20
percent of the mean values established during the initial
calibration (Sec. 7.7.1.4.5).
7.7.4.2 The measured RFs [RFm for the labeled standards]
obtained during the routine calibration runs must be within
+ 30 percent of the mean values established during the initial
calibration (Sec. 7.7.1.4.7).
7.7.4.3 The ion abundance ratios (Table 8) must be within
the allowed control limits.
7.7.4.4 If either one of the criteria in Sees. 7.7.4.1 and
7.7.4.2 is not satisfied, repeat one more time. If these criteria
are still not satisfied, the entire routine calibration process
(Sec. 7.7.1) must be reviewed. It is realized that it may not
always be possible to achieve all RF criteria. For example, it has
occurred that the RF criteria for 13C12-HpCDD and 13C12-OCDD were not
met, however, the RF values for the corresponding unlabeled
compounds were routinely within the criteria established in the
method. In these cases, 24 of the 26 RF parameters have met the QC
criteria, and the data quality for the unlabeled HpCDD and OCDD
values were not compromised as a result of the calibration event.
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In these situations, the analyst must assess the effect on overall
data quality as required for the data quality objectives and decide
on appropriate action. Corrective action would be in order, for
example, if the compounds for which the RF criteria were not met
included both the unlabeled and the corresponding internal standard
compounds. If the ion abundance ratio criterion (Sec. 7.7.4.3) is
not satisfied, refer to the note in Sec. 7.7.1.4.2 for resolution.
NOTE: An initial calibration must be carried out
whenever the HRCC-3, the sample fortification, or
the recovery standard solution is replaced by a
new solution from a different lot.
7.8 Analysis
7.8.1 Remove the sample or blank extract (from Sec. 7.5.3.6) from
storage. With a stream of dry, purified nitrogen, reduce the extract
volume to 10 pi to 50 fj,l.
NOTE: A final volume of 20 pi or more should be used whenever
possible. A 10 /A final volume is difficult to handle, and
injection of 2 juL out of 10 juL leaves little sample for
confirmations and repeat injections, and for archiving.
7.8.2 Inject a 2 juL aliquot of the extract into the GC, operated
under the conditions that have been established to produce acceptable
results with the performance check solution (Sees. 7.6.1 and 7.6.2).
7.8.3 Acquire SIM data according to Sees. 7.6.2 and 7.6.3. Use the
same acquisition and mass spectrometer operating conditions previously
used to determine the relative response factors (Sees. 7.7.1.4.4 through
7.7.1.4.7). Ions characteristic of polychlorinated diphenyl ethers are
included in the descriptors listed in Table 6.
NOTE: The acquisition period must at least encompass the PCDD/PCDF
overall retention time window previously determined (Sec.
8.2.1.3). Selected ion current profiles (SICP) for the lock-
mass ions (one per mass descriptor) must also be recorded and
included in the data package. These SICPs must be true
representations of the evolution of the lock-mass ions
amplitudes during the HRGC/HRMS run (see Sec. 8.2.2 for the
proper level of reference compound to be metered into the ion
chamber.) The analyst may be required to monitor a PFK ion,
not as a lock-mass, but as a regular ion, in order to meet
this requirement. It is recommended to examine the lock-mass
ion SICP for obvious basic sensitivity and stability changes
of the instrument during the GC/MS run that could affect the
measurements [Tondeur et al., 1984, 1987]. Report any
discrepancies in the case narrative.
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7.8.4 Identification Criteria - For a gas chromatographic peak to
be identified as a PCDD or PCDF, it must meet all of the following
criteria:
7.8.4.1 Retention Times
7.8.4.1.1 For 2,3,7,8-substituted congeners, which
have an isotopically labeled internal or recovery standard
present in the sample extract (this represents a total of 10
congeners including OCDD; Tables 2 and 3), the retention time
(RRT; at maximum peak height) of the sample components (i.e.,
the two ions used for quantitation purposes listed in Table
6) must be within -1 to +3 seconds of the isotopically
labeled standard.
7.8.4.1.2 For 2,3,7,8-substituted compounds that do
not have an isotopically labeled internal standard present in
the sample extract (this represents a total of six congeners;
Table 3), the retention time must fall within 0.005 retention
time units of the relative retention times measured in the
routine calibration. Identification of OCDF is based on its
retention time relative to 13C12-OCDD as determined from the
daily routine calibration results.
7.8.4.1.3 For non-2,3,7,8-substituted compounds (tetra
through octa; totaling 119 congeners), the retention time
must be within the corresponding homologous retention time
windows established by analyzing the column performance check
solution (Sec. 8.1.3).
7.8.4.1.4 The ion current responses for both ions used
for quantitative purposes (e.g., for TCDDs: m/z 319.8965 and
321.8936) must reach maximum simultaneously ( + 2 seconds).
7.8.4.1.5 The ion current responses for both ions used
for the labeled standards (e.g., for 13C12-TCOD: m/z 331.9368
and m/z 333.9339) must reach maximum simultaneously (+ 2
seconds).
NOTE: The analyst is required to verify the presence of
1,2,8,9-TCDD and 1,3,4,6,8-PeCDF (Sec. 8.1.3) in
the SICPs of the daily performance checks.
Should either one compound be missing, the
analyst is required to take corrective action as
it may indicate a potential problem with the
ability to detect all the PCDDs/PCDFs.
7.8.4.2 Ion Abundance Ratios
7.8.4.2.1 The integrated ion currents for the two ions
used for quantitation purposes must have a ratio between the
lower and upper limits established for the homologous series
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to which the peak is assigned. See Sees. 7.7.1.4.1 and
7.7.1.4.2 and Table 8 for details.
7.8.4.3 Signal-to-Noise Ratio
7.8.4.3.1 All ion current intensities must be > 2.5
times noise level for positive identification of a PCDD/PCDF
compound or a group of coeluting isomers. Figure 6 describes
the procedure to be followed for the determination of the
S/N.
7.8.4.4 Polychlorinated Diphenyl Ether Interferences
7.8.4.4.1 In addition to the above criteria, the
identification of a GC peak as a PCDF can only be made if no
signal having a S/N > 2.5 is detected at the same retention
time (± 2 seconds) in the corresponding polychlorinated
diphenyl ether (PCDPE, Table 6) channel.
7.9 Calculations
7.9.1 For gas chromatographic peaks that have met the criteria
outlined in Sees. 7.8.4.1.1 through 7.8.4.3.1, calculate the concentration
of the PCDD or PCDF compounds using the formula:
Qis
Ais x W x RFn
where:
Cx = concentration of unlabeled PCDD/PCDF congeners (or group
of coeluting isomers within an homologous series) in
pg/g,
Ax = sum of the integrated ion abundances of the quantitation
ions (Table 6) for unlabeled PCDDs/PCDFs,
Ais = sum of the integrated ion abundances of the quantitation
ions (Table 6) for the labeled internal standards,
Qjs = quantity, in pg, of the internal standard added to the
sample before extraction,
W = weight, in g, of the sample (solid or organic liquid),
or volume in ml of an aqueous sample, and
RFn = calculated mean relative response factor for the analyte
[RFn with n = 1 to 17; Sec. 7.7.1.4.5].
If the analyte is identified as one of the 2,3,7,8-substituted PCDDs
or PCDFs, RFn is the value calculated using the equation in Sec. 7.7.1.4.5.
However, if it is a non-2,3,7,8-substituted congener, the RF(k) value is
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the one calculated using the equation in Sec. 7.7.1.4.6.2. [RFk k = 27
to 30].
7.9.2 Calculate the percent recovery of the nine internal standards
measured in the sample extract, using the formula:
Ais x Qre
Internal standard percent recovery = —— x 100
Qis x Are x RFm
where:
Ais = sum of the integrated ion abundances of the quantitation
ions (Table 6) for the labeled internal standard,
Ars = sum of the integrated ion abundances of the quantitation
ions (Table 6) for the labeled recovery standard; the
selection of the recovery standard depends on the type
of congeners (see Table 5, footnotes),
Qis = quantity, in pg, of the internal standard added to the
sample before extraction,
Qrs = quantity, in pg, of the recovery standard added to the
cleaned-up sample residue before HRGC/HRMS analysis, and
RFm = calculated mean relative response factor for the labeled
internal standard relative to the appropriate (see Table
5, footnotes) recovery standard. This represents the
mean obtained in Sec. 7.7.1.4.7 [RFm with m = 18 to 26].
NOTE: For human adipose tissue, adjust the percent recoveries by
adding 1 percent to the calculated value to compensate for
the 1 percent of the extract diverted for the lipid
determination.
7.9.3 If the concentration in the final extract of any of the
fifteen 2,3,7,8-substituted PCDD/PCDF compounds (Table 3) exceeds the
upper method calibration limits (MCL) listed in Table 1 (e.g., 200 pg/juL
for TCDD in soil), the linear range of response versus concentration may
have been exceeded, and a second analysis of the sample (using a one tenth
aliquot) should be undertaken. The volumes of the internal and recovery
standard solutions should remain the same as described for the sample
preparation (Sees. 7.1 to 7.9.3). For the other congeners (including
OCDD), however, report the measured concentration and indicate that the
value exceeds the MCL.
7.9.3.1 If a smaller sample size would not be
representative of the entire sample, one of the following options is
recommended:
(1) Re-extract an additional aliquot of sufficient size to insure
that it is representative of the entire sample. Spike it with a
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higher concentration of internal standard. Prior to GC/MS analysis,
dilute the sample so that it has a concentration of internal
standard equivalent to that present in the calibration standard.
Then, analyze the diluted extract.
(2) Re-extract an additional aliquot of sufficient size to insure
that it is representative of the entire sample. Spike it with a
higher concentration of internal standard. Immediately following
extraction, transfer the sample to a volumetric flask and dilute to
known volume. Remove an appropriate aliquot and proceed with
cleanup and analysis.
(3) Use the original analysis data to quantitate the internal
standard recoveries. Respike the original extract (note that no
additional cleanup is necessary) with 100 times the usual quantity
of internal standards. Dilute the re-spiked extract by a factor of
100. Reanalyze the diluted sample using the internal standard
recoveries calculated from the initial analysis to correct the
results for losses during isolation and cleanup.
7.9.4 The total concentration for each homologous series of PCDD and
PCDF is calculated by summing up the concentrations of all positively
identified isomers of each homologous series. Therefore, the total should
also include the 2,3,7,8-substituted congeners. The total number of GC
signals included in the homologous total concentration value must be
specified in the report. If an isomer is not detected, use zero (0) in
this calculation.
7.9.5 Sample Specific Estimated Detection Limit - The sample
specific estimated detection limit (EDL) is the concentration of a given
analyte required to produce a signal with a peak height of at least 2.5
times the background signal level. An EDL is calculated for each
2,3,7,8-substituted congener that is not identified, regardless of whether
or not other non-2,3,7,8-substituted isomers are present. Two methods of
calculation can be used, as follows, depending on the type of response
produced during the analysis of a particular sample.
7.9.5.1 Samples giving a response for both quantitation
ions (Tables 6 and 9) that is less than 2.5 times the background
level.
7.9.5.1.1 Use the expression for EDL (specific
2,3,7,8-substituted PCDD/PCDF) below to calculate an EDL for
each absent 2,3,7,8-substituted PCDD/PCDF (i.e., S/N < 2.5).
The background level is determined by measuring the range of
the noise (peak to peak) for the two quantitation ions (Table
6) of a particular 2,3,7,8-substituted isomer within an
homologous series, in the region of the SICP trace
corresponding to the elution of the internal standard (if the
congener possesses an internal standard) or in the region of
the SICP where the congener is expected to elute by
comparison with the routine calibration data (for those
congeners that do not have a 13C-labeled standard),
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multiplying that noise height by 2.5, and relating the
product to an estimated concentration that would produce that
peak height.
Use the formula:
2.5 x Hx x Qis
H=. x W x
•is
EDL (specific 2,3,7,8-subst. PCDD/PCDF) =
where:
EDL = estimated detection limit for homologous
2,3,7,8-substituted PCDDs/PCDFs.
Hx = sum of the height of the noise level for each
quantitation ion (Table 6) for the unlabeled
PCDDs/PCDFs, measured as shown in Figure 6.
His = sum of the height of the noise level for each
quantitation ion (Table 6) for the labeled
internal standard, measured as shown in Figure 6.
W, RFn, and Qis retain the same meanings as defined in
Sec. 7.9.1.
7.9.5.2 Samples characterized by a response above the
background level with a S/N of at least 2.5 for both quantitation
ions (Tables 6 and 9) .
7.9.5.2.1 When the response of a signal having the
same retention time as a 2,3,7,8-substituted congener has a
S/N in excess of 2.5 and does not meet any of the other
qualitative identification criteria listed in Sec. 7.8.4,
calculate the "Estimated Maximum Possible Concentration"
(EMPC) according to the expression shown in Sec. 7.9.1,
except that Ax in Sec. 7.9.1 should represent the sum of the
area under the smaller peak and of the other peak area
calculated using the theoretical chlorine isotope ratio.
7.9.6 The relative percent difference (RPD) of any duplicate sample
results are calculated as follows:
RPD = - x 100
(S, + S2 ) / 2
S, and S2 represent sample and duplicate sample results.
7.9.7 The 2,3,7,8-TCDD toxicity equivalents (TE) of PCDDs and PCDFs
present in the sample are calculated, if requested by the data user,
according to the method recommended by the Chlorinated Dioxins Workgroup
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(CDWG) of the EPA and the Center for Disease Control (CDC). This method
assigns a 2,3,7,8-TCDD toxicity equivalency factor (TEF) to each of the
fifteen 2,3,7,8-substituted PCDDs and PCDFs (Table 3) and to OCDD and
OCDF, as shown in Table 10. The 2,3,7,8-TCDD equivalent of the PCDDs and
PCDFs present in the sample is calculated by summing the TEF times their
concentration for each of the compounds or groups of compounds listed in
Table 10. The exclusion of other homologous series such as mono-, di-,
and tri- chlorinated dibenzodioxins and dibenzofurans does not mean that
they are non-toxic. However, their toxicity, as known at this time, is
much lower than the toxicity of the compounds listed in Table 10. The
above procedure for calculating the 2,3,7,8-TCDD toxicity equivalents is
not claimed by the CDWG to be based on a thoroughly established scientific
foundation. The procedure, rather, represents a "consensus recommendation
on science policy". Since the procedure may be changed in the future,
reporting requirements for PCDD and PCDF data would still include the
reporting of the analyte concentrations of the PCDD/PCDF congener as
calculated in Sees. 7.9.1 and 7.9.4.
7.9.7.1 Two GC Column TEF Determination
7.9.7.1.1 The concentration of 2,3,7,8-TCDD (see note
below), is calculated from the analysis of the sample extract
on the 60 m DB-5 fused silica capillary column. The
experimental conditions remain the same as the conditions
described previously in Sec. 7.8, and the calculations are
performed as outlined in Sec. 7.9. The chromatographic
separation between the 2,3,7,8-TCDD and its close eluters
(1,2,3,7/1,2,3,8-TCDD and 1,2,3,9-TCDD) must be equal or less
than 25 percent valley.
7.9.7.1.2 The concentration of the 2,3,7,8-TCDF is
obtained from the analysis of the sample extract on the 30 m
DB-225 fused silica capillary column. However, the GC/MS
conditions must be altered so that: (1) only the first three
descriptors (i.e., tetra-, penta-, and hexachlorinated
congeners) of Table 6 are used; and (2) the switching time
between descriptor 2 (pentachlorinated congeners) and
descriptor 3 (hexachlorinated congeners) takes place
following the elution of 13Cl2-l,2,3,7,8-PeCDD. The
concentration calculations are performed as outlined in Sec.
7.9. The chromatographic separation between the 2,3,7,8-TCDF
and its close eluters (2,3,4,7-TCDF and 1,2,3,9-TCDF) must be
equal or less than 25 percent valley.
NOTE: The confirmation and quantitation of 2,3,7,8-TCDD
(Sec. 7.9.7.1.1) may be accomplished on the SP-
2330 GC column instead of the DB-5 column,
provided the criteria listed in Sec. 8.2.1 are
met and the requirements described in Sec.
8.3.2 are followed.
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7.9.7.1.3 For a gas chromatographic peak to be
identified as a 2,3,7,8-substituted PCDD/PCDF congener, it
must meet the ion abundance and signal-to-noise ratio
criteria listed in Sees. 7.8.4.2 and 7.8.4.3, respectively.
In addition, the retention time identification criterion
described in Sec. 7.8.4.1.1 applies here for congeners for
which a carbon-labeled analogue is available in the sample
extract. However, the relative retention time (RRT) of the
2,3,7,8-substituted congeners for which no carbon-labeled
analogues are available must fall within 0.006 units of the
carbon-labeled standard RRT. Experimentally, this is
accomplished by using the attributions described in Table 11
and the results from the routine calibration run on the
SP-2330 column.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for specific quality control (QC) procedures.
Quality control to validate sample extraction is covered in Method 3500. If
extract cleanup was performed, follow the QC in Method 3600 and in the specific
cleanup method.
8.2 System Performance Criteria - System performance criteria are
presented below. The laboratory may use the recommended GC column described in
Sec. 4.2. It must be documented that all applicable system performance criteria
(specified in Sees. 8.2.1 and 8.2.2) were met before analysis of any sample is
performed. Sec. 7.6.1 provides recommended GC conditions that can be used to
satisfy the required criteria. Figure 3 provides a typical 12-hour analysis
sequence, whereby the response factors and mass spectrometer resolving power
checks must be performed at the beginning and the end of each 12-hour period of
operation. A GC column performance check is only required at the beginning of
each 12-hour period during which samples are analyzed. An HRGC/HRMS method blank
run is required between a calibration run and the first sample run. The same
method blank extract may thus be analyzed more than once if the number of samples
within a batch requires more than 12 hours of analyses.
8.2.1 GC Column Performance
8.2.1.1 Inject 2 /ut- (Sec. 4.1.1) of the column performance
check solution (Sec. 5.7) and acquire selected ion monitoring (SIM)
data as described in Sec. 7.6.2 within a total cycle time of < 1
second (Sec. 7.6.3.1).
8.2.1.2 The chromatographic separation between 2,3,7,8-
TCDD and the peaks representing any other unlabeled TCDD isomers
must be resolved with a valley of < 25 percent (Figure 4), where:
Valley percent = (x/y) (100)
x = measured as in Figure 4 from the 2,3,7,8-closest TCDD
eluting isomer, and
y = the peak height of 2,3,7,8-TCDD.
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It is the responsibility of the laboratory to verify the
conditions suitable for the appropriate resolution of 2,3,7,8-TCDD
from all other TCDD isomers. The GC column performance check
solution also contains the known first and last PCDD/PCDF eluters
under the conditions specified in this protocol. Their retention
times are used to determine the eight homologue retention time
windows that are used for qualitative (Sec. 7.8.4.1) and
quantitative purposes. All peaks (that includes 13C12-2,3,7,8-TCDD)
should be labeled and identified on the chromatograms. Furthermore,
all first eluters of a homologous series should be labeled with the
letter F, and all last eluters of a homologous series should be
labeled with the letter L (Figure 4 shows an example of peak
labeling for TCDD isomers). Any individual selected ion current
profile (SICP) (for the tetras, this would be the SICP for m/z 322
and m/z 304) or the reconstructed homologue ion current (for the
tetras, this would correspond to m/z 320 + m/z 322 + m/z 304 + m/z
306) constitutes an acceptable form of data presentation. An SICP
for the labeled compounds (e.g., m/z 334 for labeled TCDD) is also
required.
8.2.1.3 The retention times for the switching of SIM ions
characteristic of one homologous series to the next higher
homologous series must be indicated in the SICP. Accurate switching
at the appropriate times is absolutely necessary for accurate
monitoring of these compounds. -Allowable tolerance on the daily
verification with the GC performance check solution should be better
than 10 seconds for the absolute retention times of all the
components of the mixture. Particular caution should be exercised
for the switching time between the last tetrachlorinated congener
(i.e., 1,2,8,9-TCDD) and the first pentachlorinated congener (i.e.,
1,3,4,6,8-PeCDF), as these two compounds elute within 15 seconds of
each other on the 60 m DB-5 column. A laboratory with a GC/MS
system that is not capable of detecting both congeners (1,2,8,9-TCDD
and 1,3,4,6,8-PeCDF) within one analysis must take corrective
action. If the recommended column is not used, then the first and
last eluting isomer of each homologue must be determined
experimentally on the column which is used, and the appropriate
isomers must then be used for window definition and switching times.
8.2.2 Mass Spectrometer Performance
8.2.2.1 The mass spectrometer must be operated in the
electron ionization mode. A static resolving power of at least
10,000 (10 percent valley definition) must be demonstrated at
appropriate masses before any analysis is performed (Sec. 7.8).
Static resolving power checks must be performed at the beginning and
at the end of each 12 hour period of operation. However, it is
recommended that a check of the static resolution be made and
documented before and after each analysis. Corrective action must
be implemented whenever the resolving power does not meet the
requirement.
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8.2.2.2 Chromatography time for PCDDs and PCDFs exceeds
the long term mass stability of the mass spectrometer. Because the
instrument is operated in the high-resolution mode, mass drifts of
a few ppm (e.g., 5 ppm in mass) can have serious adverse effects on
instrument performance. Therefore, a mass drift correction is
mandatory. To that effect, it is recommended to select a lock-mass
ion from the reference compound (PFK is recommended) used for tuning
the mass spectrometer. The selection of the lock-mass ion is
dependent on the masses of the ions monitored within each
descriptor. Table 6 offers some suggestions for the lock-mass ions.
However, an acceptable lock-mass ion at any mass between the
lightest and heaviest ion in each descriptor can be used to monitor
and correct mass drifts. The level of the reference compound (PFK)
metered into the ion chamber during HRGC/HRMS analyses should be
adjusted so that the amplitude of the most intense selected lock-
mass ion signal (regardless of the descriptor number) does not
exceed 10 percent of the full scale deflection for a given set of
detector parameters. Under those conditions, sensitivity changes
that might occur during the analysis can be more effectively
monitored.
NOTE: Excessive PFK (or any other reference substance) may cause
noise problems and contamination of the ion source resulting
in an increase in downtime for source cleaning.
8.2.2.3 Documentation of the instrument resolving power
must then be accomplished by recording the peak profile of the high-
mass reference signal (m/z 380.9760) obtained during the above peak
matching experiment by using the low-mass PFK ion at m/z 304.9824 as
a reference. The minimum resolving power of 10,000 must be
demonstrated on the high-mass ion while it is transmitted at a lower
accelerating voltage than the low-mass reference ion, which is
transmitted at full sensitivity. The format of the peak profile
representation (Figure 5) must allow manual determination of the
resolution, i.e., the horizontal axis must be a calibrated mass
scale (amu or ppm per division). The result of the peak width
measurement (performed at 5 percent of the maximum, which
corresponds to the 10 percent valley definition) must appear on the
hard copy and cannot exceed 100 ppm at m/z 380.9760 (or 0.038 amu at
that particular mass).
8.3 Quality Control Samples
8.3.1 Performance Evaluation Samples - Included among the samples
in all batches may be samples (blind or double blind) containing known
amounts of unlabeled 2,3,7,8-substituted PCDDs/PCDFs or other PCDD/PCDF
congeners.
8.3.2 Performance Check Solutions
8.3.2.1 At the beginning of each 12-hour period during
which samples are to be analyzed, an aliquot of the 1) GC column
performance check solution and 2) high-resolution concentration
calibration solution No. 3 (HRCC-3; see Table 5) shall be analyzed
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to demonstrate adequate GC resolution and sensitivity, response
factor reproducibility, and mass range calibration, and to establish
the PCDD/PCDF retention time windows. A mass resolution check shall
also be performed to demonstrate adequate mass resolution using an
appropriate reference compound (PFK is recommended). If the
required criteria are not met, remedial action must be taken before
any samples are analyzed.
8.3.2.2 To validate positive sample data, the routine or
continuing calibration (HRCC-3; Table 5) and the mass resolution
check must be performed also at the end of each 12-hour period
during which samples are analyzed. Furthermore, an HRGC/HRMS method
blank run must be recorded following a calibration run and the first
sample run.
8.3.2.2.1 If the laboratory operates only during one
period (shift) each day of 12 hours or less, the GC
performance check solution must be analyzed only once (at the
beginning of the period) to validate the data acquired during
the period. However, the mass resolution and continuing
calibration checks must be performed at the beginning as well
as at the end of the period.
8.3.2.2.2 If the laboratory operates during
consecutive 12-hour periods (shifts), analysis of the GC
performance check solution must be performed at the beginning
of each 12-hour period. The mass resolution and continuing
calibration checks from the previous period can be used for
the beginning of the next period.
8.3.2.3 Results of at least one analysis of the GC column
performance check solution and of two mass resolution and continuing
calibration checks must be reported with the sample data collected
during a 12 hour period.
8.3.2.4 Deviations from criteria specified for the GC
performance check or for the mass resolution check invalidate all
positive sample data collected between analyses of the performance
check solution, and the extracts from those positive samples shall
be reanalyzed.
If the routine calibration run fails at the beginning of a 12
hour shift, the instructions in Sec. 7.7.4.4 must be followed. If
the continuing calibration check performed at the end of a 12 hour
period fails by no more than 25 percent RPD for the 17 unlabeled
compounds and 3_5 percent RPD for the 9 labeled reference compounds,
use the mean RFs from the two daily routine calibration runs to
compute the analyte concentrations, instead of the RFs obtained from
the initial calibration. A new initial calibration (new RFs) is
required immediately (within two hours) following the analysis of
the samples, whenever the RPD from the end-of-shift routine
calibration exceeds 25 percent or 35 percent, respectively. Failure
to perform a new initial calibration immediately following the
analysis of the samples will automatically require reanalysis of all
8290 - 40 Revision 0
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positive sample extracts analyzed before the failed end-of-shift
continuing calibration check.
8.3.3 The GC column performance check mixture, high-resolution
concentration calibration solutions, and the sample fortification
solutions may be obtained from the EMSL-CIN. However, if not available
from the EMSL-CIN, standards can be obtained from other sources, and
solutions can be prepared in the laboratory. Concentrations of all
solutions containing 2,3,7,8-substituted PCDDs/PCDFs, which are not
obtained from the EMSL-CIN, must be verified by comparison with the EPA
standard solutions that are available from the EMSL-CIN.
8.3.4 Field Blanks - Each batch of samples usually contains a field
blank sample of uncontaminated soil, sediment or water that is to be
fortified before analysis according to Sec. 8.3.4.1. In addition to this
field blank, a batch of samples may include a rinsate, which is a portion
of the solvent (usually trichloroethylene) that was used to rinse sampling
equipment. The rinsate is analyzed to assure that the samples were not
contaminated by the sampling equipment.
8.3.4.1 Fortified Field Blank
8.3.4.1.1 Weigh a 10 g portion or use 1 L (for aqueous
samples) of the specified field blank sample and add 100 juL
of the solution containing the nine internal standards
(Table 2) diluted with 1.0 mL acetone (Sec. 7.1).
8.3.4.1.2 Extract by using the procedures beginning
in Sees. 7.4.5 or 7.4.6, as applicable, add 10 /uL of the
recovery standard solution (Sec. 7.5.3.6) and analyze a 2 juL
aliquot of the concentrated extract.
8.3.4.1.3 Calculate the concentration (Sec. 7.9.1) of
2,3,7,8-substituted PCDDs/PCDFs and the percent recovery of
the internal standards (Sec. 7.9.2).
8.3.4.1.4 Extract and analyze a new simulated
fortified field blank whenever new lots of solvents or
reagents are used for sample extraction or for column
chromatographic procedures.
8.3.4.2 Rinsate Sample
8.3.4.2.1 The rinsate sample must be fortified like
a regular sample.
8.3.4.2.2 Take a 100 mL (± 0.5 mL) portion of the
sampling equipment rinse solvent (rinsate sample), filter, if
necessary, and add 100 fj,L of the solution containing the nine
internal standards (Table 2).
8.3.4.2.3 Using a KD apparatus, concentrate to
approximately 5 mL.
8290 - 41 Revision 0
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NOTE: As an option, a rotary evaporator may be used in
place of the KD apparatus for the concentration
of the rinsate.
8.3.4.2.4 Transfer the 5 ml concentrate from the KD
concentrator tube in 1 ml portions to a 1 ml minivial,
reducing the volume in the minivial as necessary with a
gentle stream of dry nitrogen.
8.3.4.2.5 Rinse the KD concentrator tube with two
0.5 ml portions of hexane and transfer the rinses to the 1 ml
minivial. Blow down with dry nitrogen as necessary.
8.3.4.2.6 Just before analysis, add 10 /zL recovery
standard solution (Table 2) and reduce the volume to its
final volume, as necessary (Sec. 7.8.1). No column
chromatography is required.
8.3.4.2.7 Analyze an aliquot following the same
procedures used to analyze samples.
8.3.4.2.8 Report percent recovery of the internal
standard and the presence of any PCDD/PCDF compounds in /ug/L
of rinsate solvent.
8.3.5 Duplicate Analyses
8.3.5.1 In each batch of samples, locate the sample
specified for duplicate analysis, and analyze a second 10 g soil or
sediment sample portion or 1 L water sample, or an appropriate
amount of the type of matrix under consideration.
8.3.5.1.1 The results of the laboratory duplicates
(percent recovery and concentrations of 2,3,7,8-substituted
PCDD/PCDF compounds) should agree within 25 percent relative
difference (difference expressed as percentage of the mean).
Report all results.
8.3.5.1.2 Recommended actions to help locate problems:
8.3.5.1.2.1 Verify satisfactory instrument
performance (Sees. 8.2 and 8.3).
8.3.5.1.2.2 If possible, verify that no error was
made while weighing the sample portions.
8.3.5.1.2.3 Review the analytical procedures with
the performing laboratory personnel.
8.3.6 Matrix Spike and Matrix Spike Duplicate
8.3.6.1 Locate the sample for the MS and MSD analyses (the
sample may be labeled "double volume").
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8.3.6.2 Add an appropriate volume of the matrix spike
fortification solution (Sec. 5.10) and of the sample fortification
solution (Sec. 5.8), adjusting the fortification level as specified
in Table 1 under IS Spiking Levels.
8.3.6.3 Analyze the MS and MSD samples as described in
Sec. 7.
8.3.6.4 The results obtained from the MS and MSD samples
(concentrations of 2,3,7,8-substituted PCDDs/PCDFs) should agree
within 20 percent relative difference.
8.4 Percent Recovery of the Internal Standards - For each sample, method
blank and rinsate, calculate the percent recovery (Sec. 7.9.2). The percent
recovery should be between 40 percent and 135 percent for all 2,3,7,8-substituted
internal standards.
NOTE: A low or high percent recovery for a blank does not require
discarding the analytical data but it may indicate a
potential problem with future analytical data.
8.5 Identification Criteria
8.5.1 If either one of the identification criteria appearing in
Sees. 7.8.4.1.1 through 7.8.4.1.4 is not met for an homologous series, it
is reported that the sample does not contain unlabeled 2,3,7,8-substituted
PCDD/PCDF isomers for that homologous series at the calculated detection
limit (Sec. 7.9.5)
8.5.2 If the first initial identification criteria (Sees. 7.8.4.1.1
through 7.8.4.1.4) are met, but the criteria appearing in Sees. 7.8.4.1.5
and 7.8.4.2.1 are not met, that sample is presumed to contain interfering
contaminants. This must be noted on the analytical report form, and the
sample should be rerun or the extract reanalyzed.
8.6 Unused portions of samples and sample extracts should be preserved
for six months after sample receipt to allow further analyses.
8.7 Reuse of glassware is to be minimized to avoid the risk of
contamination.
9.0 METHOD PERFORMANCE
9.1 Data are currently not available.
10.0 REFERENCES
1. "Control of Interferences in the Analysis of Human Adipose Tissue for
2,3,7,8-Tetrachlorodibenzo-p-dioxin". D. G. Patterson, J.S. Holler, D.F.
Grote, L.R. Alexander, C.R. Lapeza, R.C. O'Connor and J.A. Liddle.
Environ. Toxicol. Chem. 5, 355-360 (1986).
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2. "Method 8290: Analytical Procedures and Quality Assurance for Multimedia
Analysis of Polychlorinated Dibenzo-p-Dioxins and Dibenzofurans by High-
Resolution Gas Chromatography/High-Resolution Mass Spectrometry". Y.
Tondeur and W.F. Beckert. U.S. Environmental Protection Agency,
Environmental Monitoring Systems Laboratory, Las Vegas, NV.
3. "Carcinogens - Working with Carcinogens", Department of Health, Education,
and Welfare, Public Health Service, Center for Disease Control. National
Institute for Occupational Safety and Health. Publication No. 77-206,
August 1977.
4. "OSHA Safety and Health Standards, General Industry", (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206 (revised January
1976).
5. "Safety in Academic Chemistry Laboratories", American Chemical Society
Publication, Committee on Chemical Safety (3rd Edition, 1979.)
6. "Hybrid HRGC/MS/MS Method for the Characterization of Tetrachlorinated
Dibenzo-p-dioxins in Environmental Samples." Y. Tondeur, W.J. Niederhut,
S.R. Missler, and J.E. Campana, Mass Spectrom. 14, 449-456 (1987).
7. USEPA National Dioxin Study - Phase II, "Analytical Procedures and Quality
Assurance Plan for the Determination of PCDD/PCDF in Fish", EPA-Duluth,
October 26, 1987.
11.0 SAFETY
11.1 The following safety practices are excerpts from EPA Method 613,
Sec. 4 (July 1982 version) and amended for use in conjunction with this method.
The 2,3,7,8-TCDD isomer has been found to be acnegenic, carcinogenic, and
teratogenic in laboratory animal studies. Other PCDDs and PCDFs containing
chlorine atoms in positions 2,3,7,8 are known to have toxicities comparable to
that of 2,3,7,8-TCDD. The analyst should note that finely divided dry soils
contaminated with PCDDs and PCDFs are particularly hazardous because of the
potential for inhalation and ingestion. It is recommended that such samples be
processed in a confined environment, such as a hood or a glove box. Laboratory
personnel handling these types of samples should wear masks fitted with charcoal
filters to prevent inhalation of dust.
11.2 The toxicity or carcinogenicity of each reagent used in this method
is not precisely defined; however, each chemical compound should be treated as
a potential health hazard. From this viewpoint, exposure to these chemicals must
be kept to a minimum. The laboratory is responsible for maintaining a current
awareness file of OSHA regulations regarding the safe handling of the chemicals
specified in this method. A reference file of material safety data sheets should
be made available to all personnel involved in the chemical analysis of samples
suspected to contain PCDDs and/or PCDFs. Additional references to laboratory
safety are given in references 3, 4 and 5.
11.3 Each laboratory must develop a strict safety program for the handling
of PCDDs and PCDFs. The laboratory practices listed below are recommended.
8290 - 44 Revision 0
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11.3.1 Contamination of the laboratory will be minimized by
conducting most of the manipulations in a hood.
11.3.2 The effluents of sample splitters for the gas
chromatograph and roughing pumps on the HRGC/HRMS system should pass
through either a column of activated charcoal or be bubbled through a trap
containing oil or high boiling alcohols.
11.3.3 Liquid waste should be dissolved in methanol or ethanol
and irradiated with ultraviolet light at a wavelength less than 290 nm for
several days (use F 40 BL lamps, or equivalent). Using this analytical
method, analyze the irradiated liquid wastes and dispose of the solutions
when 2,3,7,8-TCDD and -TCDF congeners can no longer be detected.
11.4 The following precautions were issued by Dow Chemical U.S.A. (revised
11/78) for safe handling of 2,3,7,8-TCDD in the laboratory and amended for use
in conjunction with this method.
11.4.1 The following statements on safe handling are as complete
as possible on the basis of available toxicological information. The
precautions for safe handling and use are necessarily general in nature
since detailed, specific recommendations can be made only for the
particular exposure and circumstances of each individual use. Assistance
in evaluating the health hazards of particular plant conditions may be
obtained from certain consulting laboratories and from State Departments
of Health or of Labor, many of which have an industrial health service.
The 2,3,7,8-TCDD isomer is extremely toxic to certain kinds of laboratory
animals. However, it has been handled for years without injury in
analytical and biological laboratories. Many techniques used in handling
radioactive and infectious materials are applicable to 2,3,7,8-TCDD.
11.4.1.1 Protective Equipment: Throw away plastic gloves,
apron or lab coat, safety glasses and laboratory hood adequate for
radioactive work. However, PVC gloves should not be used.
11.4.1.2 Training: Workers must be trained in the proper
method of removing contaminated gloves and clothing without
contacting the exterior surfaces.
11.4.1.3 Personal Hygiene: Thorough washing of hands and
forearms after each manipulation and before breaks (coffee, lunch,
and shift).
11.4.1.4 Confinement: Isolated work area, posted with
signs, segregated glassware and tools, plastic backed absorbent
paper on benchtops.
11.4.1.5 Waste: Good technique includes minimizing
contaminated waste. Plastic bag liners should be used in waste
cans.
11.4.1.6 Disposal of Hazardous Wastes: Refer to the
November 7, 1986 issue of the Federal Register on Land Ban Rulings
for details concerning the handling of dioxin containing wastes.
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11.4.1.7 Decontamination: Personnel - apply a mild soap
with plenty of scrubbing action. Glassware, tools and surfaces -
Chlorothene NU Solvent (Trademark of the Dow Chemical Company) is
the least toxic solvent shown to be effective. Satisfactory
cleaning may be accomplished by rinsing with Chlorothene, then
washing with a .detergent and water. Dish water may be disposed to
the sewer after percolation through a charcoal bed filter. It is
prudent to minimize solvent wastes because they require special
disposal through commercial services that are expensive.
11.4.1.8 Laundry: Clothing known to be contaminated should
be disposed with the precautions described under "Disposal of
Hazardous Wastes". Laboratory coats or other clothing worn in
2,3,7,8-TCDD work area may be laundered. Clothing should be
collected in plastic bags. Persons who convey the bags and launder
the clothing should be advised of the hazard and trained in proper
handling. The clothing may be put into a washer without contact if
the launderer knows the problem. The washer should be run through
one full cycle before being used again for other clothing.
11.4.1.9 Wipe Tests: A useful method for determining
cleanliness of work surfaces and tools is to wipe the surface with
a piece of filter paper, extract the filter paper and analyze the
extract.
NOTE: A procedure for the collection, handling,
analysis, and reporting requirements of wipe
tests performed within the laboratory is
described in Attachment A. The results and
decision making processes are based on the
presence of 2,3,7,8-substituted PCDDs/PCDFs.
11.4.1.10 Inhalation: Any procedure that may generate
airborne contamination must be carried out with good ventilation.
Gross losses to a ventilation system must not be allowed. Handling
of the dilute solutions normally used in analytical and animal work
presents no significant inhalation hazards except in case of an
accident.
11.4.1.11 Accidents: Remove contaminated clothing
immediately, taking precautions not to contaminate skin or other
articles. Wash exposed skin vigorously and repeatedly until medical
attention is obtained.
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Attachment A
PROCEDURES FOR THE COLLECTION, HANDLING, ANALYSIS, AND
REPORTING OF WIPE TESTS PERFORMED WITHIN THE LABORATORY
This procedure is designed for the periodic evaluation of potential con-
tamination by 2,3,7,8-substituted PCDD/PCDF congeners of the working areas inside
the laboratory.
A.I Perform the wipe tests on surface areas of two inches by one foot
with glass fiber paper saturated with distilled in glass acetone using a pair of
clean stainless steel forceps. Use one wiper for each of the designated areas.
Combine the wipers to one composite sample in an extraction jar containing 200
mL distilled in glass acetone. Place an equal number of unused wipers in 200 mL
acetone and use this as a control. Add 100 fj,l of the sample fortification
solution to each jar containing used or unused wipers (Sec. 5.8).
A.1.1 Close the jar containing the wipers and the acetone and
extract for 20 minutes using a wrist action shaker. Transfer the extract
into a KD apparatus fitted with a concentration tube and a three ball
Snyder column. Add two Teflon™ or Carborundum™ boiling chips and
concentrate the extract to an apparent volume of 1.0 mL on a steam bath.
Rinse the Snyder column and the KD assembly with two 1 mL portions of
hexane into the concentrator tube, and concentrate its contents to near
dryness with a gentle stream of nitrogen. Add 1.0 mL hexane to the
concentrator tube and swirl the solvent on the walls.
A.1.2 Prepare a neutral alumina column as described in Sec. 7.5.2.2
and follow the steps outlined in Sees. 7.5.2.3 through 7.5.2.5.
A. 1.3 Add 10 jLtL of the recovery standard solution as described in
Sec. 7.5.3.6.
A.2 Concentrate the contents of the vial to a final volume of 10 /*L
(either in a minivial or in a capillary tube). Inject 2 /uL of each extract
(wipe and control) onto a capillary column and analyze for 2,3,7,8-substituted
PCDDs/PCDFs as specified in the analytical method in Sec. 7.8. Perform
calculations according to Sec. 7.9.
A.3 Report the presence of 2,3,7,8-substituted PCDDs and PCDFs as a
quantity (pg or ng) per wipe test experiment (WTE). Under the conditions out-
lined in this analytical protocol, a lower limit of calibration of 10 pg/WTE is
expected for 2,3,7,8-TCDD. A positive response for the blank (control) is
defined as a signal in the TCDD retention time window at any of the masses
monitored which is equivalent to or above 3 pg of 2,3,7,8-TCDD per WTE. For
other congeners, use the multiplication factors listed in Table 1, footnote (a)
(e.g., for OCDD, the lower MCL is 10 x 5 = 50 pg/WTE and the positive response
for the blank would be 3 x 5 = 15 pg). Also, report the recoveries of the
internal standards during the simplified cleanup procedure.
A.4 At a minimum, wipe tests should be performed when there is evidence
of contamination in the method blanks.
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A.5 An upper limit of 25 pg per TCDD isomer and per wipe test experiment
is allowed (use multiplication factors listed in footnote (a) from Table 1 for
other congeners). This value corresponds to 2? times the lower calibration limit
of the analytical method. Steps to correct the contamination must be taken
whenever these levels are exceeded. To that effect, first vacuum the working
places (hoods, benches, sink) using a vacuum cleaner equipped with a high
efficiency particulate absorbent (HEPA) filter and then wash with a detergent.
A new set of wipes should be analyzed before anyone is allowed to work in the
dioxin area of the laboratory after corrective action has been taken.
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Figure 1.
8
o
Dibenzodioxin
8
Dibenzofuran
General structures of dibenzo-p-dioxin and dibenzofuran.
8290 - 49
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Figure 2.
M/AM
5,600
5,600
8,550
Peak profile displays demonstrating the effect of the detector zero on the
measured resolving power. In this example, the true resolving power is 5,600.
A) The zero was set too high; no effect is observed upon the
measurement of the resolving power.
B) The zero was adjusted properly.
C) The zero was set too low; this results in overestimating the actual
resolving power because the peak-to-peak noise cannot be measured
accurately.
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Figure 3.
Analytical Procedure
8:00 AM
Mass Resolution
Mass Accuracy
Thaw Sample Extract
1
Concentrate to 10 uL
1
9:00 AM
Initial or
Routine
Calibration
GC Column
Performance
11:00 AM
Samples
Method
Blank
8:00 PM
Mass
Resolution
Routine
Calibration
Typical 12 hour analysis sequence of events.
8290 - 51
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Figure 4.
100-1
c
0)
•«-»
£
0)
o>
CC
00
u>
to
I
22:30
24:00
Time
25:30
I T »
27:00
Selected ion current profile for m/z 322 (TCDDs) produced by MS analysis of
the GC performance check solution on a 60 m DB-5 fused silica capillary column
under the conditions listed in Sec. 7.6.
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Figure 5.
Ref. mass 304.9824 Peak top
Span. 200 ppm
80~1 ™- System file name YVES150
60-j i^H Data file name A:S5Z567
Resolution 10000
40-| '
Group number 1
20-| l^^^^^^li lonization mode El +
Switching VOLTAGE
Ref. masses 304.9824
80-, i IIMI 380.9260
60-| .^^,
^^" M/AM—10.500
40-I '^^^
20-
Channel B 380.9260 Lock mass
Span 200 ppm
Peak profiles representing two PFK reference ions at m/z 305 and 381. The
resolution of the high-mass signal is 95 ppm at 5 percent of the peak height;
this corresponds to a resolving power M/£iM of 10,500 (10 percent valley
definition).
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Figure 6.
20:00
22:00
26:00
28:00
30:00
Manual determination of S/N.
The peak height (S) is measured between the mean noise {lines C and D).
These mean signal values are obtained by tracing the line between the
baseline average noise extremes, El and E2, and between the apex average
noise extremes, E3 and E4, at the apex of the signal.
NOTE:
It is imperative that the instrument interface amplifier
electronic zero offset be set high enough so that negative
going baseline noise is recorded.
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Table 1.
Types of Matrices, Sample Sizes and 2,3,7,8-TCDD-Based
Method Calibration Limits (Parts per Trillion)
Lower MCLa
Upper MCLa
Weight (g)
IS Spiking
Levels (ppt)
Final Extr.
Vol. (ML)d
Water
0.
2
1000
1
10-50
Soil
Sediment
Paper Pulpb
01 1.0
200
10
100
10-50
Fly
Ash
1.0
200
10
100
50
Fish
Tissue
1.0
200
20
100
10-50
Human
Adipose
0 Tissue
1.0
200
10
100
10-50
Sludges,
Fuel Oil
5.0
1000
2
500
50
Still-
Bottom
10
2000
1
1000
50
a For other congeners multiply the values by 1 for TCDF/PeCDD/PeCDF, by 2.5
for HxCDD/HxCDF/HpCDD/HpCDF, and by 5 for OCDD/OCDF.
b Sample dewatered according to Sec. 6.5.
c One half of the extract from the 20 g sample is used for determination of
lipid content (Sec. 7.2.2).
d See Sec. 7.8.1, Note.
NOTE: Chemical reactor residues are treated as still bottoms if their
appearances so suggest.
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Table 2.
Composition of the Sample Fortification
and Recovery Standard Solutions8
Analyte
Sample Fortification
Solution
Concentration
(pg/AtL; Solvent:
Nonane)
Recovery Standard
Solution
Concentration
(pg//iL; Solvent:
Nonane)
13C12-2,3,7,8-TCDD
13C,,-2,3,7,8-TCDF
13,
13,
C12-1,2,3,4-TCDD
13C12-l,2,3,7,8-PeCDD
C12-l,2,3,7,8-PeCDF
C12-l,2,3,6,7,8-HxCDD
C12-l,2,3,4,7,8-HxCDF
C12-l,2,3,7,8,9-HxCDD
13,
13|
13,
13
13
'12
C12-l,2,3,4,6,7,8-HpCDD
,2-1,2,3,4,6,7,8-HpCDF
C12-OCDD
10
10
10
10
25
25
25
25
50
50
50
(a) These solutions should be made freshly every day because of the possibility
of adsorptive losses to glassware. If these solutions are to be kept for more
than one day, then the sample fortification solution concentrations should be
increased ten fold, and the recovery standard solution concentrations should be
doubled. Corresponding adjustments of the spiking volumes must then be made.
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Table 3.
The Fifteen 2,3,7,8-Substituted PCDD and PCOF Congeners
PCDD PCDF
2,3,7,8-TCDD(*) 2,3,7,8-TCDF(*)
l,2,3,7,8-PeCDD(*) 1,2,3,7,8-PeCDF(*)
l,2,3,6,7,8-HxCDO(*) 2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDD 1,2,3,6,7,8-HxCDF
l,2,3,7,8,9-HxCDD(+) 1,2,3,7,8,9-HxCDF
1,2,3,4,6,7,8-HpCDD(*) 1, 2,3,4,7,8-HxCDF(*)
2,3,4,6,7,8-HxCDF
1.2,3,4,6,7,8-HpCDF(*)
1,2,3,4,7,8,9-HpCDF
(*) The ISC-labeled analogue is used as an internal standard.
(+) The 13C-labeled analogue is used as a recovery standard.
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Table 4.
Isomers of Chlorinated Dioxins and Furans as a
Function of the Number of Chlorine Atoms
Number of
Chlorine
Atoms
1
2
3
4
5
6
7
8
Total
Number of
Dioxin
Isomers
2
10
14
22
14
10
2
1
75
Number of
2,3,7,8
Isomers
—
—
—
1
1
3
1
1
7
Number of
Furan
Isomers
4
16
28
38
28
16
4
1
135
Number of
2,3,7,8
Isomers
—
—
—
1
2
4
2
1
10
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Table 5.
High-Resolution Concentration Calibration Solutions
Concentration (pq/uL, in Nonane)
Compound
HRCC
Unlabeled Analytes
2,3,7,8-TCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDD
1,2,3,7,8-PeCDF
2,3,4
1,2,3
1,2,3
1,2,3
1,2,3
1,2,3
1,2,3
2,3,4
1,2,3
1,2,3
1,2,3
OCDD
OCDF
,7,
,4,
,6,
,7,
,4,
,6,
,7,
,6,
,4,
,4,
,4,
8-PeCDF
7
7
8
7
7
8
7
6
6
7
Internal
13C .
13p12_
12
13C12-
13C12-
13C -
,,12
13p
13C12_
°12
13r
,, 12~
2,3
2,3
1,2
1,2
1,2
1,2
1,2
1,2
,
,
,
?
,
,
,
,
,
?
,
,
,
5
J
,
8-HxCDD
8-HxCDD
9-HxCDD
8-HxCDF
8-HxCDF
9-HxCDF
8-HxCDF
7,8-HpCDD
7,8-HpCDF
8,9-HpCDF
200
200
500
500
500
500
500
500
500
500
500
500
500
500
500
1,000
1,000
50
50
125
125
125
125
125
125
125
125
125
125
125
125
125
250
250
10
10
25
25
25
25
25
25
25
25
25
25
25
25
25
50
50
2
2
6
6
6
6
6
6
6
6
6
6
6
6
6
12
12
.5
.5
.25
.25
.25
.25
.25
.25
.25
.25
.25
.25
.25
.25
.25
.5
.5
1
1
2.
2.
2.
2.
2.
2.
2.
2.
2.
2.
2.
2.
2.
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Standards
7
7
3
3
3
3
3
3
,8-TCDD
,8-TCDF
,7,8-PeCDD
,7,8-PeCDF
,6,7,8-HxCDD
,4,7,8-HxCDF
,4,6,7,8-HpCDD
,4,6,7,8-HpCDF
13C12-OCDD
Recovery
13r
13p12
1,2
1,2
'
50
50
50
50
125
125
125
125
250
50
50
50
50
125
125
125
125
250
50
50
50
50
125
125
125
125
250
50
50
50
50
125
125
125
125
250
50
50
50
50
125
125
125
125
250
Standards
3
3
,4-TCDD(a|
,7,8,9-HxCDD(bl
50
125
50
125
50
125
50
125
50
125
(a) Used for recovery determinations of TCDD, TCDF, PeCDD and PeCDF internal
standards.
(b) Used for recovery determinations of HxCDD, HxCDF, HpCDD, HpCDF and OCDD
internal standards.
8290 - 59
Revision 0
September 1994
-------
Table 6.
Ions Monitored for HRGC/HRMS Analysis of PCDDs/PCDFs
Descriptor
1
2
3
Accurate1"1
Mass
303.9016
305.8987
315.9419
317.9389
319.8965
321.8936
331.9368
333.9338
375.8364
[354.9792]
339.8597
341.8567
351.9000
353.8970
355.8546
357.8516
367.8949
369.8919
409.7974
[354.9792]
373.8208
375.8178
383,8639
385.8610
389.8156
391.8127
401.8559
403.8529
445.7555
[430.9728]
Ion
ID
M
M+2
M
M+2
M
M+2
M
M+2
M+2
LOCK
M+2
M+4
M+2
M+4
M+2
M+4
M+2
M+4
M+2
LOCK
M+2
M+4
M
M+2
M+2
M+4
M+2
M+4
M+4
LOCK
Elemental
Composition
C12H435C140
C12H436C1337C10
13C12H435C140
13C12H43BC1337C10
C12H435C1402
CI2H435C1337C102
13f II 35n n
L12n4 L I 4U2
13C12H435C1337C102
C12H435C1537C10
C9F13
C12H335C1437C10
C12H335C1337C120
13C12H336C1437C10
13C12H335C1337C120
C12H335C1437C102
C12H336C1337C1202
13C12H335C1437C102
13C12H335C1337C1202
C12H335C1637C10
C9F13
C12H235C1537C10
C12H235C1437C120
13C12H235C160
13C12H235C1537C10
C12H235C1537C102
C12H235C1437C1202
13C12H235C1537C102
13C12H235C1437C1202
C12H235C1637C120
C9F17
Analyte
TCDF
TCDF
TCDF (S)
TCDF (S)
TCDD
TCDD
TCDD (S)
TCDD (S)
HxCDPE
PFK
PeCDF
PeCDF
PeCDF (S)
PeCDF (S)
PeCDD
PeCDD
PeCDD (S)
PeCDD (S)
HpCDPE
PFK
HxCDF
HxCDF
HxCDF (S)
HxCDF (S)
HxCDD
HxCDD
HxCDD (S)
HxCDD (S)
OCDPE
PFK
8290 - 60
Revision 0
September 1994
-------
Table 6.
Continued
Descriptor Accurate'8' Ion
Mass ID
4 407.7818 M+2
409.7788 M+4
417.8250 M
419.8220 M+2
423.7767 M+2
425.7737 M+4
435.8169 M+2
437.8140 M+4
479.7165 M+4
[430.9728] LOCK
5 441.7428 M+2
443.7399 M+4
457.7377 M+2
459.7348 M+4
469.7780 M+2
471.7750 M+4
513.6775 M+4
[442.9728] LOCK
Elemental
Composition
C12H35C1637C10
C12H35C1537C120
13C12H35C170
13C12H35C1637C10
C12H35C1637C102
C12H35C1537C1202
13C12H35C1637C102
13C12H35C1537C1202
C12H35C1737C120
CgM?
C1235C1737C10
C1235C1637C120
C1235C1737C102
12 6 22
13C1235C1737C102
13C1235C1637C1202
C1235C1837C120
ClOM7
Analyte
HpCDF
HpCDF
HpCDF (S)
HpCDF
HpCDD
HpCDD
HpCDD (S)
HpCDD (S)
NCDPE
PFK
OCDF
OCDF
OCDD
OCDD
OCDD (S)
OCDD (S)
DCDPE
PFK
(al The following nuclidic masses were used:
H = 1.007825 0
C = 12.000000 3SC1
13C = 13.003355 37C1
F = 18.9984
15.994915
34.968853
36.965903
S = internal/recovery standard
8290 - 61
Revision 0
September 1994
-------
Table 7.
PCDD and PCDF Congeners Present in the GC Performance
Evaluation Solution and Used for Defining the
Homologous GC Retention Time Windows on a
60 m DB-5 Column
No. of
Chlorine
Atoms
4<«>
5
6
7
8
PCDD Positional
First
Eluter
1,3,6,8
1,2,4,6,8/
1,2,4,7,9
1,2,4,6,7,97
1,2,4,6,8,9
1,2,3,4,6,7,9
Isomer
Last
Eluter
1,2,8,9
1,2,3,8,9
1,2,3,4,6,
1,2,3,4,6,
1,2,3,4,6,
PCDF Positional
First
Eluter
1,3,6,8
1,3,4,6,8
7 1,2,3,4,6,8
7,8 1,2,3,4,6,7,8
7,8,9
Isomer
Last
Eluter
1,2,8,9
1,2,3,8,9
1,2,3,4,8,9
1,2,3,4,7,8,9
1,2,3,4,6,7,8,9
181 In addition to these two TCDD isomers, the 1,2,3,4-, 1,2,3,7-, 1,2,3,8-, 2,3,7,8-,
13C12-2,3,7,8-, and 1,2,3,9-TCDD isomers must also be present as a check of column
resolution.
8290 - 62 Revision 0
September 1994
-------
Table 8.
Theoretical Ion Abundance Ratios and Their Control Limits
for PCDDs and PCDFs
Number of
Chlorine Ion
Atoms Type
4
5
6
gla)
ylb)
7
8
M/M+2
M+2/M+4
M+2/M+4
M/M+2
M/M+2
M+2/M+4
M+2/M+4
Theoretical
Ratio
0.77
1.55
1.24
0.51
0.44
1.04
0.89
Control
lower
0.65
1.32
1.05
0.43
0.37
0.88
0.76
Limits
upper
0.89
1.78
1.43
0.59
0.51
1.20
1.02
la) Used only for 13C-HxCDF (IS).
(bl Used only for 13C-HpCDF (IS).
8290 - 63
Revision 0
September 1994
-------
Table 9.
Relative Response Factor [RF (number)] Attributions
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2,3,7
2,3,7
1,2,3
1,2,3
2,3,4
1,2,3
1,2,3
1,2,3
1,2,3
1,2,3
1,2,3
2,3,4
1,2,3
1,2,3
1,2,3
OCDD
OCDF
13C -
L12
13C -
,,12
13f _
,,12
13f _
i, 12
13p
,, 12~
13p
,o 12"
13f _
,, 12
13f _
,, 12
Specific Congener Name
,8-TCDD (and total TCDDs)
,8-TCDF (and total TCDFs)
,7,8-PeCDD (and total PeCDDs)
,7,8-PeCDF
,7,8-PeCDF
,4,7,8-HxCDD
,6,7,8-HxCDD
,7,8,9-HxCDD
,4,7,8-HxCDF
,6,7,8-HxCDF
,7,8,9-HxCDF
,6,7,8-HxCDF
,4,6,7,8-HpCDD (and total HpCDDs)
,4,6,7,8-HpCDF
,4,7,8,9-HpCDF
2,3,7,8-TCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDD
1,2,3,7,8-PeCDF
1,2,3,6,7,8-HxCDD
1,2,3,4,7,8-HxCDF
1,2,3,4,6,7,8-HpCDD
1,2,3,4,6,7,8-HpCDF
13C12-OCDD
Total
Total
Total
Total
PeCDFs
HxCDFs
HxCDDs
HpCDFs
8290 - 64 Revision 0
September 1994
-------
Table 10.
2,3,7,8-TCDD Toxicity Equivalency Factors (TEFs) for the
Polychlorinated Dibenzodioxins and Dibenzofurans
Number Compound(s) TEF"
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
1,2,3,6,7,8-HxCDD
1,2,3,7, 8, 9-HxCDD
1,2,3,4,7,8-HxCDD
1,2,3,4,6,7,8-HpCDD
1,2,3,4,6,7,8,9-OCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
1,2,3,4,7,8-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
1,2,3,4,6,7,8,9-OCDF
1.00
0.50
0.10
0.10
0.10
0.01
0.001
0.1
0.05
0.5
0.1
0.1
0.1
0.1
0.01
0.01
0.001
Taken from "Interim Procedures for Estimating Risks Associated with Exposures
to Mixtures of Chlorinated Dibenzo-p-Dioxin and -Dibenzofurans (CDDs and CDFs)
and 1989 Update", (EPA/625/3-89/016, March 1989).
8290 - 65 Revision 0
September 1994
-------
Table 11.
Analyte Relative Retention Time Reference Attributions
Analyte Analyte RRT Reference'"
1,2,3,4,7,8-HxCDD 13C12-l,2,3,6,7,8-HxCDD
1,2,3,6,7,8-HxCDF 13C12-l,2,3,4,7,8-HxCDF
1,2,3,7,8,9-HxCDF 13C12-l,2,3,4,7,8-HxCDF
2,3,4,6,7,8-HxCDF 13C12-l,2,3,4,7,8-HxCDF
tal The retention time of 2,3,4,7,8-PeCDF on the DB-5 column is measured relative
to 13Cl2-l,2,3,7,8-PeCDF and the retention time of 1,2,3,4,7,8,9-HpCDF relative
to 13C12-l,2,3,4,6,7,8-HpCDF.
8290 - 66 Revision 0
September 1994
-------
METHOD 8290
POLYCHLORINATED DIBENZODIOXINS (PCDDs) AND POLYCHLORINATED DIBENZOFURANS (PCDFs)
BY HIGH-RESOLUTION GAS CHROMATOGRAPHY/HIGH-RESOLUTION MASS SPECTROMETRY
(HRGC/HRMS)
7.1 Internal Standard Addition
7.1.1 Sample size of 1 to 1000
grams, see Section 7.4 & Table 1.
Determine wt. on tared flask
7.1.2 Spike samples w/100 uL
fortification mixture yielding internal
standard cones, ol Table 1, except
for adipose tissue
7.1.2.1 For soil, sediment, fly ash,
water, and fish tissue, mix 1 mL
acetone with 100 uL mixture
7.1.2.2 Do not dilute for other
sample matrices
7.2 Fish and Paper Pulp
7.2.1 Mix 60 gr sodium sulfate
and 20 gr sample; place
mix in Soxhtet; add 200 ml
1:1 hexane/MeCI; reflux
12 hours
7.2.2 Transfer extract to a
KD apparatus with a Snyder
column
7.2.3 Add Teflon boiling
chip; concentrate to 10 mL
in water bath; cool for 5 mins.
7.2.4 Add new chip. 50 mL
hexane to flask; concentrate
to 5 mL; cool for 5 mins.;
assure MeCI out before next
step
7.2.5 Rinse apparatus with
hexane; transfer contents
to a separatory funnel; do
cleanup procedure
7.2 Sample Extraction and Purification
7.3 Human Adipose Tissue
7.3.1 Store samples at or
below -20 C, care taken
in handling
7.3.2 Extraction
.1 Weigh out sample
.2 Let stand to room Temp
.3 Add MeCI, fortification
sola, homogenize
.4 Separate MeCI layer,
filter, dry, transfer to
vol. flask
.5 Redo step 3, add to
vol. flask
.6 Rinse sample train,
add to vol. flask
.7 Adjust to mark w/MeCI
7.3.3 Determine Upid Content
.1 Preweigh 1 gram
glass vial
.2 Transfer and reduce 1
mL extract to vial until
weight constant
.3 Calculate weight dried
extract
.4 Calculate % lipid
content from eqn.
.5 Record lipid extract wt.
and % lipid content
L
•©•
8290 - 67
1
7.4 Environmental and Waste
•0
7.3.4 Extract Concentration
.1 Transfer and rinse vol.
flask contents of 7.3.2.7
to round bottom
.2 Concentrate on rotovap
at40C
I
7.3.5 Extract Cleanup
.1 Dissolve Section 4 extract
with hexane
.2 Add acid impregnated
silica, stir for 2 hours
.3 Decant and dry liquid
with sodium sulfate
.4 Rinse silica 2x w/hexane,
dry w/sodium sulfate,
combine rinses w/step 3
.5 Rinse sodkim sulfate,
combine rinse w/step 4
.6 Prepare acidic silica
column
.7 Pass hexane extract
through column, collect
eluate in 500 mL KD assembly
.8 Rinse column w/hexane,
combine eluate w/step 7,
concentrate total eluate
tolOOuL
Note: If column discolored repeat
cleanup (7.3.5.1)
.9 Extract ready for column
cleanup
J
Revision 0
September 1994
-------
METHOD 8290
continued
[ 7.4 Environmental and Waste Samples |
7.4.1 Sludge/Wet Fuel Oil
.1 Extract sample with toluene
using Dean-Stark water
separator
.2 Cool sample, filter through
glass fiber filter
.3 Rinse fitter w/toluene,
combine w/extract
.4 Concentrate to near dryness
using rotovap
Note: Sample dissolves in toluene,
treat as in Section 7.4.2;
sample from pulp, treat as
in Section 7.2
7.4.2 Still Bottom/OII
.1 Extract sample w/toluene.
filter through glass fiber
filter into round bottom
.2 Concentrate on rotovap
atSOC
7.4.4 Transfer concentrate to sep.
funnel using hexane; rinse
container, add to funnel;
add 5% NaCI sola, shake
2 minutes; discard aqueous
layer
7.4.5 Aqueous
.1 Let sample stand to room Temp;
mark meniscus on bottle; add
fortification soln.
.2 Filter sample: centrifuge first
If needed
.3 Combine fittered/centrifuged
solids along w/filter; do Soxtilet
extraction of Section 7.4.6.1;
rinse assembly & combine
.4 Transfer aqueous phase to sep
funnel; rinse sample bottles
w/MeCI & transfer to funnel;
shake and extract water
.5 Let phases separate, use
mechanical means if needed
.6 Pass MeCI layer through drying
agent, collect in KD assembly
w/conoentrator tube
.7 Repeat step 4-6 2x. rinse
drying agent, combine all
in KD assembly
Note: Continuous liquid-liquid
extractor may be used if
emulsion problems occur
.8 Attach Snyder column,
concentrate on water bath
til 5 ml left; remove KD
assembly, allow to drain & cool
.9 Remove column; add hexane,
extraction concentrate of solids,
& new boiling chip; attach column,
concentrate to 5 mL
. 10 Rinse flask and assembly to final
volume 15 mL
.11 Determine original sample volume
by transferring meniscus volume
to graduated cylinder
L
7.4.3 Fly Ash
.1 Weigh sample; add
fortification soln. in acetone,
1 M HCI; shake in extraction
jar for 3 hours
.2 Filter mix in Buchner funnel:
rinse filter cake w/water; dry
filter cake at room Temp.
.3 Add sodium sulfate to cake,
mix and let stand for 1 hr,
mix again and let stand
.4 Place sample in extraction
thimble; extract in Soxhlet
for 16 hours w/toluene
.5 Cool and filter extract; rinse
containers & combine:
rotovap to near dryness
atSOC
7.4.6 Soil
.1 Add sodium sulfate, mix; transfer mixture to
Soxhlet assembly atop glass wool plug
.2 Add toluene, reflux for 24 hours
Note: Add more sodium sulfate if sample does not
flow freely
.3 Transfer extract to round bottom
.4 Concentrate to 10 mL on rotovap, allow to cool
.5 Transfer concentrate and hexane rinses to KD
assembly; concentrate to 10 mL, allow to cool
.6 Rinse Snydor column into KD; transfer KD
& concentrator tube liquids to sep funnel;
rinse KD assembly w/hexane & add to funnel
8290A.UP2
8290 - 68
Revision 0
September 1994
-------
METHOD 8290
continued
7.5 Cleanup
7.5.1 Partition
.1 Partition extract w/ concentrated
sutfurtc acid; shake, discard
add layer; repeat add wash till
no color present or done 4x
.2 OMIT FOR FISH SAMPLES. Partition
extract w/NaCI soln.; shake,
discard aqueous layer
.3 OMIT FOR FISH SAMPLES. Partition
extract w/KOH sotn.; shake,
discard base layer; repeat base
wash till no color obtained in wash
or done 4x
.4 Partition extract w/NaCI sotr..;
shake, discard aqueous layer.
Dry extract w/sodium surfate
into round bottom flask; rinse
sodium sultate w/hexane,
concentrate hexane soln. in
rotovap
7.5.2 Silica/Alumina Column
.1 Pack a gravity column w/silica gel; fill
w/hexane. elute to top of bed;
check (or channeling
.2 Pack a gravity column w/alumina. fill
w/hexane, elute to top of bed, check
for channeling
Note: Acidic alumina may be used instead of
neutral alumina.
.3 Dissolve residue of Section 7.5 1.4
in hexane; transfer soln. to top of
silica column
.4 Elute silica column w/hexane
directly onto alumina column
.5 Add hexane to alumina column;
elute to top of sodium sultate in
collect and save eluted hexane
.6 Add MeCI/hexane soln. to alumina
column: collect eluate in concentrator
tube
7.S.3 Carbon Column
. 1 Prepare AX-21/Ce)ite 545 column:
activate mixture at 130 C for 6 hours;
store in dessicator
.2 Pack a 10 mL serotogical pipet
w/prepared AX-21/Celite 545 mix
Note: Each batch of AX-21/Celite 545
must be checked for % recovery
of analytes.
.3 Concentrate MeCI/hexane fraction
of Section 7.5.2.6 to 2 mL
w/nitrogen; rinse column
w/several solns ; add sample
concentrate and rinses to top
of column
.4 Elute column sequentially
w/cydohexane/MeCI; MeCI/
melhanol/toluene; combine eluates
.5 Turn column upside down, elute
PCDD/PCDF fraction w/tduene;
filter if carbon fines present
.6 Concentrate toluene fraction on
rotovap; further concentrate to
100 uL in minivial using nitrogen
at 50 C; rinse flask 3x w/1%
toluene in MeCI; add tridecane
recovery std.; store room temp.
in the dark
8290 - 69
Revision 0
September 1994
-------
METHOD 8290
continued
7.6 Chromatographic, Mass Spectrometric, and
__DateAcgotei«on Parameters
7.6.1 Gas Chfomatograph
Select correct dimensions and parameters
of column, and set-up Chromatographic
conditions.
I
7.6.2 Mass Spectrometer
. 1 Operate mass spectrometer in selected
ion monitoring (SIM) mode; monitor ions
of five SIM descriptors
.2 Tune mass spectrometer based on ions
of SIM descriptors
7.6.3 Data Aquisition
. 1 Total cycle time of < or - 1 second
.2 Acquire SIM data for ions of 5
descriptors
L
7.7.2 Criteria for Acceptable Calibration
Criteria listed must be met before analysis
.1 The % RSD for unlabeted stds. must
be within +/- 20%; for labeled, +/- 30%
.2 S/N ratio for GC signals > - 2.5
.3 Table 8 isotcpic ratios within limits
Note: When criteria for acceptable calibration
are met, mean RRF's used for calculations
until routine calibration criteria are not met
L
i
I 7.7 CaltoratJon |
T
7.7.1 Initial Calibration
Required before any sample analysis.
and if routine calibration does not
meet criteria
.1 All 5 calibration solns. must be
used for initial calibration
.2 Tune mass spectrometer w/PFK as
described in Section 7.7.3
.3 Inject 2 uL of GC column performance
check sola and acquire SIM data;
assure Section 8.1.2 criteria are met
.4 Analyze each of 5 calibration standards
using the same conditions, with the
following MS operating parameters:
.1 Ratio of integrated ion current for
Table 8 ions within control limits
.2 Ratio of integrated ion current for
carbon labeled internal and recovery
standards within control limits
Note: Control limits must be achieved in
one run for all ions.
.3 Signal to noise (S/N) ratio for each
target analyte and labeled std. selected
ion current profiles (SICP) and
GC signals > 2.5
7.7.1.4
.4 Calculate relative response factors (RRF)
for unlabeted and labeled target analytes
relative to internal stds. (Table 5)
.5 Calculate average and relative standard
deviation for the 5 calibration solutions
.6 RRFs for concentration determination of
total isomers in a homologous series
are calculated as:
.1 Congeners in a homologous series w/one
isomer. mean RRF used is same as
Section 7.7.1.4.5
Note: Calibration solns. do not contain
labeled OCDF; therefore, RRF OCDF
relative to labeled OCDO
.2 Calculation for mean RRF for congeners
in a homologous series w/more than one
isomer
Note: Isomers in homologous series w/o .
2,3.7,8 substitution pattern alloted
same response factor as other 2,3,
7, 8 isomers in series
.7 Calculation of RRF's used to determine
% recoveries of nine internal standards
7.7.3 Routine Calibration
Performed at 12 hour periods after
successful resolution checks
.1 Inject 2 uL calibration soln. HRCC-3;
use same HRGC/HRMS conditions of
Sections 7.6.1 and 7.6.2; document
an acceptable calibration
L
7.7.4 Criteria for Acceptable Routine Calibration
.1 Measured unlabeled RRFs must be w/in
+/- 20% of initial calibration values
.2 Measured labeled RRFs must be w/in
+/- 30% of initial calibration values
.3 Table 8 ion abundance ratios must be
w/in limits
.4 Review routine calibration process if
criteria of steps 1 and 2 are not satisfied
Note: An initial calibration must be done when
new HRCC-3, sample fortification, or
recovery std. soln. from another lot is used
8290 - 70
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METHOD 8290
continued
JL
| 7.8Analysis}
""^
7.8.1 Reduce extract or blank
volume to 10 or 50 uL
±
7.8.2 Inject 2 ul aliquot of the
sample into the GC
7.8.3 Acquire SIM data according
to Section 7.6.2 and 7.6.3
Note: Acquisition period must at
least encompass PCDD/PCDF
overall retention time window
1
7.8.4 GC Identification Criteria
.1 Relative Retention Times
.1 2,3,7,8 sub: Sample components
relative retention time (RRT) w/in
•1 to 3 seconds of retention
time of labeled internal or
recovery std.
.2 2,3,7,8 sub: Sample RRTs
w/in homologous retention
time windows if w/o labeled
internal std.
.3 non 2,3,7,8 sub: Retention
time w/in homologous
retention time window
4 Ion current responses for
quantitation must reach maximum
w/in 2 seconds
.5 Ion current responses for labeled
stds. must reach maximum w/in
2 seconds
Note: Verify presence of 1,2,8,9-TCDD and
1.3,4.6.8-PeCDFinSICPs
.2 Ion Abundance Ratios
.1 Ratio of integrated ion current for
two ions used for quantification
w/in limits of homologous series
.3 Signal-to-Noise Ratio
.1 AH ion current intensities > =2.5
.4 Polychlorinated Diphenyl Ether
Interferences
.1 Corresponding PCDPE channel
dear of signal > . S/N 2 5 at
same retention time
17.9 Calculations
JL
7.9.1 Calculate concentration of
PCDD or PCDF compounds
w/formula
7.9.2 Calculate % recovery of nine
internal stds. using formula
Note: Add 1% recovery for human
adipose tissue samples
7.9.3 Use smaller sample amt. if
calculated concentration
exceeds method calibration limits
7.9.4 Sum of isomer concentration
is total concentration for a
homologous series
,7.9.5 Sample-Specific-Estimated Detection
Limit (EDI) ..
EDL: Anafyte concentration yielding
peak ht. 2.5x noise level. EDLs calculated
for non-identified 2.3.7.8-sub congeners
Two methods of calculation:
.1 Samples w/response <2.5x noise for
both quantification ions
.1 Use EDL expression to
calculate for absent
2,3,7,8 substituted PCDD/PCDF
.2 Samples w/response >2.5x noise for
at least 1 quantification ion
.1 Calculate "Estimated Maximum Possible
Concentration" (EMPC) when signal >
2.5x noise and retention time the same
I
7.9.6 Relative percent difference (RPD) formula I
7.9.7 Calculation of 2,3,7.8-TCDD toxicity
equivalent factors (TEF) of PCDDs and PCDFs
.1 Two GC Column TEF Determination:
Reanalyze sample extract on 60 meter
SP-2330 column
.1 Concentrations of specified congeners
calculated from analysis done on DB-5
column
.2 Concentrations of specified congeners
calculated from analysis done on
SP-2330 column w/different GC/MS
conditions
Confirmation and quantification of 2,3,7.8-
TCDD done on either column as long as
Section 8.1.2 criteria met
.3 GC peak must meet criteria of Sections
7.8.4.2, 7.8.4.3, and/or 7.8.4.1.1 RRTs
of 2.3,7,8-sub congeners w/no carbon-
labeled analogues referred to w/in 0.006
RRT units of carbon-labeled std.
Note:
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00
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METHOD 8315
DETERMINATION OF CARBONYL COMPOUNDS
BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC^
1.0 SCOPE AND APPLICATION
1.1 This method provides procedures for the determination of free
carbonyl compounds in various matrices by derivatization with
2,4-dinitrophenylhydrazine (DNPH). The method utilizes high performance liquid
chromatography (HPLC) with ultraviolet/visible (UV/vis) detection to identify and
quantitate the target analytes using two different sets of conditions: Option 1
and Option 2. Option 1 has been shown to perform well for one set of target
analytes for aqueous samples, soil or waste samples, and stack samples collected
by Method 0011. Option 2 has been shown to work well for another set of target
analytes in indoor air samples collected by Method 0100. The two sets of target
analytes overlap for some compounds. Refer to the Analysis Option listed in the
following table to determine which analytes may be analyzed by which option. The
following compounds may be determined by this method:
Compound Name
CAS No.a
Chemical Abstract Services Registry Number.
This list of target analytes contains an
compounds that have been evaluated using
Analysis Option13
Acetaldehyde
Acetone
Acrolein
Benzaldehyde
Butanal (butyraldehyde)
Crotonaldehyde
Cyclohexanone
Decanal
2 , 5 -Dimethyl benzal dehyde
Formaldehyde
Heptanal
Hexanal (hexaldehyde)
Isovaleraldehyde
Nonanal
Octanal
Pentanal (valeraldehyde)
Propanal (propionaldehyde)
m-Tolualdehyde
o-Tolualdehyde
p-Tolualdehyde
75-07-0
67-64-1
107-02-8
100-52-7
123-72-8
123-73-9
108-94-1
112-31-2
5779-94-2
50-00-0
111-71-7
66-25-1
590-86-3
124-19-6
124-13-0
110-62-3
123-38-6
620-23-5
529-20-4
104-87-0
1,2
2
2
2
1,2
1,2
1
1
2
1,2
1
1,2
2
1
1
1,2
1,2
2
2
2
apping list of
modifications of the
8315 - 1
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analysis. Refer to the respective option number when choosing the
appropriate analysis technique for a particular compound.
1.2 The Option 1 method detection limits (MDL) are listed in Tables 1 and
2. The sensitivity data for sampling and analysis using Method 0100 (Option 2)
are given in Table 3. The MDL for a specific sample may differ from that listed,
depending upon the nature of interferences in the sample matrix and the amount
of sample used in the procedure.
1.3 The extraction procedure for solid samples is similar to that
specified in Method 1311. Thus, a single sample may be extracted to measure the
analytes included in the scope of other appropriate methods. The analyst is
allowed the flexibility to select chromatographic conditions appropriate for the
simultaneous measurement of combinations of these analytes.
1.4 When this method is used to analyze unfamiliar sample matrices,
compound identification should be supported by at least one additional
qualitative technique. A gas chromatograph/mass spectrometer (GC/MS) may be used
for the qualitative confirmation of results for the target analytes, using the
extract produced by this method.
1.5 This method is restricted to use by, or under the supervision of,
analysts experienced in the use of chromatography and in the interpretation of
chromatograms. Each analyst must demonstrate the ability to generate acceptable
results with this method, using the procedure described in Sec. 7.0.
2.0 SUMMARY OF METHOD
2.1 Liquid and Solid Samples (Option 1)
2.1.1 For wastes comprised of solids, or for aqueous wastes
containing significant amounts of solid material, the aqueous phase, if
any, is separated from the solid phase and stored for later analysis. If
necessary, the particle size of the solids in the waste is reduced. The
solid phase is extracted with an amount of extraction fluid equal to 20
times the weight of the solid phase. The extraction fluid employed is a
function of the alkalinity of the solid phase of the waste. A special
extractor vessel is used when testing for volatiles. Following extraction,
the aqueous extract is separated from the solid phase by filtration
employing 0.6 to 0.8 pm glass fiber filter.
2.1.2 If compatible (i.e., multiple phases will not form on
combination), the initial aqueous phase of the waste is added to the
aqueous extract, and these liquids are analyzed together. If
incompatible, the liquids are analyzed separately and the results are
mathematically combined to yield a volume-weighted average concentration.
2.1.3 A measured volume of aqueous sample (approx. 100 mL) or an
appropriate amount of solids extract (approx. 25 g), is buffered to pH 3
and derivatized with 2,4-dinitrophenylhydrazine (DNPH), using either the
liquid-solid or a liquid-liquid extraction option. If the liquid-solid
8315 - 2 Revision 0
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option is used, the derivative is extracted using solid sorbent
cartridges, followed by elution with ethanol. If the liquid-liquid option
is used, the derivative is extracted from the sample with three (3)
portions of methylene chloride. The methylene chloride extracts are
concentrated using the Kuderna-Danish (K-D) procedure and exchanged with
acetonitrile prior to HPLC analysis. Liquid chromatographic conditions
are described which permit the separation and measurement of various
carbonyl compounds in the extract by absorbance detection at 360 nm.
2.1.4 If formaldehyde is the only analyte of interest, the aqueous
sample or solids extract should be buffered to pH 5.0 to minimize artifact
formaldehyde formation.
2.2 Stack Gas Samples Collected by Method 0011 (Option 1) - The entire
sample returned to the laboratory is extracted with methylene chloride and the
methylene chloride extract is brought up to a known volume. An aliquot of the
methylene chloride extract is solvent exchanged and concentrated or diluted as
necessary. Liquid chromatographic conditions are described that permit the
separation and measurement of various carbonyl compounds in the extract by
absorbance detection at 360 nm.
2.3 Indoor Air Samples by Method 0100 (Option 2) - The sample cartridges
are returned to the laboratory and backflushed with acetonitrile into a 5 ml
volumetric flask. The eluate is brought up to volume with more acetonitrile.
Two (2) aliquots of the acetonitrile extract are pipetted into two (2) sample
vials having Teflon-lined septa. Liquid chromatographic conditions are described
that permit the separation and measurement of the various carbonyl compounds in
the extract by absorbance detection at 360 nm.
3.0 INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware that lead to discrete
artifacts and/or elevated baselines in the chromatograms. All of these materials
must be routinely demonstrated to be free from interferences under the conditions
of the analysis by analyzing laboratory reagent blanks as described in Sec. 8.5.
3.1.1 Glassware must be scrupulously cleaned. Clean all glassware
as soon as possible after use by rinsing with the last solvent used. This
should be followed by detergent washing with hot water, and rinses with
tap water and organic-free reagent water. It should then be drained,
dried, and heated in a laboratory oven at 130°C for several hours before
use. Solvent rinses with acetonitrile may be substituted for the oven
heating. After drying and cooling, glassware should be stored in a clean
environment to prevent any accumulation of dust or other contaminants.
NOTE: Do not use acetone or methanol. These solvents react with
DNPH to form interfering compounds.
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3.1.2 The use of high purity reagents and solvents helps to minimize
interference problems. Purification of solvents by distillation in all
glass systems may be required.
3.1.3 Polyethylene gloves must be worn when handling the silica gel
cartridges to reduce the possibility of contamination.
3.2 Formaldehyde contamination of the DNPH reagent is a frequently
encountered problem due to its widespread occurrence in the environment. The
DNPH reagent in Option 2 must be purified by multiple recrystallizations in UV-
grade acetonitrile. Recrystallization is accomplished, at 40-60°C, by slow
evaporation of the solvent to maximize crystal size. The purified DNPH crystals
are stored under UV-grade acetonitrile until use. Impurity levels of carbonyl
compounds in the DNPH are determined prior to the analysis of the samples and
should be less than 25 mg/L. Refer to Appendix A for the recrystall ization
procedure.
3.3 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences will vary
considerably from source to source, depending upon the nature and diversity of
the matrix being sampled. Although the HPLC conditions described allow for a
resolution of the specific compounds covered by this method, other matrix
components may interfere. If interferences occur in subsequent samples,
modification of the mobile phase or some additional cleanup may be necessary.
3.4 In Option 1, acetaldehyde is generated during the derivatization step
if ethanol is present in the sample. This background will impair the measurement
of acetaldehyde at levels below 0.5 ppm (500 ppb).
3.5 For Option 2, at the stated two column analysis conditions, the
identification and quantitation of butyraldehyde may be difficult due to
coelution with isobutyraldehyde and methyl ethyl ketone. Precautions should be
taken and adjustment of the analysis conditions should be done, if necessary, to
avoid potential problems.
4.0 APPARATUS AND MATERIALS
4.1 High performance liquid chromatograph (modular)
4.1.1 Pumping system - Gradient, with constant flow control capable
of 1.50 mL/min.
4.1.2 High pressure injection valve with 20 /xL loop.
4.1.3 Column - 250 mm x 4.6 mm ID, 5 jum particle size, C18 (Zorbax
or equivalent).
4.1.4 Absorbance detector - 360 nm.
4.1.5 Strip-chart recorder compatible with detector - Use of a data
system for measuring peak areas and retention times is recommended.
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4.1.6 Helium - for degassing mobile phase solvents. (Options
1 and 2)
4.1.7 Mobile Phase Reservoirs and Suction Filtration Apparatus - For
holding and filtering HPLC mobile phase. Filtering system should be all
glass and Teflon and use a 0.22 pm polyester membrane filter. (Option 2)
4.1.8 Syringes - for HPLC injection loop loading, with capacity at
least four times the loop volume.
4.2 Apparatus and Materials for Option 1
4.2.1 Reaction vessel - 250 ml Florence flask.
4.2.2 Separatory funnel - 250 tnL, with Teflon stopcock.
4.2.3 Kuderna-Danish (K-D) apparatus.
4.2.3.1 Concentrator tube - 10 mL graduated (Kontes
K-570050-1025 or equivalent). A ground glass stopper is used to
prevent evaporation of extracts.
4.2.3.2 Evaporation flask - 500 mL (Kontes K-570001-500 or
equivalent). Attach to concentrator tube with springs, clamps, or
equivalent.
4.2.3.3 Snyder column - Three ball macro (Kontes
K-503000-0121 or equivalent).
4.2.3.4 Snyder column - Two ball micro (Kontes
K-569001-0219 or equivalent).
4.2.3.5 Springs - 1/2 inch (Kontes K-662750 or
equivalent).
4.2.4 Boiling chips - Solvent extracted with methylene chloride,
approximately 10/40 mesh (silicon carbide or equivalent).
4.2.5 pH meter - Capable of measuring to the nearest 0.01 units.
4.2.6 Glass fiber filter paper - 1.2 /urn pore size (Fisher Grade G4
or equivalent).
4.2.7 Solid sorbent cartridges - Packed with 2 g CIS (Baker or
equivalent).
4.2.8 Vacuum manifold - Capable of simultaneous extraction of up to
12 samples (Supelco or equivalent).
4.2.9 Sample reservoirs - 60 mL capacity (Supelco or equivalent).
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4.2.10 Pipet - Capable of accurately delivering 0.10 ml
solution (Pipetman or equivalent).
4.2.11 Water bath - Heated, with concentric ring cover, capable
of temperature control (+ 2°C). The bath should be used under a hood.
4.2.12 Sample shaker - Controlled temperature incubator (± 2°C)
with orbital shaking (Lab-Line Orbit Environ-Shaker Model 3527 or
equivalent).
4.2.13 Syringes - 5 mL, 500 /xL, 100 jzL, (Luer-Lok or
equivalent).
4.2.14 Syringe Filters - 0.45 jum filtration disks (Gelman
Acrodisc 4438 or equivalent).
4.3 Apparatus and Materials for Option 2
4.3.1 Syringes - 10 mL, with Luer-Lok type adapter, used to
backflush the sample cartridges by gravity feed.
4.3.2 Syringe Rack - made of an aluminum plate with adjustable legs
on all four corners. Circular holes of a diameter slightly larger than
the diameter of the 10 mL syringes are drilled through the plate to allow
batch processing of cartridges for cleaning, coating, and sample elution.
A plate (0.16 x 36 x 53 cm) with 45 holes in a 5x9 matrix is recommended.
See Figure 2 in Method 0100.
4.4 Volumetric Flasks - 5 mL, 10 mL, and 250 or 500 mL.
4.5 Vials - 10 or 25 mL, glass with Teflon-lined screw caps or crimp
tops.
4.6 Balance - Analytical, capable of accurately weighing to 0.0001 g.
4.7 Glass Funnels
4.8 Polyethylene Gloves - used to handle silica gel cartridges.
5.0 REAGENTS
5.1 Reagent grade inorganic chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Organic-free reagent water - Water in which an interferant is not
observed at the method detection limit for the compounds of interest.
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5.3 Formalin - Solution of formaldehyde (CH20) in organic-free reagent
water, nominally 37.6 percent (w/w). Exact concentration will be determined for
the stock solution in Sec. 5.7.1.1.
5.4 Aldehydes and ketones - analytical grade, used for preparation of
DNPH derivative standards of target analytes other than formaldehyde. Refer to
the target analyte list.
5.5 Option 1 Reagents
5.5.1 Methylene chloride, CH2C12 - HPLC grade or equivalent.
5.5.2 Acetonitrile, CH3CN - HPLC grade or equivalent.
5.5.3 Sodium hydroxide solutions, NaOH, 1.0 N and 5 N.
5.5.4 Sodium chloride, NaCl, saturated solution - Prepare by
dissolving an excess of the reagent grade solid in organic-free reagent
water.
5.5.5 Sodium sulfite solution, Na2S03, 0.1 M.
5.5.6 Sodium sulfate, Na2S04 - granular, anhydrous.
5.5.7 Citric Acid, C8H807, 1.0 M solution.
5.5.8 Sodium Citrate, C6H5Na307.2H20, 1.0 M trisodium salt dihydrate
solution.
5.5.9 Acetic acid (glacial), CH3C02H.
5.5.10 Sodium acetate, CH3C02Na.
5.5.11 Hydrochloric Acid, HC1, 0.1 N.
5.5.12 Citrate buffer, 1 M, pH 3 - Prepare by adding 80 ml of 1 M
citric acid solution to 20 ml of 1 M sodium citrate solution. Mix
thoroughly. Adjust pH with NaOH or HC1 as needed.
5.5.13 pH 5.0 Acetate buffer (5M) - Formaldehyde analysis only.
Prepared by adding 40 ml 5M acetic acid solution to 60 ml 5M sodium
acetate solution. Mix thoroughly. Adjust pH with NaOH or HC1 as needed.
5.5.14 2,4-Dinitrophenylhydrazine, 2,4-(02N)2C6H3]NHNH2, (DNPH), 70%
in organic-free reagent water (w/w).
5.5.14.1 DNPH (3.00 mg/mL) - Dissolve 428.7 mg of 707. (w/w)
DNPH solution in 100 ml acetonitrile.
5.5.15 Extraction fluid for Option 1 - Dilute 64.3 ml of 1.0 N NaOH
and 5.7 ml glacial acetic acid to 900 ml with organic-free reagent water.
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The pH should be 4.93
Dilute to 1 liter with organic-free reagent water.
± 0.02.
5.6 Option 2 Reagents
5.6.1 Acetonitrile, CH3CN - UV grade.
5.6.2 2,4-Dinitrophenylhydrazine, C6H6N404, (DNPH) - recrystallize
at least twice with UV grade acetonitrile using the procedure in Appendix
A.
5.7 Stock Standard Solutions for Option 1
5.7.1 Stock formaldehyde (approximately 1000 mg/L) - Prepare by
diluting an appropriate amount of the certified or standardized
formaldehyde (approximately 265 /nL) to 100 ml with organic-free reagent
water. If a certified formaldehyde solution is not available or there is
any question regarding the quality of a certified solution, the solution
may be standardized using the procedure in Sec. 5.7.1.1.
5.7.1.1 Standardization of formaldehyde stock solution -
Transfer a 25 ml aliquot of a 0.1 M Na2S03 solution to a beaker and
record the pH. Add a 25.0 ml aliquot of the formaldehyde stock
solution (Sec. 5.18.1) and record the pH. Titrate this mixture back
to the original pH using 0.1 N HC1. The formaldehyde concentration
is calculated using the following equation:
Concentration (mg/L)
(30.03)(N HCl)(mL HC1
25.0 ml
where:
N HC1
ml HC1
30.03
Normality of HC1 solution used (in milli-
equivalents/mL) (1 mmole of HCl = 1 mini-
equivalent of HCl)
ml of standardized HCl solution used
Molecular of weight of formaldehyde (in
mg/mmole)
5.7.2 Stock aldehyde(s) and ketone(s) - Prepare by adding an
appropriate amount of the pure material to 90 ml of acetonitrile and
dilute to 100 ml, to give a final concentration of 1000 mg/L.
5.8 Stock Standard Solutions for Option 2
5.8.1 Preparation of the DNPH Derivatives for HPLC analysis
5.8.1.1 To a portion of the recrystallized DNPH, add
sufficient 2N HCl to obtain an approximately saturated solution.
Add to this solution the target analyte in molar excess of the DNPH.
Filter the DNPH derivative precipitate, wash it with 2N HCl, wash it
again with water, and allow it to dry in air.
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5.8.1.2 Check the purity of the DNPH derivative by melting
point determination or HPLC analysis. If the impurity level is not
acceptable, recrystalllze the derivative in acetonitrile. Repeat
the purity check and recrystallization as necessary until 99% purity
is achieved.
5.8.2 Preparation of DNPH Derivative Standards and Calibration
Standards for HPLC analysis
5.8.2.1 Stock Standard Solutions - Prepare individual
stock standard solutions for each of the target analyte DNPH
derivatives fay dissolving accurately weighed amounts in
acetonitrile. Individual stock solutions of approximately 100 mg/L
may be prepared by dissolving 0.010 g of the solid derivative in
100 mi of acetonitrile.
5.8.2.2 Secondary Dilution Standard(s) - Using the
individual stock standard solutions, prepare secondary dilution
standards in acetonitrile containing the DNPH derivatives from the
target analytes mixed together. Solutions of 100 jug/L may be
prepared by placing 100 /iL of a 100 mg/L solution in a 100 mL
volumetric flask and diluting to the mark with acetonitrile.
5.8.2.3 Calibration Standards - Prepare a working
calibration standard mix from the secondary dilution standard, using
the mixture of DNPH derivatives at concentrations of 0.5-2.0 p,g/l
(which spans the concentration of interest for most indoor air
work). The concentration of the DNPH derivative in the standard mix
solutions may need to be adjusted to reflect relative concentration
distribution in a real sample.
5.9 Standard Storage - Store all standard solutions at 4°C in a glass
vial with a Teflon-lined cap, with minimum headspace, and in the dark. They
should be stable for about 6 weeks. All standards should be checked frequently
for signs of degradation or evaporation, especially just prior to preparing
calibration standards from them.
5.10 Calibration Standards
5.10.1 Prepare calibration solutions at a minimum of 5
concentrations for each analyte of interest in organic-free reagent water
(or acetonitrile for Option 2} from the stock standard solution. The
lowest concentration of each analyte should be at, or just above, the MDLs
listed in Tables 1 or 2. The other concentrations of the calibration
curve should correspond to the expected range of concentrations found in
real samples.
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6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this Chapter, Organic Analytes, Sec.
4.1.
6.2 Samples must be refrigerated at 4°C. Aqueous samples must be
derivatized and extracted within 3 days of sample collection. The holding times
of leachates of solid samples should be kept at a minimum. All derivatized
sample extracts should be analyzed within 3 days after preparation.
6.3 Samples collected by Methods 0011 or 0100 must be refrigerated at
4°C. It is recommended that samples be extracted and analyzed within 30 days of
collection.
7.0 PROCEDURE
7.1 Extraction of Solid Samples (Option 1)
7.1.1 All solid samples should be made as homogeneous as possible
by stirring and removal of sticks, rocks, and other extraneous material.
When the sample is not dry, determine the dry weight of the sample, using
a representative aliquot. If particle size reduction is necessary,
proceed as per Method 1311.
7.1.1.1 Determination of dry weight - In certain cases,
sample results are desired based on a dry weight basis. When such
data are desired or required, a portion of sample for dry weight
determination should be weighed out at the same time as the portion
used for analytical determination.
WARNING: The drying oven should be contained in a hood or
vented. Significant laboratory contamination may
result from drying a heavily contaminated
hazardous waste sample.
7.1.1.2 Immediately after weighing the sample for
extraction, weigh 5-10 g of the sample into a tared crucible.
Determine the % dry weight of the sample by drying overnight at
105°C. Allow to cool in a desiccator before weighing:
g of dry sample
% dry weight = x 100
g of sample
7.1.2 Measure 25 g of solid into a 500 mL bottle with a Teflon
lined screw cap or crimp top, and add 500 mL of extraction fluid (Sec.
5.5.15). Extract the solid by rotating the bottle at approximately 30 rpm
for 18 hours. Filter the extract through glass fiber filter paper and
store in sealed bottles at 4°C. Each mL of extract represents 0.050 g
solid. Smaller quantities of solid sample may be used with
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correspondingly reduced volumes of extraction fluid maintaining the 1:20
mass to volume ratio.
7.2 Cleanup and Separation (Option 1)
7.2.1 Cleanup procedures may not be necessary for a relatively
clean sample matrix. The cleanup procedures recommended in this method
have been used for the analysis of various sample types. If particular
samples demand the use of an alternative cleanup procedure, the analyst
must determine the elution profile and demonstrate that the recovery of
formaldehyde from a spiked sample is greater than 85%. Recovery may be
lower for samples which form emulsions.
7.2.2 If the sample is not clear, or the complexity is unknown, the
entire sample should be centrifuged at 2500 rpm for 10 minutes. Decant
the supernatant liquid from the centrifuge bottle, and filter through
glass fiber filter paper into a container which can be tightly sealed.
7.3 Derivatization (Option 1)
7.3.1 For aqueous samples, measure an aliquot of sample which is
appropriate to the anticipated analyte concentration range (nominally
100 ml). Quantitatively transfer the sample aliquot to the reaction
vessel (Sec. 4.2).
7.3.2 For solid samples, 1 to 10 ml of extract (Sec. 7.1) will
usually be required. The amount used for a particular sample must be
determined through preliminary experiments.
NOTE: In cases where the selected sample or extract volume is less
than 100 ml, the total volume of the aqueous layer should be
adjusted to 100 ml with organic-free reagent water. Record
original sample volume prior to dilution.
7.3.3 Derivatization and extraction of the target analytes may be
accomplished using the liquid-solid (Sec. 7.3.4) or liquid-liquid (Sec.
7.3.5) procedures.
7.3.4 Liquid-Solid Derivatization and Extraction
7.3.4.1 For analytes other than formaldehyde, add 4 ml of
citrate buffer and adjust the pH to 3.0 + 0.1 with 6M HC1 or 6M
NaOH. Add 6 ml of DNPH reagent, seal the container, and place in a
heated (40°C), orbital shaker (Sec. 4.2.12) for 1 hour. Adjust the
agitation to produce a gentle swirling of the reaction solution.
7.3.4.2 If formaldehyde is the only analyte of interest,
add 4 ml acetate buffer and adjust pH to 5.0 ± 0.1 with 6M HC1 or 6M
NaOH. Add 6 ml of DNPH reagent, seal the container, and place in a
heated (40°C), orbital shaker (Sec. 4.2.12) for 1 hour. Adjust the
agitation to produce a gentle swirling of the reaction solution.
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7.3.4.3 Assemble the vacuum manifold and connect to a
water aspirator or vacuum pump. Attach a 2 g sorbent cartridge to
the vacuum manifold. Condition each cartridge by passing 10 ml
dilute citrate buffer (10 ml of 1 M citrate buffer dissolved in 250
ml of organic-free reagent water) through each sorbent cartridge.
7.3.4.4 Remove the reaction vessel from the shaker
immediately at the end of the one hour reaction period and add 10 ml
saturated NaCl solution to the vessel.
7.3.4.5 Quantitatively transfer the reaction solution to
the sorbent cartridge and apply a vacuum so that the solution is
drawn through the cartridge at a rate of 3 to 5 mL/min. Continue
applying the vacuum for about 1 minute after the liquid sample has
passed through the cartridge.
7.3.4.6 While maintaining the vacuum conditions described
in Sec. 7.3.4.4, elute each cartridge train with approximately 9 ml
of acetonitrile directly into a 10 ml volumetric flask. Dilute the
solution to volume with acetonitrile, mix thoroughly, and place in
a tightly sealed vial until analyzed.
NOTE:
Because this method uses an excess of DNPH, the
cartridges will remain a yellow color after
completion of Sec. 7.3.4.5. The presence of this
color is not indicative of the loss of the
analyte derivatives.
7.3.5 Liquid-Liquid Derivatization and Extraction
7.3.5.1 For analytes other than formaldehyde, add 4 mL of
citrate buffer and adjust the pH to 3.0 + 0.1 with 6M HC1 or 6M
NaOH. Add 6 mL of DNPH reagent, seal the container, and place in a
heated (40°C), orbital shaker for 1 hour. Adjust the agitation to
produce a gentle swirling of the reaction solution.
7.3.5.2 If formaldehyde is the only analyte of interest,
add 4 mL acetate buffer and adjust pH to 5.0 + 0.1 with 6M HC1 or 6M
NaOH. Add 6 mL of DNPH reagent, seal the container, and place in a
heated (40°C), orbital shaker for 1 hour. Adjust the agitation to
produce a gentle swirling of the reaction solution.
7.3.5.3 Serially extract the solution with three 20 mL
portions of methylene chloride using a 250 mL separatory funnel. If
an emulsion forms upon extraction, remove the entire emulsion and
centrifuge at 2000 rpm for 10 minutes. Separate the layers and
proceed with the next extraction. Combine the methylene chloride
layers in a 125 mL Erlenmeyer flask containing 5.0 grams of
anhydrous sodium sulfate. Swirl contents to complete the extract
drying process.
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7.3.5.4 Assemble a Kuderna-Danish (K-D) concentrator by
attaching a 10 ml concentrator tube to a 500 ml evaporator flask.
Pour the extract into the evaporator flask being careful to minimize
transfer of sodium sulfate granules. Wash the Erlenmeyer flask with
30 ml of methylene chloride and add wash to the evaporator flask to
complete quantitative transfer.
7.3.5.5 Add one to two clean boiling chips to the
evaporative flask and attach a three ball Snyder column. Prewet the
Snyder column by adding about 1 ml methylene chloride to the top.
Place the K-D apparatus on a hot water bath (80-90°C) so that the
concentrator tube is partially immersed in the hot water and the
entire lower rounded surface of the flask is bathed with hot vapor.
Adjust the vertical position of the apparatus and the water
temperature, as required, to complete the concentration in 10-15
min. At the proper rate of distillation the balls of the column
will actively chatter, but the chambers will not flood with
condensed solvent. When the apparent volume of liquid reaches 5 ml,
remove the K-D apparatus and allow it to drain and cool for at least
10 min.
7.3.5.6 Prior to liquid chromatographic analysis, the
extract solvent must be exchanged to acetonitrile. The analyst must
ensure quantitative transfer of the extract concentrate. The
exchange is performed as follows:
7.3.5.6.1 Remove the three-ball Snyder column and
evaporator flask. Add 5 ml of acetonitrile , a new glass
bead or boiling chip, and attach the micro-Snyder column to
the concentrator tube. Concentrate the extract using 1 ml
of acetonitrile to prewet the Snyder column. Place the K-D
apparatus on the water bath so that the concentrator tube is
partially immersed in the hot water. Adjust the vertical
position of the apparatus and the water temperature, as
required, to complete concentration. At the proper rate of
distillation the balls of the column will actively chatter,
but the chambers will not flood. When the apparent volume
of liquid reaches less than 5 ml, remove the K-D apparatus
and allow it to drain and cool for at least 10 minutes.
7.3.5.6.2 Remove the Snyder column and rinse the flask
and its lower joint with 1-2 ml of acetonitrile and add to
concentrator tube. Quantitatively transfer the sample to a
10 ml volumetric flask using a 5 ml syringe with an attached
Acrodisc 0.45 pm filter cassette. Adjust the extract volume
to 10 mi. Stopper the flask and store refrigerated at 4°C
if further processing will not be performed immediately. If
the extract will be stored longer than two (2) days, it
should be transferred to a vial with a Teflon lined screw
cap or crimp top. Proceed with HPLC chromatographic
analysis if further cleanup is not required.
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7.4 Extraction of Samples from Methods 0011 and 0100 (Options 1 and 2)
7.4.1 Stack gas samples collected by Method 0011 (Option 1)
7.4.1.1 Measure the volume of the aqueous phase of the
sample prior to extraction (for moisture determination in case the
volume was not measured in the field). Pour the sample into a
separatory funnel and drain the methylene chloride into a volumetric
flask.
7.4.1.2 Extract the aqueous solution with two or three
aliquots of methylene chloride. Add the methylene chloride extracts
to the volumetric flask.
7.4.1.3 Fill the volumetric flask to the line with
methylene chloride. Mix well and remove an aliquot.
7.4.1.4 If high concentrations of formaldehyde are
present, the extract can be diluted with mobile phase, otherwise the
extract solvent must be exchanged as described in Sec. 7.3.5.5. If
low concentrations of formaldehyde are present, the sample should be
concentrated during the solvent exchange procedure.
7.4.1.5 Store the sample at 4°C. If the extract will be
stored longer than two days, it should be transferred to a vial with
a Teflon-lined screw cap, or a crimp top with a Teflon-lined septum.
Proceed with HPLC chromatographic analysis if further cleanup is not
required.
7.4.2 Ambient air samples collected by Method 0100 (Option 2)
7.4.2.1 The samples will be received by the laboratory in
a friction-top can containing 2 to 5 cm of granular charcoal, and
should be stored in this can, in a refrigerator, until analysis.
Alternatively, the samples may also be stored alone in their
individual glass containers. The time between sampling and analysis
should not exceed 30 days.
7.4.2.2 Remove the sample cartridge from the labeled
culture tube. Connect the sample cartridge (outlet or long end
during sampling) to a clean syringe.
NOTE: The liquid flow during desorption should be in
the opposite direction from the air flow during
sample collection (i.e, backflush the cartridge).
7.4.2.3 Place the cartridge/syringe in the syringe rack.
7.4.2.4 Backflush the cartridge (gravity feed) by passing
6 ml of acetonitrile from the syringe through the cartridge to a
graduated test tube, or to a 5 ml volumetric flask.
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NOTE: A dry cartridge has an acetonitrile holdup volume
slightly greater than 1 ml. The eluate flow may
stop before the acetonitrile in the syringe is
completely drained into the cartridge because of
air trapped between the cartridge filter and the
syringe Luer-Lok tip. If this happens, displace
the trapped air with the acetonitrile in the
syringe using a long-tip disposable Pasteur
pipet.
7.4.2.5 Dilute to the 5 ml mark with acetonitrile. Label
the flask with sample identification. Pipet two aliquots into
sample vials having Teflon-lined septa.
7.4.2.6 Store the sample at 4°C. Proceed with HPLC
chromatographic analysis of the first aliquot if further cleanup is
not required. Store the second aliquot in the refrigerator until
the results of the analysis of the first aliquot are complete and
validated. The second aliquot can be used for confirmatory
analysis, if necessary.
7.5 Chromatographic Conditions (Recommended):
7.5.1 Option 1 - For aqueous samples, soil or waste samples, and
stack gas samples collected by Method 0011.
Column: CIS, 4.6 mm x 250 mm ID, 5 ^m particle size
Mobile Phase Gradient: 70%/30% acetonitrile/water (v/v), hold for
20 min.
70%/30% acetonitrile/water to 100%
acetonitrile in 15 min.
100% acetonitrile for 15 min.
Flow Rate: 1.2 mL/min
Detector: Ultraviolet, operated at 360 nm
Injection Volume: 20 /iL
7.5.2 Option 2 - For ambient air samples collected by Method 0100.
Column: Two HPLC columns, 4.6 mm x 250 mm ID,
(Zorbax ODS, or equivalent) in series
Mobile Phase Gradient: 60%/40% CH3CN/H20, hold for 0 min.
60%/40% to 75%/25% CH3CN/H20, linearly in 30
min.
75%/25% to 100%/0% CH3CN/H20, linearly in 20
min.
100% CH3CN for 5 minutes.
100%/0% to 60%/40% CH3CN/H20, linearly in 1
min.
60%/40% CH3CN/H20 for 15 minutes.
Detector: Ultraviolet, operated at 360 nm
Flow Rate: 1.0 mL/min
Sample Injection volume:25 ^L (suggested)
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NOTE: For Options 1 and 2, analysts are advised to adjust their
HPLC systems to optimize chromatographic conditions for
their particular analytical needs. The separation of
acrolein, acetone, and propionaldehyde should be a minimum
criterion of the optimization in Option 2.
7.5.3 Filter and degas the mobile phase to remove dissolved gasses,
using the following procedure:
7.5.3.1 Filter each solvent (water and acetonitrile)
through a 0.22 ym polyester membrane filter, in an all glass and
Teflon suction filtration apparatus.
7.5.3.2 Degas each filtered solution by purging with
helium for 10-15 minutes (100 mL/min) or by heating to 60°C for 5-10
minutes in an Erlenmeyer flask covered with a watch glass. A
constant back pressure restrictor (350 kPa) or 15-30 cm of 0.25 mm
ID Teflon tubing should be placed after the detector to eliminate
further mobile phase outgassing.
7.5.3.3 Place the mobile phase components in their
respective HPLC solvent reservoirs, and program the gradient system
according to the conditions listed in Sec. 7.5.2. Allow the system
to pump for 20-30 minutes at a flow rate of 1.0 mL/min with the
initial solvent mixture ratio .(60%/40% CH3CN/H20). Display the
detector output on a strip chart recorder or similar output device
to establish a stable baseline.
7.6 Calibration
7.6.1 Establish liquid chromatographic operating conditions to
produce a retention time similar to that indicated in Table 1 for the
liquid-solid derivatization and extraction or in Table 2 for liquid-liquid
derivatization and extraction. For determination of retention time
windows, see Sec. 7.5 of Method 8000. Suggested chromatographic
conditions are provided in Sec. 7.5.
7.6.2 Process each calibration standard solution through
derivatization and extraction, using the same procedure employed for
sample processing (Sees. 7.3.4 or 7,3.5).
7.6.3 Analyze a solvent blank to ensure that the system is clean
and interference free.
NOTE: The samples and standards must be allowed to come to ambient
temperature before analysis.
7.6.4 Analyze each processed calibration standard using the
chromatographic conditions listed in Sec. 7.5, and tabulate peak area
against calibration solution concentration in /^g/L.
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7.6.5 Tabulate the peak area along with standard concentration
injected to determine the response factor (RF) for the analyte at each
concentration (see Sec. 7.8.1 for equations). The percent relative
standard deviation (%RSD) of the mean RF of the calibration standards
should be no greater than + 20 percent or a system check will have to be
performed. If a calibration check after the system check does not meet
the criteria, a recal ibration will have to be performed. If the
recalibration does not meet the established criteria, new calibration
standards must be made.
7.6.6 The working calibration curve must be verified each day,
before and after analyses are performed, by analyzing one or more
calibration standards. The response obtained should fall within + 15
percent of the initially established response or a system check will have
to be performed. If a calibration check after the system check does not
meet the criteria, the system must be recalibrated.
7.6.7 After 10 sample runs, or less, one of the calibration
standards must be reanalyzed to ensure that the DNPH derivative response
factors remain within +15% of the original calibration response factors.
7.7 Sample Analysis
7.7.1 Analyze samples by HPLC, using conditions established in Sec.
7.5. For analytes to be analyzed by Option 1, Tables 1 and 2 list the
retention times and MDLs that were obtained under these conditions. For
Option 2 analytes, refer to Figure 3 for the sample chromatogram.
7.7.2 If the peak area exceeds the linear range of the calibration
curve, a smaller sample injection volume should be used. Alternatively,
the final solution may be diluted with acetonitrile and reanalyzed.
7.7.3 After elution of the target analytes, calculate the
concentration of analytes found in the samples using the equations found
in Sec. 7.8 or the specific sampling method used.
7.7.4 If the peak area measurement is prevented by the presence of
observed interferences, further cleanup is required.
7.8 Calculations
7.8.1 Calculate each response factor, mean response factor, and
percent relative standard deviation as follows:
Concentration of standard injected,
RF =
Area of signal
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__
Mean RF = RF
I (RF, - RF)" /N-l
%RSD = — x 100%
RF
where:
RF = Mean response factor or mean of the response factors
using the 5 calibration concentrations.
RFj = Response factor for calibration standard i (i = 1-5).
%RSD = Percent relative standard deviation of the response
factors.
N = Number of calibration standards.
7.8.2 Calculate the analyte concentrations in liquid samples as
follows:
Concentration of aldehydes in /jg/L = (RT)(Area of signal)(100/V8)
where:
RF = Mean response factor for a particular analyte.
V6 = Number of ml of sample (unitless).
7.8.3 Calculate the analyte concentration in solid samples as
follows:
Concentration of aldehydes in /xg/g = (RF)(Area of signal)(20/ Vex)
where:
RF = Mean response factor for a particular analyte.
Vex = Number of ml extraction fluid aliquot (unitless).
7.8.4 Calculate the concentration of formaldehyde in stack gas
samples (Method 0011) as follows: (Option 1)
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7.8.4.1 Calculation of Total Formaldehyde: To determine
the total formaldehyde in mg, use the following equation:
[g/mole formaldehyde]
Total mg formaldehyde = Cd x V x DF x x 10'3 mg/jug
[g/mole DNPH derivative]
where:
Cd = measured concentration of DNPH-formaldehyde
derivative, mg/L
V = organic extract volume, ml
DF = dilution factor
7.8.4.2 Formaldehyde concentration in stack gas: Determine
the formaldehyde concentration in the stack gas using the following
equation:
C, = K [total formaldehyde, mg] / Vm(8tdl
where:
K = 35.31 ft3/m3, if Vm(std) is expressed in
English units
1.00 m3/m3, if Vm(std) is expressed in metric
units
Vm(stdl = volume of gas sample as measured by dry gas
meter, corrected to standard conditions,
dscm (dscf)
7.8.5 Calculation of the Concentration of Formaldehyde and Other
Carbonyls from Indoor Air Sampling by Method 0100. (Option 2)
7.8.5.1 The concentration of target analyte "a" in air at
standard conditions (25°C and 101.3 kPa), Concastd in ng/L, may be
calculated using the following equation:
(Area.) (RF)(Vola)(MWa)( 1000 ng/Mg)
Conca = x DF
(MWd)(VTotStd)(1000 ml/I)
where:
Areaa = Area of the sample peak for analyte "a"
RF = Mean response factor for analyte "a" from
the calibration in M9/L. (See Sec. 7.8.1)
Vola = Total volume of the sample cartridge eluate
(ml)
MWa = Molecular weight of analyte "a" in g/mole
MWd = Molecular weight of the DNPH derivative of
analyte "a" in g/mole
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VTotStd = Total volume of air sampled converted to
standard conditions in liters (L). (To
calculate the concentration at sampling
conditions use Vtot.)(See Sec. 9.1.3 of
Method 0100)
DF = Dilution Factor for the sample cartridge
eluate, if any. If there is no dilution,
DF = 1
7.8.5.2 The target analyte "a" concentration at standard
conditions may be converted to parts per billion by volume, Conca in
ppbv, using the following equation:
(Cone.) (22.4)
Conca in ppbv = -
(MWJ
where:
Conca = Concentration of analyte "a" in ng/L
22.4 = Ideal gas law volume (22.4 nl_ of gas = 1
nmole at standard conditions)
MWa = Molecular weight of analyte "a" in g/mole
(or ng/nmole)
8.0 QUALITY CONTROL
8.1 Refer to Chapter One and Method 8000 for specific quality control
procedures. Refer to Table 4 for QC acceptance limits derived from the
interlaboratory method validation study on Method 8315.
9.0 METHOD PERFORMANCE
9.1 The MDLs for Option 1 listed in Table 1 were obtained using organic-
free reagent water and liquid-solid extraction. The MDLs for Option 1 listed in
Table 2 were obtained using organic-free reagent water and methylene chloride
extraction. Results reported in Tables 1 and 2 were achieved using fortified
reagent water volumes of 100 mL. Lower detection limits may be obtained using
larger sample volumes.
9.1.1 Option 1 of this method has been tested for linearity of
recovery from spiked organic-free reagent water and has been demonstrated
to be applicable over the range 50-1000
9.1.2 To generate the MDL and precision and accuracy data reported
in this section, analytes were segregated into two spiking groups, A and
B. Representative chromatograms using liquid-solid and liquid-liquid
extraction are presented in Figures 1 (a and b) and 2 (a and b),
respectively.
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9.2 The Sensitivity of Option 2 sampling (Method 0100) and analysis is
listed in Table 3.
9.3 Method 8315, Option 1, was tested by 12 laboratories using reagent
water and ground waters spiked at six concentration levels over the range 30-2200
/ig/L. Method accuracy and precision were found to be directly related to the
concentration of the analyte and independent of the sample matrix. Mean recovery
weighted linear regression equations, calculated as a function of spike
concentration, as well as overall and single-analyst precision regression
equations, calculated as functions of mean recovery, are presented in Table 5.
These equations can be used to estimate mean recovery and precision at any
concentration value within the range tested.
10.0 REFERENCES
1. "OSHA Safety and Health Standards, General Industry", (29CRF1910).
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
11.0 SAFETY
11.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound should be treated
as a potential health hazard. From this viewpoint, exposure to these chemicals
must be reduced to the lowest possible level by whatever means available. The
laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this
method. A reference file of material safety data sheets should also be made
available to all personnel involved in the chemical analysis. Additional
references to laboratory safety are available.
11.2 Formaldehyde has been tentatively classified as a known or suspected,
human or mammalian carcinogen.
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TABLE 1.
OPTION 1 - METHOD DETECTION LIMITS" USING
LIQUID-SOLID EXTRACTION
Analyte Retention Time MDL
(minutes)
Formaldehyde
Acetaldehyde
Propanal
Crotonaldehyde
Butanal
Cyclohexanone
Pentanal
Hexanal
Heptanal
Octanal
Nonanal
Decanal
5.3
7.4
11.7
16.1
18.1
27.6
28.4
34.1
35.0
40.1
40.4
44.1
6.2
43. 7b
11.0
5.9
6.3
5.8
15.3
10.7
10.0
6.9
13.6
4.4
The method detection limit (MDL) is defined as the minimum
concentration that can be measured with 99% confidence that the
value is above background level. With the exception of
acetaldehyde, all reported MDLs are based upon analyses of 6 to 8
replicate blanks spiked at 25 M9/L. The MDL was computed as
follows:
MDL = Wo.o^Std. Dev.)
where:
t(N-i.o.oi) = The upper first percentile point of the
t-distribution with n-1 degrees of freedom.
Std. Dev. = Standard deviation, calculated using n-1
degrees of freedom.
The reported MDL is based upon analyses of 3 replicate, fortified
blanks at 250
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TABLE 2.
OPTION 1 - METHOD DETECTION LIMITS" USING
LIQUID-LIQUID EXTRACTION
Analyte Retention Time MDL
(minutes)
Formaldehyde
Acetaldehyde
Propanal
Crotonaldehyde
Butanal
Cyclohexanone
Pentanal
Hexanal
Heptanal
Octanal
Nonanal
Decanal
5.3
7.4
11.7
16.1
18.1
27.6
28.4
34.1
35.0
40.1
40.4
44.1
23.2
110. 2b
8.4
5.9
7.8
6.9
13.4
12.4
6.6
9.9
7.4
13.1
8 The method detection limit (MDL) is defined as the minimum
concentration that can be measured with 99% confidence that the value
is above background level. With the exception of acetaldehyde, all
reported MDLs are based upon analyses of 6 to 8 replicate blanks
spiked at 25 M9/L. The MDL was computed as follows:
MDL = V,.0.01l(Std. Dev.)
where:
t|N-i.o.cm = The upper first percentile point of the t-
distribution with n-1 degrees of freedom.
Std. Dev. = Standard deviation, calculated using n-1 degrees of
freedom.
b The reported MDL is based upon analyses of 3 replicate, fortified
blanks at 250 /xg/L.
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TABLE 3.
OPTION 2 - SENSITIVITY (ppb, v/v) OF SAMPLING AND ANALYSIS FOR
CARBONYL COMPOUNDS IN AMBIENT AIR USING AN ADSORBENT CARTRIDGE
FOLLOWED BY GRADIENT HPLC"
Sample Volume (L)b
Compound 10 20 30 40 50 100 200 300 400 500
Acetaldehyde
Acetone
Acrolein
Benzaldehyde
Butyraldehyde
Crotonaldehyde
2,5-Dimethyl-
benzaldehyde
Formaldehyde
Hexanal
Isovaleraldehyde
Propionaldehyde
m-Tolualdehyde
o-Tolualdehyde
p-Tolualdehyde
Valeraldehyde
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
.36
.28
.29
.07
.21
.22
.97
.45
.09
.15
.28
.02
.02
.02
.15
0.68
0.64
0.65
0.53
0.61
0.61
0.49
0.73
0.55
0.57
0.64
0.51
0.51
0.51
0.57
0.45
0.43
0.43
0.36
0.40
0.41
0.32
0.48
0.36
0.38
0.43
0.34
0.34
0.34
0.38
0.34
0.32
0.32
0.27
0.30
0.31
0.24
0.36
0.27
0.29
0.32
0.25
0.25
0.25
0.29
0.27
0.26
0.26
0.21
0.24
0.24
0.19
0.29
0.22
0.23
0.26
0.20
0.20
0.20
0.23
0.14
0.13
0.13
0.11
0.12
0.12
0.10
0.15
0.11
0.11
0.13
0.10
0.10
0.10
0.11
0.07
0.06
0.06
0.05
0.06
0.06
0.05
0.07
0.05
0.06
0.06
0.05
0.05
0.05
0.06
0.05
0.04
0.04
0.04
0.04
0.04
0.03
0.05
0.04
0.04
0.04
0.03
0.03
0.03
0.04
0.03
0.03
0.03
0.03
0.03
0.03
0.02
0.04
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.02
0.02
0.02
0.02
0.03
0.02
0.02
0.03
0.02
0.02
0.02
0.02
The ppb values are measured at 1 atm and 25°C. The sample cartridge is
eluted with 5 mL acetonitrile and 25 juL is injected into the HPLC. The
maximum sampling flow through a DNPH-coated Sep-Pak is about 1.5 L/minute.
A sample volume of 1000 L was also analyzed. The results show a
sensitivity of 0.01 ppb for all the target analytes.
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TABLE 4.
PERFORMANCE-BASED QC ACCEPTANCE LIMITS CALCULATED
USING THE COLLABORATIVE STUDY DATA
Spike
Analyte Concentration8
Formaldehyde
Propanal
Crotonaldehyde
Butanal
Cyclohexanone
Hexanal
Octanal
Decanal
160
160
160
160
160
160
160
160
Xb
154
148
160
151
169
151
145
153
C C
^R
30.5
22.4
34.8
22.7
39.2
34.6
40.1
40.0
Acceptance
Limits, %d
39-153
50-134
35-165
52-137
32-179
30-159
15-166
21-171
a Spike concentration, /ug/L.
" Mrt r» it v*f*sr\\tf\v*\t ^rilyiilri + ft/J *io-iMn 4" k» Q v»a ^ n Art4" ui a t A v* m A ^ ft VQ^*n\/av^w "1 -i n a D fc*
regression equation,
Overall standard deviation calculated using the reagent water, overall
standard deviation linear regression equation, M9/L-
Acceptance limits calculated as (X + 3sR)100/spike concentration.
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TABLE 5.
WEIGHTED LINEAR REGRESSION EQUATIONS FOR MEAN RECOVERY AND PRECISION
Analyte
Formaldehyde
Propanal
Crotonaldehyde
Butanal
Cyclohexanone
Hexanal
Octanal
Decanal
Applicable
Cone. Range
39.2-2450
31.9-2000
32.4-2030
35.4-2220
31.6-1970
34.1-2130
32.9-2050
33.2-2080
Reagent Water
X 0.909C + 8.79
SR 0.185X + 1.988
sr 0.093X + 5.79
X 0.858C + 10.49
SR 0.140X + 1.63
sr 0.056X + 2.76
X 0.975C + 4.36
SR 0.185X + 5.15
sr 0.096X + 1.85
X 0.902C + 6.65
SR 0.149X + 0.21
sr 0:086X - 0.71
X 0.962C + H.97
SR 0.204X + 4.73a
sr 0.187X + 3.46
X 0.844C + 15.81
SR 0.169X + 9.07
sr 0.098X + 0.378
X 0.856C + 7.88
SR 0.200X + 11.17
sr 0.092X + 1.71"
X 0.883C + 12.00
SR 0.225X + 5.52
sr 0.088X + 2.28a
0 Variance is not constant over concentration range.
X Mean recovery, jug/L, exclusive of outliers.
SR Overall standard deviation, M9/U exclusive of outl
sr Single-analyst standard deviation, /ug/L, exclusive
Ground Water
0.870C +14.84
0.177X + 13.85
0.108X + 6.24
0.892C + 22.22
0.180X + 12.37
0.146X + 2.088
0.971C + 2.94
0.157X + 6.09
0.119X - 2.27
0.925C + 12.71
0.140X + 6.89
0.108X - 1.63*
0.946C + 28.95
0.345X + 5.02
0.123X + 7.64
0.926C + 9.16
0.132X + 8.31
0.074X - 0.40a
0.914C + 13.09
0.097X + 12.41
0.039X + 1.14
0.908C + 6.46
0.153X + 2.23
0.052X + 0.37
iers.
of outliers.
8315 - 26
Revision 0
September 1994
-------
FIGURE la.
OPTION 2 - LIQUID-SOLID PROCEDURAL STANDARD OF GROUP A ANALYTES AT 625 M9/L
-0.80
-1.00-
.-1.20-
s
7-1.40-
x ,
-1.80-
-1.80-
-2.1
a
I
i.oo
a. oo
3.00
4.00
10*
Retention Time
(minutes)
5.33
11.68
18.13
27.93
36.60
42.99
Analyte
Derivative
Formaldehyde
Propanal
Butanal
Cyclohexanone
Heptanal
Nonanal
8315 - 27
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September 1994
-------
FIGURE Ib.
OPTION 1 - LIQUID-SOLID PROCEDURAL STANDARD OF GROUP B ANALYTES AT 625
-O.TO-
-0.80-
-l.SO-
1.00
. oo
3.00
4.00
10*
Retention Time
(minutes)
7.50
16.68
26.88
32.53
40.36
45.49
Analyte
Derivative
Acetaldehyde
Crotonaldehyde
Pentanal
Hexanal
Octanal
Decanal
8315 - 28
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September 1994
-------
FIGURE 2a.
OPTION 1 - LIQUID-LIQUID PROCEDURAL STANDARD OF GROUP A ANAIYTES AT 625 /ig/L
-1.40-
5-1. to
«•
K*
-2.00-
2.00
3.00
x 10* ainutM
4.00
Retention Time
(minutes)
5.82
13.23
20.83
29.95
37.77
43.80
Analyte
Derivative
Formaldehyde
Propanal
Butanal
Cyclohexanone
Heptanal
Nonanal
8315 - 29
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-------
FIGURE 2b.
OPTION I - LIQUID-LIQUID PROCEDURAL STANDARD OF GROUP B ANALYTES AT 625
-8.00-1
1.00
10*
Retention Time
(minutes)
7.79
17.38
27.22
32.76
40.51
45.62
Analyte
Derivative
Acetaldehyde
Crotonaldehyde
Pentanal
Hexanal
Octanal
Decanal
8315 - 30
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September 1994
-------
FIGURE 3.
OPTION 2 - CHROMATOGRAPHIC SEPARATION OF THE DNPH DERIVATIVES
OF 15 CARBONYL COMPOUNDS
DNPH
10
20
TIME, mln
Peak Identification
40
Number Compound
Concentrationfnq/ I)
1 Formaldehyde
2 Acetaldehyde
3 Acrolein
4 Acetone
5 Propanal
6 Crotonaldehyde
7 Butanal
8 Benzaldehyde
9 Isovaleraldehyde
10 Pentanal
11 o-Tolualdehyde
12 m-Tolualdehyde
13 p-Tolualdehyde
14 Hexanal
15 2,4-Dimethylbenzaldehyde
1.140
1.000
1.000
1.000
1.000
1.000
0.905
1.000
0.450
0.485
0.515
0.505
0.510
1.000
0.510
8315 - 31
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METHOD 8315
DETERMINATION OF CARBONYL COMPOUNDS
BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
0
Ambient Air (Option 2)
7.1.1-7.1.1.1
Homogenize sample
and determine dry
weight
Solid
7.1.2 Extract
sample tor 18
hours; filter and
store extract
7.3.2 Measure 1-10
mL extract; adjust
volume to 100 ml
with water
SoJkL
7.0 What is
the sample
matrix?
Stack Qas (Option
1 Media (Option 1)
7.0 te media
solid or
aqueous?
Is sample
dear or sample
complexity
Known?
•©
No
7.2.2 Centrifuge sample
at 2500 rpm for 10
minutes; decant
and filter
7.3.1 Is
medta solid
or aqueous?
Aqueous
7.3.1 Measure
aliquot of sample;
adjust volume to
100 mL with water
7.3.5.5 Exchange
solvent to metnanol
0
8315 - 32
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-------
METHOD 8315
continued
7.4.1.1 Measure volume
of aqueous phase of
sample; pour sample into
sepaiatory funnel and
drata mefnytene chloride
(from Method 0011) Into
volumetric flask
7.4.1.2 Extract aqueous
solution with metnytene
chtoride; add methytene
chloride extracts to
volumetric flask
7.4.1.3 Diute to volume
with metTvtene chloride;
mix weM; remove aliquot
7.4.1. 5 Store
sample at 4C
i
t
7.4.1.4
sample have
a high concentration
of formaldehyde?
7.4.1.4 Dilute
extract with mobile
phase
7.4.1.4 Exchange
solvent with methand
as In 7.3.5.5
7.4.1.4
Does sample
have a low
concentration of
7.4.1.4 Concentrate
extract during
solvent exchange
process
o
8315 - 33
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METHOD 8315
continued
O
1
7.4.2.2 • 7.4.2.3
Connect sample cartridge
to dean syringe and
place In syringe rack
1
7.4.2.4 Backflush
carWdgewtth
acetonltrtte
7.4.2.4
Doesekjate
flow become
btocted?
7.4.2.4 Displace
trapped air wit)
acctonftrttem
syringe using a long-tip
disposable Pasteur pipet
7.4.2.5 Dilute to 5
mLwtthacetonitrite;
label flask; pipet 2
atfquoteinto
sample vials
I
7.4.2.6 Store
sample at 4C
8315 - 34
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METHOD 8315
continued
7.5.2 Set LC conditions
to produce appropriate
retention tiroes
i
7.5.2.1 Filter and
degas mobile phase
7.5.1 Set LC
condWons to produce
appropriate retention
times
7.5.1 Option
lor 21C
conditions?
7.6.2 Process calibration
standards through same
processing steps as samples
7.6.3 - 7.6.4
Analyze solvent blank
and calibration standards:
tabulate peak areas
7.6.5 Determine response
factor at each concentration
7.6.5
Does
calibration
check meet
criteria?
7.6.5 Recalibrate
7.6.5
Does
calibration
check meet
criteria?
7.6.5 Prepare new
calibration
standards
8315 - 35
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September 1994
-------
METHOD 8315
continued
O
1
r
7.6.6 - 7.6.7 Verity
calibration curve every day;
reanalyze 1 calibration
standard after 10
sample runs or less
7.7 Analyze samples
byHPLC
7.7.2 Inject a smaller
volume or dilute sample
7.7.4 Further
cleanup Is required
7.7.2
Does peak
area exceed
calibration
curve?
7.7.4 Are
Interferences
present?
7.8.1 Calculate each
response factor, mean
response factor, and
percent RSO
\
7.8.2 - 7.8.5
Calculate analyte
concentrations
'
Stop
8315 - 36
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September 1994
-------
APPENDIX A
RECRYSTALLIZATION OF 2,4-DINITROPHENYLHYDRAZINE (DNPH)
NOTE: This procedure should be performed under a properly ventilated hood.
Inhalation of acetonitrile can result in nose and throat irritation (brief
exposure at 500 ppm) or more serious effects at higher concentration
and/or longer exposures.
A.I Prepare a saturated solution of DNPH by boiling excess DNPH in 200 ml
of acetonitrile for approximately 1 hour.
A.2 After 1 hour, remove and transfer the supernatant to a covered beaker
on a hot plate and allow gradual cooling to 40 to 60°C. Maintain this
temperature range until 95% of the solvent has evaporated, leaving crystals.
A.3 Decant the solution to waste and rinse the remaining crystals twice
with three times their apparent volume of acetonitrile.
A.4 Transfer the crystals to a clean beaker, add 200 ml of acetonitrile,
heat to boiling, and again let the crystals grow slowly at 40 to 60°C until 95%
of the solvent has evaporated. Repeat the rinsing process as in Sec. A.3.
A.5 Take an aliquot of the second rinse, dilute 10 times with
acetonitrile, acidify with 1 ml of 3.8 M perchloric acid per 100 ml of DNPH
solution, and analyze with HPLC as in Sec. 7.0 for Option 2. An acceptable
impurity level is less than 0.025 ng//iL of formaldehyde in recrystallized DNPH
reagent or below the sensitivity (ppb, v/v) level indicated in Table 3 for the
anticipated sample volume.
A.6 If the impurity level is not satisfactory, pipet off the solution to
waste, repeat the recrystallization as in Sec. A.4 but rinse with two 25 ml
portions of acetonitrile. Prep and analyze the second rinse as in Sec. A.5.
A.7 When the impurity level is satisfactory, place the crystals in an
all-glass reagent bottle, add another 25 ml of acetonitrile, stopper, and shake
the bottle. Use clean pipets when removing the saturated DNPH stock solution to
reduce the possibility of contamination of the solution. Maintain only a minimum
volume of the saturated solution adequate for day to day operation to minimize
waste of the purified reagent.
8315 - 37 Revision 0
September 1994
-------
00
-------
METHOD 8316
ACRYLAMIDE, ACRYLONITRILE AND ACROLEIN BY HIGH PERFORMANCE
LIQUID CHRQMATOGRAPHY (HPLC)
1.0 SCOPE AND APPLICATION
1.1 The following compounds can be determined by this method:
Compound Name CAS No."
Acrylamide 79-06-1
Acrylonitrile 107-13-1
Acrolein (Propenal) 107-02-8
8 Chemical Abstract Services Registry Number.
1.2 The method detection limits (MDLs) for the target analytes in
organic-free reagent water are listed in Table 1. The method may be applicable
to other matrices.
1.3 This method is restricted to use by or under the supervision of
analysts experienced in the use of high performance liquid chromatographs and
skilled in the interpretation of high performance liquid chromatograms. Each
analyst must demonstrate the ability to generate acceptable results with this
method.
2.0 SUMMARY OF METHOD
2.1 Water samples are analyzed by high performance liquid chromatography
(HPLC). A 200 juL aliquot is injected onto a C-18 reverse-phase column, and
compounds in the effluent are detected with an ultraviolet (UV) detector.
3.0 INTERFERENCES
3.1 Contamination by carryover can occur whenever high-concentration and
low-concentration samples are sequentially analyzed. To reduce carryover, the
sample syringe must be rinsed out between samples with solvent. Whenever an
unusually concentrated sample is encountered, it should be followed by the
analysis of solvent to check for cross contamination.
8316 - 1 Revision 0
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4.0 APPARATUS AND MATERIALS
4.1 HPLC system
4.1.1 One high pressure pump.
4.1.2 Octadecyl Silane (ODS, C-18) reverse phase HPLC column,
25 cm x 4.6 mm, 10 /xm, (Zorbax, or equivalent).
4.1.3 Variable wavelength UV detector.
4.1.4 Data system.
4.2 Other apparatus
4.2.1 Water degassing unit - 1 liter filter flask with stopper and
pressure tubing.
4.2.2 Analytical balance - + 0.0001 g.
4.2.3 Magnetic stirrer and magnetic stirring bar.
4.2.4 Sample filtration unit - syringe filter with 0.45 /zm filter
membrane, or equivalent disposable filter unit.
4.3 Materials
4.3.1 Syringes - 10, 25, 50 and 250 juL and 10 mL.
4.3.2 Volumetric pipettes, Class A, glass - 1, 5 and 10 mL.
4.3.3 Volumetric flasks - 5, 10, 50 and 100 mL.
4.3.4 Vials - 25 mL, glass with Teflon lined screw caps or crimp
tops.
5.0 REAGENTS
5.1 Reagent grade inorganic chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Acrylamide, CH2:CHCONH2, 99+% purity, electrophoresis reagent grade.
5.3 Acrylonitrile, H2C:CHCN, 99+% purity.
5.4 Acrolein, CH2:CHCHO, 99+% purity.
8316 - 2 Revision 0
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5.5 Organic-free reagent water. All references to water in this method
refer to organic-free reagent water, as defined in Chapter One. Sparge with He
to eliminate 02 to prevent significant absorption interference from 02 at the 195
nm wavelength.
5.6 Stock standard solutions - Can be prepared from pure standard
materials or can be purchased as certified solutions. Commercially prepared
stock standards can be used if they are certified by the manufacturer and
verified against a standard made from pure material.
5.6.1 Acrylamide
5.6.1.1 Weigh 0.0100 g of acrylamide neat standard into a
100 ml volumetric flask, and dilute to the mark with organic-free
reagent water. Calculate the concentration of the standard solution
from the actual weight used. When compound purity is assayed to be
96% or greater, the weight can be used without correction to
calculate the concentration of the stock standard.
5.6.1.2 Transfer the stock solution into vials with Teflon
lined screw caps or crimp tops. Store at 4°C, protected from light.
5.6.1.3 Stock solutions must be replaced after six months,
or sooner if comparison with the check standards indicates a
problem.
5.6.2 Acrylonitrile and Acrolein - Prepare separate stock solutions
for acrylonitrile and acrolein.
5.6.2.1 Place about 9.8 ml of organic-free reagent water
into a 10 ml volumetric flask before weighing the flask and stopper.
Weigh the flask and record the weight to the nearest 0.0001 g. Add
two drops of neat standard, using a 50 /xL syringe, to the flask.
The liquid must fall directly into the water, without contacting the
inside wall of the flask.
CAUTION: Acrylonitrile and acrolein are toxic. Standard
preparation should be performed in an laboratory
fume hood.
5.6.2.2 Stopper the flask and then reweigh. Dilute to
volume with organic-free reagent water. Calculate the concentration
from the net gain in weight. When compound purity is assayed to be
96% or greater, the weight can be used without correction to
calculate the concentration of the stock standard.
5.6.2.3 Stock solutions must be replaced after six months,
or sooner if comparison with the check standards indicates a
problem.
8316 - 3 Revision 0
September 1994
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5.7 Calibration standards
5.7.1 Prepare calibration standards at a minimum of five
concentrations by diluting the stock solutions with organic-free reagent
water.
5.7.2 One calibration standard should be prepared at a concentration
near, but above, the method detection limit; the remaining standards should
correspond to the range of concentrations found in real samples, but should not
exceed the working range of the HPLC system (1 mg/L to 10 mg/L).
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this chapter, Organic Analytes,
Sec. 4.1.
7.0 PROCEDURE
7.1 HPLC Conditions
Mobile Phase: Degassed organic-free reagent water
Injection Volume: 200 /iL
Flow Rate: 2.0 mL/min
Pressure: 38 atm
Temperature: 25°C
Detector UV wavelength: 195 nm
7.2 Calibration:
7.2.1 Prepare standard solutions of acrylamide as described in Sec.
5.7.1. Inject 200 /uL aliquots of each solution into the chromatograph.
See Method 8000 for additional guidance on calibration by the external
standard method.
7.3 Chromatographic analysis:
7.3.1 Analyze the samples using the same chromatographic conditions
used to prepare the standard curve. Suggested chromatographic conditions
are given in Sec. 7.1. Table 1 provides the retention times that were
obtained under these conditions during method development.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for specific quality control procedures.
8.2 Before processing any samples, the analyst must demonstrate, through
the analysis of a method blank, that all glassware and reagents are interference
free.
8316 - 4 Revision 0
September 1994
-------
9.0 METHOD PERFORMANCE
9.1 Method performance data are not available.
10.0 REFERENCES
1. Hayes, Sam; "Acrylamide, Acrylonitrile, and Acrolein Determination in
Water by High Pressure Liquid Chromatography," USEPA.
8316 - 5 Revision 0
September 1994
-------
TABLE 1
ANALYTE RETENTION TIMES AND METHOD DETECTION LIMITS
Retention MDL
Compound Time (min) (/^9/L)
Acrylamide 3.5 10
Acrylonitrile 8.9 20
Acrolein (Propenal) 10.1 30
8316 - 6 Revision 0
September 1994
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METHOD 8316
ACRYLAMIDE. ACRYLQNITRILE AND ACROLEIN BY HIGH PERFORMANCE
LIQUID CHRQMATOGRAPHY (HPLC)
7.1 Set by
HPLC
Conditions.
7.2 Calibrate
Chromatograph.
7.3
Chromatographic
analysis.
f Stop J
8316 - 7
Revision 0
September 1994
-------
00
u>
M
00
-------
METHOD 8318
N-METHYLCARBAMATES BY HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
1.0 SCOPE AND APPLICATION
1.1 Method 8318 is used to determine the concentration of
N-methylcarbamates in soil, water and waste matrices. The following compounds can
be determined by this method:
Compound Name CAS No.
a
Aldicarb (Temik) 116-06-3
Aldicarb Sulfone 1646-88-4
Carbaryl (Sevin) 63-25-2
Carbofuran (Furadan) 1563-66-2
Dioxacarb 6988-21-2
3-Hydroxycarbofuran 16655-82-6
Methiocarb (Mesurol) 2032-65-7
Methomyl (Lannate) 16752-77-5
Promecarb 2631-37-0
Propoxur (Baygon) 114-26-1
a Chemical Abstract Services Registry Number.
1.2 The method detection limits (MDLs) of Method 8318 for determining the
target analytes in organic-free reagent water and in soil are listed in Table 1.
1.3 This method is restricted to use by, or under the supervision of,
analysts experienced in the use of high performance liquid chromatography (HPLC)
and skilled in the interpretation of chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 N-methylcarbamates are extracted from aqueous samples with methylene
chloride, and from soils, oily solid waste and oils with acetonitrile. The
extract solvent is exchanged to methanol/ethylene glycol, and then the extract
is cleaned up on a C-18 cartridge, filtered, and eluted on a C-18 analytical
column. After separation, the target analytes are hydrolyzed and derivatized
post-column, then quantitated fluorometrically.
2.2 Due to the specific nature of this analysis, confirmation by a
secondary method is not essential. However, fluorescence due to post-column
derivatization may be confirmed by substituting the NaOH and o-phthalaldehyde
solutions with organic-free reagent water and reanalyzing the sample. If
8318 - 1 Revision 0
September 1994
-------
fluorescence is still detected, then a positive interference is present and care
should be taken in the interpretation of the results.
2.3 The sensitivity of the method usually depends on the level of
interferences present, rather than on the instrumental conditions. Waste samples
with a high level of extractable fluorescent compounds are expected to yield
significantly higher detection limits.
3.0 INTERFERENCES
3.1 Fluorescent compounds, primarily alkyl amines and compounds which
yield primary alkyl amines on base hydrolysis, are potential sources of
interferences.
3.2 Coeluting compounds that are fluorescence quenchers may result in
negative interferences.
3.3 Impurities in solvents and reagents are additional sources of
interferences. Before processing any samples, the analyst must demonstrate
daily, through the analysis of solvent blanks, that the entire analytical system
is interference free.
4.0 APPARATUS AND MATERIALS
4.1 HPLC system
4.1.1 An HPLC system capable of injecting 20 yL aliquots and
performing multilinear gradients at a constant flow. The system must also
be equipped with a data system to measure the peak areas.
4.1.2 C-18 reverse phase HPLC column, 25 cm x 4.6 mm (5 /im).
4.1.3 Post Column Reactor with two solvent delivery systems (Kratos
PCRS 520 with two Kratos Spectroflow 400 Solvent Delivery Systems, or
equivalent).
4.1.4 Fluorescence detector (Kratos Spectroflow 980, or equivalent).
4.2 Other apparatus
4.2.1 Centrifuge.
4.2.2 Analytical balance - + 0.0001 g.
4.2.3 Top loading balance - + 0.01 g.
4.2.4 Platform shaker.
4.2.5 Heating block, or equivalent apparatus, that can accommodate
10 mL graduated vials (Sec. 4.3.11).
8318 - 2 Revision 0
September 1994
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4.3 Materials
4.3.1 HPLC injection syringe - 50 juL.
4.3.2 Filter paper, (Whatman #113 or #114, or equivalent).
4.3.3 Volumetric pipettes, Class A, glass, assorted sizes.
p
4.3.4 Reverse phase cartridges, (C-18 Sep-Pak [Waters Associates],
or equivalent).
4.3.5 Glass syringes - 5 mL.
4.3.6 Volumetric flasks, Class A - Sizes as appropriate.
4.3.7 Erlenmeyer flasks with teflon-lined screw caps, 250 ml.
4.3.8 Assorted glass funnels.
4.3.9 Separatory funnels, with ground glass stoppers and teflon
stopcocks - 250 ml.
4.3.10 Graduated cylinders - 100 mL.
4.3.11 Graduated glass vials - 10 mL, 20 ml.
4.3.12 Centrifuge tubes - 25C ml.
4.3.13 Vials - 25 mL, glass with Teflon lined screw caps or
crimp tops.
4.3.14 Positive displacement micro-pipettor, 3 to 25 yl
displacement, (Gilson Microman [Rainin #M-25] with tips, [Rainin #CP-25],
or equivalent).
4.3.15 Nylon filter unit, 25 mm diameter, 0.45 /^m pore size,
disposable (Alltech Associates, #2047, or equivalent).
5.0 REAGENTS
5.1 HPLC grade chemicals shall be used in all tests. It is intended that
all reagents shall conform to the specifications of the Committee on Analytical
Reagents of the American Chemical Society, where such specifications are
available. Other grades may be used, provided it is first ascertained that the
reagent is of sufficiently high purity to permit its use without lowering the
accuracy of the determination.
5.2 General
5.2.1 Acetonitrile, CH3CN - HPLC grade - minimum UV cutoff at 203 nm
(EM Omnisolv #AX0142-1, or equivalent).
8318 - 3 Revision 0
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5.2.2 Methanol, CH3OH - HPLC grade - minimum UV cutoff at 230 nm (EM
Omni solv 0MX0488-1, or equivalent).
5.2.3 Methylene chloride, CH-Cl, - HPLC grade - minimum UV cutoff at
230 nm (EM Omnisolv #0X0831-1, or 'equivalent).
5.2.4 Hexane, CgH14 - pesticide grade - (EM Omnisolv #HX0298-1, or
equivalent).
5.2.5 Ethylene glycol, HOCH2CH2OH - Reagent grade - (EM Science, or
equivalent).
5.2.6 Organic-free reagent water - All references to water in this
method refer to organic-free reagent water, as defined in Chapter One.
5.2.7 Sodium hydroxide, NaOH - reagent grade - 0.05N NaOH solution.
5.2.8 Phosphoric acid, HgPO^ - reagent grade.
5.2.9 pH 10 borate buffer (J.T. Baker #5609-1, or equivalent).
5.2.10 o-Phthalaldehyde, o-CfiHd(CHO)? - reagent grade (Fisher
#0-4241, or equivalent). D 4
5.2.11 2-Mercaptoethanol, HSCH^CH^OH - reagent grade (Fisher
#0-3446, or equivalent).
5.2.12 N-methylcarbamate neat standards (equivalence to EPA
standards must be demonstrated for purchased solutions).
5.2.13 Chloroacetic acid, CICHgCOOH, 0.1 N.
5.3 Reaction solution
5.3.1 Dissolve 0.500 g of o-phthalaldehyde in 10 mL of methanol, in
all volumetric flask. To this solution, add 900 ml of organic-free
reagent water, followed by 50 ml of the borate buffer (pH 10). After
mixing well, add 1 ml of 2-mercaptoethanol, and dilute to the mark with
organic-free reagent water. Mix the solution thoroughly. Prepare fresh
solutions on a weekly basis, as needed. Protect from light and store
under refrigeration.
5.4 Standard solutions
5.4.1 Stock standard solutions: prepare individual 1000 mg/L
solutions by adding 0.025 g of carbamate to a 25 ml volumetric flask, and
diluting to the mark with methanol. Store solutions, under refrigeration,
in glass vials with Teflon lined screw caps or crimp tops. Replace every
six months.
5.4.2 Intermediate standard solution: prepare a mixed 50.0 mg/L
solution by adding 2.5 mL of each stock solution to a 50 mL volumetric
flask, and diluting to the mark with methanol. Store solutions, under
8318 - 4 Revision 0
September 1994
-------
refrigeration, in glass vials with Teflon lined screw caps or crimp tops.
Replace every three months.
5.4.3 Working standard solutions: prepare 0.5, 1.0, 2.0, 3.0 and 5.0
mg/L solutions by adding 0.25, 0.5, 1.0, 1.5 and 2.5 mL of the
intermediate mixed standard to respective 25 ml volumetric flasks, and
diluting each to the mark with methanol. Store solutions, under
refrigeration, in glass vials with Teflon lined screw caps or crimp tops.
Replace every two months, or sooner if necessary.
5.4.4 Mixed QC standard solution: prepare a 40.0 mg/L solution from
another set of stock standard solutions, prepared similarly to those
described in Sec. 5.4.1. Add 2.0 ml of each stock solution to a 50 mL
volumetric flask and dilute to the mark with methanol. Store the
solution, under refrigeration, in a glass vial with a Teflon lined screw
cap or crimp top. Replace every three months.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 Due to the extreme instability of N-methylcarbamates in alkaline
media, water, waste water and leachates should be preserved immediately after
collection by acidifying to pH 4-5 with 0.1 N chloroacetic acid.
6.2 Store samples at 4'C and out of direct sunlight, from the time of
collection through analysis. N-methylcarbamates are sensitive to alkaline
hydrolysis and heat.
6.3 All samples must be extracted within seven days of collection, and
analyzed within 40 days of extraction.
7.0 PROCEDURE
7.1 Extraction
7.1.1 Water, domestic wastewater, aqueous industrial wastes, and
leachates
7.1.1.1 Measure 100 mL of sample into a 250 mL separatory
funnel and extract by shaking vigorously for about 2 minutes with 30
mL of methylene chloride. Repeat the extraction two more times.
Combine all three extracts in a 100 mL volumetric flask and dilute
to volume with methylene chloride. If cleanup is required, go to
Sec. 7.2. If cleanup is not required, proceed directly to Sec.
7.3.1.
7.1.2 Soils, solids, sludges, and heavy aqueous suspensions
7.1.2.1 Determination of sample % dry weight - In certain
cases, sample results are desired based on dry-weight basis. When
such data is desired, a portion of sample for this determination
should be weighed out at the same time as the portion used for
analytical determination.
8318 - 5 Revision 0
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WARNING: The drying oven should be contained in a hood or
vented. Significant laboratory contamination may
result from a heavily contaminated hazardous
waste sample.
7.1.2.1.1 Immediately after weighing the sample for
extraction, weigh 5-10 g of the sample into a tared crucible.
Determine the % dry weight of the sample by drying overnight
at 105°C. Allow to cool in a desiccator before weighing:
% dry weight = q of dry sample x 100
g of sample
7.1.2.2 Extraction - Weigh out 20 + 0.1 g of sample into
a 250 ml Erlenmeyer flask with a Teflon-lined screw cap. Add 50 ml
of acetonitrile and shake for 2 hours on a platform shaker. Allow
the mixture to settle (5-10 min), then decant the extract into a 250
ml centrifuge tube. Repeat the extraction two more times with 20 ml
of acetonitrile and 1 hour shaking each time. Decant and combine
all three extracts. Centrifuge the combined extract at 200 rpm for
10 min. Carefully decant the supernatant into a 100 ml volumetric
flask and dilute to volume with acetonitrile. (Dilution factor = 5)
Proceed to Sec. 7.3.2.
7.1.3 Soils heavily contaminated with non-aqueous substances, such
as oils
7.1.3.1 Determination of sample % dry weight - Follow
Sees. 7.1.2.1 through 7.1.2.1.1.
7.1.3.2 Extraction - Weigh out 20 + 0.1 g of sample into
a 250 ml Erlenmeyer flask with a Teflon-lined screw cap. Add 60 ml
of hexane and shake for 1 hour on a platform shaker. Add 50 ml of
acetonitrile and shake for an additional 3 hours. Allow the mixture
to settle (5-10 min), then decant the solvent layers into a 250 ml
separatory funnel. Drain the acetonitrile (bottom layer) through
filter paper into a 100 ml volumetric flask. Add 60 ml of hexane and
50 ml of acetonitrile to the sample extraction flask and shake for
1 hour. Allow the mixture to settle, then decant the mixture into
the separatory funnel containing the hexane from the first
extraction. Shake the separatory funnel for 2 minutes, allow the
phases to separate, drain the acetonitrile layer through filter
paper into the volumetric flask, and dilute to volume with
acetonitrile. (Dilution factor = 5) Proceed to Sec. 7.3.2.
7.1.4 Non-aqueous liquids such as oils
7.1.4.1 Extraction - Weigh out 20 + 0.1 g of sample into
a 125 ml separatory funnel. Add 40 mL of hexane and 25 ml of
acetonitrile and vigorously shake the sample mixture for 2 minutes.
Allow the phases to separate, then drain the acetonitrile (bottom
layer) into a 100 ml volumetric flask. Add 25 ml of acetonitrile to
the sample funnel, shake for 2 minutes, allow the phases to
8318 - 6 Revision 0
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Repeat the extraction with another 25 ml portion of acetonitrile,
combining the extracts. Dilute to volume with acetonitrile.
(Dilution factor = 5). Proceed to Sec. 7.3.2.
7.2 Cleanup - Pipet 20.0 ml of the extract into a 20 ml glass vial
containing 100 /iL of ethylene glycol. Place the vial in a heating block set at
50 C, and gently evaporate the extract under a stream of nitrogen (in a fume
hood) until only the ethylene glycol keeper remains. Dissolve the ethylene
glycol residue in 2 ml of methanol, pass the extract through a pre-washed C-18
reverse phase cartridge, and collect the eluate in a 5 ml volumetric flask.
Elute the cartridge with methanol, and collect the eluate until the final volume
of 5.0 ml is obtained. (Dilution factor = 0.25) Using a disposable 0.45 urn
filter, filter an aliquot of the clean extract directly into a properly labelled
autosampler vial. The extract is now ready for analysis. Proceed to Sec. 7.4.
7.3 Solvent Exchange
7.3.1 Water, domestic wastewater, aqueous industrial wastes, and
leachates:
Pipet 10.0 ml of the extract into a 10 ml graduated glass vial
containing 100 juL of ethylene glycol. Place the vial in a heating block
set at 50 C, and gently evaporate the extract under a stream of nitrogen
(in a fume hood) until only the ethylene glycol keeper remains. Add
methanol to the ethylene glycol residue, dropwise, until the total volume
is 1.0 ml. (Dilution factor = 0.1). Using a disposable 0.45 jum filter,
filter this extract directly into a properly labelled autosampler vial.
The extract is now ready for analysis. Proceed to Sec. 7.4.
7.3.2 Soils, solids, sludges, heavy aqueous suspensions, and non-
aqueous liquids:
Elute 15 ml of the acetonitrile extract through a C-18 reverse phase
cartridge, prewashed with 5 ml of acetonitrile. Discard the first 2 ml of
eluate and collect the remainder. Pipet 10.0 ml of the clean extract into
a 10 ml graduated glass vial containing 100 /zL of ethylene glycol. Place
the vial in a heating block set at 50° C, and gently evaporate the extract
under a stream of nitrogen (in a fume hood) until only the ethylene glycol
keeper remains. Add methanol to the ethylene glycol residue, dropwise,
until the total volume is 1.0 ml. (Additional dilution factor = 0.1;
overall dilution factor = 0.5). Using a disposable 0.45 /urn filter, filter
this extract directly into a properly labelled autosampler vial. The
extract is now ready for analysis. Proceed to Sec. 7.4.
7.4 Sample Analysis
7.4.1 Analyze the samples using the chromatographic conditions,
post-column reaction parameters and instrument parameters given in Sees.
7.4.1.1, 7.4.1.2, 7.4.1.3 and 7.4.1.4. Table 2 provides the retention
times that were obtained under these conditions during method development.
A chromatogram of the separation is shown in Figure 1.
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7.4.1.1 Chromatographic Conditions (Recommended)
Solvent A: Organic-free reagent water, acidified with
0.4 mL of phosphoric acid per liter of
water
Solvent B: Methanol/acetonitrile (1:1, v/v)
Flow rate: 1.0 mL/min
Injection Volume: 20 ^L
Solvent delivery system program:
Function
FR
B%
BX
BX
BX
B%
BX
ALARM
Value
1.0
10%
80%
100%
100%
10%
10%
Duration
jmin)
20
5
5
3
7
0.01
File
0
0
0
0
0
0
0
0
7.4.1.2 Post-column Hydrolysis Parameters (Recommended)
Solution:
Flow Rate:
Temperature:
Residence Time:
0.05 N aqueous sodium hydroxide
0.7 mL/min
&
7.4.1.3
(Recommended)
Solution:
35 seconds (1 mL reaction coil)
Post-column Derivatization Parameters
Flow Rate:
Temperature:
Residence time:
o-phthalaldehyde/2-mercaptoethanol
5.3.1)
0.7 mL/min
40b C
25 seconds (1 mL reaction coil)
(Sec.
7.4.1.4
Fluorometer Parameters (Recommended)
Cell: 10 Mi-
Excitation wavelength: 340 nm
Emission wavelength: 418 nm cutoff filter
Sensitivity wavelength: 0.5 /uA
PMT voltage: -800 V
Time constant: 2 sec
7.4.2 If the peak areas of the sample signals exceed the calibration
range of the system, dilute the extract as necessary and reanalyze the
diluted extract.
8318 - 8
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7.5 Calibration:
7.5.1 Analyze a solvent blank (20 IJ.L of methanol) to ensure that the
system is clean. Analyze the calibration standards (Sec. 5.4.3), starting
with the 0.5 mg/L standards and ending with the 5.0 mg/L standard. If the
percent relative standard deviation (%RSD) of the mean response factor
(RF) for each analyte does not exceed 20%, the system is calibrated and
the analysis of samples may proceed. If the %RSD for any analyte exceeds
20%, recheck the system and/or recalibrate with freshly prepared
calibration solutions.
7.5.2 Using the established calibration mean response factors, check
the calibration of the instrument at the beginning of each day by
analyzing the 2.0 mg/L mixed standard. If the concentration of each
analyte falls within the range of 1.70 to 2.30 mg/L (i.e., within + 15% of
the true value), the instrument is considered to be calibrated and the
analysis of samples may proceed. If the observed value of any analyte
exceeds its true value by more than + 15%, the instrument must be
recalibrated (Sec. 7.5.1).
7.5.3 After 10 sample runs, or less, the 2.0 mg/L standards must be
analyzed to ensure that the retention times and response factors are still
within acceptable limits. Significant variations (i.e., observed
concentrations exceeding the true concentrations by more than + 15%) may
require a re-analysis of the samples.
7.6 Calculations
7.6.1 Calculate each response factor as follows (mean value based on
5 points):
RF
concentration of standard
area of the signal
5
(Z RF.)
i
mean RF = RF =
[(I RFi - RF)2]1/2 / 4
%RSD of RF =
X 100%
RF
7.6.2 Calculate the concentration of each N-methylcarbamate as
follows:
jug/g or mg/L = (RF) (area of signal) (dilution factor)
8318 - 9
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8.0 QUALITY CONTROL
8.1 Before processing any samples, the analyst must demonstrate, through
the analysis of a method blank for each matrix type, that all glassware and
reagents are interference free. Each time there is a change of reagents, a
method blank must be processed as a safeguard against laboratory contamination.
8.2 A QC check solution must be prepared and analyzed with each sample
batch that is processed. Prepare this solution, at a concentration of 2.0 mg/L
of each analyte, from the 40.0 mg/L mixed QC standard solution (Sec. 5.4.4). The
acceptable response range is 1.7 to 2.3 mg/L for each analyte.
8.3 Negative interference due to quenching may be examined by spiking the
extract with the appropriate standard, at an appropriate concentration, and
examining the observed response against the expected response.
8.4 Confirm any detected analytes by substituting the NaOH and OPA
reagents in the post column reaction system with deionized water, and reanalyze
the suspected extract. Continued fluorescence response will indicate that a
positive interference is present (since the fluorescence response is not due to
the post column derivatization). Exercise caution in the interpretation of the
chromatogram.
9.0 METHOD PERFORMANCE
9.1 Table 1 lists the single operator method detection limit (MDL) for
each compound in organic-free reagent water and soil. Seven/ten replicate
samples were analyzed, as indicated in the table. See reference 7 for more
details.
9.2 Tables 2, 3 and 4 list the single operator average recoveries and
standard deviations for organic-free reagent water, wastewater and soil. Ten
replicate samples were analyzed at each indicated spike concentration for each
matrix type.
9.3 The method detection limit, accuracy and precision obtained will be
determined by the sample matrix.
10.0 REFERENCES
1. California Department of Health Services, Hazardous Materials Laboratory,
"N-Methylcarbamates by HPLC", Revision No. 1.0, September 14, 1989.
2. Krause, R.T. Journal of Chromatographic Science, 1978, vol. 16, pg 281.
3. Klotter, Kevin, and Robert Cunico, "HPLC Post Column Detection of
Carbamate Pesticides", Varian Instrument Group, Walnut Creek, CA 94598.
4. USEPA, "Method 531. Measurement of N-Methylcarbomyloximes and N-
Methylcarbamates in Drinking Water by Direct Aqueous Injection HPLC with
Post Column Derivatization", EPA 600/4-85-054, Environmental Monitoring
and Support Laboratory, Cincinnati, OH 45268.
8318 - 10 Revision 0
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5. USEPA, "Method 632. The Determination of Carbamate and Urea Pesticides in
Industrial and Municipal Wastewater", EPA 600/4-21-014, Environmental
Monitoring and Support Laboratory, Cincinnati, OH 45268.
6. Federal Register, "Appendix B to Part 136 - Definition and Procedure for
the Determination of the Method Detection Limit - Revision 1.11", Friday,
October 26, 1984, 49, No. 209, 198-199.
7. Okamoto, H.S., D. Wijekoon, C. Esperanza, J. Cheng, S. Park, J. Garcha, S.
Gill, K. Perera "Analysis for N-Methylcarbamate Pesticides by HPLC in
Environmental Samples", Proceedings of the Fifth Annual USEPA Symposium on
Waste Testing and Quality Assurance, July 24-28, 1989, Vol. II, 57-71.
8318 - 11 Revision 0
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TABLE 1
ELUTION ORDER, RETENTION TIMES3 AND
SINGLE OPERATOR METHOD DETECTION LIMITS
Method Detection Limits
Compound
Aldicarb Sulfone
Methomyl (Lannate)
3-Hydroxycarbofuran
Dioxacarb
Aldicarb (Temlk)
Propoxur (Baygon)
Carbofuran (Furadan)
Carbaryl (Sevin)
a-Naphthol°
Methiocarb (Mesurol)
Promecarb
a See Sec. 7.4 for
Mill •f r\v> r\ v* si *\ m. A f*
Retention
Time
(min)
9.59
9.59
12.70
13.50
16.05
18.06
18.28
19.13
20.30
22.56
23.02
chromatographic
_ •Pv*rt/\ v» rt 11 /f Q r» 4* t*
Organic-free
Reagent Water
(MA)
1.9C
1.7
2.6
2.2r
9.4C
2.4
2.0
1.7
-
3.1
2.5
conditions
ira-f- n v c a rtf\ cr\ i 1 UIQV^O
Soil
UgAg)
44C
12c
loj
>50C
12C
17
22
31
-
32
17
Af^i- avm -i nar\
c
d
analyzing 10 low concentration spike replicate for each matrix type
(except where noted). See reference 7 for more details.
MDL determined by analyzing 7 spiked replicates.
Breakdown product of Carbaryl.
8318 - 12
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TABLE 2
SINGLE OPERATOR AVERAGE RECOVERY AND
PRECISION DATAa FOR ORGANIC-FREE REAGENT WATER
Compound
Aldicarb Sulfone
Methomyl (Lannate)
3-Hydroxycarbofuran
Dioxacarb
Aldicarb (Temik)
Propoxur (Baygon)
Carbofuran (Furadan)
Carbaryl (Sevin)
Methiocarb (Mesurol }
Promecarb
Recovered
225
244
210
241
224
232
239
242
231
111
% Recovery
75.0
81.3
70.0
80.3
74.7
77.3
79.6
80.7
77.0
75.7
SD
7.28
8.34
7.85
8.53
13.5
10.6
9.23
8.56
8.09
9.43
%RSD
3.24
3.42
3.74
3.54
6.03
4.57
3.86
3.54
3.50
4.1
Spike Concentration = 300 /xg/L of each compound, n = 10
8318 - 13 Revision 0
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TABLE 3
SINGLE OPERATOR AVERAGE RECOVERY AND
PRECISION DATA* FOR WASTEWATER
Compound
Aldicarb Sulfone
Methomyl (Lannate)
3-Hydroxycarbofuran
Dioxacarb
Aldicarb (Temik)
Propoxur (Baygon)
Carbofuran (Furadan)
Carbaryl (Sevin)
Methiocarb (Mesurol)
Promecarb
a Spike Concentration
** \t\f\ u*r*f+r\\ti~\\fi%\t
Recovered
235
247
2ll
u
258
263
262
262
254
263
= 300 jug/L of each
% Recovery
78.3
82.3
83.7
-
86.0
87.7
87.3
87.3
84.7
87.7
compound, n = 10
SD
17.6
29.9
25.4
-
16.4
16.7
15.7
17.2
19.9
15.1
%RSD
7.49
12.10
10.11
-
6.36
6.47
5.99
6.56
7.83
5.74
8318 - 14
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TABLE 4
SINGLE OPERATOR AVERAGE RECOVERY AND
PRECISION DATA3 FOR SOIL
Compound
Aldicarb Sulfone
Methomyl (Lannate)
3-Hydroxycarbofuran
Dioxacarb
Aldicarb (Temik)
Propoxur (Baygon)
Carbofuran (Furadan)
Carbaryl (Sevin)
Methiocarb (Mesurol)
Promecarb
Recovered
1.57
1.48
1.60
1.51
1.29
1.33
1.46
1.53
1.45
1.29
% Recovery
78.5
74.0
80.0
75.5
64.5
66.5
73.0
76.5
72.5
64.7
SD
0.069
0.086
0.071
0.073
0.142
0.126
0.092
0.076
0.071
0.124
%RSD
4.39
5.81
4.44
4.83
11.0
9.47
6.30
4.90
4.90
9.61
Spike Concentration = 2.00 mg/kg of each compound, n = 10
8318 - 15
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FIGURE 1
100
R
E
S
P
0
N
S
E
12
TIME (HIM)
1.00 jug/raL EACH OF:
1. ALDICARB SULFONE
2. METHOMYL
3. 3-HYDROXYCARBOFURAN
4. DIOXACARB
5. ALDICARB
24
6. PROPOXUR
7. CARBOFURAN
8. CARBARYL
9. METHIOCARB
10. PROMECARB
30
8318 - 16
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METHOD 8318
N-METHYLCARBAMATES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
7.1 Extraction
71 1 Water domestic
wastewater. aqueous
industrial wastes and
leachates.
1 Extract 100 ml sample
w/30 ml MeCI 3x in sep.
funnel: combine extracts in
lOOmLvol Bask and dilute
to mark
No
7.2 Cleanup
Combine 20 mL extract
and 100 uL ethytene glycol
in a glass vial: blowdown
mixture w/N2 in heating
block set at 50 C. dissolve
residue in 2 mL MeOH.
pass soln through pro
washed C18 cartridge, collect
plute in 5 ml vol flask, elule
cartridge w/MeOH into vol fla
up to mark, tiller MeOH soln
through 0 45 um titter into
aulosample vial
7 1.2 Soils, solids, sludges, and heavy
aqueous suspensions
1 Determine % dry wt:
1 Weigh 5 10 gr sample into crucible:
oven dry overnight at 105 C. cool in
dessicator: reweigh
2 Extraction
Weigh 20 g sample into 250 ml
Ertenmeyer; add 50 ml acetonilrile.
shake for 2 hrs.: decant extract into
centrifuge tube: repeat extraction 2x
w/20 ml acetonitrile. shake 1 hr.
combine extracts and centrifuge
10 mins at 200 rpm. decant supernatant
to 100 ml vol flask and dilute to mark
:t
lycd
wn
ofve
1.
»•
.collect
. elute
) vol flask
Hsoln
into
1
7.3 Solvent Exchange
7.3 1 Water, domestic, wastewater.
aqueous industrial wastes.
and leachates Combine
10 mL extract and 100uL
ethylene glycol in a glass
vial: blowdown mixture w/N2
in heating block at 50 C: add
MeOH to residue to total
volume of 1 mL. filler
MeOH soln through 045 um
filter into autosampler vial
713 Soils heavily contaminated with
non-aqueous substances, such as oils
1 Determine % dry wt Follow Section 7 1 2 1
2 Extraction: Weigh 20 gr sample into 250 ml
Erlenmeyer: add 60 mL hexane. shake
I hr: add 50 mL acenlonitrile. shake
3 hrs : let settle, decant extract layers
to 250 mL sep funnel: filter bottom
acelonitrile layer into too mL vol flask:
repeat sample flask extraction w/same
volumes, decant extract layers on top of
first hexane layer: shake funnel, filter bottom
layer into vol flask: dilute to mark
7 1 4 Non aqueous liquids such as oils
1 Extraction Weigh 20 gr sample into
125 mL sep funnel, add 40 mL
hexane and 25 mL acetonitrile. shake.
sprtle and drain bottom acetonilrile
layer into 100 ml vol flask: repeat
extraction 2x by adding 25 mL
acetonitrile to initial flask mix.
combine acetoniliile layers into vol
flask: dilute to mark
73 Solvent Exchange
732 Soils, solids, sludges, heavy
aqueous suspensions, and non-
aqueous liquids Elule 15 mL extract
through acetonitrile prpwashed C16
cartridge, collect latter 13 mL: combine
10 mL cleaned extract and 100 uL
ethylnne glycol in glass vial: blowdown
mixture w/N2 in heating block at
50 C: add MeOH to residue to
total volume of 1 mL: filter MtOH
soln through 0 45 um filter into
autosampler vial
8318 - 17
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September 1994
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METHOD 8318
(continued)
| 7 4 Sample Analysis
*
7 41 Initialize Instrumentation
f Set chromatographic parameters
.2 Set Post-column Hydrolysis parameters
3 Set Post-column Derivatization parameters
.4 Set Fluorometer parameters
7 4.2 Dilute sample extract and reanalyze if
calibrator! range is exceeded
75 Calibration
7.5.1 Analyze a solvent blank then the calibration
stds of Section 54 3: ensure hat %RSD of
each analyte respxxise factor (RF) is <20%;
recheck system and recalibrate w/fresh
sdns il %RSD > 20%
7.5 2 Check calibration daily w/2 ug/ml std :
ensure that indrvickial analyte cones ta'll
w/in w- 15% ot true value; recalibrate
if observed difference? 15%
753 Check calibration every 10 samples or less
w/2 ug/mL sld : variations > 15% may
require re-analysis ot samples
-»•{ 7 6 Calculations |
761 Calculate response factors and % RSD
according to equation
762 Calculate sample analyte cones according
to equation
8318 - 18
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00
-------
METHOD 8321
SOLVENT EXTRACTABLE NON-VOLATILE COMPOUNDS BY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/THERMOSPRAY/MASS SPECTRQMETRY
(HPLC/TSP/MS) OR ULTRAVIOLET (UV) DETECTION
1.0 SCOPE AND APPLICATION
1.1 This method covers the use of high performance liquid chromatography
(HPLC), coupled with either thermospray-mass spectrometry (TSP-MS), and/or
ultraviolet (UV), for the determination of disperse azo dyes, organophosphorus
compounds, and Tris-(2,3-dibromopropyl)phosphate in wastewater, ground water,
sludge, and soil/sediment matrices, and chlorinated phenoxyacid compounds and
their esters in wastewater, ground water, and soil/sediment matrices. Data are
also provided for chlorophenoxy acid herbicides in fly ash (Table 15), however,
recoveries for most compounds are very poor indicating poor extraction efficiency
for these analytes using the extraction procedure included in this method.
Additionally, this method may apply to other non-volatile compounds that are
solvent extractable, are amenable to HPLC, and are ionizable under thermospray
introduction for mass spectrometric detection. The following compounds can be
determined by this method:
Compound Name
CAS No.'
Azo Dyes
Disperse Red 1
Disperse Red 5
Disperse Red 13
Disperse Yellow 5
Disperse Orange 3
Disperse Orange 30
Disperse Brown 1
Solvent Red 3
Solvent Red 23
Anthraquinone Dyes
Disperse Blue 3
Disperse Blue 14
Disperse Red 60
Coumarin Dyes
(Fluorescent Briqhteners)
Fluorescent Brightener 61
Fluorescent Brightener 236
Alkaloids
Caffeine
Strychnine
2872-
3180-
2832-
6439-
730-
5261-
17464-
6535-
85-
52-8
81-2
40-8
53-8
40-5
31-4
91-4
42-8
86-9
2475-46-9
2475-44-7
17418-58-5
8066-05-5
63590-17-0
58-08-2
57-24-9
8321 - 1
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Compound Name CAS No.'
Organophosphorus Compounds
Methomyl 16752-77-5
Thiofanox 39196-18-4
Famphur 52-85-7
Asulam 3337-71-1
Dichlorvos 62-73-7
Dimethoate 60-51-5
Disulfoton 298-04-4
Fensulfothion 115-90-2
Merphos 150-50-5
Methyl parathion 298-00-0
Monocrotophos 919-44-8
Naled 300-76-5
Phorate 298-02-2
Trichlorfon 52-68-6
Tris-(2,3-Dibromopropyl) phosphate, (Tris-BP) 126-72-7
Chlorinated Phenoxvacid Compounds
Dalapon 75-99-0
Dicamba 1918-00-9
2,4-0 94-75-7
MCPA 94-74-6
MCPP 7085-19-0
Dichlorprop 120-36-5
2,4,5-T 93-76-5
Silvex (2,4,5-TP) 93-72-1
Dinoseb 88-85-7
2,4-DB 94-82-6
2,4-D, butoxyethanol ester 1929-73-3
2,4-D, ethylhexyl ester 1928-43-4
2,4,5-T, butyl ester 93-79-8
2,4,5-T, butoxyethanol ester 2545-59-7
0 Chemical Abstract Services Registry Number.
1.2 This method may be applicable to the analysis of other non-volatile
or semivolatile compounds.
1.3 Tris-BP has been classified as a carcinogen. Purified standard
material and stock standard solutions should be handled in a hood.
1.4 Method 8321 is designed to detect the chlorinated phenoxyacid
compounds (free acid form) and their esters without the use of hydrolysis and
esterification in the extraction procedure.
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1.5 The compounds were chosen for analysis by HPLC/MS because they have
been designated as problem compounds that are hard to analyze by traditional
chromatographic methods (e.g. gas chromatography). The sensitivity of this
method is dependent upon the level of interferants within a given matrix, and
varies with compound class and even with compounds within that class.
Additionally, the limit of detection (LOD) is dependent upon the mode of
operation of the mass spectrometer. For example, the LOD for caffeine in the
selected reaction monitoring (SRM) mode is 45 pg of standard injected (10 /iL
injection), while for Disperse Red 1 the LOD is 180 pg. The LOD for caffeine
under single quadrupole scanning is 84 pg and is 600 pg for Disperse Red 1 under
similar scanning conditions.
1.6 The experimentally determined limits of detection (LOD) for the
target analytes are presented in Tables 3, 10, 13, and 14. For further compound
identification, MS/MS (CAD - collision activated dissociation) can be used as an
optional extension of this method.
1.7 This method is restricted to use by or under the supervision of
analysts experienced in the use of high performance liquid chromatographs/mass
spectrometers and skilled in the interpretation of liquid chromatograms and mass
spectra. Each analyst must demonstrate the ability to generate acceptable
results with this method.
2.0 SUMMARY OF METHOD
2.1 This method provides reverse phase high performance liquid
chromatographic (RP/HPLC) and thermospray (TSP) mass spectrometric (MS)
conditions for the detection of the target analytes. Quantitative analysis is
performed by TSP/MS, using an external standard approach. Sample extracts can
be analyzed by direct injection into the thermospray or onto a liquid
chromatographic-thermospray interface. A gradient elution program is used on the
chromatograph to separate the compounds. Detection is achieved both by negative
ionization (discharge electrode) and positive ionization, with a single
quadrupole mass spectrometer. Since this method is based on an HPLC technique,
the use of ultraviolet (UV) detection is optional on routine samples.
2.2 Prior to the use of this method, appropriate sample preparation
techniques must be used.
2.2.1 Samples for analysis of chlorinated phenoxyacid compounds are
prepared by a modification of Method 8151 (see Sec. 7.1.2). In general,
one liter of aqueous sample or fifty grams of solid sample are pH
adjusted, extracted with diethyl ether,- concentrated and solvent exchanged
to acetonitrile.
2.2.2 Samples for analysis of the other target analytes are prepared
by established extraction techniques. In general, water samples are
extracted at a neutral pH with methylene chloride, using a separatory
funnel (Method 3510) or a continuous liquid-liquid extractor (Method
3520). Soxhlet (Methods 3540/3541) or ultrasonic (Method 3550) extraction
using methylene chloride/acetone (1:1) is used for solid samples. A
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micro-extraction technique is included for the extraction of Tris-BP from
aqueous and non-aqueous matrices.
2.3 An optional thermospray-mass spectrometry/mass spectrometry
(TS-MS/MS) confirmatory method is provided. Confirmation is obtained by using
MS/MS collision activated dissociation (CAD) or wire-repeller CAD.
3.0 INTERFERENCES
3.1 Refer to Methods 3500, 3600, 8000 and 8150/8151.
3.2 The use of Florisil Column Cleanup (Method 3620) has been
demonstrated to yield recoveries less than 85% for some of the compounds in this
method, and is therefore not recommended for all compounds. Refer to Table 2 of
Method 3620 for recoveries of organophosphorus compounds as a function of
Florisil fractions.
3.3 Compounds with high proton affinity may mask some of the target
analytes. Therefore, an HPLC must be used as a chromatographic separator, for
quantitative analysis.
3.4 Analytical difficulties encountered with specific organophosphorus
compounds, as applied in this method, may include (but are not limited to) the
following:
3.4.1 Methyl parathion shows some minor degradation upon analysis.
3.4.2 Naled can undergo debromination to form dichlorvos.
3.4.3 Merphos often contains contamination from merphos oxide.
Oxidation of merphos can occur during storage, and possibly upon
introduction into the mass spectrometer.
Refer to Method 8141 for other compound problems as related to the
various extraction methods.
3.5 The chlorinated phenoxy acid compounds, being strong organic acids,
react readily with alkaline substances and may be lost during analysis.
Therefore, glassware and glass wool must be acid-rinsed, and sodium sulfate must
be acidified with sulfuric acid prior to use to avoid this possibility.
3.6 Due to the reactivity of the chlorinated herbicides, the standards
must be prepared in acetonitrile. Methylation will occur if prepared in
methanol.
3.7 Solvents, reagents, glassware, and other sample processing hardware
may yield discrete artifacts or elevated baselines, or both, causing
misinterpretation of chromatograms or spectra. All of these materials must be
demonstrated to be free from interferences under the conditions of the analysis
by running reagent blanks. Specific selection of reagents and purification of
solvents by distillation in all-glass systems may be required.
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3.8 Interferants co-extracted from the sample will vary considerably
from source to source. Retention times of target analytes must be verified by
using reference standards.
3.9 The optional use of HPLC/MS/MS methods aids in the confirmation of
specific analytes. These methods are less subject to chemical noise than other
mass spectrometric methods.
4.0 APPARATUS AND MATERIALS
4.1 HPLC/MS
4.1.1 High Performance Liquid Chromatograph (HPLC) - An analytical
system with programmable solvent delivery system and all required
accessories including 10 /zL injection loop, analytical columns, purging
gases, etc. The solvent delivery system must be capable, at a minimum, of
a binary solvent system. The chromatographic system must be capable of
interfacing with a Mass Spectrometer (MS).
4.1.1.1 HPLC Post-Column Addition Pump - A pump for post-
column addition should be used. Ideally, this pump should be a
syringe pump, and does not have to be capable of solvent
programming.
4,1.1.2 Recommended HPLC Columns - A guard column and an
analytical column are required.
4.1.1.2.1 Guard Column - C18 reverse phase guard
column, 10 mm x 2.6 mm ID, 0.5 ^m frit, or equivalent.
4.1.1.2.2 Analytical Column - C18 reverse phase
column, 100 mm x 2 mm ID, 5 ^m particle size of ODS-Hypersil;
or C8 reversed phase column, 100 mm x 2 mm 10, 3 jum particle
size of MOS2-Hypersil, or equivalent.
4.1.2 HPLC/MS interface(s)
4.1.2.1 Micromixer - 10 ^L, interfaces HPLC column system
with HPLC post-column addition solvent system.
4.1.2.2 Interface - Thermospray ionization interface and
source that will give acceptable calibration response for each
analyte of interest at the concentration required. The source must
be capable of generating both positive and negative ions, and have
a discharge electrode or filament.
4.1.3 Mass spectrometer system - A single quadrupole mass
spectrometer capable of scanning from 1 to 1000 amu. The spectrometer
must also be capable of scanning from 150 to 450 amu in 1.5 sec or less,
using 70 volts (nominal) electron energy in the positive or negative
electron impact modes. In addition, the mass spectrometer must be capable
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of producing a calibrated mass spectrum for PEG 400, 600, or 800 (see Sec.
5.14).
4.1.3.1 Optional triple quadrupole mass spectrometer -
capable of generating daughter ion spectra with a collision gas in
the second quadrupole and operation in the single quadrupole mode.
4.1.4 Data System - A computer system that allows the continuous
acquisition and storage on machine-readable media of all mass spectra
obtained throughout the duration of the chromatographic program must be
interfaced to the mass spectrometer. The computer must have software that
allows any MS data file to be searched for ions of a specified mass, and
such ion abundances to be plotted versus time or scan number. This type
of plot is defined as an Extracted Ion Current Profile (EICP). Software
must also be available that allows integration of the abundances in any
EICP between specified time or scan-number limits. There must be computer
software available to operate the specific modes of the mass spectrometer.
4.2 HPLC with UV detector - An analytical system with solvent
programmable pumping system for at least a binary solvent system, and all
required accessories including syringes, 10 /uL injection loop, analytical
columns, purging gases, etc. An automatic injector is optional, but is useful
for multiple samples. The columns specified in Sec. 4.1.1.2 are also used with
this system.
4.2.1 If the UV detector is to be used in tandem with the
thermospray interface, then the detector cell must be capable of
withstanding high pressures (up to 6000 psi). However, the UV detector
may be attached to an HPLC independent of the HPLC/TS/MS and, in that
case, standard HPLC pressures are acceptable.
4.3 Purification Equipment for Azo Dye Standards
4.3.1 Soxhlet extraction apparatus.
4.3.2 Extraction thimbles, single thickness, 43 x 123 mm.
4.3.3 Filter paper, 9.0 cm (Whatman qualitative No. 1 or
equivalent).
4.3.4 Silica-gel column - 3 in. x 8 in., packed with Silica gel
(Type 60, EM reagent 70/230 mesh).
4.4 Extraction equipment for Chlorinated Phenoxyacid Compounds
4.4.1 Erlenmeyer flasks - 500-mL wide-mouth Pyrex, 500-mL Pyrex,
with 24/40 ground glass joint, 1000-mL pyrex.
4.4.2 Separatory funnel - 2000 mL.
4.4.3 Graduated cylinder - 1000 mL.
4.4.4 Funnel - 75 mm diameter.
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4.4.5 Wrist shaker - Burrell Model 75 or equivalent.
4.4.6 pH meter.
4.5 Kuderna-Danish (K-0) apparatus (optional).
4.5.1 Concentrator tube - 10 ml graduated (Kontes K-570050-1025 or
equivalent). A ground glass stopper is used to prevent evaporation of
extracts.
4.5.2 Evaporation flask - 500 ml (Kontes K-570001-500 or
equivalent). Attach to concentrator tube with springs, clamps, or
equivalent.
4.5.3 Snyder column - Two ball micro (Kontes K-569001-0219 or
equivalent).
4.5.4 Springs - 1/2 in. (Kontes K-662750 or equivalent).
4.6 Disposable serological pipets - 5 ml x 1/10, 5.5 mm ID.
4.7 Collection tube - 15 ml conical, graduated (Kimble No. 45165 or
equivalent).
4.8 Vials - 5 ml conical, glass, with Teflon lined screw-caps or crimp
tops.
4.9 Glass wool - Supelco No. 2-0411 or equivalent.
4.10 Microsyringes - 100 juL, 50 yuL, 10 ^,1 (Hamilton 701 N or equivalent),
and 50 p,i (Blunted, Hamilton 705SNR or equivalent).
4.11 Rotary evaporator - Equipped with 1000 ml receiving flask.
4.12 Balances - Analytical, 0.0001 g, Top-loading, 0.01 g.
4.13 Volumetric flasks, Class A - 10 ml to 1000 ml.
4.14 Graduated cylinder - 100 ml.
4.15 Separatory funnel - 250 ml.
5.0 REAGENTS
5.1 Reagent grade inorganic chemicals shall be used in all tests.
Unless otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
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5.2 Organic free reagent water. All references to water in this method
refer to organic-free reagent water, as defined in Chapter One.
5.3 Sodium sulfate (granular, anhydrous), Na2S04. Purify by heating at
400°C for 4 hours in a shallow tray, or by precleaning the sodium sulfate with
methylene chloride.
5.4 Ammonium acetate, NH4OOCCH3, solution (0.1 M). Filter through a 0.45
micron membrane filter (Millipore HA or equivalent).
5.5 Acetic acid, CH3C02H
5.6 Sulfuric acid solution
5.6.1 {(1:1) (v/v)) - Slowly add 50 ml H2S04 (sp. gr. 1.84) to 50
ml of water.
5.6.2 ((1:3) (v/v)) - slowly add 25 ml H2S04 (sp. gr. 1.84) to 75
ml of water.
5.7 Argon gas, 99+% pure.
5.8 Solvents
5.8.1 Methylene chloride, CH2C12 - Pesticide quality or equivalent.
5.8.2 Toluene, C6H5CH3 - Pesticide quality or equivalent.
5.8.3 Acetone, CH3COCH3 - Pesticide quality or equivalent.
5.8.4 Diethyl Ether, C2H5OC2H5 - Pesticide quality or equivalent.
Must be free of peroxides as indicated by test strips (EM Quant, or
equivalent). Procedures for removal of peroxides are provided with the
test strips. After cleanup, 20 mL of ethyl alcohol preservative must be
added to each liter of ether.
5.8.5 Methanol, CH3OH - HPLC quality or equivalent.
5.8.6 Acetonitrile, CH3CN - HPLC quality or equivalent.
5.8.7 Ethyl acetate CH3C02C2H5 - Pesticide quality or equivalent.
5.9 Standard Materials - pure standard materials or certified solutions
of each analyte targeted for analysis. Disperse azo dyes must be purified before
use according to Sec. 5.10.
5.10 Disperse Azo Dye Purification
5.10.1 Two procedures are involved. The first step is the
Soxhlet extraction of the dye for 24 hours with toluene and evaporation of
the liquid extract to dryness, using a rotary evaporator. The solid is
then recrystallized from toluene, and dried in an oven at approximately
100°C. If this step does not give the required purity, column
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chromatography should be employed. Load the solid onto a 3 x 8 inch
silica gel column (Sec. 4.3.4), and elute with diethyl ether. Separate
impurities chromatographically, and collect the major dye fraction.
5.11 Stock standard solutions - Can be prepared from pure standard
materials or can be purchased as certified solutions.
5.11.1 Prepare stock standard solutions by accurately weighing
0.0100 g of pure material. Dissolve the material in methanol or other
suitable solvent (e.g. prepare Tris-BP in ethyl acetate), and dilute to
known volume in a volumetric flask.
NOTE: Due to the reactivity of the chlorinated herbicides, the
standards must be prepared in acetonitrile. Methylation will
occur if prepared in methanol.
If compound purity is certified at 96% or greater, the weight can
be used without correction to calculate the concentration of the stock
standard. Commercially prepared stock standards can be used at any
concentration if they are certified by the manufacturer or by an
independent source.
5.11.2 Transfer the stock standard solutions into glass vials
with Teflon lined screw-caps or crimp-tops. Store at 4°C and protect from
light. Stock standard solutions should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration
standards.
5.12 Calibration standards - A minimum of five concentrations for each
parameter of interest should be prepared through dilution of the stock standards
with methanol (or other suitable solvent). One of these concentrations should
be near, but above, the MDL. The remaining concentrations should correspond to
the expected range of concentrations found in real samples, or should define the
working range of the HPLC-UV/VIS or HPLC-TSP/MS. Calibration standards must be
replaced after one or two months, or sooner if comparison with check standards
indicates a problem.
5.13 Surrogate standards - The analyst should monitor the performance of
the extraction, cleanup (when used), and analytical system, along with the
effectiveness of the method in dealing with each sample matrix, by spiking each
sample, standard, and blank with one or two surrogates (e.g., organophosphorus
or chlorinated phenoxyacid compounds not expected to be present in the sample).
5.14 HPLC/MS tuning standard - Polyethylene glycol 400 (PEG-400), PEG-600
or PEG-800. Dilute to 10 percent (v/v) in methanol. Dependent upon analyte
molecular weight range: m.w. < 500 amu, use PEG-400; m.w. > 500 amu, use PEG-600,
or PEG-800.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this Chapter, Organic Analytes,
Sec. 4.1.
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7.0 PROCEDURE
7.1 Sample preparation - Samples for analysis of disperse azo dyes and
organophosphorus compounds must be prepared by one of the following methods prior
to HPLC/MS analysis:
Matrix Methods
Water 3510, 3520
Soil/sediment 3540, 3541, 3550
Waste 3540, 3541, 3550, 3580
Samples for the analysis of Tris-(2,3-dibromopropyl )phosphate in wastewater
must be prepared according to Sec. 7.1.1 prior to HPLC/MS analysis. Samples for
the analysis of chlorinated phenoxyacid compounds and their esters should be
prepared according to Sec. 7.1.2 prior to HPLC/MS analysis.
7.1.1 Microextraction for Tris-BP:
7.1.1.1 Solid Samples
7.1.1.1.1 Weigh a 1 gram portion of the sample into
a tared beaker. If the sample appears moist, add an
equivalent amount of anhydrous sodium sulfate and mix well.
Add 100 /zL of Tris-BP (approximate concentration 1000 mg/L)
to the sample selected for spiking; the amount added should
result in a final concentration of 100 ng//A in the 1 mL
extract.
7.1.1.1.2 Remove the glass wool plug from a disposable
serological pipet. Insert a 1 cm plug of clean silane
treated glass wool to the bottom (narrow end) of the pipet.
Pack 2 cm of anhydrous sodium sulfate onto the top of the
glass wool. Wash pipet and contents with 3 - 5 mL of
methanol.
7.1.1.1.3 Pack the sample into the pipet prepared
according to Sec. 7.1.1.1.2. If packing material has dried,
wet with a few mL of methanol first, then pack sample into
the pipet.
7.1.1.1.4 Extract the sample with 3 mL of methanol
followed by 4 mL of 50% (v/v) methanol/methylene chloride
(rinse the sample beaker with each volume of extraction
solvent prior to adding it to the pipet containing the
sample). Collect the extract in a 15 mL graduated glass
tube.
7.1.1.1.5 Evaporate the extract to 1 mL using the
nitrogen blowdown technique (Sec. 7.1.1.1.6). Record the
volume. It may not be possible to evaporate some sludge
samples to a reasonable concentration.
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7.1.1.1.6 Nitrogen Slowdown Technique
7.1.1.1.6.1 Place the concentrator tube in
a warm water bath (approximately 35°C) and evaporate the
solvent volume to the required level using a gentle
stream of clean, dry nitrogen (filtered through a
column of activated carbon).
CAUTION: Do not use plasticized tubing
between the carbon trap and the
sample.
7.1.1.1.6.2 The internal wall of the tube
must be rinsed down several times with methylene
chloride during the operation. During evaporation, the
solvent level in the tube must be positioned to prevent
water from condensing into the sample (i.e., the
solvent level should be below the level of the water
bath). Under normal operating conditions, the
extract should not be allowed to become dry. Proceed
to Sec. 7.1.1.1.7.
7.1.1.1.7 Transfer the extract to a glass vial with
a Teflon lined screw-cap or crimp-top and store refrigerated
at 4°C. Proceed with HPLC analysis.
7.1.1.1.8 Determination of percent dry weight - In
certain cases, sample results are desired based on a dry
weight basis. When such data are desired, or required, a
portion of sample for this determination should be weighed
out at the same time as the portion used for analytical
determination.
WARNING: The drying oven should be contained in a
hood or vented. Significant laboratory
contamination may result from drying a
heavily contaminated hazardous waste
sample.
7.1.1.1.9 Immediately after weighing the sample for
extraction, weigh 5-10 g of the sample into a tared crucible.
Determine the % dry weight of the sample by drying overnight
at 105°C. Allow to cool in a desiccator before weighing:
% dry weight = q of dry sample x 100
g of sample
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7.1.1.2 Aqueous Samples
7.1.1.2.1 Using a 100 ml graduated cylinder, measure
100 ml of sample and transfer it to a 250 ml separatory
funnel. Add 200 /iL of Tris-BP (approximate concentration
1000 mg/L) to the sample selected for spiking; the amount
added should result in a final concentration of 200 ng//^L in
the 1 ml extract.
7.1.1.2.2 Add 10 ml of methylene chloride to the
separatory funnel. Seal and shake the separatory funnel
three times, approximately 30 seconds each time, with
periodic venting to release excess pressure. NOTE: Methylene
chloride creates excessive pressure rapidly; therefore,
initial venting should be done immediately after the
separatory funnel has been sealed and shaken once. Methylene
chloride is a suspected carcinogen, use necessary safety
precautions.
7.1.1.2.3 Allow the organic layer to separate from the
water phase for a minimum of 10 minutes. If the emulsion
interface between layers is more than one-third the size of
the solvent layer, the analyst must employ mechanical
techniques to complete phase separation. See Sec. 7.5,
Method 3510.
7.1.1.2.4 Collect the extract in a 15 ml graduated
glass tube. Proceed as in Sec. 7.1.1.1.5.
7.1.2 Extraction for chlorinated phenoxyacid compounds - Preparation
of soil, sediment, and other solid samples must follow Method 8151, with
the exception of no hydrolysis or esterification. Sec. 7.1.2.1 presents
an outline of the procedure with the appropriate changes necessary for
determination by Method 8321. Sec. 7.1.2.2 describes the extraction
procedure for aqueous samples.
7.1.2.1 Extraction of solid samples
7.1.2.1.1 Add 50 g of soil/sediment sample to a 500
ml, wide mouth Erlenmeyer. Add spiking solutions if
required, mix well and allow to stand for 15 minutes. Add 50
ml of organic-free reagent water and stir for 30 minutes.
Determine the pH of the sample with a glass electrode and pH
meter, while stirring. Adjust the pH to 2 with cold H2S04
(1:1) and monitor the pH for 15 minutes, with stirring. If
necessary, add additional H2S04 until the pH remains at 2.
7.1.2.1.2 Add 20 ml of acetone to the flask, and mix
the contents with the wrist shaker for 20 minutes. Add 80 ml
of diethyl ether to the same flask, and shake again for 20
minutes. Decant the extract and measure the volume of
solvent recovered.
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7.1.2.1.3 Extract the sample twice more using 20 ml
of acetone followed by 80 mL of diethyl ether. After
addition of each solvent, the mixture should be shaken with
the wrist shaker for 10 minutes and the acetone-ether extract
decanted.
7.1.2.1.4 After the third extraction, the volume of
extract recovered should be at least 75% of the volume of
added solvent. If this is not the case, additional
extractions may be necessary. Combine the extracts in a 2000
ml separatory funnel containing 250 ml of reagent water. If
an emulsion forms, slowly add 5 g of acidified sodium sulfate
(anhydrous) until the solvent-water mixture separates. A
quantity of acidified sodium sulfate equal to the weight of
the sample may be added, if necessary.
7.1.2.1.5 Check the pH of the extract. If it is not
at or below pH 2, add more concentrated HC1 until the extract
is stabilized at the desired pH. Gently mix the contents of
the separatory funnel for 1 minute and allow the layers to
separate. Collect the aqueous phase in a clean beaker, and
the extract phase (top layer) in a 500 mL ground-glass
Erlenmeyer flask. Place the aqueous phase back into the
separatory funnel and re-extract using 25 ml of diethyl
ether. Allow the layers to separate and discard the aqueous
layer. Combine the ether extracts in the 500 ml Erlenmeyer
flask.
7.1.2.1.6 Add 45 - 50 g acidified anhydrous sodium
sulfate to the combined ether extracts. Allow the extract to
remain in contact with the sodium sulfate for approximately
2 hours.
NOTE: The drying step is very critical. Any moisture
remaining in the ether will result in low
recoveries. The amount of sodium sulfate used is
adequate if some free flowing crystals are
visible when swirling the flask. If all of the
sodium sulfate solidifies in a cake, add a few
additional grams of acidified sodium sulfate and
again test by swirling. The 2 hour drying time is
a minimum; however, the extracts may be held
overnight in contact with the sodium sulfate.
7.1.2.1.7 Transfer the ether extract, through a funnel
plugged with acid-washed glass wool, into a 500 ml K-D flask
equipped with a 10 ml concentrator tube. Use a glass rod to
crush caked sodium sulfate during the transfer. Rinse the
Erlenmeyer flask and column with 20-30 ml of diethyl ether to
complete the quantitative transfer. Reduce the volume of the
extract using the macro K-D technique (Sec. 7.1.2.1.8).
7.1.2.1.8 Add one or two clean boiling chips to the
flask and attach a three ball macro-Snyder column. Prewet
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the Snyder column by adding about 1 ml of diethyl ether to
the top. Place the apparatus on a hot water bath (60°-65°C)
so that the concentrator tube is partially immersed in the
hot water and the entire lower rounded surface of the flask
is bathed in vapor. Adjust the vertical position of the
apparatus and the water temperature, as required, to complete
the concentration in 15-20 minutes. At the proper rate of
distillation the balls of the column will actively chatter,
but the chambers will not flood. When the apparent volume of
liquid reaches 5 ml, remove the K-D apparatus from the water
bath and allow it to drain and cool for at least 10 minutes.
7.1.2.1.9 Exchange the solvent of the extract to
acetonitrile by quantitatively transferring the extract with
acetonitrile to a blow-down apparatus. Add a total of 5 ml
acetonitrile. Reduce the extract volume according to Sec.
7.1.1.1.6, and adjust the final volume to 1 ml.
7.1.2.2
Preparation of aqueous samples
7.1.2.2.1 Using a 1000 ml graduated cylinder, measure
1 liter (nominal) of sample, record the sample volume to the
nearest 5 ml, and transfer it to a separatory funnel. If
high concentrations are anticipated, a smaller volume may be
used and then diluted with organic-free reagent water to 1
liter. Adjust the pH to less than 2 with sulfuric acid (1:1).
7.1.2.2.2 Add 150 ml of diethyl ether to the sample
bottle, seal, and shake for 30 seconds to rinse the walls.
Transfer the solvent wash to the separatory funnel and
extract the sample by shaking the funnel for 2 minutes with
periodic venting to release excess pressure. Allow the
organic layer to separate from the water layer for a minimum
of 10 minutes. If the emulsion interface between layers is
more than one-third the size
analyst must employ mechanical
phase separation. The optimum
sample, and may
through glass
methods.
flask.
of the solvent layer, the
techniques to complete the
technique depends upon the
include stirring, filtration of the emulsion
wool, centrifugation, or other physical
Drain the aqueous phase into a 1000 ml Erlenmeyer
7.1.2.2.3 Repeat the extraction two more times using
100 ml of diethyl ether each time. Combine the extracts in
a 500 ml Erlenmeyer flask. (Rinse the 1000 ml flask with
each additional aliquot of extracting solvent to make a
quantitative transfer.)
7.1.2.2.4
concentration,
adjustment).
Proceed to Sec. 7.1.2.1.6 (drying, K-D
solvent exchange, and final volume
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7.2 Prior to HPLC analysis, the extraction solvent must be exchanged to
methanol or acetonitrile {Sec. 7.1.2.1.9). The exchange is performed using the
K-D procedures listed in all of the extraction methods.
7.3 HPLC Chromatographic Conditions:
7.3.1 Analyte-specific chromatographic conditions are shown in
Table 1. Chromatographic conditions which are not analyte-specific are as
follows:
Flow rate: 0.4 mL/min
Post-column mobile phase: 0.1 M ammonium acetate (1% methanol)
(0.1 M ammonium acetate for
phenoxyacid compounds)
Post-column flow rate: 0.8 mL/min
7.3.2 If there is a chromatographic problem from compound retention
when analyzing for disperse azo dyes, organophosphorus compounds, or
Tris-(2,3-dibromopropyl)phosphate, a 2% constant flow of methylene
chloride may be applied as needed. Methylene chloride/aqueous methanol
solutions must be used with caution as HPLC eluants. Acetic acid (1%),
another mobile phase modifier, can be used with compounds with acid
functional groups.
7.3.3 A total flow rate of 1.0 to 1.5 mL/min is necessary to
maintain thermospray ionization.
7.3.4 Retention times for organophosphorus compounds on the
specified analytical column are presented in Table 9.
7.4 Recommended HPLC/Thermospray/MS operating conditions:
7.4.1 Positive Ionization mode
Repeller (wire or plate, optional): 170 to 250 v (sensitivity
optimized). See Figure 2 for schematic of source with wire repeller.
Mass range: 150 to 450 amu (compound dependent, expect 1 to 18 amu
higher than molecular weight of the compound).
Scan time: 1.50 sec/scan.
7.4.2 Negative Ionization mode
Discharge electrode: on
Filament: off
Mass Range: 135 to 450 amu
Scan time: 1.50 sec/scan.
7.4.3 Thermospray temperatures:
Vaporizer control 110°C to 130°C.
Vaporizer tip 200°C to 215°C.
8321 - 15 Revision 0
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Jet 210°C to 220°C.
Source block 230°C to 265°C. (Some compounds may degrade in
the source block at higher temperatures, the
operator should use knowledge of chemical
properties to estimate proper source
temperature).
7.4.4 Sample injection volume: 20 /uL is necessary in order to
overfill the 10 /nL injection loop. If solids are present in the extract,
allow them to settle or centrifuge the extract and withdraw the injection
volume from the clear layer.
7.5 Calibration:
7.5.1 Thermospray/MS system - Must be hardware-tuned on quadrupole
1 (and quadrupole 3 for triple quadrupoles) for accurate mass assignment,
sensitivity, and resolution. This is accomplished using polyethylene
glycol (PEG) 400, 600, or 800 (see Sec. 5.14) which have average molecular
weights of 400, 600, and 800, respectively. A mixture of these PEGs can
be made such that it will approximate the expected working mass range for
the analyses. Use PEG 400 for analysis of chlorinated phenoxyacid
compounds. The PEG is introduced via the thermospray interface,
circumventing the HPLC.
7.5.1.1 The mass calibration parameters are as follows:
for PEG 400 and 600 for PEG 800
Mass range: 15 to 765 amu Mass range: 15 to 900 amu
Scan time: 5.00 sec/scan Scan time: 5.00 sec/scan
Approximately 100 scans should be acquired, with 2 to 3
injections made. The scan with the best fit to the accurate mass
table (see Tables 7 and 8) should be used as the calibration table.
7.5.1.2 The low mass range from 15 to 100 amu is covered
by the ions from the ammonium acetate buffer used in the thermospray
process: NH/ (18 amu), NH4+H20 (36), CH3OHNH/ (50) (methanol),
or CH3CNNH4+ (59) (acetonitrile), and CH3COOH'NH4+ (78) (acetic
acid). The appearance of the m/z 50 or 59 ion depends upon the use
of methanol or acetonitrile as the organic modifier. The higher
mass range is covered by the ammonium ion adducts of the various
ethylene glycols (e.g. H(OCH2CH2)nOH where n=4, gives the
H(OCH2CH2)4OH-NH4+ ion at m/z 212).
7.5.2 Liquid Chromatograph
7.5.2.1 Prepare calibration standards as outlined in Sec.
5.12.
7.5.2.2 Choose the proper ionization conditions, as
outlined in Sec. 7.4. Inject each calibration standard onto the
HPLC, using the chromatographic conditions outlined in Table 1.
Calculate the area under the curve for the mass chromatogram of each
8321 - 16 Revision 0
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quantitation ion. For example, Table 9 lists the retention times
and the major ions (>5%) present in the positive ionization
thermospray single quadrupole spectra of the organophosphorus
compounds. In most cases the (M+H)"1" and (M+NH4)+ adduct ions are
the only ions of significant abundance. Plot these ions as area
response versus the amount injected. The points should fall on a
straight line, with a correlation coefficient of at least 0.99 (0.97
for chlorinated phenoxyacid analytes).
7.5.2.3 If HPLC-UV detection is also being used,
calibrate the instrument by preparing calibration standards as
outlined in Sec. 5.12, and injecting each calibration standard onto
the HPLC using the chromatographic conditions outlined in Table 1.
Integrate the area under the full chromatographic peak for each
concentration. Quantitation by HPLC-UV may be preferred if it is
known that sample interference and/or analyte coelution are not a
problem.
7.5.2.4 For the methods specified in Sec. 7.5.2.2 and
7.5.2.3, the retention time of the chromatographic peak is an
important variable in analyte identification. Therefore, the ratio
of the retention time of the sample analyte to the standard analyte
should be 1.0 - 0.1.
7.5.2.5 The concentration of the sample analyte will be
determined by using the calibration curves determined in Sees.
7.5.2.2 and 7.5.2.3. These calibration curves must be generated on
the same day as each sample is analyzed. At least duplicate
determinations should be made for each sample extract. Samples
whose concentrations exceed the standard calibration range should
be diluted to fall within the range.
7.5.2.6 Refer to Method 8000 for further information on
calculations.
7.5.2.7 Precision can also be calculated from the ratio
of response (area) to the amount injected; this is defined as the
calibration factor (CF) for each standard concentration. If the
percent relative standard deviation (%RSD) of the CF is less than
20 percent over the working range, linearity through the origin can
be assumed, and the average calibration factor can be used in place
of a calibration curve. The CF and %RSD can be calculated as
follows:
CF = Total Area of Peak/Mass injected (ng)
%RSD = SD/CF x 100
where:
SD = Standard deviation between CFs
CF = Average CF
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7.6 Sample Analysis
7.6.1 Once the LC/MS system has been calibrated as outlined in Sec.
7.5, it is ready for sample analysis. It is recommended that the samples
initially be analyzed in the negative ionization mode. If low levels of
compounds are suspected, then the samples should also be screened in the
positive ionization mode.
7.6.1.1 A blank 20 juL injection (methanol) must be
analyzed after the standard(s) analyses, in order to determine any
residual contamination of the Thermospray/HPLC/MS system.
7.6.1.2 Take a 20 ^L aliquot of the sample extract from
Sec. 7.4.4. Start the HPLC gradient elution, load and inject the
sample aliquot, and start the mass spectrometer data system
analysis.
7.7 Calculations
7.7.1 Using the external standard calibration procedure (Method
8000), determine the identity and quantity of each component peak in the
sample reconstructed ion chromatogram which corresponds to the compounds
used for calibration processes. See Method 8000 for calculation
equations.
7.7.2 The retention time of the chromatographic peak is an important
parameter for the identity of the analyte. However, because matrix
interferences can change chromatographic column conditions, the retention
times are not as significant, and the mass spectra confirmations are
important criteria for analyte identification.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One and Method 8000 for specific quality control
procedures.
8.2 Tables 4, 5, 6, 11, 12, and 15 indicate the single operator accuracy
and precision for this method. Compare the results obtained with the results in
the tables to determine if the data quality is acceptable. Tables 4, 5, and 6
provide single lab data for Disperse Red 1, Table 11 with organophoshorus
pesticides, Table 12 with Tris-BP and Table 15 with chlorophenoxyacid herbicides.
8.2.1 If recovery is not acceptable, check the following:
8.2.1.1 Check to be sure that there are no errors in the
calculations, surrogate solutions or internal standards. If errors
are found, recalculate the data accordingly.
8.2.1.2 Check instrument performance. If an instrument
performance problem is identified, correct the problem and
re-analyze the extract.
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8.2.1.3 If no problem is found, re-extract and re-analyze
the sample.
8.2.1.4 If, upon re-analysis, the recovery is again not
within limits, flag the data as "estimated concentration".
8.3 Instrument performance - Check the performance of the entire
analytical system daily using data gathered from analyses of blanks, standards,
and replicate samples.
8.3.1 See Sec. 7.5.2.7 for required HPLC/MS parameters for standard
calibration curve %RSD limits.
8.3.2 See Sec. 7.5.2.4 regarding retention time window QC limits.
8.3.3 If any of the chromatographic QC limits are not met, the
analyst should examine the LC system for:
• Leaks,
• Proper pressure delivery,
• A dirty guard column; may need replacing or repacking, and
• Possible partial thermospray plugging.
Any of the above items will necessitate shutting down the HPLC/TSP
system, making repairs and/or replacements, and then restarting the
analyses. The calibration standard should be reanalyzed before any sample
analyses, as described in Sec. 7.5.
8.3.4 The experience of the analyst performing liquid
chromatography is invaluable to the success of the method. Each day that
analysis is performed, the daily calibration standard should be evaluated
to determine if the chromatographic system is operating properly. If any
changes are made to the system (e.g. column change), the system must be
recalibrated.
8.4 Optional Thermospray HPLC/MS/MS confirmation
8.4.1 With respect to this method, MS/MS shall be defined as the
daughter ion collision activated dissociation acquisition with quadrupole
one set on one mass (parent ion), quadrupole two pressurized with argon
and with a higher offset voltage than normal, and quadrupole three set to
scan desired mass range.
8.4.2 Since the thermospray process often generates only one or two
ions per compound, the use of MS/MS is a more specific mode of operation,
yielding molecular structural information. In this mode, fast screening
of samples can be accomplished through direct injection of the sample into
the thermospray.
8.4.3 For MS/MS experiments, the first quadrupole should be set to
the protonated molecule or ammoniated adduct of the analyte of interest.
The third quadrupole should be set to scan from 30 amu to just above the
mass region of the protonated molecule.
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8.4.4 The collision gas pressure (Ar) should be set at about 1.0
mTorr and the collision energy at 20 eV. If these parameters fail to give
considerable fragmentation, they may be raised above these settings to
create more and stronger collisions.
8.4.5 For analytical determinations, the base peak of the collision
spectrum shall be taken as the quantification ion. For extra specificity,
a second ion should be chosen as a backup quantification ion.
8.4.6 Generate a calibration curve as outlined in Sec. 7.5.2.
8.4.7 For analytical determinations, calibration blanks must be run
in the MS/MS mode to determine specific ion interferences. If no
calibration blanks are available, chromatographic separation must be
performed to assure no interferences at specific masses. For fast
screening, the MS/MS spectra of the standard and the analyte could be
compared and the ratios of the three major (most intense) ions examined.
These ratios should be approximately the same, unless there is an
interference. If an interference appears, chromatography must be
utilized.
8.4.8 For unknown concentrations, the total area of the quantitation
ion(s) is calculated and the calibration curves generated as in Sec. 7.5
are used to attain an injected weight number.
8.4.9 MS/MS techniques can also be used to perform structural
analysis on ions represented by unassigned m/z ratios. The procedure for
compounds of unknown structures is to set up a CAD experiment on the ion
of interest. The spectrum generated from this experiment will reflect the
structure of the compound by its fragmentation pattern. A trained mass
spectroscopist and some history of the sample are usually needed to
interpret the spectrum. (CAD experiments on actual standards of the
expected compound are necessary for confirmation or denial of that
substance.)
8.5 Optional wire-repeller CAD confirmation
8.5.1 See Figure 3 for the correct position of the wire-repeller in
the thermospray source block.
8.5.2 Once the wire-repeller is inserted into the thermospray flow,
the voltage can be increased to approximately 500 - 700 v. Enough voltage
is necessary to create fragment ions, but not so much that shorting
occurs.
8.5.3 Continue as outlined in Sec. 7.6.
9.0 METHOD PERFORMANCE
9.1 Single operator accuracy and precision studies have been conducted
using spiked sediment, wastewater, sludge, and water samples. The results are
presented in Tables 4, 5, 6, 11, 12, and 15. Tables 4, 5, and 6 provide single
8321 - 20 Revision 0
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lab data for Disperse Red 1, Table 11 for organophoshorus pesticides, Table 12
for Tris-BP and Table 15 with chlorophenoxyacid herbicides.
9.2 LODs should be calculated for the known analytes, on each instrument
to be used. Tables 3, 10, and 13 list limits of detection (LOD) and/or estimated
quantitation limits (EQL) that are typical with this method.
9.2.1 The LODs presented in this method were calculated by analyzing
three replicates of four standard concentrations, with the lowest
concentration being near the instrument detection limit. A linear
regression was performed on the data set to calculate the slope and
intercept. Three times the standard deviation (3a) of the lowest standard
amount, along with the calculated slope and intercept, were used to find
the LOD. The LOD was not calculated using the specifications in Chapter
One, but according to the ACS guidelines specified in Reference 4.
9.2.2 Table 17 presents a comparison of the LODs from Method 8151
and Method 8321 for the chlorinated phenoxyacid compounds.
9.3 Table 16 presents multilaboratory accuracy and precision data for
the chlorinated phenoxyacid herbicides. The data summary is based on data from
three laboratories that analyzed duplicate solvent solutions at each
concentration specified in the Table.
10.0 REFERENCES
1. Voyksner, R.D.; Haney, C.A. "Optimization and Application of Thermospray
High-Performance Liquid Chromatography/Mass Spectrometry"; Anal. Chem.
1985, 57, 991-996.
2. Blakley, C.R.; Vestal, M.L. "Thermospray Interface for Liquid
Chromatography/Mass Spectrometry"; Anal. Chem. 1983, 55, 750-754.
3. Taylor, V.; Hickey, D. M., Marsden, P. J. "Single Laboratory Validation of
EPA Method 8140"; EPA-600/4-87/009, U.S. Environmental Protection Agency,
Las Vegas, NV, 1987, 144 pp.
4. "Guidelines for Data Acquisition and Data Quality Evaluation in
Environmental Chemistry"; Anal. Chem. 1980, 52, 2242-2249.
5. Betowski, L. D.; Jones, T. L. "The Analysis of Organophosphorus Pesticide
Samples by HPLC/MS and HPLC/MS/MS"; Environmental Science and Technology.
1988,
8. EPA: 2nd Annual Report on Carcinogens, NTP 81-43, Dec. 1981, pp. 236-237.
9. Blum, A.; Ames, B. N. Science 195, 1977, 17.
10. Zweidinger, R. A.; Cooper, S. D.; Pellazari, E. D., Measurements of
Organic Pollutants in Water and Wastewater, ASTM 686.
11. Cremlyn, R. Pesticides: Preparation and mode of Action; John Wiley and
Sons: Chichester, 1978; p. 142.
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12. Cotterill, E. 6.; Byast, T. H. "HPLC of Pesticide Residues in
Environmental Samples." in Liquid Chromatoqraphy in Environmental
Analysis; Laurence, J. F., Ed.; Humana Press: Clifton, NJ, 1984.
13. Voyksner, R. D. "Thermospray HPLC/MS for Monitoring the Environment." In
Applications of New Mass Spectrometry Techniques in Pesticide Chemistry;
Rosen, J. D., Ed., John Wiley and Sons: New York, 1987.
H. Yinon, J.; Jones, T. L.; Betowski, L. D. Rap. Comm. Mass Spectrom. 1989,
3, 38.
15. Shore, F. L.; Amick, E. N., Pan, S. T., Gurka, D. F. "Single Laboratory
Validation of EPA Method 8150 for the Analysis of Chlorinated Herbicides
in Hazardous Waste"; EPA/600/4-85/060, U.S. Environmental Protection
Agency, Las Vegas, NV, 1985.
16. "Development and Evaluations of an LC/MS/MS Protocol", EPA/600/X-86/328,
Dec. 1986.
17. "An LC/MS Performance Evaluation Study of Organophosphorus Pesticides",
EPA/600/X-89/006, Jan. 1989.
18. "A Performance Evaluation Study of a Liquid Chromatography/Mass
Spectrometry Method for Tris-(2,3-Dibromopropyl) Phosphate",
EPA/600/X-89/135, June 1989.
19. "Liquid Chromatography/Mass Spectrometry Performance Evaluation of
Chlorinated Phenoxyacid Herbicides and Their Esters", EPA/600/X-89/176,
July 1989.
20. "An Interlaboratory Comparison of an SW-846 Method for the Analysis of the
Chlorinated Phenoxyacid Herbicides by LC/MS", EPA/600/X-90/133, June 1990.
8321 - 22 Revision 0
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TABLE 1.
RECOMMENDED HPLC CHROMATOGRAPHIC CONDITIONS
Analytes
Organophosphorus
Compounds
Initial
Mobile
Phase
(%)
50/50
(water/
methanol )
Initial
Time
(min)
0
Gradient
(linear)
(min)
10
Final
Mobile
Phase
(%)
100
(methanol)
Final
Time
(min)
5
Azo Dyes (e.g.
Disperse Red 1)
50/50
(water/CH3CN)
100 5
(CH3CN)
Tris-(2,3-dibromo-
propyl)phosphate
50/50 0
(water/methanol)
10
100 5
(methanol)
Chlorinated
phenoxyacid
compounds
* Where A = 0.01
75/25
(A/methanol)
40/60
(A/methanol)
M ammonium acetate
2 15
3 5
(1% acetic acid)
40/60
(A/methanol)*
75/25
(A/methanol)*
10
8321 - 23
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September 1994
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TABLE 2.
COMPOUNDS AMENABLE TO THERMOSPRAY MASS SPECTROMETRY
Disperse Azo Dyes Alkaloids
Methine Dyes Aromatic ureas
Arylmethane Dyes Amides
Coumarin Dyes Amines
Anthraquinone Dyes Amino acids
Xanthene Dyes Organophosphorus Compounds
Flame retardants Chlorinated Phenoxyacid Compounds
TABLE 3.
LIMITS OF DETECTION (LOD) AND METHOD SENSITIVITIES
FOR DISPERSE RED 1 AND CAFFEINE
Compound
Disperse Red 1
Caffeine
Mode
SRM
Single Quad
CAD
SRM
Single Quad
CAD
LOD
(P9)
180
600
2,000
45
84
240
EQL(7s)
(P9)
420
1400
4700
115
200
560
EQL(lOs)
(P9)
600
2000
6700
150
280
800
EQL = Estimated Quantitation Limit
Data from Reference 16.
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September 1994
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TABLE 4.
PRECISION AND ACCURACY COMPARISONS OF MS AND MS/MS WITH
HPLC/UV FOR ORGANIC-FREE REAGENT WATER SPIKED WITH DISPERSE RED 1
Percent Recovery
Sample
Spike 1
Spike 2
RPD
HPLC/UV
82.2 ± 0.2
87.4 ± 0.6
6.1%
MS
92.5 ± 3.7
90.2 ± 4.7
2 . 5%
CAD
87.6 ± 4.6
90.4 + 9.9
3 . 2%
SRM
95.5 ± 17.1
90.0 ± 5.9
5.9%
Data from Reference 16.
TABLE 5.
PRECISION AND ACCURACY COMPARISONS OF MS AND MS/MS WITH
HPLC/UV FOR MUNICIPAL WASTEWATER SPIKED WITH DISPERSE RED 1
Percent Recovery
Sample
Spike 1
Spike 2
RPD
HPLC/UV
93.4 ± 0.3
96.2 ± 0.1
3.0%
MS
102.0 + 31
79.7 + 15
25%
CAD
82.7
83.7
1
+ 13
+ 5.2
.2%
Data from Reference 16.
8321 - 25
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September 1994
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TABLE 6.
RESULTS FROM ANALYSES OF ACTIVATED SLUDGE PROCESS WASTEWATER
Sample
5 mg/L Spiking
Concentration
1
1-D
2
3
RPD
Unspiked
Sample
1
1-D
2
3
RPD
Recovery
HPLC/UV
0.721 ± 0.003
0.731 ± 0.021
0.279 ± 0.000
0.482 ± 0.001
1.3%
0.000
0.000
0.000
0.000
--
of Disperse Red 1
MS
0.664 ± 0.030
0.600 ± 0.068
0.253 ± 0.052
0.449 ± 0.016
10.1%
0.005 ± 0.0007
0.006 ± 0.001
0.002 ± 0.0003
0.003 ± 0.0004
18.2%
(mq/L)
CAD
0.796 + 0.008
0.768 + 0.093
0.301 + 0.042
0.510 + 0.091
3.6%
<0.001
<0.001
<0.001
<0.001
--
Data from Reference 16.
8321 - 26 Revision 0
September 1994
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TABLE 7.
CALIBRATION MASSES AND % RELATIVE ABUNDANCES
OF PEG 400
Mass
18.0
35.06
36.04
50.06
77.04
168.12
212.14
256.17
300.20
344.22
388.25
432.28
476.30
520.33
564.35
608.38
652.41
653.41
696.43
697.44
% Relative
Abundances8
32.3
13.5
40.5
94.6
27.0
5.4
10.3
17.6
27.0
45.9
64.9
100
94.6
81.1
67.6
32.4
16.2
4.1
8.1
2.7
Intensity is normalized to mass 432.
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September 1994
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TABLE 8.
CALIBRATION MASSES AND % RELATIVE ABUNDANCES
OF PEG 600
Mass
18.0
36.04
50.06
77.04
168.12
212.14
256.17
300.20
344.22
388.25
432.28
476.30
520.33
564.35
608.38
652.41
653.41
696.43
% Relative
Abundances"
4.7
11.4
64.9
17.5
9.3
43.9
56.1
22.8
28.1
38.6
54.4
64.9
86.0
100
63.2
17.5
5.6
1.8
Intensity is normalized to mass 564
8321 - 28 Revision 0
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TABLE 9.
RETENTION TIMES AND THERMOSPRAY MASS SPECTRA
OF ORGANOPHOSPHORUS COMPOUNDS
Compound
Monocrotophos
Trichlorfon
Dimethoate
Dichlorvos
Naled
Fensulfothion
Methyl parathion
Phorate
Disulfoton
Merphos
Retention Time
(minutes)
1:09
1:22
1:28
4:40
9:16
9:52
10:52
13:30
13:55
18:51
Mass Spectra
(% Relative Abundance)3
241 (100), 224 (14)
274 (100), 257 (19), 238 (19)
230 (100), 247 (20)
238 (100), 221 (40)
398 (100), 381 (23), 238 (5),
221 (2)
326 (10), 309 (100)
281 (100), 264 (8), 251 (21),
234 (48)
278 (4), 261 (100)
292 (10), 275 (100)
315 (100), 299 (15)
a For molecules containing Cl, Br and S, only the base peak of the isotopic
cluster is listed.
Data from Reference 17.
8321 - 29
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TABLE 10.
PRECISION AND METHOD DETECTION LIMITS (MDLs) FOR
ORGANOPHOSPHORUS COMPOUND STANDARDS
Compound
Dichlorvos
Dimethoate
Phorate
Disulfoton
Fensulfothion
Naled
Merphos
Methyl
parathion
Ion
238
230
261
275
309
398
299
281
Standard
Quantitation
Concentration
(ng/W
2
12.5
25
50
2
12.5
25
50
2
12.5
25
50
2
12.5
25
50
2
12.5
25
50
2
12.5
25
50
2
12.5
25
50
2
12.5
25
50
%RSD
16
13
5.7
4.2
2.2
4.2
13
7.3
0.84
14
7.1
4.0
2.2
14
6.7
3.0
4.1
9.2
9.8
2.5
9.5
9.6
5.2
6.3
5.5
17
3.9
5.3
7.1
4.8
1.5
MDL (ng)
4
2
2
1
0.4
0.2
1
30
Data from Reference 17.
8321 - 30
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September 1994
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TABLE 11.
SINGLE OPERATOR ACCURACY AND PRECISION FOR LOW CONCENTRATION DRINKING
WATER (A), LOW CONCENTRATION SOIL (B), MEDIUM CONCENTRATION DRINKING
WATER (C), MEDIUM CONCENTRATION SEDIMENT (D)
Average
Recovery
Compound (%)
A
Dimethoate
Dichlorvos
Naled
Fensulfothion
Methyl parathion
Phorate
Disulfoton
Merphos
B
Dimethoate
Dichlorvos
Naled
Fensulfothion
Methyl parathion
Phorate
Disulfoton
Merphos
C
Dimethoate
Dichlorvos
Naled
Fensulfothion
Methyl parathion
Phorate
Disulfoton
Merphos
D
Dimethoate
Dichlorvos
Naled
Fensulfothion
Methyl parathion
Phorate
Disulfoton
Merphos
70
40
0.5
112
50
16
3.5
237
16
ND
ND
45
ND
78
36
118
52
146
4
65
85
10
2
101
74
166
ND
72
84
58
56
78
Standard
Deviation
7.7
12
1.0
3.3
28
35
8
25
4
—
5
15
7
19
4
29
3
7
24
15
1
13
8.5
25
8.6
9
6
5
4
Spike
Amount
uq/L
5
5
5
5
10
5
5
5
ua/kg
50
50
50
50
100
50
50
50
uq/L
50
50
50
50
100
50
50
50
mq/kq
2
'2
2
2
3
2
2
2
Range of
Recovery
(%)
54
14
0
106
0
0
0
187
7
-
-
34
-
48
22
81
43
89
0
51
37
0
0
75
57
115
-
55
66
46
47
70
- 85
- 64
- 2
- 119
- 105
- 86
- 19
-287
- 24
-
-
- 56
-
- 109
- 49
- 155
- 61
- 204
- 9
- 79
- 133
- 41
- 4
- 126
- 91
- 216
-
- 90
- 102
- 70
- 66
- 86
Number
of
Analyses
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
12
12
12
12
12
12
12
12
15
15
15
15
15
15
15
12
Data from Reference 17.
8321 - 31
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September 1994
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TABLE 12
SINGLE OPERATOR ACCURACY AND PRECISION FOR MUNICIPAL WASTE
WATER (A), DRINKING WATER (B), CHEMICAL SLUDGE WASTE (C)
Average
Recovery
Compound (%)
Tris-BP (A) 25
(B) 40
(C) 63
Spike Range
Standard Amount of % Number of
Deviation (ng/^L) Recovery Analyses
8.0 2 41 - 9.0 15
5.0 2 50-30 12
11 100 84-42 8
Data from Reference 18.
8321 - 32
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September 1994
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TABLE 13.
SINGLE OPERATOR ESTIMATED QUANTITATION LIMIT (EQL) TABLE FOR TRIS-BP
Concentration Average Standard 3*Std 7*Std 10*Std Lower Upper
Area Deviation Dev. Dev. Dev. LOD EQL EQL
(ng/ML)
50 2675 782 2347 5476 7823 33 113 172
100 5091 558
150 7674 2090
200 8379 2030
Data from Reference 18.
8321 - 33 Revision 0
September 1994
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TABLE 14
LIMITS OF DETECTION (LOD) IN THE POSITIVE AND NEGATIVE ION MODES
FOR THE CHLORINATED PHENOXYACID HERBICIDES AND FOUR ESTERS
Compound
Dalapon
Dicamba
2,4-D
MCPA
Dichlorprop
MCPP
2,4,5-T
2,4,5-TP (Silvex)
Dinoseb
2,4-DB
2,4-D,Butoxy
ethanol ester
2,4,5-T,Butoxy
ethanol ester
2,4,5-T, Butyl
ester
2,4-D,ethyl-
hexyl ester
Positive Mode
Quantitation
Ion
Not detected
238 (M+NHJ +
238 (M+NH4)+
218 (M+NH4)+
252 (M+NH4)+
232 (M+NHJ +
272 (M+NH4)+
286 (M+NH4)+
228 (M+NH4-NO)+
266 (M+NH4)+
321 (M+H)+
372 (M+NH4)+
328 (M+NH4)+
350 (M+NH4)+
LOD
(ng)
13
2.9
120
2.7
5.0
170
160
24
3.4
1.4
0.6
8.6
1.2
Negative Mode
Quantitation
Ion
141 (M-H)'
184 (M-HC1)'
184 (M-HC1)'
199 (M-l)-
235 (M-l)-
213 {M-l}-
218 (M-HC1)
269 (M-iy
240 (M)-
247 (M-l)-
185 (M-CgH^OJ-
195 (M-C8H1503)'
195 (M-C6Hn02)'
161 (M-C10H1903)-
LOD
(ng)
11
3.0
50
28
25
12
6.5
43
19
110
Data from Reference 19.
8321 - 34
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September 1994
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TABLE 15
SINGLE LABORATORY OPERATOR ACCURACY AND PRECISION
FOR THE CHLORINATED PHENOXYACID HERBICIDES
Compound
Dicamba
2,4-D
MCPA
MCPP
Dichlorprop
2,4, 5-T
Silvex
2,4-DB
Dinoseb
Dalapon
2,4-D, ester
Dicamba
2,4-D
MCPA
MCPP
Dichlorprop
2,4,5-T
Silvex
2,4-DB
Dinoseb
Dalapon
2,4-D, ester
Dicamba
2,4-D
MCPA
MCPP
Dichlorprop
2,4,5-T
Silvex
2,4-DB
Dinoseb
Dalapon
2,4-D, ester
(a)
Average Standard
Recovery(%) Deviation
LOW LEVEL
63
26
60
78
43
72
62
29
73
NO
73
HIGH LEVEL
54
60
67
66
66
61
74
83
91
43
97
LOW
117
147
167
142
ND
134
121
199
76
ND
180
DRINKING WATER
22
13
23
21
18
31
14
24
11
ND
17
DRINKING WATER
30
35
41
33
33
23
35
25
10
9.6
19
LEVEL SAND
26
23
79
39
ND
27
23
86
74
ND
58
Spike
Amount
M9/L
5
5
5
5
5
5
5
5
5
5
5
M9/L
50
50
50
50
50
50
50
50
50
50
50
M9/9
.1
.1
.1
.1
.1
.1
.1
.1
.1
.1
.1
Range of
Recovery
(%)
33
0
37
54
0
43
46
0
49
48
26
35
32
35
27
44
45
52
76
31
76
82
118
78
81
99
85
0
6
59
- 86
- 37
- 92
- 116
- 61
- 138
- 88
- 62
- 85
ND
- 104
- 103
- 119
- 128
- 122
- 116
- 99
- 132
- 120
- 102
- 56
- 130
- 147
- 180
- 280
- 192
ND
- 171
- 154
- 245
- 210
ND
- 239
Number
of
Analyses
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
6
9
10
10
10
10
10
10
10
10
10
10
7
'"'All recoveries are in negative ionization mode, except for 2,4-D,ester.
ND = Not Detected.
8321 - 35
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September 1994
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TABLE 15 (cent.)
SINGLE LABORATORY OPERATOR ACCURACY AND PRECISION
FOR THE CHLORINATED PHENOXYACIO HERBICIDES
Compound
la)
Average
Recovery(%)
Standard
Deviation
Spike
Amount
Range of
Recovery
{%)
Number
of
Analyses
Dicamba
2,4-D
MCPA
MCPP
Dichlorprop
2,4,5-T
Silvex
2,4-DB
Dinoseb
Dalapon
2,4-D,ester
Dicamba
2,4-D
MCPA
MCPP
Dichlorprop
2,4,5-T
Silvex
2,4-DB
Dinoseb
Dalapon
2,4-D,ester
Dicamba
2,4-D
MCPA
MCPP
Dichlorprop
2,4,5-T
Silvex
2,4-DB
Dinoseb
Dalapon
2,4-D,ester
HIGH LEVEL SAND
M9/9
153
218
143
158
92
160
176
145
114
287
20
LOW LEVEL
83
ND
ND
ND
ND
27
68
ND
44
ND
29
HIGH LEVEL
66
8.7
3.2
10
ND
2.9
6.0
ND
16
ND
1.9
33
27
30
34
37
29
34
22
28
86
3.6
MUNICIPAL ASH
22
ND
ND
ND
ND
25
38
ND
13
ND
23
MUNICIPAL ASH
21
4.8
4.8
4.3
ND
1.2
3.1
ND
6.8
ND
1.7
1
1
1
1
1
1
1
1
1
1
1
M9/9
.1
.1
.1
.1
.1
.1
.1
.1
.1
.1
.1
M9/9
1
1
3
1
1
1
1
1
1
1
1
119
187
111
115
51
131
141
110
65
166
17
- 209
- 276
- 205
- 226
- 161
- 204
- 225
- 192
- 140
- 418
- 25
48 - 104
ND
ND
ND
ND
0 - 60
22 - 128
ND
26 - 65
ND
0 - 53
41 - 96
5 - 21
0 - 10
4.7 - 16
ND
0 - 3.6
2.8 - 12
ND
0 - 23
ND
0 - 6.7
9
9
9
9
9
9
9
9
9
9
7
9
9
9
9
9
9
9
9
9
9
6
9
9
9
9
9
9
9
9
9
9
6
'"'All recoveries are in negative ionization mode, except for 2,4-D,ester.
ND = Not Detected.
8321 - 36
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September 1994
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TABLE 16
MULTILABORATORY ACCURACY AND PRECISION DATA
FOR THE CHLORINATED PHENOXYACID HERBICIDES
Compounds
Spiking
Concentration
Mean
(% Recovery)8
% Relative
Standard Deviation6
500 mq/L
2,4,5-T
2,4,5-T,butoxy
2,4-D
2,4-DB
Dalapon
Dicamba
Dichlorprop
Dinoseb
MCPA
MCPP
Silvex
50 mq/L
2,4,5-T
2,4,5-T,butoxy
2,4-D
2,4-DB
Dalapon
Dicamba
Dichlorprop
Dinoseb
MCPA
MCPP
Silvex
5 mq/L
2,4,5-T
2,4,5-T,butoxy
2,4-D
2,4-DB
Dalapon
Dicamba
Dichlorprop
Dinoseb
MCPA
MCPP
Silvex
90
90
86
95
83
77
84
78
89
86
96
62
85
64
104
121
90
96
86
96
76
65
90
99
103
96
150
105
102
108
94
98
87
23
29
17
22
13
25
20
15
11
12
27
68
9
80
28
99
23
15
57
20
74
71
28
17
31
21
4
12
22
30
18
15
15
Data from Reference 20.
8 Mean of duplicate data from 3 laboratories.
b % RSD of duplicate data from 3 laboratories.
8321 - 37
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September 1994
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TABLE 17
COMPARISON OF LODs: METHOD 8151 vs. METHOD 8321
Compound
Method 8151
LOD(Mg/L)
Method 8321
LOD {/ig/L}
lonization
Mode
Dalapon
Dicamba
2,4-D
MCPA
Dichloroprop
MCPP
2,4,5-T
2,4,5-TP (Silvex)
2,4-DB
Dinoseb
1.3
0.8
0.2
0.06
0.26
0.09
0.08
0.17
0.8
0.19
1.1
0.3
0.29
2.8
0.27
0.50
0.65
4.3
0.34
1.9
8321 - 38
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September 1994
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FIGURE 1.
SCHEMATIC OF THE THERMOSPRAY PROBE AND ION SOURCE
Flange
To
Trip
& ~—
Mechanical
Pump
tx3
*x
^
2
i
Ion Sampling
Cone
Source
Mounting . Ions EN
Plate | t a
(1 ~~
fa
t
t
II
Source I
Block
^* /
Vapor
*" Temperature
T
ictron Vaporizer
earn s Probe
* *
Heater Vi
3
**
'x
X
$
$
K4
iporizer
X
^
1
Vaporizar
Controlar
Coupling '" T
— LC
Block
Temperature
T.
8321 - 39
Revision 0
September 1994
-------
FIGURE 2.
THERMOSPRAY SOURCE WITH WIRE-REPELLER
(High sensitivity configuration)
CERAMIC INSULATOR
WIRE REPELLER
8321 - 40
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September 1994
-------
FIGURE 3
THERMOSPRAY SOURCE WITH WIRE-REPELLER
(CAD configuration)
CERAMIC INSULATOR
WIRE REPELLER
8321 - 41
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September 1994
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METHOD 8321
SOLVENT EXTRACTABLE NON-VOLATILE COMPOUNDS BY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/THERMOSPRAY/MASS SPECTROMETRY
(HPLC/TSP/MS) OR ULTRAVIOLET (UV) DETECTION
7.3 Sat HPLC
Chromatographic
condition!.
8321 - 42
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September 1994
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00
OJ
OJ
o
-------
METHOD 8330
NITROAROMATICS AND NITRAMINES BY HIGH
PERFORMANCE LIQUID CHROHATOGRAPHY (HPLC)
1.0 SCOPE AND APPLICATION
1.1 Method 8330 is intended for the trace analysis of explosives residues
by high performance liquid chromatography using a UV detector. This method is
used to determine the concentration of the following compounds in a water, soil,
or sediment matrix:
Compound
Abbreviation
CAS No"
Octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine HMX
Hexahydro-l,3,5-trinitro-l,3,5-triazine RDX
1,3,5-Trinitrobenzene 1,3,5-TNB
1,3-Dinitrobenzene 1,3-DNB
Methyl-2,4,6-trinitrophenylnitramine Tetryl
Nitrobenzene NB
2,4,6-Trinitrotoluene 2,4,6-TNT
4-Amino-2,6-dinitrotoluene 4-Am-DNT
2-Amino-4, 6-dinitrotoluene 2-Am-DNT
2,4-Dinitrotoluene 2,4-DNT
2,6-Dinitrotoluene 2,6-DNT
2-Nitrotoluene 2-NT
3-Nitrotoluene 3-NT
4-Nitrotoluene 4-NT
2691-41-0
121-82-4
99-35-4
99-65-0
479-45-8
98-95-3
118-96-7
1946-51-0
355-72-78-2
121-14-2
606-20-2
88-72-2
99-08-1
99-99-0
a Chemical Abstracts Service Registry number
1.2 Method 8330 provides a salting-out extraction procedure for low
concentration (parts per trillion, or nanograms per liter) of explosives residues
in surface or ground water. Direct injection of diluted and filtered water
samples can be used for water samples of higher concentration (See Table 1).
1.3 All of these compounds are either used in the manufacture of
explosives or are the degradation products of compounds used for that purpose.
When making stock solutions for calibration, treat each explosive compound with
caution. See NOTE in Sec. 5.3.1 and Sec. 11 on Safety.
1.4 The estimated quantitation limits (EQLs) of target analytes
determined by Method 8330 in water and soil are presented in Table 1.
1.5 This method is restricted to use by or under the supervision of
analysts experienced in the use of HPLC, skilled in the interpretation of
chromatograms, and experienced in handling explosive materials. (See Sec. 11.0
8330 - 1
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September 1994
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on SAFETY.) Each analyst must demonstrate the ability to generate acceptable
results with this method.
2.0 SUMMARY OF METHOD
2.1 Method 8330 provides high performance liquid chromatographic (HPLC)
conditions for the detection of ppb levels of certain explosives residues in
water, soil and sediment matrix. Prior to use of this method, appropriate sample
preparation techniques must be used.
2.2 Low-Level Salting-out Method With No Evaporation: Aqueous samples
of low concentration are extracted by a salting-out extraction procedure with
acetonitrile and sodium chloride. The small volume of acetonitrile that remains
undissolved above the salt water is drawn off and transferred to a smaller
volumetric flask. It is back-extracted by vigorous stirring with a specific
volume of salt water. After equilibration, the phases are allowed to separate
and the small volume of acetonitrile residing in the narrow neck of the
volumetric flask is removed using a Pasteur pipet. The concentrated extract is
diluted 1:1 with reagent grade water. An aliquot is separated on a C-18 reverse
phase column, determined at 254 nm, and confirmed on a CN reverse phase column.
2.3 High-level Direct Injection Method: Aqueous samples of higher
concentration can be diluted 1/1 (v/v) with methanol or acetonitrile, filtered,
separated on a C-18 reverse phase column, determine at 254 nm, and confirmed on
a CN reverse phase column. If HMX is an important target analyte, methanol is
preferred.
2.4 Soil and sediment samples are extracted using acetonitrile in an
ultrasonic bath, filtered and chromatographed as in Sec. 2.3.
3.0 INTERFERENCES
3.1 Solvents, reagents, glassware and other sample processing hardware
may yield discrete artifacts and/or elevated basel ines, causing misinterpretation
of the chromatograms. All of these materials must be demonstrated to be free
from interferences.
3.2 2,4-DNT and 2,6-DNT elute at similar retention times (retention time
difference of 0.2 minutes). A large concentration of one isomer may mask the
response of the other isomer. If it is not apparent that both isomers are
present (or are not detected), an isomeric mixture should be reported.
3.3 Tetryl decomposes rapidly in methanol/water solutions, as well as
with heat. All aqueous samples expected to contain tetryl should be diluted with
acetonitrile prior to filtration and acidified to pH <3. All samples expected
to contain tetryl should not be exposed to temperatures above room temperature.
3.4 Degradation products of tetryl appear as a shoulder on the 2,4,6-TNT
peak. Peak heights rather than peak areas should be used when tetryl is present
in concentrations that are significant relative to the concentration of
2,4,6-TNT.
8330 - 2 Revision 0
September 1994
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4.0 APPARATUS AND MATERIALS
4.1 HPLC system
4.1.1 HPLC - equipped with a pump capable of achieving 4000 psi, a
100 /il loop injector and a 254 nm UV detector (Perldn Elmer Series 3, or
equivalent). For the low concentration option, the detector must be
capable of a stable baseline at 0.001 absorbance units full scale.
4.1.2 Recommended Columns:
4.1.2.1 Primary column: C-18 Reverse phase HPLC column,
25 cm x 4.6 mm (5 /Ltm), (Supelco LC-18, or equivalent).
4.1.2.2 Secondary column: CN Reverse phase HPLC column,
25 cm x 4.6 mm (5 /urn), (Supelco LC-CN, or equivalent).
4.1.3 Strip chart recorder.
4.1.4 Digital integrator (optional).
4.1.5 Autosampler (optional).
4.2 Other Equipment
4.2.1 Temperature controlled ultrasonic bath.
4.2.2 Vortex mixer.
4.2.3 Balance, + 0.0001 g.
4.2.4 Magnetic stirrer with stirring pellets.
4.2.5 Water bath - Heated, with concentric ring cover, capable of
temperature control (± 5°C). The bath should be used in a hood.
4.2.6 Oven - Forced air, without heating.
4.3 Materials
4.3.1 High pressure injection syringe - 500 /uL, (Hamilton liquid
syringe or equivalent).
4.3.2 Disposable cartridge filters - 0.45 /im Teflon filter.
4.3.3 Pipets - Class A, glass, Appropriate sizes.
4.3.4 Pasteur pipets.
4.3.5 Scintillation Vials - 20 mL, glass.
4.3.6 Vials - 15 mL, glass, Teflon-lined cap.
8330 - 3 Revision 0
September 1994
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4.3.7 Vials- 40 ml, glass, Teflon-lined cap.
4.3.8 Disposable syringes - Plastipak, 3 ml and 10 ml or equivalent.
4.3.9 Volumetric flasks - Appropriate sizes with ground glass
stoppers, Class A.
NOTE: The 100 ml and 1 L volumetric flasks used for magnetic stirrer
extraction must be round.
4.3.10 Vacuum desiccator - Glass.
4.3.11 Mortar and pestle - Steel.
4.3.12 Sieve - 30 mesh.
4.3.13 Graduated cylinders - Appropriate sizes.
4.4 Preparation of Materials
4.4.1 Prepare all materials to be used as described in Chapter 4 for
semivolatile organics.
5.0 REAGENTS
5.1 Reagent grade inorganic chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lowering the accuracy of the determination.
5.1.1 Acetonitrile, CH3CN - HPLC grade.
5.1.2 Methanol, CH3OH - HPLC grade.
5.1.3 Calcium chloride, CaCl2 - Reagent grade. Prepare an aqueous
solution of 5 g/L.
5.1.4 Sodium chloride, NaCl, shipped in glass bottles - reagent
grade.
5.2 Organic-free reagent water - All references to water in this method
refer to organic-free reagent water, as defined in Chapter One.
5.3 Stock Standard Solutions
5.3.1 Dry each solid analyte standard to constant weight in a vacuum
desiccator in the dark. Place about 0.100 g (weighed to 0.0001 g) of a
single analyte into a 100 ml volumetric flask and dilute to volume with
acetonitrile. Invert flask several times until dissolved. Store in
8330 - 4 Revision 0
September 1994
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refrigerator at 4°C in the dark. Calculate the concentration of the stock
solution from the actual weight used (nominal concentration = 1,000 mg/L).
Stock solutions may be used for up to one year.
NOTE: The HMX, RDX, Tetryl, and 2,4,6-TNT are explosives and the
neat material should be handled carefully. See SAFETY in Sec.
11 for guidance. HMX, RDX, and Tetryl reference materials
are shipped under water. Drying at ambient temperature
requires several days. DO NOT DRV AT HEATED TEMPERATURES!
5.4 Intermediate Standards Solutions
5.4.1 If both 2,4-DNT and 2,6-DNT are to be determined, prepare two
separate intermediate stock solutions containing (1) HMX, RDX, 1,3,5-TNB,
1,3-DNB, NB, 2,4,6-TNT, and 2,4-DNT and (2) Tetryl, 2,6-DNT, 2-NT, 3-NT,
and 4-NT. Intermediate stock standard solutions should be prepared at
1,000 /ig/L, in acetonitrile when analyzing soil samples, and in methanol
when analyzing aqueous samples.
5.4.2 Dilute the two concentrated intermediate stock solutions, with
the appropriate solvent, to prepare intermediate standard solutions that
cover the range of 2.5 - 1,000 jig/l. These solutions should be
refrigerated on preparation, and may be used for 30 days.
5.4.3 For the low-level method, the analyst must conduct a detection
limit study and devise dilution series appropriate to the desired range.
Standards for the low level method must be prepared immediately prior to
use.
5.5 Working standards
5,5.1 Calibration standards at a minimum of five concentration
levels should be prepared through dilution of the intermediate standards
solutions by 50% (v/v) with 5 g/L calcium chloride solution (Sec. 5.1.3).
These solutions must be refrigerated and stored in the dark, and prepared
fresh on the day of calibration.
5.6 Surrogate Spiking Solution
5.6.1 The analyst should monitor the performance of the extraction
and analytical system as well as the effectiveness of the method in
dealing with each sample matrix by spiking each sample, standard and
reagent water blank with one or two surrogates (e.g., analytes not
expected to be present in the sample).
5.7 Matrix Spiking Solutions
5.7.1 Prepare matrix spiking solutions in methanol such that the
concentration in the sample is five times the Estimated Quantitation Limit
(Table 1). All target analytes should be included.
8330 - 5 Revision 0
September 1994
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5.8 HPLC Mobile Phase
5.8.1 To prepare 1 liter of mobile phase, add 500 ml of methanol to
500 mL of organic-free reagent water.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 Follow conventional sampling and sample handling procedures as
specified for semivolatile organics in Chapter Four.
6.2 Samples and sample extracts must be stored in the dark at 4°C.
Holding times are the same as for semivolatile organics.
7.0 PROCEDURE
7.1 Sample Preparation
7.1.1 Aqueous Samples: It is highly recommended that process waste
samples be screened with the high-level method to determine if the low
level method (1-50 M9/L) is required. Most groundwater samples will fall
into the low level method.
7.1.1.1 Low-Level Method (salting-out extraction)
7.1.1.1.1 Add 251.3 g of sodium chloride to a 1 L
volumetric flask (round). Measure out 770 mL of a water
sample (using a 1 L graduated cylinder) and transfer it to the
volumetric flask containing the salt. Add a stir bar and mix
the contents at maximum speed on a magnetic stirrer until the
salt is completely dissolved.
7.1.1.1.2 Add 164 mL of acetonitrile (measured with a
250 mL graduated cylinder) while the solution is being stirred
and stir for an additional 15 minutes. Turn off the stirrer
and allow the phases to separate for 10 minutes.
7.1.1.1.3 Remove the acetonitrile (upper) layer (about
8 mL} with a Pasteur pipet and transfer it to a 100 mL
volumetric flask (round). Add 10 mL of fresh acetonitrile to
the water sample in the 1 L flask. Again stir the contents of
the flask for 15 minutes followed by 10 minutes for phase
separation. Combine the second acetonitrile portion with the
initial extract. The inclusion of a few drops of salt water
at this point is unimportant.
7.1.1.1.4 Add 84 mL of salt water (325 g NaCl per 1000
mL of reagent water) to the acetonitrile extract in the 100 mL
volumetric flask. Add a stir bar and stir the contents on a
magnetic stirrer for 15 minutes, followed by 10 minutes for
phase separation. Carefully transfer the acetonitrile phase
8330 - 6 Revision 0
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to a 10 ml graduated cylinder using a Pasteur pipet. At this
stage, the amount of water transferred with the acetonitrile
must be minimized. The water contains a high concentration of
NaCl that produces a large peak at the beginning of the
chromatogram, where it could interfere with the HMX
determination.
7.1.1.1.5 Add an additional 1.0 ml of acetonitrile to
the 100 ml volumetric flask. Again stir the contents of the
flask for 15 minutes, followed by 10 minutes for phase
separation. Combine the second acetonitrile portion with the
initial extract in the 10 ml graduated cylinder (transfer to
a 25 ml graduated cylinder if the volume exceeds 5 mL).
Record the total volume of acetonitrile extract to the nearest
0.1 ml. (Use this as the volume of total extract [Vt] in the
calculation of concentration after converting to juL). The
resulting extract, about 5 - 6 ml, is then diluted 1:1 with
organic-free reagent water (with pH <3 if tetryl is a
suspected analyte) prior to analysis.
7.1.1.1.6 If the diluted extract is turbid, filter it
through a 0.45 - urn Teflon filter using a disposable syringe.
Discard the first 0.5 ml of filtrate, and retain the remainder
in a Teflon-capped vial for RP-HPLC analysis as in Sec. 7.4.
7.1.1.2 High-level Method
7.1.1.2.1 Sample filtration: Place a 5 ml aliquot of
each water sample in a scintillation vial, add 5 ml of
acetonitrile, shake thoroughly, and filter through a 0.45-/nm
Teflon filter using a disposable syringe. Discard the first
3 ml of filtrate, and retain the remainder in a Teflon-capped
vial for RP-HPLC analysis as in Sec. 7.4. HMX quantitation
can be improved with the use of methanol rather than
acetonitrile for dilution before filtration.
7.1.2 Soil and Sediment Samples
7.1.2.1 Sample homogenization: Dry soil samples in air at
room temperature or colder to a constant weight, being careful not
to expose the samples to direct sunlight. Grind and homogenize the
dried sample thoroughly in an acetonitrile-rinsed mortar to pass a
30 mesh sieve.
NOTE: Soil samples should be screened by Method 8515 prior to
grinding in a mortar and pestle (See Safety Sec. 11.2).
7.1.2.2 Sample extraction
7.1.2.2.1 Place a 2.0 g subsample of each soil sample
in a 15 mi glass vial. Add 10.0 ml of acetonitrile, cap with
8330 - 7 Revision 0
September 1994
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Teflon-lined cap, vortex swirl for one minute, and place in a
cooled ultrasonic bath for 18 hours.
7.1.2.2.2 After sonication, allow sample to settle for
30 minutes. Remove 5.0 ml of supernatant, and combine with
5.0 ml of calcium chloride solution (Sec. 5.1.3) in a 20 ml
vial. Shake, and let stand for 15 minutes.
7.1.2.2.3 Place supernatant in a disposable syringe
and filter through a 0.45-/nm Teflon filter. Discard first 3
ml and retain remainder in a Teflon-capped vial for RP-HPLC
analysis as in Sec. 7.4.
7.2 Chromatographic Conditions (Recommended)
Primary Column: C-18 reverse phase HPLC column, 25-cm
x 4.6-mm, 5 pm, (Supelco LC-18 or equivalent).
Secondary Column: CN reverse phase HPLC column, 25-cm x
4.6-mm, 5 /xm, (Supelco LC-CN or
equivalent).
Mobile Phase: 50/50 (v/v) methanol/organic-free
reagent water.
Flow Rate: 1.5 mL/min
Injection volume: 100-juL
UV Detector: 254 nm
7.3 Calibration of HPLC
7.3.1 All electronic equipment is allowed to warm up for 30 minutes.
During this period, at least 15 void volumes of mobile phase are passed
through the column (approximately 20 min at 1.5 mL/min) and continued
until the baseline is level at the UV detector's greatest sensitivity.
7.3.2 Initial Calibration. Injections of each calibration standard
over the concentration range of interest are made sequentially into the
HPLC in random order. Peak heights or peak areas are obtained for each
analyte. Experience indicates that a linear calibration curve with zero
intercept is appropriate for each analyte. Therefore, a response factor
for each analyte can be taken as the slope of the best-fit regression
line.
7.3.3 Daily Calibration. Analyze midpoint calibration standards, at
a minimum, at the beginning of the day, singly at the midpoint of the run,
and singly after the last sample of the day (assuming a sample group of 10
samples or less). Obtain the response factor for each analyte from the
mean peak heights or peak areas and compare it with the response factor
obtained for the initial calibration. The mean response factor for the
8330 - 8 Revision 0
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daily calibration must agree within ±15% of the response factor of the
initial calibration. The same criteria is required for subsequent
standard responses compared to the mean response of the triplicate
standards beginning the day. If this criterion is not met, a new initial
calibration must be obtained.
7.4 HPLC Analysis
7.4.1 Analyze the samples using the chromatographic conditions given
in Sec. 7.2. All positive measurements observed on the C-18 column must
be confirmed by injection onto the CN column.
7.4.2 Follow Sec. 7.0 in Method 8000 for instructions on the
analysis sequence, appropriate dilutions, establishing daily retention
time windows, and identification criteria. Include a mid-level standard
after each group of 10 samples in the analysis sequence. If column
temperature control is not employed, special care must be taken to ensure
that temperature shifts do not cause peak misidentification.
7.4.3 Table 2 summarizes the estimated retention times on both C-18
and CN columns for a number of analytes analyzable using this method. An
example of the separation achieved by Column 1 is shown in Figure 1.
7.4.4 Record the resulting peak sizes in peak heights or area units.
The use of peak heights is recommended to improve reproducibility of low
level samples.
7.4.5 Calculation of concentration is covered in Sec. 7.0 of Method
8000.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for specific quality control procedures.
Quality control to validate sample extraction is covered in Method 3500.
8.2 Quality control required to validate the HPLC system operation is
found in Method 8000, Sec. 8.0.
8.3 Prior to preparation of stock solutions, acetonitrile, methanol, and
water blanks should be run to determine possible interferences with analyte
peaks. If the acetonitrile, methanol, or water blanks show contamination, a
different batch should be used.
9.0 METHOD PERFORMANCE
9.1 Table 3 presents the single laboratory precision based on data from
the analysis of blind duplicates of four spiked soil samples and four field
contaminated samples analyzed by seven laboratories.
8330 - 9 Revision 0
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9.2 Table 4 presents the rnultilaboratory error based on data from the
analysis of blind duplicates of four spiked soil samples and four field
contaminated samples analyzed by seven laboratories.
9.3 Table 5 presents the multilaboratory variance of the high
concentration method for water based on data from nine laboratories.
9.4 Table 6 presents multi laboratory recovery data from the analysis of
spiked soil samples by seven laboratories.
9.5 Table 7 presents a comparison of method accuracy for soil and aqueous
samples (high concentration method).
9.6 Table 8 contains precision and accuracy data for the salting-out
extraction method.
10.0 REFERENCES
1. Bauer, C.F., T.F. Jenkins, S.M. Koza, P.W. Schumacher, P.H. Miyares and
M.E. Walsh (1989). Development of an analytical method for the
determination of explosive residues in soil. Part 3. Collaborative test
results and final performance evaluation. USA Cold Regions Research and
Engineering Laboratory, CRREL Report 89-9.
2. Grant, C.L., A.D. Hewitt and T.F. Jenkins (1989) Comparison of low
concentration measurement capability estimates in trace analysis: Method
Detection Limits and Certified Reporting Limits. USA Cold Regions
Research and Engineering Laboratory, Special Report 89-20.
3. Jenkins, T.F., C.F. Bauer, D.C. Leggett and C.L. Grant (1984)
Reversed-phased HPLC method for analysis of TNT, RDX, HMX and 2,4-DNT in
munitions wastewater. USA Cold Regions Research and Engineering
Laboratory, CRREL Report 84-29.
4. Jenkins, T.F. and M.E. Walsh (1987) Development of an analytical method
for explosive residues in soil. USA Cold Regions Research and Engineering
Laboratory, CRREL Report 87-7.
5. Jenkins, T.F., P.H. Miyares and ME. Walsh (1988a) An improved RP-HPLC
method for determining nitroaromatics and nitramines in water. USA Cold
Regions Research and Engineering Laboratory, Special Report 88-23.
6. Jenkins, T.F. and P.H. Miyares (1992) Comparison of Cartridge and
Membrane Solid-Phase Extraction with Salting-out Solvent Extraction for
Preconcentration of Nitroaromatic and Nitramine Explosives from Water.
USA Cold Regions Research and Engineering Laboratory, Draft CRREL Special
Report.
7. Jenkins, T.F., P.W. Schumacher, M.E. Walsh and C.F. Bauer (1988b)
Development of an analytical method for the determination of explosive
residues in soil. Part II: Further development and ruggedness testing.
USA Cold Regions Research and Engineering Laboratory, CRREL Report 88-8.
8330 - 10 Revision 0
September 1994
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8. Leggett, D.C., T.F. Jenkins and P.H. Miyares (1990) Salting-out solvent
extraction for preconcentration of neutral polar organic solutes from
water. Analytical Chemistry, 62: 1355-1356.
9. Miyares, P.H. and T.F. Jenkins (1990) Salting-out solvent extraction for
determining low levels of nitroaromatics and nitramines in water. USA
Cold Regions Research and Engineering Laboratory, Special Report 90-30.
11.0 SAFETY
11.1 Standard precautionary measures used for handling other organic
compounds should be sufficient for the safe handling of the analytes targeted by
Method 8330. The only extra caution that should be taken is when handling the
analytical standard neat material for the explosives themselves and in rare cases
where soil or waste samples are highly contaminated with the explosives. Follow
the note for drying the neat materials at ambient temperatures.
11.2 It is advisable to screen soil or waste samples using Method 8515 to
determine whether high concentrations of explosives are present. Soil samples
as high as 2% 2,4,6-TNT have been safely ground. Samples containing higher
concentrations should not be ground in the mortar and pestle. Method 8515 is for
2,4,6-TNT, however, the other nitroaromatics will also cause a color to be
developed and provide a rough estimation of their concentrations. 2,4,6-TNT is
the analyte most often detected in high concentrations in soil samples. Visual
observation of a soil sample is also important when the sample is taken from a
site expected to contain explosives. Lumps of material that have a chemical
appearance should be suspect and not ground. Explosives are generally a very
finely ground grayish-white material.
8330 - 11 Revision 0
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TABLE 1
ESTIMATED QUANTITATION LIMITS
Compounds
HMX
RDX
1,3,5-TNB
1,3-DNB
Tetryl
NB
2,4,6-TNT
4-Am-DNT
2-Am-DNT
2,6-DNT
2,4-DNT
2-NT
4-NT
3-NT
Water
Low-Level
-
0.84
0.26
0.11
-
-
0.11
0.060
0.035
0.31
0.020
-
-
-
(uq/L)
High-Level
13.0
14.0
7.3
4.0
4.0
6.4
6.9
-
-
9.4
5.7
12.0
8.5
7.9
Soil (mg/kg)
2.2
1.0
0.25
0.25
0.65
0.26
0.25
-
-
0.26
0.25
0.25
0.25
0.25
8330 - 12
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TABLE 2
RETENTION TIMES AND CAPACITY FACTORS ON LC-18 AND LC-CN COLUMNS
Compound
HMX
RDX
1,3,5-TNB
1,3-DNB
Tetryl
NB
2,4,6-TNT
4-Am-DNT
2-Am-DNT
2,6-DNT
2,4-DNT
2-NT
4-NT
3-NT
Retention
(mi
LC-18
2.44
3.73
5.11
6.16
6.93
7.23
8.42
8.88
9.12
9.82
10.05
12.26
13.26
14.23
time
n)
LC-CN
8.35
6.15
4.05
4.18
7.36
3.81
5.00
5.10
5.65
4.61
4.87
4.37
4.41
4.45
Capacity
(k)
LC-18
0.49
1.27
2.12
2.76
3.23
3.41
4.13
4.41
4.56
4.99
5.13
6.48
7.09
7.68
factor
*
LC-CN
2.52
1.59
0.71
0.76
2.11
0.61
1.11
1.15
1.38
0.95
1.05
0.84
0.86
0.88
* Capacity factors are based on an unretained peak for nitrate at 1.71 min on
LC-18 and at 2.00 min on LC-CN.
8330 - 13 Revision 0
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TABLE 3
SINGLE LABORATORY PRECISION OF METHOD FOR SOIL SAMPLES
Spiked Soils
RDX
1,3,5-TNB
1,3-DNB
Tetryl
2,4,6-TNT
2,4-DNT
Mean Cone.
(mg/kg) SD
%RSD
Field-Contaminated Soils
Mean Cone.
(mg/kg) SD %RSD
HMX
46
1.7
3.7
14
153
1.8
21.6
12.8
14.1
60
8.6
46
3.5
17
40
5.0
1.4
0.4
1.9
0.14
3.1
1.4
0.17
2.3
4.6
4.1
4.0
17.9
3.5
3.4
104
877
2.8
72
1.1
2.3
7.0
669
1.0
12
29.6
0.2
6.0
0.11
0.41
0.61
55
0.44
11.5
3.4
7.1
8.3
9.8
18.0
9.0
8.2
42.3
8330 - 14
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TABLE 4
MULTILABORATORY ERROR OF METHOD FOR SOIL SAMPLES
Spiked Soils
HMX
RDX
1,3,5-TNB
1,3-DNB
Tetryl
2,4,6-TNT
2,4-DNT
Mean
(mg/kg)
46
60
8.6
46
3.5
17
40
5.0
Cone.
SO
2.6
2.6
0.61
2.97
0.24
5.22
1.88
0.22
%RSD
5.7
4.4
7.1
6.5
6.9
30.7
4.7
4.4
Field-Contaminated Soils
(mg/kg)
14
153
104
877
2.8
72
1.1
2.3
7.0
669
1.0
Mean Cone.
SD %RSD
3.7
37.3
17.4
67.3
0.23
8.8
0.16
0.49
1.27
63.4
0.74
26.0
24.0
17.0
7.7
8.2
12.2
14.5
21.3
18.0
9.5
74.0
TABLE 5
MULTILABORATORY VARIANCE OF METHOD FOR WATER SAMPLES"
Compounds
HMX
RDX
2,4-DNT
2,4,6-TNT
Mean Cone.
(M9/L)
203
274
107
107
SD
14.8
20.8
7.7
11.1
%RSD
7.3
7.6
7.2
10.4
a Nine Laboratories
8330 - 15
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TABLE 6
MULTILABORATORY RECOVERY DATA FOR SPIKED SOIL SAMPLES
Laboratory
1
3
4
5
6
7
8
True Cone
Mean
Std Dev
% RSD
% Diff*
Mean %
Recovery
HMX
44.97
50.25
42.40
46.50
56.20
41.50
52.70
50.35
47.79
5.46
11.42
5.08
95
Concentration (jig/g)
1,3,5- 1,3-
RDX TNB DNB
48.78
48.50
44.00
48.40
55.00
41.50
52.20
50.20
48.34
4.57
9.45
3.71
96
48.99
45.85
43.40
46.90
41.60
38.00
48.00
50.15
44.68
3.91
8.75
10.91
89
49.94
45.96
49.50
48.80
46.30
44.50
48.30
50.05
47.67
2.09
4.39
4.76
95
Tetryl
32.48
47.91
31.60
32.10
13.20
2.60
44.80
50.35
29.24
16.24
55.53
41.93
58
2,4,6-
TNT
49.73
46.25
53.50
55.80
56.80
36.00
51.30
50.65
49.91
7.11
14.26
1.46
98
2,4-
DNT
51.05
48.37
50.90
49.60
45.70
43.50
49.10
50.05
48.32
2.78
5.76
3.46
96
* Between true value and mean determined value.
8330 - 16
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September 1994
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Analyte
TABLE 7
COMPARISON OF METHOD ACCURACY FOR SOIL AND AQUEOUS SAMPLES
(HIGH CONCENTRATION METHOD)
Recovery (%)
Soil Method*
Aqueous Method
**
2,4-DNT
2,4,6-TNT
RDX
HMX
96.0
96.8
96.8
95.4
98.6
94.4
99.6
95.5
* Taken from Bauer et al. (1989), Reference 1.
** Taken from Jenkins et al. (1984), Reference 3.
8330 - 17
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September 1994
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TABLE 8
PRECISION AND ACCURACY DATA FOR THE SALTING-OUT EXTRACTION METHOD
Analyte
HMX
RDX
1,3,5-TNB
1,3-DNB
Tetryl
2,4,6-TNT
2-Atn-DNT
2,4-DNT
1,2-NT
1,4-NT
1,3-NT
No. of Samples1
20
20
20
20
20
20
20
20
20
20
20
Precision
(% RSD)
10.5
8.7
7.6
6.6
16.4
7.6
9.1
5.8
9.1
18.1
12.4
Ave. Recovery
(%)
106
106
119
102
93
105
102
101
102
96
97
Cone. Range
(M9/L)
0-1.14
0-1.04
0-0.82
0-1.04
0-0.93
0-0.98
0-1.04
0-1.01
0-1.07
0-1.06
0-1.23
1Reagent water
8330 - 18
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September 1994
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EXPLOSIVES ON A
CIS COLUMN
X
CD
2
EXPLOSIVES ON A
CM COLUMN
FIGURE 1
CHROMATOGRAMS FOR COLUMNS DESCRIBED IN Sec. 4.1.2.
COURTESY OF U.S. ARMY CORPS OF ENGINEERS, OMAHA, NE.
8330 - 19
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September 1994
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METHOD 8330
NITROAROMATICS AND NITRAMINES BY HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
SaHino Oifl
71111 Add 251 3 g ot sat
and 1 L ot water sample to a
t L vol. flasK Mix the contents
I '
711 1 2 Add 164 mL ot
acatoniinle (ACN) and stir
lor 1 5 mins
i
7 i.\ VI Transfer ACN layer
(o tOOmL tot. (task. Add to ml.
ot Iresh ACN to 1 L lUisK and
stir Transfer 2nd portion and
combine wnn 1 st in 1 00 mL flask.
1
71 1 1 .4 Add 84 ml. ot sail
water to the ACN extratf and stir
Transtsr ACN extract lo 10 mL
qrad cylinder
(
r
7 1 1 1 5 Add 1 mlolACNlo
too ml vol. flasK. Stir and
transfer to the 10 mL grad.
cylinder. Record volume.
Dilute 1 1 with reagent water.
1
7 t 1 1 6 FilteMf tu
-------
METHOD 8330
(continued)
7 1 2. i Sample riomoggnizanon
Air dry sample at room Temp
or Below Avoid exposure to
direct sunligm. Grind sample
in an acetonitrile rinsed mortar
T 1 a 2 Sample Extraction
7 1 22.1
Place 2 g soil sjbsample.
10 mis acetontnle in 15 mi.
glass vial Cap. vortex swirl.
place in room Temp or below
ultrasonic bath tor 18 hrs.
1 1 2.2.2
Lsrsoln sente AddSmL
supernatant to 5 rnL calcium
chloride sotn in 20 ml. vial
Shake let stand 15 mins
7 1 22.3
Filter supernatant through
05 urn filter. Discard initial
3 mL. retain remainder
lor analysis.
7 2 Set Chroma tographx Conditions
7 3 Calibration ot HPLC
732
Run working stds in tnplicate.
Plot ng vs peak area or ht
Curve should be linear with
zero intercept.
733
Analyze midrange calibration
std at beginning, middle.
and end ot sample analyses.
Rodo Section 7 3 1 it peaK
areas or hts do not agree
to w/in */• 20% of inidai
calibrator values.
7 4 Sample Analysis
74 1
Analyze samples Confirm
measurment w/mjection onto
CN column.
743
Refer to Table 2 tor typical
analyte retention Dmes
8330 - 21
Revision 0
September 1994
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00
u>
OJ
-------
METHOD 8331
TETRAZENE BY REVERSE PHASE
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
1.0 SCOPE AND APPLICATION
1.1 This method is intended for the analysis of tetrazene, an explosive
residue, in soil and water. This method is limited to use by analysts
experienced in handling and analyzing explosive materials. The following
compounds can be determined by this method:
Compound CAS No"
Tetrazene 31330-63-9
8 Chemical Abstracts Service Registry number
1.2 Tetrazene degrades rapidly in water and methanol at room temperature.
Special care must be taken to refrigerate or cool all solutions throughout the
analytical process.
1.3 Tetrazene, in its dry form, is extremely explosive. Caution must be
taken during preparation of standards.
1.4 The estimated quant itat ion 1 imit (EQL) of Method 8331 for determining
the concentration of tetrazene is approximately 7 M9/L in water and
approximately 1 mg/kg in soil.
1.5 This method is restricted to use by or under the supervision of
analysts experienced in the use of HPLC, skilled in the interpretation of
chromatograms, and experienced in handling explosive materials. Each analyst
must demonstrate the ability to generate acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 A 10 mL water sample is filtered, eluted on a C-18 column using ion
pairing reverse phase HPLC, and quantitated at 280 nm.
2.2 2 g of soil are extracted with 55:45 v/v methanol-water and
1-decanesulfonic acid on a platform shaker, filtered, and eluted on a C-18 column
using ion pairing reverse phase HPLC, and quantitated at 280 nm.
8331 - 1 Revision 0
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3.0 INTERFERENCES
3.1 No interferences are known. Tetrazene elutes early, however, and if
a computing integrator is used for peak quantification, the baseline setting may
have to be set to exclude baseline aberrations. Baseline setting is particularly
important at low concentrations of analyte.
4.0 APPARATUS AND MATERIALS
4.1 HPLC system
4.1.1 HPLC - Pump capable of achieving 4000 psi.
4.1.2 100 /XL loop injector.
4.1.3 Variable or fixed wavelength detector capable of reading
280 nm.
4.1.4 C-18 reverse phase HPLC column, 25 cm x 4.6 mm (5 /zm)
(Supelco LC-18, or equivalent).
4.1.5 Digital integrator - HP 3390A (or equivalent)
4.1.6 Strip chart recorder.
4.2 Other apparatus
4.2.1 Platform orbital shaker.
4.2.2 Analytical balance - ± 0,0001 g.
4.2.3 Desiccator.
4.3 Materials
4.3.1 Injection syringe - 500 /zL.
4.3.2 Filters - 0.5 /im Millex-SR and 0.5 /urn Millex-HV, disposable,
or equivalent.
4.3.3 Pipets - volumetric, glass, Class A.
4.3.4 Scintillation vials - 20 mL, glass.
4.3.5 Syringes - 10 mL.
4.3.6 Volumetric flasks, Class A - 100 mL, 200 mL.
4.3.7 Erlenmeyer flasks with ground glass stoppers - 125 mL.
8331 - 2 Revision 0
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4,4 Preparation
4.4.1 Prepare all materials as described in Chapter 4 for volatile
organics.
5.0 REAGENTS
5.1 HPLC grade chemicals shall be used in all tests. It is intended that
all reagents shall conform to the specifications of the Committee on Analytical
Reagents of the American Chemical Society, where such specifications are
available. Other grades may be used, provided it is first ascertained that the
reagent is of sufficiently high purity to permit its use without lowering the
accuracy of the determination.
5.2 General
5.2.1 Methanol, CH3OH - HPLC grade.
5.2.2 Organic-free reagent water - All references to water in this
method refer to organic-free reagent water, as defined in Chapter One.
5.2.3 1-Decanesulfonic acid, sodium salt, C10H21S03Na - HPLC grade.
5.2.4 Acetic acid (glacial), CH3COOH - reagent grade.
5.3 Standard Solutions
5.3.1 Tetrazene - Standard Analytical Reference Material.
5.3.2 Stock standard solution - Dry tetrazene to constant weight
in a vacuum desiccator in the dark. (Tetrazene is extremely explosive in
the dry state. Do not dry more reagent than is necessary to prepare stock
solutions.) Place about 0.0010 g (weighed to 0.0001 g) into a 100-ml
volumetric flask and dilute to volume with methanol. Invert flask several
times until tetrazene is dissolved. Store in freezer at -10°C. Stock
solution is about 100 mg/L. Replace stock standard solution every week.
5.3.3 Intermediate standard solutions
5.3.3.1 Prepare a 4 mg/L standard by diluting the stock
solution 1/25 v/v with methanol.
5.3.3.2 Pipet 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 mL of the
4 mg/L standard solution into 6 separate 100 mL volumetric flasks,
and make up to volume with methanol. Pipet 25.0 mL of the 4 mg/L
standard solution into a 50 mL volumetric flask, and make up to
volume with methanol. This results in intermediate standards of
about 0.02, 0.04, 0.08, 0.2, 0.4, 0.8, 2 and 4 mg/L.
5.3.3.3 Cool immediately on preparation in refrigerator or
ice bath.
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5.3.4 Working standard solutions
5.3.4.1 Inject 4 ml of each of the intermediate standard
solutions into 6.0 mL of water. This results in concentrations of
about 0.008, 0.016, 0.032, 0.08, 0.16, 0.3, 0.8 and 1.6 mg/L.
5.3.4.2 Cool immediately on preparation in refrigerator or
ice bath.
5.5 QC spike concentrate solution
5.5.1 Dry tetrazene to constant weight in a vacuum desiccator in
the dark. (Tetrazene is extremely explosive in the dry state. Do not dry
any more than necessary to prepare standards.) Place about 0.0011 g
(weighed to 0.0001 g) into a 200-ml volumetric flask and dilute to volume
with methanol. Invert flask several times until tetrazene is dissolved.
Store in freezer at -10"C. QC spike concentrate solution is about 55
mg/L. Replace stock standard solution every week.
5.5.2 Prepare spiking solutions, at concentrations appropriate to
the concentration range of the samples being analyzed, by diluting the QC
spike concentrate solution with methanol. Cool on preparation in
refrigerator or ice bath.
5.6 Eluent
5.6.1 To make about 1 liter of eluent, add 2.44 g of
1-decanesulfonic acid, sodium salt to 400/600 v/v methanol/water, and add
2.0 ml of glacial acetic acid.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this Chapter, Organic Analytes,
Section 4.1.
6.2 Samples must be collected and stored in glass containers. Follow
conventional sampling procedures.
6.3 Samples must be kept below 4°C from the time of collection through
analysis.
7.0 PROCEDURE
7.1 Sample Preparation
7.1.1 Filtration of Water Samples
7.1.1.1 Place a 10 mL portion of each water sample in a
syringe and filter through a 0.5 (j,m Millex-HV filter unit. Discard
first 5 mL of filtrate, and retain 5 mL for analysis.
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7.1.2 Extraction and Filtration of Soil Samples
7.1.2.1 Determination of sample % dry weight - In certain
cases, sample results are desired based on dry-weight basis. When
such data is desired, a portion of sample for this determination
should be weighed out at the same time as the portion used for
analytical determination.
WARNING: The drying oven should be contained in a hood or
vented. Significant laboratory contamination may
result from a heavily contaminated hazardous
waste sample.
7.1.2.1.1 Immediately after weighing the sample for
extraction, weigh 5-10 g of the sample into a tared
crucible. Determine the % dry weight of the sample by
drying overnight at 105°C. Allow to cool in a desiccator
before weighing:
% dry weight = q of dry sample x 100
g of sample
7.1.2.2 Weigh 2 g soil subsamples into 125 ml Erlenmeyer
flasks with ground glass stoppers.
7.1.2.3 Add 50 mL of 55/45 v/v methane! -water with
1-decanesulfonic acid, sodium salt added to make a 0.1 M solution.
7.1.2.4 Vortex for 15 seconds.
7.1.2.5 Shake for 5 hr at 2000 rpm on platform shaker.
7.1.2.6 Place a 10 ml portion of each soil sample extract
in a syringe and filter through a 0.5 /^m Millex-SR filter unit.
Discard first 5 mi of filtrate, and retain 5 ml for analysis.
7.2 Sample Analysis
7.2.1 Analyze the samples using the chromatographic conditions
given in Section 7.2.1.1. Under these conditions, the retention time of
tetrazene is 2.8 min. A sample chromatogram, including other compounds
likely to be present in samples containing tetrazene, is shown in
Figure 1.
7.2.1.1 Chromatographic Conditions
Solvent: 0.01 M 1-decanesulfonic acid, in
acidic methanol/water (Section 5.5)
Flow rate: 1.5 mL/min
Injection volume: 100 fj,L
UV Detector: 280 nm
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7.3 Calibration of HPLC
7.3.1 Initial Calibration - Analyze the working standards
(Section 5.3.4), starting with the 0.008 mg/L standards and ending with
the 0.30 mg/L standard. If the percent relative standard deviation (%RSD)
of the mean response factor (RF) for each analyte does not exceed 20%, the
system is calibrated and the analysis of samples may proceed. If the %RSD
for any analyte exceeds 20%, recheck the system and/or recalibrate with
freshly prepared calibration solutions.
7.3.2 Continuing Calibration - On a daily basis, inject 250 pi of
stock standard into 20 mL water. Keep solution in refrigerator until
analysis. Analyze in triplicate (by overfilling loop) at the beginning of
the day, singly after each five samples, and singly after the last sample
of the day. Compare response factors from the mean peak area or peak
height obtained over the day with the response factor at initial
calibration. If these values do not agree within 10%, recalibrate.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for specific quality control procedures.
8.2 Prior to preparation of stock solutions, methanol should be analyzed
to determine possible interferences with the tetrazene peak. If the methanol
shows contamination, a different batch of methanol should be used.
8.3 Method Blanks
8.3.1 Method blanks for the analysis of water samples should be
organic-free reagent water carried through all sample storage and handling
procedures.
8.3.2 Method blanks for the analysis of soil samples should be
uncontaminated soil carried through all sample storage, extraction, and
handling procedures.
9.0 METHOD PERFORMANCE
9.1 Method 8331 was tested in a laboratory over a period of four days.
Spiked organic-free reagent water and standard soil were analyzed in duplicate
each day for four days. The HPLC was calibrated daily according to the
procedures given in Section 7.1. Method performance data are presented in Tables
1 and 2.
10.0 REFERENCES
1. Walsh, M.E., and T.F. Jenkins, "Analytical Method for Determining
Tetrazene in Water," U.S. Army Corps of Engineers, Cold Regions Research
& Engineering Laboratory, Special Report 87-25, 1987.
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2. Walsh, M.E., and T.F. Jenkins, "Analytical Method for Determining
Tetrazene in Soil," U.S. Army Corps of Engineers, Cold Regions Research &
Engineering Laboratory, Special Report 88-15, 1988.
11.0 SAFETY
11.1 Standard precautionary measures used for handling other organic
compounds should be sufficient for safe handling of the analytes targeted by
Method 8331.
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FIGURE 1
16
TNT
12
£
8 8
0.064
Absorbonca Units
8331 - 8
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TABLE 1.
METHOD PERFORMANCE, WATER MATRIX
Spike
Cone,
(M9/L)
0.00
7.25
14.5
29
72.5
145
290
725
OVERALL
Avq % Recovery
Repl icate
Replicate 1
% Recovery
Replicate 2
% Recovery
Replicate 1
% Recovery
Repl icate 2
% Recovery
Replicate 1
% Recovery
Replicate 2
% Recovery
Replicate 1
% Recovery
Replicate 2
% Recovery
Replicate 1
% Recovery
Replicate 2
% Recovery
Replicate 1
% Recovery
Replicate 2
% Recovery
Replicate 1
% Recovery
Replicate 2
% Recovery
Replicate 1
% Recovery
Replicate 2
% Recovery
Day 1
0.0
NA
0.0
NA
8.9
122
6.6
91
14.6
101
14.8
102
31.8
110
29.5
102
71.1
98
71.2
98
140.6
97
138.5
96
289.4
100
282.0
97
737.6
102
700.2
97
Day 2
0.0
NA
0.0
NA
7.8
108
9.9
137
14.6
101
14.1
97
30.0
103
29.7
102
73.6
102
71.3
98
143.8
99
140.8
97
288.5
99
284.2
98
707.2
98
695.8
96
Day 3
0.0
NA
0.0
NA
7.4
102
8.5
117
13.8
95
14.1
98
30.8
106
30.4
105
75.7
104
70.7
98
144.7
100
140.9
97
291.0
100
281.9
97
714.3
99
714.2
99
Average
Day 4
0.0
NA
0.0
NA
9.4
130
6.7
92
14.6
101
15.2
105
28.7
99
30.7
106
73.9
102
71.6
99
142.1
98
136.9
94
289.8
100
282.5
97
722.0
100
716.3
99
% Recovery
NA
NA
116
109
99
100
105
104
101
98
98
96
100
97
99
97
102
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TABLE 2
METHOD PERFORMANCE, SOIL MATRIX
Spike
Cone.
(M9/L)
0.00
1.28
2.56
5.12
12.8
25.6
OVERALL
Avq % Recovery
Replicate
Replicate 1
% Recovery
Replicate 2
% Recovery
Replicate 1
% Recovery
Replicate 2
% Recovery
Replicate 1
% Recovery
Replicate 2
% Recovery
Replicate 1
% Recovery
Replicate 2
% Recovery
Replicate 1
% Recovery
Replicate 2
% Recovery
Replicate 1
% Recovery
Replicate 2
% Recovery
Day 1
0.0
NA
0.0
NA
0.6
49
1.2
92
1.4
56
1.5
59
2.9
57
3.0
58
7.8
61
8.0
62
17.2
67
16.7
65
Day 2
0.0
NA
0.0
NA
0.9
73
0.7
56
1.5
58
2.0
79
3.0
58
3.0
59
7.6
59
8.4'
66
16.7
65
16.8
66
Day 3
0.0
NA
0.0
NA
0.6
48
0.8
63
1.6
61
1.4
56
2.9
56
3.5
69
7.8
61
7.7
60
17.4
68
17.6
69
Average
Day 4
0.0
NA
0.0
NA
1.0
74
0.7
56
1.6
61
1.3
50
2.9
56
3.1
60
8.1
63
8.2
64
17.3
68
17.2
67
% Recovery
NA
NA
61
67
59
61
57
61
61
63
67
67
62
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METHOD 8331
TETRAZENE BY REVERSE PHASE
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Start
7.1 .1 Filter 10 mL
water sample; discard
first 5 mL; analyze last 5.
7.1 .2.1 Determine
% dry weight.
7.1 .2.2 • 7.1.2.5
Extract 2 g soil
with 50 mL solvent.
7.1.2.6 Filter 10 ml
extract; discard 5 mL;
analyze last 5 mL.
7.2 Analyze samples
using chromatographic
conditions in
Section 7.2.1.1.
7.3.1 Initial calibration:
Analyze working
standards
(Section 5.3.3).
7.3.1 is % RSD
of mean RF
>20%?
7.3.1 Recheck system/
recalibrate with new
calibration solution.
7.3.2
Continuing
Calibration.
^
r
Stop
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00
A.
»««*
o
-------
METHOD 8410
GAS CHROMATOGRAPHY/FOURIER TRANSFORM INFRARED
(GC/FT-IR) SPECTROMETRY FOR SEMIVOLATILE ORGANICS:
CAPILLARY COLUMN
1.0 SCOPE AND APPLICATION
1.1 This method covers the automated identification, or compound class
assignment of unidentifiable compounds, of solvent extractable semivolatile
organic compounds which are amenable to gas chromatography, by GC/FT-IR.
GC/FT-IR can be a useful complement to GC/MS analysis (Method 8270). It is
particularly well suited for the identification of specific isomers that are not
differentiated using GC/MS. Compound class assignments are made using infrared
group absorption frequencies. The presence of an infrared band in the
appropriate group frequency region may be taken as evidence of the possible
presence of a particular compound class, while its absence may be construed as
evidence that the compound class in question is not present. This evidence will
be further strengthened by the presence of confirmatory group frequency bands.
Identification limits of the following compounds have been demonstrated by this
method.
Compound Name
CAS
Acenaphthene
Acenaphthylene
Anthracene
Benzo( a) anthracene
Benzo(a)pyrene
Benzoic acid
Bis(2-chloroethoxy) methane
Bis(2-chloroethyl ) ether
Bis(2-chloroisopropyl ) ether
Bis(2-ethylhexyl) phthalate
4-Bromophenyl phenyl ether
Butyl benzyl phthalate
4-Chloroaniline
4- Chi oro-3 -methyl phenol
2-Chloronaphthalene
2-Chlorophenol
4-Chlorophenol
4-Chlorophenyl phenyl ether
Chrysene
Dibenzofuran
Di-n-butyl phthalate
1,2-Dichlorobenzene
1 ,3-Dichlorobenzene
1,4-Dichlorobenzene
2,4-Dichlorophenol
83-32-9
208-96-8
120-12-7
56-55-3
50-32-8
65-85-0
111-91-1
111-44-4
39638-32-9
117-81-7
101-55-3
85-68-7
106-47-8
59-50-7
91-58-7
95-57-8
106-48-9
7005-72-3
218-01-9
132-64-9
84-74-2
95-50-1
541-73-1
106-46-7
120-83-2
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Compound Name CAS No.8
Dimethyl phthalate 131-11-3
Diethyl phthalate 84-66-2
4,6-Dinitro-2-methyl phenol 534-52-1
2,4-Dinitrophenol 51-28-5
2,4-Dinitrotoluene 121-14-2
2,6-Dinitrotoluene 606-20-2
Di-n-octyl phthalate 117-84-0
Di-n-propyl phthalate 131-16-8
Fluoranthene 206-44-0
Fluorene 86-73-7
Hexachlorobenzene 118-74-1
1,3-Hexachlorobutadiene 87-68-3
Hexachlorocyclopentadiene 77-47-4
Hexachloroethane 67-72-1
Isophorone 78-59-1
2-Methylnaphthalene 91-57-6
2-Methyl phenol 95-48-7
4-Methylphenol 106-44-5
Naphthalene 91-20-3
2-Nitroaniline 88-74-4
3-Nitroaniline 99-09-2
4-Nitroaniline 100-01-6
Nitrobenzene 98-95-3
2-Nitrophenol 88-75-5
4-Nitrophenol 100-02-7
N-Nitrosodimethylamine 62-75-9
N-Nitrosodiphenylamine 86-30-9
N-Nitroso-di-n-propylamine 621-64-7
Pentachlorophenol 87-86-5
Phenanthrene 85-01-8
Phenol 108-95-2
Pyrene 129-00-0
1,2,4-Trichlorobenzene 120-82-1
2,4,5-Trichlorophenol 95-95-4
2,4,6-Trichlorophenol 88-06-2
" Chemical Abstract Services Registry Number.
1.2 This method is applicable to the determination of most extractable,
semi volatile-organic compounds in wastewater, soils and sediments, and solid
wastes. Benzidine can be subject to losses during solvent concentration and GC
analysis; a-BHC, /8-BHC, Endosulfan 1 and II, and Endrin are subject to
decomposition under the alkaline conditions of the extraction step; Endrin is
subject to decomposition during GC analysis; and Hexachlorocyclopentadiene and
N-Nitrosodiphenylamine may decompose during extraction and GC analysis. Other
extraction and/or instrumentation procedures should be considered for unstable
analytes.
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1.3 The identification limit of this method may depend strongly upon the
level and type of gas chromatographable (GC) semi volatile extractants. The
values listed in Tables 1 and 2 represent the minimum quantities of semivolatile
organic compounds which have been identified by the specified GC/FT-IR system,
using this method and under routine environmental analysis conditions. Capillary
GC/FT-IR wastewater identification limits of 25 /ig/L may be achieved for weak
infrared absorbers with this method, while the corresponding identification
limits for strong infrared absorbers is 2 ng/L. Identification limits for other
sample matrices can be calculated from the wastewater values after choice of the
proper sample workup procedure (see Sec. 7.1).
2.0 SUMMARY OF METHOD
2.1 Prior to using this method, the samples should be prepared for
chromatography using the appropriate sample preparation and cleanup methods.
This method describes chromatographic conditions that will allow for the
separation of the compounds in the extract and uses FT-IR for detection and
quantitation of the target analytes.
3.0 INTERFERENCES
3.1 Glassware and other sample processing hardware must be thoroughly
cleaned to prevent contamination and misinterpretation. All of these materials
must be demonstrated to be free from interferences under the conditions of the
analysis by running method blanks. Specific selection of reagents or
purification of solvents by distillation in all-glass systems may be required.
3.2 Matrix interference will vary considerably from source to source,
depending upon the diversity of the residual waste being sampled. While general
cleanup techniques are provided as part of this method, unique samples may
require additional cleanup to isolate the analytes of interest from interferences
in order to achieve maximum sensitivity.
3.3 4-Chlorophenol and 2-nitrophenol are subject to interference from co-
el uting compounds.
3.4 Clean all glassware as soon as possible after use by rinsing with the
last solvent used. Glassware should be sealed/stored in a clean environment
immediately after drying to prevent any accumulation of dust or other
contaminants.
4.0 APPARATUS AND MATERIALS
4.1 Gas Chromatographic/Fourier Transform Infrared Spectrometric
Equipment
4.1.1 Fourier Transform-Infrared Spectrometer - A spectrometer
capable of collecting at least one scan set per second at 8 cm"1 resolution
is required. In general, a spectrometer purchased after 1985, or
retrofitted to meet post-1985 FT-IR improvements, will be necessary to
8410 - 3 Revision 0
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meet the detection limits of this protocol. A state-of-the-art A/D
converter is required, since it has been shown that the signal-to-
noise ratio of single beam GC/FT-IR systems is A/D converter
1imited.
4.1.2 GC/FT-IR Interface - The interface should be lightpipe volume-
optimized for the selected chromatographic conditions (lightpipe volume of
100-200 p.L for capillary columns). The shortest possible inert transfer
line (preferably fused silica) should be used to interface the end of the
chromatographic column to the lightpipe. If fused silica capillary
columns are employed, the end of the GC column can serve as the transfer
line if it is adequately heated. It has been demonstrated that the
optimum lightpipe volume is equal to the full width at half height of the
GC eluate peak.
4.1.3 Capillary Column - A fused silica DB-5 30 m x 0.32 mm
capillary column with 1.0 jum film thickness (or equivalent).
4.1.4 Data Acquisition - A computer system dedicated to the GC/FT-IR
system to allow the continuous acquisition of scan sets for a full
chromatographic run. Peripheral data storage systems should be available
(magnetic tape and/or disk) for the storage of all acquired data.
Software should be available to allow the acquisition and storage of every
scan set to locate the file numbers and transform high S/N scan sets, and
to provide a real time reconstructed chromatogram.
4.1.5 Detector - A cryoscopic, medium-band HgCdTe (MCT) detector
with the smallest practical focal area. Typical narrow-band MCT detectors
operate from 3800-800 cm"1, but medium-band MCT detectors can reach
650 cm'1. A 750 cm"1 cutoff (or lower) is desirable since it allows the
detection of typical carbon-chlorine stretch and aromatic out-of-plane
carbon-hydrogen vibrations of environmentally important organo-chlorine
and polynuclear aromatic compounds. The MCT detector sensitivity (D)'
should be > 1 x 1010 cm.
4.1.6 Lightpipe - Constructed of inert materials, gold coated, and
volume-opt i mi zeid for the desired chromatographic conditions (see Sec.
7.3).
4.1.7 Gas Chromatograph - The FT-IR spectrometer should be
interfaced to a temperature programmable gas chromatograph equipped with
a Grob-type (or equivalent) purged splitless injection system suitable for
capillary glass columns or an on-column injector system.
A short, inert transfer line should interface the gas chromatograph
to the FT-IR lightpipe and, if applicable, to the GC detector. Fused
silica GC columns may be directly interfaced to the lightpipe inlet and
outlet.
4.2 Dry Purge Gas - If the spectrometer is the purge-type, provisions
should be made to provide a suitable continuous source of dry purge-gas to the
FT-IR spectrometer.
8410 - 4 Revision 0
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4.3 Dry Carrier Gas - The carrier gas should be passed through an
efficient cartridge-type drier.
4.4 Syringes - 1-juL, 10-juL.
5.0 REAGENTS
5.1 Reagent grade inorganic chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Organic-free reagent water. All references to water in this method
refer to organic-free reagent water, as defined in Chapter One.
5.3 Solvents
5.3.1 Acetone, CH3COCH3 - Pesticide quality, or equivalent.
5.3.2 Methylene chloride, CH2C12 - Pesticide quality, or equivalent.
5.4 Stock Standard Solutions (1000 mg/L) - Standard solutions can be
prepared from pure standard materials or purchased as a certified solution.
5.4.1 Prepare stock standard solutions by accurately weighing 0.1000
± 0.0010 g of pure material. Dissolve the material in pesticide quality
acetone or other suitable solvent and dilute to volume in a 100 ml
volumetric flask. Larger volumes can be used at the convenience of the
analyst. When compound purity is assayed to be 96 percent or greater, the
weight may be used without correction to calculate the concentration of
the stock standard. Commercially prepared stock standards may be used at
any concentration if they are certified by the manufacturer or by an
independent source.
5.4.2 Transfer the stock standard solutions into bottles with Teflon
lined screw-caps. Store at 4°C and protect from light. Stock standard
solutions should be checked frequently for signs of degradation or
evaporation, especially just prior to preparing calibration standards from
them.
5.4.3 Stock standard solutions must be replaced after 6 months or
sooner if comparison with quality control reference samples indicates a
problem.
5.5 Calibration Standards and Internal Standards - For use in situations
where GC/FT-IR will be used for primary quantitation of analytes rather than
confirmation of GC/MS identification.
5.5.1 Prepare calibration standards that contain the compounds of
interest, either singly or mixed together. The standards should be
8410 - 5 Revision 0
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prepared at concentrations that will completely bracket the working range
of the chromatographic system (at least one order of magnitude is
suggested).
5.5.2 Prepare internal standard solutions. Suggested internal
standards are 1-Fluoronaphthalene, Terphenyl, 2-Chlorophenol, Phenol,
Bis(2-chloroethoxy)methane, 2,4-Dichlorophenol, Phenanthrene, Anthracene,
and Butyl benzyl phthalate. Determine the internal standard concentration
levels from the minimum identifiable quantities. See Tables 1 and 2.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this chapter, Organic Analytes, Sec.
4.1.
7.0 PROCEDURE
7.1 Sample Preparation - Samples must be prepared by one of the following
methods prior to GC/FT-IR analysis.
Matrix Methods
Water 3510, 35.20
Soil/sediment 3540, 3541, 3550
Waste 3540, 3541, 3550, 3580
7.2 Extracts may be cleaned up by Method 3640, Gel-Permeation Cleanup.
7.3 Initial Calibration - Recommended GC/FT-IR conditions:
Scan time: At least 2 scan/sec.
Initial column temperature and hold time: 40°C for 1 minute.
Column temperature program: 40-280°C at 10°C/min.
Final column temperature hold: 280°C.
Injector temperature: 280-300°C.
Transfer line temperature: 270°C.
Lightpipe: 280°C.
Injector: Grob-type, splitless or on-
column.
Sample volume: 2-3 ^L.
Carrier gas: Dry helium at about 1 mL/min.
7.4 With an oscilloscope, check the detector centerburst intensity versus
the manufacturer's specifications. Increase the source voltage, if necessary,
to meet these specifications. For reference purposes, laboratories should
prepare a plot of time versus detector voltage over at least a 5 day period.
7.5 Capillary Column Interface Sensitivity Test - Install a 30 m x
0.32 mm fused silica capillary column coated with 1.0 /urn of DB-5 (or
equivalent). Set the lightpipe and transfer lines at 280°C, the injector at
225°C and the GC detector at 280°C (if used). Under splitless Grob-type or on-
8410 - 6 Revision 0
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column injection conditions, inject 25 ng of nitrobenzene, dissolved in 1 /uL of
methylene chloride. The nitrobenzene should be identified by the on-line library
software search within the first five hits (nitrobenzene should be contained
within the search library).
7.6 Interferometer - If the interferometer is air-driven, adjust the
interferometer drive air pressure to manufacturer's specifications.
7.7 MCT Detector Check - If the centerburst intensity is 75 percent or
less of the mean intensity of the plot maximum obtained by the procedure of Sec.
7.4, install a new source and check the MCT centerburst with an oscilloscope
versus the manufacturer's specifications (if available). Allow at least five
hours of new source operation before data acquisition.
7.8 Frequency Calibration - At the present time, no consensus exists
within the spectroscopic community on a suitable frequency reference standard for
vapor-phase FT-IR. One reviewer has suggested the use of indene as an on-the-fly
standard.
7.9 Minimum Identifiable Quantities - Using the GC/FT-IR operating
parameters specified in Sec. 7.3, determine the minimum identifiable quantities
for the compounds of interest.
7.9.1 Prepare a plot of lightpipe temperature versus MCT centerburst
intensity (in volts or other vertical height units). This plot should
span the temperature range between ambient and the lightpipe thermal limit
in increments of about 20CC. Use this plot for daily QA/QC (see Sec. 8.4).
Note that modern GC/FT-IR interfaces (1985 and later) may have eliminated
most of this temperature effect.
7.10 GC/FT-IR Extract Analysis
7.10.1 Analysis - Analyze the dried methylene chloride extract
using the chromatographic conditions specified in Sec. 7.3 for capillary
column interfaces.
7.10.2 GC/FT-IR Identification - Visually compare the analyte
infrared (IR) spectrum versus the search library spectrum of the most
promising on-line library search hits. Report, as identified, those
analytes with IR frequencies for the five (maximum number) most intense IR
bands (S/N > 5) which are within + 5.0 cm"1 of the corresponding bands in
the library spectrum. Choose IR bands which are sharp and well resolved.
The software used to locate spectral peaks should employ the peak "center
of gravity" technique. In addition, the IR frequencies of the analyte and
library spectra should be determined with the same computer software.
7.10.3 Retention Time Confirmation - After visual comparison of
the analyte and library spectrum as described in Sec. 7.10.2, compare the
relative retention times (RRT) of the analyte and an authentic standard of
the most promising library search hit. The standard and analyte RRT
should agree within + 0.01 RRT units when both are determined at the same
chromatographic conditions.
8410 - 7 Revision 0
September 1994
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7.10.4 Compound Class or Functionality Assignment - If the
analyte cannot be unequivocally identified, report its compound class or
functionality. See Table 3 for gas-phase group frequencies to be used as
an aid for compound class assignment. It should be noted that FT-IR gas-
phase group stretching frequencies are 0-30 cm"1 higher in frequency than
those of the condensed phase.
7.10.5 Quantitation - This protocol can be used to confirm GC/MS
identifications, with subsequent quantitation. Two FT-IR quantitation and
a supplemental GC detector technique are also provided.
7.10.5.1 Integrated Absorbance Technique - After analyte
identification, construct a standard calibration curve of
concentration versus integrated infrared absorbance. For this
purpose, choose for integration only those FT-IR scans which are at
or above the peak half-height. The calibration curve should span at
least one order of magnitude and the working range should bracket
the analyte concentration.
7.10.5.2 Maximum Absorbance Infrared Band Technique -
Following analyte identification, construct a standard calibration
curve of concentration versus maximum infrared band intensity. For
this purpose, choose an intense, symmetrical and well resolved IR
absorbance band.
(Note that IR transmission is not proportional to concentra-
tion). Select the FT-IR scan with the highest absorbance to plot
against concentration. The calibration curve should span at least
one order of magnitude and the working range should bracket the
analyte concentration. This method is most practical for
repetitive, target compound analyses. It is more sensitive than the
integrated absorbance technique.
7.10.5.3 Supplemental GC Detector Technique - If a GC
detector is used in tandem with the FT-IR detector, the following
technique may be used: following analyte identification, construct
a standard calibration curve of concentration versus integrated peak
area. The calibration curve should span at least one order of
magnitude and the working range should bracket the analyte
concentration. This method is most practical for repetitive, target
compound analyses.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for specific quality control procedures.
Quality control to validate sample extraction is covered in Method 3500 and in
the extraction method utilized. If extract cleanup was performed, follow the QC
in Method 3600 and in the specific cleanup method.
8.2 One Hundred Percent Line Test - Set the GC/FT-IR operating conditions
to those employed for the Sensitivity Test (see Sec. 7.5). Collect 16 scans over
the entire detector spectral range. Plot the test and measure the peak-to-peak
8410 - 8 Revision 0
September 1994
-------
noise between 1800 and 2000 cm"1. This noise should be < 0.15%. Store this plot
for future reference.
8.3 Single Beam Test - With the GC/FT-IR at analysis conditions, collect
16 scans in the single beam mode. Plot the co-added file and compare with a
subsequent file acquired in the same fashion several minutes later. Note if the
spectrometer is at purge equilibrium. Also check the plot for signs of
deterioration of the lightpipe potassium bromide windows. Store this plot for
future reference.
8.4 Align Test - With the lightpipe and MCT detector at thermal
equilibrium, check the intensity of the centerburst versus the signal temperature
calibration curve. Signal intensity deviation from the predicted intensity may
mean thermal equilibrium has not yet been achieved, loss of detector coolant,
decrease in source output, or a loss in signal throughput resulting from
lightpipe deterioration.
8.5 Mirror Alignment - Adjust the interferometer mirrors to attain the
most intense signal. Data collection should not be initiated until the
interferogram is stable. If necessary, align the mirrors prior to each GC/FT-IR
run.
8.6 Lightpipe - The lightpipe and lightpipe windows should be protected
from moisture and other corrosive substances at all times. For this purpose,
maintain the lightpipe temperature above the maximum GC program temperature but
below its thermal degradation limit. When not in use, maintain the lightpipe
temperature slightly above ambient. At all times, maintain a flow of dry, inert,
carrier gas through the lightpipe.
8.7 Beamsplitter - If the spectrometer is thermostated, maintain the
beamsplitter at a temperature slightly above ambient at all times. If the
spectrometer is not thermostated, minimize exposure of the beamsplitter to
atmospheric water vapor.
9.0 METHOD PERFORMANCE
9.1 Method 8410 has been in use at the U.S. Environmental Protection
Agency Environmental Monitoring Systems Laboratory for more than two years.
Portions of it have been reviewed by key members of the FT-IR spectroscopic
community (9). Side-by-side comparisons with GC/MS sample analyses indicate
similar demands upon analytical personnel for the two techniques. Extracts
previously subjected to GC/MS analysis are generally compatible with GC/FT-IR.
However, it should be kept in mind that lightpipe windows are typically water
soluble. Thus, extracts must be vigorously dried prior to analysis.
9.2 Table 4 provides performance data for this method.
8410 - 9 Revision 0
September 1994
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10.0 REFERENCES
1. Handbook for Analytical Quality Control in Water and Wastewater
Laboratories; U.S. Environmental Protection Agency. Environmental
Monitoring and Support Laboratory. ORD Publication Offices of Center for
Environmental Research Information: Cincinnati, OH, March 1979; Sec. 4,
EPA-600/4-79-019.
2. Freeman, R.R. Hewlett Packard Application Note: Quantitative Analysis
Using a Purged Splitless Injection Technique; ANGC 7-76.
3. Cole, R.H. Tables of Wavenumbers for the Calibration of Infrared
Spectrometers; Pergamon: New York, 1977.
4. Grasselli, J.G.; Griffiths, P.R.; Hannah, R.W. "Criteria for Presentation
of Spectra from Computerized IR Instruments"; Appl. Spectrosc. 1982, 35,
87.
5. Nyquist, R.A. The Interpretation of Vapor-Phase Infrared Spectra. Group
Frequency Data; Volume I. Sadtler Laboratories: Philadelphia, PA, 1984.
6. Socrates, G. Infrared Characteristic Group Frequencies; John Wiley and
Sons: New York, NY, 1980.
7. Bellamy, L.J. The Infrared Spectra of .Complex Organic Molegjles; 2nd ed.;
John Wiley and Sons: New York, NY, 1958.
8. Szymanski, H.A. Infrared Band Handbook, Volumes I and II; Plenum: New
York, NY, 1965.
9. Gurka, D.F. "Interim Protocol for the Automated Analysis of Semivolatile
Organic Compounds by Gas Chromatography/Fourier Transform-Infrared
Spectrometry"; Appl. Spectrosc. 1985, 39, 826.
10. Griffiths, P.R.; de Haseth, J.A.; Azarraga, L.V. "Capillary GC/FT-IR";
Anal. Chem. 1983, 55, 1361A.
11. Griffiths, P.R.; de Haseth, J.A. Fourier Transform-Infrared Spectrometry;
Wiley-Interscience: New York, NY, 1986.
12. Gurka, D. F.; Farnham, I.; Potter, B. B.; Pyle, S.; Titus, R. and Duncan,
W. "Quantitation Capability of a Directly Linked Gas
Chromatography/Fourier Transform Infrared/Mass Spectrometry System"; Anal.
Chem., 1989, 61, 1584.
8410 - 10 Revision 0
September 1994
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TABLE 1.
FUSED SILICA CAPILLARY COLUMN GAS CHROMATOGRAPHIC/FOURIER TRANSFORM
INFRARED IDENTIFICATION LIMITS FOR BASE/NEUTRAL EXTRACTABLES
Compound
Acenaphthene
Acenaphthylene
Anthracene
Benzo (a) anthracene
Benzo(a)pyrene
Bis(2-chloroethyl) ether
Bis(2-chloroethoxy)methane
Bis(2-chloroisopropyl) ether
Butyl benzyl phthalate
4-Bromophenyl phenyl ether
2-Chloronaphthalene
4-Chloroaniline
4-Chlorophenyl phenyl ether
Chrysene
Di-n-butyl phthalate
Dibenzofuran
Diethyl phthalate
Dimethyl phthalate
Di-n-octyl phthalate
Di-n-propyl phthalate
1,2-Dichlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
2,4-Dinitrotoluene
2,6-Dinitrotoluene
Bis-(2-ethylhexyl) phthalate
Fluoranthene
Fluorene
Hexachlorobenzene
Hexachlorocyclopentadiene
Hexachloroethane
1,3-Hexachlorobutadiene
Isophorone
2-Methylnaphthalene
Naphthalene
Nitrobenzene
N- Ni trosodi methyl ami ne
N-Nitrosodi -n-propyl amine
N-Ni trosodi phenyl ami ned
2-Nitroanil ine
3-Nitroanil ine
Identification
ng injected"
40{25)
50(50)
40(50)
(50)
(100)
70(10)
50(10)
50(10)
25(10)
40(5)
110
40
20(5)
(100)
20(5)
40
20(5)
20(5)
25(10)
25(5)
50
50
50
20
20
25(10)
100(50)
40(50)
40
120
50
120
40
110
40(25)
25
20(5)
50(5)
40
40
40
Limit
M9/L6
20(12.5)
25(25)
20(25)
(25)
(50)
35(5)
25(5)
25(5)
12.5(5)
20(2.5)
55
20
10(2.5)
(50)
10(2.5)
20
10(2.5)
10(2.5)
12.5(5)
12.5(2.5)
25
25
25
10
10
12.5(5)
50(25)
20(25)
20
60
25
60
20
55
20(12.5)
12.5
10(2.5)
25(2.5)
20
20
20
/'max, cm"
799
799
874
745
756
1115
1084
1088
1748
1238
851
1543
1242
757
1748
1192
1748
1751
1748
1748
1458
779
1474
1547
1551
1748
773
737
1346
814
783
853
1690
3069
779
1539
1483
1485
1501
1564
1583
8410 - 11
Revision 0
September 1994
-------
TABLE 1.
(Continued)
Compound
Identification Limit
ng injected8
*>max, cm
4-Nitroaniline
Phenanthrene
Pyrene
1,2,4-Trichlorobenzene
40
50(50}
100(50)
50(25)
20
25(25}
50(25)
25(12.5)
1362
729
820
750
Determined using on-column injection and the conditions of Sec. 7.3. A medium
band HgCdTe detector [3800-700 cm"1; D'value (/(peak 1000 Hz, 1) 4.5 x 1010 cm
Hz1'^"1] type with a 0.25 mm2 focal chip was used. The GC/FT-IR system is a
1976 retrofitted model. Values in parentheses were determined with a new
-1
(1986) GC/FT-IR system. A narrow band HgCdTe detector [3800-750cm ; D value
(Mpeak 1000 Hz, 1) 4 x
are those of Sec. 7.3.
10 cm Hz W ] was used. Chromatographic conditions
b Based on a 2 juL injection of a one liter sample that has been extracted and
concentrated to a volume of 1.0 mL. Values in parentheses were determined
with a new (1986) GC/FT-IR system. A narrow band HgCdTe detector [3800-750cm~
1; D'value (Apeak 1000 Hz, 1) 4 x 1010 cm Hz1/2W'1] was used. Chromatographic
conditions are those of Sec. 7.3.
c Most intense IR peak and suggested quantitation peak.
d Detected as diphenylamine.
8410 - 12
Revision 0
September 1994
-------
TABLE 2.
FUSED SILICA CAPILLARY COLUMN GAS CHROMATOGRAPHIC/FOURIER TRANSFORM
INFRARED ON-LINE AUTOMATED IDENTIFICATION LIMITS FOR ACIDIC EXTRACTABLES
Compound
Identification Limit
ng injected8
cm
,-lc
Benzoic acid
2-Chlorophenol
4-Chlorophenold
4-Chloro-3-methylphenol
2-Methylphenol
4-Methylphenol
2,4-Dichlorophenol
2,4-Dinitrophenol
4,6-Dinitro-2-methylphenol
2-Nitrophenold
4-Nitrophenol
Pentachlorophenol
Phenol
2,4,6-Trichlorophenol
2,4,5-Trichlorophenol
70
50
100
25
50
50
50
60
60
40
50
50
70
120
120
35
25
50
12.
25
25
25
30
30
20
25
25
35
60
60
1751
1485
1500
1177
748
1177
1481
1346
1346
1335
1350
1381
1184
1470
1458
a Operating conditions are the same as those cited in Sec. 7.3.
b Based on a 2 juL injection of a one liter sample that has been extracted and
concentrated to a volume of 1.0 mL.
c Most intense IR peak and suggested quantitation peak.
d Subject to interference from co-eluting compounds.
8410 - 13
Revision 0
September 1994
-------
TABLE 3.
GAS-PHASE GROUP FREQUENCIES
Number of
Functionality Class Compounds
Ether
Ester
Nitro
Nitrile
Ketone
Amide
Al kyne
Acid
Phenol
Aryl , Al kyl
Benzyl, Alkyl
Diaryl
Dial kyl
Alkyl, Vinyl
Unsubstituted Aliphatic
Aromatic
Monosubstituted Acetate
Aliphatic
Aromatic
Aliphatic
Aromatic
Aliphatic (acyclic)
(a,/3 unsaturated)
Aromatic
Substituted Acetamides
Aliphatic
Aliphatic
Dimerized-Aliphatic
Aromatic
1,4-Disubstituted
1,3-Disubstituted
1,2-Disubstituted
14
3
5
12
3
29
11
34
5
18
9
9
13
2
16
8
8
24
22
2
10
10
15
15
15
10
10
10
6
Frequency
Range, j>cm"1
1215-1275
1103-1117
1238-1250
1084-1130
1204-1207
1128-1142
1748-1761
1703-1759
1753-1788
1566-1594
1548-1589
1377-1408
1327-1381
1535-1566
1335-1358
2240-2265
2234-2245
1726-1732
1638-1699
1701-1722
1710-1724
3323-3329
3574-3580
1770-1782
3586-3595
3574-3586
1757-1774
3645-3657
1233-1269
1171-1190
3643-3655
1256-1315
1157-1198
3582-3595
1255-1274
(continued)
8410 - 14
Revision 0
September 1994
-------
TABLE 3.
(Continued)
Functional ity
Alcohol
Amine
Alkane
Aldehyde
Benzene
Class
Primary Aliphatic
Secondary Aliphatic
Tertiary Aliphatic
Primary Aromatic
Secondary Aromatic
Al iphatic
Aromatic
Al iphatic
Monosubstituted
Number of
Compounds
20
11
16
17
10
10
6
15
5
10
14
12
12
12
6
6
6
7
24
24
11
23
25
Frequency
Range, j>cm"1
3630-3680
1206-1270
1026-1094
3604-3665
1231-1270
3640-3670
1213-1245
3480-3532
3387-3480
760- 785
2930-2970
2851-2884
1450-1475
1355-1389
1703-1749
2820-2866
2720-2760
1742-1744
2802-2877
2698-2712
1707-1737
1582-1630
1470-1510
831- 893
735- 790
675- 698
8410 - 15
Revision 0
September 1994
-------
TABLE 4. FUSED SILICA CAPILLARY COLUMN GC/FT-IR QUANTITATION RESULTS
Compound
Concentration
Range, and
Identification
Limit, nga
Maximum
Absorbanceb
Correlation
Coefficient11
Integrated
Absorbancec
Correlation
Coefficient11
Acenaphthene
Acenaphthylene
Anthracene
Benzo(a)anthracene
Benzoic acid
Benzo(a)pyrene
Bis(2-chloroethoxy)methane
Bis{2-chloroethyl) ether
Bis{2-chloroisopropyl) ether
4-Bromophenyl phenyl ether
Butyl benzyl phthalate
4-Chloroaniline
4- Chi oro-3 -methyl phenol
2-Chloronaphthalene
2-Chlorophenol
4-Chlorophenol"
4-Chlorophenyl phenyl ether
Chrysene
Dibenzofuran
Di-n-butyl phthalate
1,2-Dichlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
2,4-Dichlorophenol
Dimethyl phthalate
Dimethyl phthalate
Dinitro-2 -methyl phenol
2,4-Dinitrophenol
2,4-Dinitrotoluene
2,5-Dinitrotoluene
Di-n-octyl phthalate
Bis(2-ethylhexyl) phthalate
Fluoranthene
Fluorene
Hexachlorobenzene
1,3-Hexachlorobutadiene
Hexachlorocyclopentadiene
Hexachloroethane
Isophorone
2-Methyl naphthalene
25-250
25-250
50-250
50-250
50-250
100-250
25-250
25-250
50-250
25-250
25-250
25-250
25-250
100-250
25-250
25-250
100-250
25-250
25-250
25-250
25-250
25-250
25-250
25-250
25-250
50-250
50-250
25-250
25-250
25-250
25-250
25-250
25-250
50-250
50-250
100-250
25-250
25-250
50-250
0.9995
0.9959
0.9969
0.9918
0.9864
0.9966
0.9992
0.9955
0.9981
0.9995
0.9999
0.9991
0.9975
0.9897
0.9976
0.9999
0.9985
0.9697
0.9998
0.9937
0.9985
0.9994
0.9964
0.9998
0.9998
0.9936
0.9920
0.9966
0.9947
0.9983
0.9991
0.9983
0.9987
0.9981
0.9960
0.9862
0.9986
0.9984
0.9981
0.9985
0.9985
0.9971
0.9921
0.9892
0.9074
0.9991
0.9992
0.9998
0.9996
0.9994
0.9965
0.9946
0.9988
0.9965
0.9997
0.9984
0.8579
0.9996
0.9947
0.9950
0.9994
0.9969
0.9996
0.9997
0.9967
0.9916
0.9928
0.9966
0.9991
0.9993
0.9966
0.9989
0.9995
0.9979
0.9845
0.9992
0.9990
0.9950
(continued)
8410 - 16
Revision 0
September 1994
-------
TABLE 4. (Continued)
Compound
Concentration
Range, and
Identification
Limit, nga
Maximum
Absorbanceb
Correlation
Coefficient1*
Integrated
Absorbancec
Correlation
Coefficient11
2-Methylphenol
4-Methylphenol
Naphthalene
2-Nitroaniline
3-Nitroaniline
4-Nitroaniline
Nitrobenzene
2-Nitrophenole
4-Nitrophenol
N-Nitrosodi methyl ami ne
N-Ni trosodiphenyl amine
N-Nitrosodi-n-propylamine
Pentachlorophenol
Phenanthrene
Phenol
Pyrene
1,2,4-Trichlorobenzene
2,4,5-Trichlorophenol
2,4,6-Trichlorophenol
25-250
25-250
25-250
25-250
25-250
25-250
25-250
50-250
25-250
25-250
25-250
50-250
25-250
25-250
50-250
50-250
25-250
25-250
0.9972
0.9972
0.9956
0.9996
0.9985
0.9936
0.9997
0.9951
0.9982
0.9994
0.9991
0.9859
0.9941
0.9978
0.9971
0.9969
0.9952
0.9969
0.9964
0.9959
0.9954
0.9994
0.9990
0.9992
0.9979
0.9953
0.9993
0.9971
0.9995
0 . 9883
0.9989
0.9966
0.9977
0.991
0.9966
0.9965
* Lower end of range is at or near the identification limit.
b FT-IR scan with highest absorbance plotted against concentration.
c Integrated absorbance of combined FT-IR scans which occur at or above the
chromatogram peak half-height.
d Regression analysis carried out at four concentration levels. Each level
analyzed in duplicate. Chromatographic conditions are stated in Sec. 7.3.
6 Subject to interference from co-eluting compounds.
8410 - 17
Revision 0
September 1994
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METHOD 8410
GAS CHROMATOGRAPHY/FOURIER TRANSFORM INFRARED (GC/FT-IR)
SPECTROMETRY FOR SEMIVOLATILE ORGANICS: CAPILLARY COLUMN
7.1 Sample
preparation
prior to
GC/FT-IR
analysis.
7.6 Adjust
interferometer
drive air
pressure.
7.2 Optional
Gel
Permeation
Cleanup of
extracts.
7.3 Initial
Calibration;
recommended
GC/FT-IR
conditions.
/7.7 MCI
Detector;
centerburst
intensity <75%,
plot max of,
. Section
No
.Yes
7.4 Check
detector
centerburst
intensity.
7.7 Replace
Source.
7.5 Column
Interface
Sensitivity.
7.8 Frequency
Calibration.
7.9 Determine
min. identifiable
quantities of
analyte of
interest.
7.9.1 Prepare
plot of
lightpipe T vs.
MCT centerburst
intensity.
7.10.1 Analyze
extracts using
conditions of
Section 7.3.
7.10.2 GC/FT-IR
Identification;
compare analyte
IR spectrum;
report.
7.10.3
Retention Time;
compare RRT of
analyte with
standard.
7.10.4 Report
compound class
if no library
match is found.
7.10.5
Quantitation
desired.
7.10.6 Standard
calibration curve
of cone. vs.
integrated IR
absorbance.
7.10.6
Quantitation
by integrated
absorbance?
7.10.7 Standard
calibration
curve of cone.
vs. max. IR band
intensity.
7.10.8 Is
GC Detector
used in tandem
with FT-IR
detector?
C Stop \4
r\ Yes
/
U — ...
7.10.8
Supplemental
GC Detector
Technique.
8410 - 18
Revision 0
September 1994
-------
o
N*
O
-------
METHOD 9010A
TOTAL AND AMENABLE CYANIDE
1.0 SCOPE AND APPLICATION
1.1 Method 9010 is used to determine the concentration of inorganic
cyanide (CAS Registry Number 57-12-5) in wastes or leachate. The method detects
inorganic cyanides that are present as either soluble salts or complexes. It is
used to determine values for both total cyanide and cyanide amenable to
chlorination. The "reactive" cyanide content of a waste, that is, the cyanide
content that could generate toxic fumes when exposed to mild acidic conditions,
is not distilled by Method 9010 (refer to Chapter Seven). However, Method 9010
is used to quantify the concentration of cyanide from the reactivity test.
1.2 The titration procedure using silver nitrate with p-dimethylamino-
benzal-rhodanine indicator is used for measuring concentrations of cyanide
exceeding 0.1 mg/L (0.025 mg/250 mL of absorbing liquid).
1.3 The colorimetric procedure is used for concentrations below 1 mg/L
of cyanide and is sensitive to about 0.02 mg/L.
1.4 This method was designed to address the problem of "trace" analyses
(<1000 ppm). The method may also be used for "minor" (1000 ppm - 10,000 ppm) and
"major" (>10,000 ppm) analyses by adapting the sample preparation techniques or
cell path length. However, the amount of sodium hydroxide in the standards and
the sample analyzed must be the same.
2.0 SUMMARY OF METHOD
2.1 The cyanide, as hydrocyanic acid (HCN), is released from samples
containing cyanide by means of a reflux-distillation operation under acidic
conditions and absorbed in a scrubber containing sodium hydroxide solution. The
cyanide in the absorbing solution is then determined colorimetrically or
titrametrically.
2.2 In the colorimetric measurement, the cyanide is converted to
cyanogen chloride (CNC1) by reaction of cyanide with chloramine-T at a pH less
than 8. After the reaction is complete, color is formed on the addition of
pyridine-barbituric acid reagent. The absorbance is read at 578 nm for the
complex formed with pyridine-barbituric acid reagent and CNC1. To obtain colors
of comparable intensity, it is essential to have the same salt content in both
the sample and the standards.
2.3 The titration measurement uses a standard solution of silver
nitrate to titrate cyanide in the presence of a silver sensitive indicator.
3.0 INTERFERENCES
3.1 Interferences are eliminated or reduced by using the distillation
procedure. Chlorine and sulfide are interferences in Method 9010.
9010A - 1 Revision 1
July 1992
-------
3.2 Oxidizing agents such as chlorine decompose most cyanides.
Chlorine interferences can be removed by adding an excess of sodium arsenite to
the waste prior to preservation and storage of the sample to reduce the chlorine
to chloride which does not interfere.
3.3 Sulfide interference can be removed by adding an excess of bismuth
nitrate to the waste (to precipitate the sulfide) before distillation. Samples
that contain hydrogen sulfide, metal sulfides, or other compounds that may
produce hydrogen sulfide during the distillation should be treated by the
addition of bismuth nitrate.
3.4 High results may be obtained for samples that contain nitrate
and/or nitrite. During the distillation, nitrate and nitrite will form nitrous
acid, which will react with some organic compounds to form oximes. These
compounds once formed will decompose under test conditions to generate HCN. The
possibility of interference of nitrate and nitrite is eliminated by pretreatment
with sulfamic acid just before distillation. Nitrate and nitrite are
interferences when present at levels higher than 10 mg/L and in conjunction with
certain organic compounds.
3.5 Thiocyanate is reported to be an interference when present at very
high levels. Levels of 10 mg/L were not found to interfere.
3.6 Fatty acids, detergents, surfactants, and other compounds may cause
foaming during the distillation when they are present in large concentrations and
will make the endpoint of the titration difficult to detect. They may be
extracted at pH 6-7,
4.0 APPARATUS AND MATERIALS
4.1 Reflux distillation apparatus such as shown in Figure 1 or Figure
2. The boiling flask should be of one liter size with inlet tube and provision
for condenser. The gas scrubber may be a 270-mL Fisher-Milligan scrubber
(Fisher,'Part No. 07-513) or equivalent. The reflux apparatus may be a Wheaton
377160 distillation unit or equivalent.
4.2 Spectrophotometer - Suitable for measurements at 578 nm with a
1.0 cm cell or larger.
4.3 Hot plate stirrer/heating mantle.
4.4 pH meter.
4.5 Amber light.
4.6 Vacuum source.
4.7 Refrigerator.
4.8 5 mL microburette
4.9 7 Class A volumetric flasks - 100 and 250 mL
4.10 Erlenmeyer flask - 500 mL
9010A - 2 Revision 1
July 1992
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5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Reagents for sample collection, preservation, and handling
5.3.1 Sodium arsenite (0.1N), NaAs02. Dissolve 3.2 g NaAs02 in
250 ml water.
5.3.2 Ascorbic acid, C6H806.
5.3.3 Sodium hydroxide solution (50%), NaOH. Commercially
available.
5.3.4 Acetic acid (1.6M) CH3COOH. Dilute one part of
concentrated acetic acid with 9 parts of water.
5.3.5 2,2,4-Trimethylpentane, C8H18.
5.3.6 Hexane, C6H14.
5.3.7 Chloroform, CHC13.
5.4 Reagents for cyanides amenable to chlorination
5.4.1 Calcium hypochlorite solution (0.35M), Ca(OCl)2. Combine
5 g of calcium hypochlorite and 100 ml of water. Shake before using.
5.4.2 Sodium hydroxide solution (1.25N), NaOH. Dissolve 50 g of
NaOH in 1 liter of water.
5.4.3 Sodium arsenite (0.1N). See Step 5.3.1.
5.4.4 Potassium iodide starch paper.
5.5 Reagents for distillation
5.5.1 Sodium hydroxide (1.25N). See Step 5.4.2.
5.5.2 Bismuth nitrate (0.062M), Bi(NO)3 • 5H,0. Dissolve 30 g
Bi(NO)3 • 5H20 in 100 ml of water. While stirring, add 250 mL of glacial
acetic acid, CH3COOH. Stir until dissolved and dilute to 1 liter with
water.
5.5.3 Sulfamic acid (0.4N), H2NS03H. Dissolve 40 g H2NS03H in
1 liter of water.
9010A - 3 Revision 1
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5.6.2
138 g of
5.5.4 Sulfuric acid (18N), H2S04. Slowly and carefully add 500
ml of concentrated H2S04 to 500 ml of water.
5.5.5 Magnesium chloride solution (2.5M), MgCl2 • 6H20. Dissolve
510 g of MgCl2 • 6H20 in 1 liter of water.
5.5.6 Lead acetate paper.
5.6 Reagents for spectrophotometric determination
5.6.1 Sodium hydroxide solution (0.25N), NaOH. Dissolve 10 g
NaOH in 1 liter of water.
6.2 Sodium phosphate monobasic (1M), NaH2P04 • H20. Dissolve
NaH2P04 • H20 in 1 liter of water. Refrigerate this solution.
5.6.3 Chloramine-T solution (0.44%), C^ClNNaC^S. Dissolve
1.0 g of white, water soluble chloramine-T in 100 ml of water and
refrigerate until ready to use.
5.6.4 Pyridine-Barbituric acid reagent, C5H5N • C,H4N203. Place
15 g of barbituric acid in a 250-mL volumetric flask and add just enough
water to wash the sides of the flask and wet the barbituric acid. Add 75
mi of pyridine and mix. Add 15 mL of concentrated hydrochloric acid
(HC1), mix, and cool to room temperature. Dilute to 250 ml with water.
This reagent is stable for approximately six months if stored in a cool,
dark place.
5.6.5 Stock potassium cyanide solution (1 ml = 1000 /Ltg CN"), KCN.
Dissolve 2.51 g of KCN and 2 g KOH in 900 ml of water. Standardize with
0.0192N silver nitrate, AgNOj. Dilute to appropriate concentration to
achieve 1 ml = 1000 p.g of CN .
NOTE: Detailed procedure for AgN03 standardization is described in
"Standard Methods for the Examination of Water and Wastewater",
16th Edition, (1985), Methods 412C and 407A.
5.6.6 Intermediate standard potassium cyanide solution, (1 ml =
100 fj,g CN'), KCNi Dilute 100 ml of stock potassium cyanide solution (1 ml
= 1000 /Ltg CN") to 1000 ml with water.
5.6.7 Working standard potassium cyanide solution (1 ml = 10 /xg
CN"), KCN. Prepare fresh daily by diluting 100 ml of intermediate standard
potassium cyanide solution and 10 ml of IN NaOH to 1 liter with water.
5.7 Reagents for titration procedure
5.7.1 Rhodanine indicator - Dissolve 20 mg of p-dimethylamino-
benzal-rhodanine, C12H12N2OS2, in 100 ml of acetone.
5.7.2 Standard silver nitrate solution (0.0192N), AgN03. Prepare
by crushing approximately 5 g AgN03 and drying to constant weight at 40'C.
Weigh out 3.2647 g of dried AgN03. Dissolve in 1 liter of water.
9010A - 4 Revision 1
July 1992
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NOTE: Detailed procedure for AgN03 standardization is described in
"Standard Methods for the Examination of Water and Wastewater",
16th Edition, (1985), Methods 412C and 407A.
6.0 SAMPLE COLLECTION, PRESERVATION AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed in Chapter Nine.
6.2 Samples should be collected in plastic or glass containers. All
containers must be thoroughly cleaned and rinsed.
6.3 Oxidizing agents such as chlorine decompose most cyanides. To
determine whether oxidizing agents are present, test a drop of the sample with
potassium iodide-starch test paper. A blue color indicates the need for
treatment. Add 0.1N sodium arsenite solution a few mL at a time until a drop of
sample produces no color on the indicator paper. Add an additional 5 mL of
sodium arsenite solution for each liter of sample. Ascorbic acid can be used as
an alternative although it is not as effective as arsenite. Add a few crystals
of ascorbic acid at a time until a drop of sample produces no color on the
indicator paper. Then add an additional 0.6 g of ascorbic acid for each liter
of sample volume.
6.4 Aqueous samples must be preserved by adding 50% sodium hydroxide
until the pH is greater than or equal to 12 at the time of collection.
6.5 Samples should be chilled to 4'C.
6.6 When properly preserved, cyanide samples can be stored for up to
14 days prior to sample preparation steps.
6.7 Solid and oily wastes may be extracted prior to analysis by method
9013. It uses a dilute NaOH solution (pH = 12) as the extractant. This yields
extractable cyanide.
6.8 If fatty acids, detergents, and surfactants are a problem, they may
be extracted using the following procedure. Acidify the sample with acetic acid
(1.6M) to pH 6.0 to 7.0.
CAUTION: This procedure can produce lethal HCN gas.
Extract with isooctane, hexane, or chloroform (preference in order named) with
solvent volume equal to 20% of the sample volume. One extraction is usually
adequate to reduce the compounds below the-interference level. Avoid multiple
extractions or a long contact time at low pH in order to keep the loss of HCN at
a minimum. When the extraction is completed, immediately raise the pH of the
sample to above 12 with 50% NaOH solution.
9010A - 5 Revision 1
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7.0 PROCEDURE
7.1 Pretreatment for cyanides amenable to chlorination
7.1.1 This test must be performed under amber light. K3[Fe-
(CN)^] may decompose under UV light and hence will test positive for
cyanide amenable to chlorination if exposed to fluorescent lighting or
sunlight. Two identical sample aliquots are required to determine cyanides
amenable to chlorination.
7.1.2 To one 500 mL sample or to a sample diluted to 500 ml, add
calcium hypochlorite solution dropwise while agitating and maintaining the
pH between 11 and 12 with 1.25N sodium hydroxide until an excess of
chlorine is present as indicated by Kl-starch paper turning blue. The
sample will be subjected to alkaline chlorination by this step.
CAUTION: The initial reaction product of alkaline chlorination is the very
toxic gas cyanogen chloride; therefore, it is necessary that this
reaction be performed in a hood.
7.1.3 Test for excess chlorine with Kl-starch paper and maintain
this excess for one hour with continuous agitation. A distinct blue color
on the test paper indicates a sufficient chlorine level. If necessary,
add additional calcium hypochlorite solution.
7.1.4 After one hour, add 1 mL portions of 0.1N sodium arsenite
until Kl-starch paper shows no residual chlorine. Add 5 ml of excess
sodium arsenite to ensure the presence of excess reducing agent.
7 1.5 Test for total cyanide as described below in hoth the
chlorinated and the unchlorinated samples. The difference of total
cyanide in the chlorinated and unchlorinated samples is the cyanide
amenable to chlorination.
7.2 Distillation Procedure
7 ?.l Place 500 ml of sample, or sample diluted to 500 mL in the
one liter boiling flask. Pipet 50 mL of 1.25N sodium hydroxide into the
gas scrubber. If the apparatus in Figure 1 is used, add water until the
spiral is covered. Connect the boiling flask, condenser, gas scrubber and
vacuum trap.
7.2.2 Start a slow stream of air entering the boiling flask by
adjusting the vacuum source. Adjust the vacuum so that approximately two
bubbles of air per second enter the boiling flask through the air inlet
tube.
7.2.3 If samples are known or suspected to contain sulfide, add
50 mL of 0.062M bismuth nitrate solution through the air inlet tube. Mix
for three minutes. Use lead acetate paper to check the sample for the
presence of sulfide. A positive test is indicated by a black color on the
paper.
9010A - 6 Revision 1
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7.2.4 If samples are known or suspected to contain nitrate or
nitrite, or if bismuth nitrate was added to the sample, add 50 ml of 0.4N
sulfamic acid solution through the air inlet tube. Mix for three minutes.
Note: Excessive use of sulfamic acid could create method bias.
7.2.5 Slowly add 50 ml of 18N sulfuric acid through the air inlet
tube. Rinse the tube with water and allow the airflow to mix the flask
contents for three minutes. Add 20 ml of 2.5M magnesium chloride through
the air inlet and wash the inlet tube with a stream of water.
7.2.6 Heat the solution to boiling. Reflux for one hour. Turn
off heat and continue the airflow for at least 15 minutes. After cooling
the boiling flask, and closing the vacuum source, disconnect the gas
scrubber.
7.2.7 Transfer the solution from the scrubber into a 250-mL
volumetric flask. Rinse the scrubber into the volumetric flask. Dilute
to volume with water.
7.2.8 If the manual spectrophotometric determination will be
performed, proceed to Step 7.3.1. If the titration procedure will be
performed, proceed to Step 7.7.
7.3 Manual spectrophotometric determination
7.3.1 Pipet 50 ml of the scrubber solution into a 100-mL
volumetric flask. If the sample is later found to be beyond the linear
range of the colorimetric determination and redistillation of a smaller
sample is not feasible, a smaller aliquot may be taken. If less than
50 ml is taken, dilute to 50 ml with 0.25N sodium hydroxide solution.
NOTE: Temperature of reagents and spiking solution can affect the
response factor of the colorimetric determination. The reagents
stored in the refrigerator should be warmed to ambient temperature
before use. Samples should not be left in a warm instrument to
develop color, but instead they should be aliquoted to a cuvette
immediately prior to reading the absorbance.
7.3.2 Add 15 mL of 1M sodium phosphate solution and mix. Add 2
ml of chloramine-T and mix. Some distillates may contain compounds that
have chlorine demand. One minute after the addition of chloramine-T, test
for excess chlorine with Kl-starch paper. If the test is negative, add
0.5 ml chloramine-T. After one mfnute recheck with Kl-starch paper.
Continue to add chloramine-T in 0.5 ml increments until an excess is
maintained. After 1 to 2 minutes, add 5 ml of pyridine-barbituric acid
solution and mix.
7.3.3 Dilute to 100 ml with water and mix again. Allow 8 minutes
for color development and then read the absorbance at 578 nm in a 1-cm
cell within 15 minutes. The sodium hydroxide concentration will be
0.125N.
9010A - 7 Revision 1
July 1992
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7.4 Standard curve for samples without sulfide
7.4.1 Prepare a series of standards by pipetting suitable volumes
of working standard potassium cyanide solution into 250-mL volumetric
flasks. To each flask, add 50 ml of 1.25N sodium hydroxide and dilute to
250 ml with water. Prepare using the following table. The sodium
hydroxide concentration will be 0.25N.
ml of Working Standard Solution Concentration
_ (1 ml = 10 ua CN") _ (uq CN'/L)
0 Blank
1.0 40
2.0 80
5.0 200
10.0 400
15.0 600
20.0 800
7.4.2 After the standard solutions have been prepared according
to the table above, pipet 50 mL of each standard solution into a 100-mL
volumetric flask and proceed to Steps 7.3.2 and 7.3.3 to obtain absorbance
values for the standard curve. The final concentrations for the standard
curve will be one half of the amounts in the above table (final
concentrations ranging from 20 to 400 M9/L}-
7.4.3 It is recommended that at least two standards (a high and
a low) be distilled and compared to similar values on the curve to ensure
that the distillation technique is reliable. If distilled standards do
not agree within + 10% of the undistilled standards, the analyst should
find the cause of the apparent error before proceeding.
7.4.4 Prepare a standard curve ranging from 20 to 400 ^g/L by
plotting absorbance of standard versus the cyanide concentration
7.5 Standard curve for samples with sulfide
7.5.1 It is imperative that all standards be distilled in the
same manner as the samples using the method of standard additions.
Standards distilled by this method will give a linear curve, at low
concentrations, but as the concentration increases, the recovery
decreases. It is recommended that at least five standards be distilled.
7.5.2 Prepare a series of standards similar in concentration to
those mentioned in Step 7.4.1 and analyze as in Step 7.3. Prepare a
standard curve by plotting absorbance of standard versus the cyanide
concentration.
7.6 Calculation - If the spectrophotometric procedure is used,
calculate the cyanide, in M9/L, in the original sample as follows.
CN" (Mg/L) = A x B x C
D x E
9010A - 8 Revision 1
July 1992
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where:
A = jug/L CN' read from standard curve.
B = ml of sample after preparation of colorimetric analysis
(100 mL recommended).
C = mL of sample after distillation (250 ml recommended).
D = ml of original sample for distillation (500 mL
recommended) .
E = mL used for colorimetric analysis (50 mL recommended).
7.7 Titration Procedure
7.7.1 Transfer the gas scrubber solution or a suitable aliquot
from the 250-mL volumetric flask to a 500-mL Erlenmeyer flask. Add 10-12
drops of the rhodanine indicator.
7.7.2 Titrate with standard 0.0192N silver nitrate to the first
change in color from yellow to brownish-pink. The titration must be
performed slowly with constant stirring. Titrate a water blank using the
same amount of sodium hydroxide and indicator as in the sample. The
analyst should be familiar with the endpoint of the titration and the
amount of indicator to be used before actually titrating the samples. A
5-mL buret may be conveniently used to obtain a precise titration.
NOTE: The titration is based on the following reaction:
Ag+ + 2CN -» [Ag(CN)2r
When all of the cyanide has complexed and more silver nitrate is
added, the excess silver combines with the rhodanine indicator to turn the
solution yellow and then brownish-pink.
7.7.3 Calculation - If the titrimetric procedure is used,
calculate concentration of CN" in ng/l in the original sample as follows:
Of- (ug/L) - {A -V xDxIx 2 ™le CN~ x 26. 02 gar x
F 1 e
-------
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for
easy reference or inspection.
8.2 Employ a minimum of one reagent blank per analytical batch or one
in every 20 samples to determine if contamination or any memory effects are
occurring.
8.3 Analyze check standards with every analytical batch of samples.
If the standards are not within 15% of the expected value, then the samples must
be reanalyzed.
8.4 Run one replicate sample for every 20 samples. A replicate sample
is a sample brought through the entire sample preparation and analytical process.
The CV of the replicates should be 20% or less. If this criterion is not met,
the samples should be reanalyzed.
8.5 Run one matrix spiked sample every 20 samples to check the
efficiency of sample distillation by adding cyanide from the working standard or
intermediate standard to 500 ml of sample to ensure a concentration of
approximately 40 /zg/L. The matrix spiked sample is brought through the entire
sample preparation and analytical process.
8.6 The method of standard additions shall be used for the analysis of
all samples that suffer from matrix interferences such as samples which contain
sulfides.
9.0 METHOD PERFORMANCE
9.1 The titration procedure using silver nitrate is used for measuring
concentrations of cyanide exceeding 0.1 mg/L. The colorimetric procedure is used
for concentrations below 1 mg/L of cyanide and is sensitive to about 0.02 mg/L.
9.2 EPA Method 335.2 (sample distillation with titration) reports that
in a single laboratory using mixed industrial and domestic waste samples at
concentrations of 0.06 to 0.62 mg/L CM", the standard deviations for precision
were ± 0.005 to + 0.094, respectively. In a single laboratory using mixed
industrial and domestic waste samples at concentrations of 0.28 and 0.62 mg/L
CN", recoveries (accuracy) were 85% and 102%, respectively.
9.3 In two additional studies using surface water, ground water, and
landfill leachate samples, the titration procedure was further evaluated. The
concentration range used in these studies was 0.5 to 10 mg/L cyanide. The
detection limit was found to be 0.2 mg/L for both total and amenable cyanide
determinations. The precision (CV) was 6.9 and 2.6 for total cyanide
determinations and 18.6 and 9.1 for amenable cyanide determinations. The mean
recoveries were 94% and 98.9% for total cyanide, and 86.7% and 97.4% for amenable
cyanide.
9010A - 10 Revision 1
July 1992
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10.0 REFERENCES
1. 1985 Annual Book of ASTH Standards, Vol. 11.01; "Standard Specification for
Reagent Water"; ATSM: Philadelphia, PA, 1985,; D1193-77.
2. 1982 Annual Book ASTM Standards. Part 19; "Standard Test Methods for
Cyanide in Water"; ASTM: Philadelphia, PA, 1982; 2036-82.
3. Bark, L.S.; Higson, H.G. Talanta 1964, 2, 471-479.
4. Britton, P.; Winter, J.; Kroner, R.C. "EPA Method Study 12, Cyanide in
Water"; final report to the U.S. Environmental Protection Agency. National
Technical Information Service: Springfield, VA, 1984; PB80-196674.
5. Casey, J.P.; Bright, J.W.; Helms, B.D. "Nitrosation Interference in
Distillation Tests for Cyanide"; Gulf Coast Waste Disposal Authority: Houston,
Texas.
6. Egekeze, J.O.; Oehne, F.W. J. Anal. Toxicology 1979, 3, 119.
7. Elly, C.T. jL. Water Pollution Control Federation 1968, 40. 848-856.
8. Fuller, W. Cyanide in the Environment; Van Zyl, D., Ed.; Proceedings of
Symposium; December, 1984.
9. Gottfried, G.J. "Precision, Accuracy, and MDL Statements for EPA Methods
9010, 9030, 9060, 7520, 7521,7550, 7551, 7910, and 7911"; final report to the
U.S. Environmental Protection Agency. Environmental Monitoring and Support
Laboratory. Biospheric: Cincinnati, OH, 1984.
10. Methods for Chemical Analysis of Water and Wastes; U.S. Environmental
Protection Agency. Office of Research and Development. Environmental Monitoring
and Support Laboratory. ORD Publication Offices of Center for Environmental
Research Information: Cincinnati, OH, 1983; EPA-600/4-79-020.
11. Rohrbough, W.G.; et al. Reagent Chemicals. American Chemical Society
Specifications, 7th ed.; American Chemical Society: Washington, DC, 1986.
12. Standard Methods for the Examination of Water and Wastewater, 16th ed.;
Greenberg, A.E.; Trussell, R.R.; Clesceri, L.S., Eds.; American Water Works
Association, Water Pollution Control Federation, American Public Health
Association: Washington, DC, 1985.
13. Umana, M.; Beach, J.; Sheldon, L. "Revisions to Method 9010"; final report
to the U.S. Environmental Protection Agency. Office of Solid Waste. Research
Triangle Institute: Research Triangle Park, NC, 1986.
14. Umana, M.; Sheldon, L. "Interim Report: Literature Review"; interim report
to the U.S. Environmental Protection Agency. Office of Solid Waste. Research
Triangle Institute: Research Triangle Park, NC, 1986.
9010A - 11 Revision 1
July 1992
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FIGURE 1.
APPARATUS FOR CYANIDE DISTILLATION
Cooling Water
Inlet Tube *
Heater ••
Screw Clamp
To Low Vacuum Source
Gas Scrubber
Distilling Rask
O
9010A - 12
Revision 1
July 1992
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FIGURE 2.
APPARATUS FOR CYANIDE DISTILLATION
Connecting Tubing
Allihn Condenser
Air Inlet Tube
c
One-Liter
Boiling Flask
Gas Scrubber
O
t
Sucdon
¥L
9010A - 13
Revision 1
July 1992
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METHOD 9010A
TOTAL AND AMENABLE CYANIDE
START
7 I Pretceat sample
to determine
cyanides amenable
to ch L orination
721 Place sample
in found bottom
flask, transfer
NaOH lolution into
scrubber; construct
distillation
assembly
'22 Turn vaccum
on and adjust
bubble rale
Yes / 7 2 3 D
samples contain
sulfide?
723 Add bitmutr
nitrate sol-jtian to
boiI ing flask
724 Nitrate
or nitrite in
samples''
7 2 4 Add sulfamic
acid solution to
boiling flask
7 2 S Add sulfurio
acid; rinse inlet
tube with water;
add magnesium
chloride; rinse
inlet tube with
water
7 2 6 Botl
solution; reflux;
cool, close vacuum
source
7 2 7 Dram
scrubber solution
into Crlenmeyer
flask
7 2 8 Which
ana 1ysis
method?
7 3 Perform
colorimet ric
analysis of aaropl
9010A - 14
Revision 1
July 1992
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METHOD 9010A
(Continued)
1 4 1 Prepare a
series of cyanide
standards through
diIution
7 S 1 Distill
standard* in same
manner as samples
"I 7 Transfer sample
to flask, add
rhodanine indicator
?42 Perform
co1o rimet ric
analysis of
standards
752 Prepare
standard curve of
absorbances
743 Distill at
least two standards
to check
distillation
recovery
744 Prepare
standard curve of
abs orbances
7 4 S Check
ef ficiency of
sample distillation
7 6 Compute
concent rat ions
7 7 2 Titrate
sample and vater
blank with silver
nitra te
773 Calculate
concentration of
cyanide in sample
STOP
STOP
9010A - 15
Revision 1
July 1992
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METHOD 9012
TOTAL AND AMENABLE CYANIDE (COLORIHETRIC, AUTOMATED UV)
1.0 SCOPE AND APPLICATION
1.1 Method 9012 1s used to determine the concentration of Inorganic
cyanide 1n an aqueous waste or leachate. The method detects Inorganic
cyanides that are present as either simple soluble salts or complex radicals.
It 1s used to determine values for both total cyanide and cyanide amenable to
chlorination. Method 9012 1s not Intended to determine 1f a waste is
hazardous by the characteristic of reactivity.
2.0 SUMMARY OF METHOD
2.1 The cyanide, as hydrocyanic add (HCN), 1s released by refluxlng the
sample with strong acid and distillation of the HCN Into an absorber-scrubber
containing sodium hydroxide solution. The cyanide 1on 1n the absorbing
solution 1s then determined by automated UV colorimetry.
2.2 In the colorlmetrlc measurement, the cyanide 1s converted to
cyanogen chloride (CNC1) by reaction with Chloramine-T at a pH less than 8
without hydrolyzlng to the cyanate. After the reaction Is complete, color 1s
formed on the addition of pyr1d1ne-barb1tur1c acid reagent. The concentration
of NaOH must be the same 1n the standards, the scrubber solutions, and any
dilution of the original scrubber solution to obtain colors of comparable
Intensity.
3.0 INTERFERENCES
3.1 Interferences are eliminated or reduced by procedures described 1n
Paragraphs 7.2.3, 7.2.4, and 7.2.5.
3.2 Sulfldes adversely affect the colorlmetrlc procedures. Samples that
contain hydrogen sulflde, metal sulfides, or other compounds that may produce
hydrogen sulflde during the distillation should be treated by addition of
bismuth nitrate prior to distillation as described 1n Paragraph 7.2.3.
3.3 High results may be obtained for samples that contain nitrate and/or
nitrite. During the distillation, nitrate and nitrite will form nitrous add,
which will react with some organic compounds to form oxlmes. These compounds
will decompose under test conditions to generate HCN. The possible
Interference of nitrate and nitrite is eliminated by pretreatment with
sulfamic add.
9012 - 1
Revision 0
Date September 1986
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4.0 APPARATUS AND MATERIALS
4.1 Reflux distillation apparatus; Such as shown 1n Figure 1 or 2. The
boiling flask should be of 1-liter size with Inlet tube and provision for
condenser. The gas absorber 1s a F1sher-M1ll1gan scrubber (Fisher Catalog
107-513) or equivalent.
4.2 Potassium Iodide-starch test paper.
4.3 Automated continuous-flow analytical Instrument with;
4.3.1 Sampler.
4.3.2 Manifold with UV digester.
4.3.3 Proportioning pump.
4.3.4 Heating bath with distillation coll.
4.3.5 Distillation head.
4.3.6 Colorimeter equipped with a 15-mm flowcell and 570 nm filter.
4.3.7 Recorder.
5.0 REAGENTS
5.1 ASTM Type II water (ASTM D1193) : Water should be monitored for
Impurities.
5.2 Sodium hydroxide solution, 1.25 N: Dissolve 50 g of NaOH 1n Type II
water and dilute to 1 liter with Type II water.
5.3 Bismuth nitrate solution; Dissolve 30.0 g of 81(1^)3)3 1n 100 mL of
Type II water. While stirring, add 250 roL of glacial acetic add. Stir until
dissolved. Dilute to 1 liter with Type II water.
5.4 Sulfurlc add. 1:1: Slowly add 500 ml of concentrated H2S04 to
500 ml of Type II water.
CAUTION: this 1s an exothermic reaction.
5.5 Sodium dlhydroqenphosphate. 1 M: Dissolve 138 g of Na^PCV^O in
1 liter of Type II water.
5.6 Stock cyanide solution; Dissolve 2.51 g of KCN and 2 g KOH in
900 mL of Type II water. Standardize with 0.0192 N AgN03. Dilute to
appropriate concentration so that 1 mL = 1 mg CN.
5.7 Intermediate standard cyanide solution; Dilute 100.0 mL of stock
(1 mL = 1 mg CN) to 1,000 mL with Type II water (1 mL = 100 ug CN) .
5.8 Working standard cyanide solution; Prepare fresh daily by diluting
100.0 mL of intermediate cyanide solution to 1,000 mL with Type II water
(1 mL - 10.0 ug CN) . Store 1n a glass-stoppered bottle.
9012 - 2
Revision 0
Date September 1986
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Connecting Tubing
Allihn Condenser
Air Inlet Tube
One-Liter
Boiling Flask
Suction
Figure 1. Apparatus for cyanide distillation.
9012 - 3
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Date September 1986
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COOLING WATER
INLET
SCREW CLAMP
J
A
HEATER*
TO LOW VACUUM
SOURCE
- ABSORBER
CONDENSER
DISTILLING FLASK
Figure 2. Cyanide distillation apparatus.
9012 - 4
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Date September 1986
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5.9 Magnesium chloride solution; Weigh 510 g of MgCl2'6H20 Into a
1,000-mL flask, dissolve, and dilute to 1 liter with Type II water.
5.10 Sulfamlc add solution; Dissolve 40 g of sulfamlc add 1n Type II
water. Dilute to l liter.
5.11 Calcium hypochlorite solution; Dissolve 5 g of calcium hypo-
chlorite [CalOCI)2] In 100 ml of Type II water.
5.12 Reagents for automated colorimetric determination;
5.12.1 Pyrid1ne-barb1tur1c acid reagent: Place 15 g of barbituric
add 1n a 250-mL volumetric flask, add just enough Type II water to wash
the sides of the flask, and wet the barbituric add. Add 75 ml of
pyrldine and mix. Add 15 ml of concentrated HC1, mix, and cool to room
temperature. Dilute to 250 ml with Type II water and mix. This reagent
1s stable for approximately six months 1f stored in a cool, dark place.
5.12.2 Chloram1ne-T solution: Dissolve 2.0 g of white, water
soluble chloram1ne-T 1n 500 ml of Type II water and refrigerate until
ready to use.
5.12.3 Sodium hydroxide, 1 N: Dissolve 40 g of NaOH In Type II
water, and dilute to 1 liter.
5.12.4 All working standards should contain 2 ml of 1 N NaOH
(Paragraph 5.12.3) per 100 ml.
5.12.5 Dilution water and receptacle wash water (NaOH, 0.25N):
Dissolve 10.0 g NaOH 1n 500 ml of Type II water. Dilute to 1 liter.
5.13 Ascorbic acid; Crystals.
5.14 Phosphate buffer, pH 5.2: Dissolve 13.6 g of potassium dihydrogen
phosphate and 0.28 g ofdTsodium phosphate in 900 ml of Type II water and
dilute to 1 liter.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed 1n Chapter Nine of this manual,
6.2 Samples should be collected 1n plastic or glass bottles of 1-liter
size or larger. All bottles must be thoroughly cleaned and thoroughly rinsed
to remove soluble materials from containers.
6.3 Oxidizing agents such as chlorine decompose most cyanides. To
determine whether oxidizing agents are present, test a drop of the sample with
acidified potassium Iodide (Kl)-starch test paper at the time the sample 1s
collected; a blue color Indicates the need for treatment. Add ascorbic acid a
9012 - 5
Revision
Date September 1986
-------
few crystals at a time until a drop of sample produces no color on the
Indicator. Then add an additional 0.6 g of ascorbic add for each liter of
water.
6.4 Samples must be preserved by addition of 10 N sodium hydroxide until
sample pH 1s greater than or equal to 12 at the time of collection.
6.5 Samples should be refrigerated at 4*C, when possible, and analyzed
as soon as possible.
7.0 PROCEDURE
7.1 Pretreatment for cyanides amenable to chlorlnation:
7.1.1 Two sample allquots are required to determine cyanides
amenable to chlorlnation. To one 500-mL aliquot, or to a volume diluted
to 500 ml, add calcium hypochlorlte solution (Paragraph 5.11) dropwlse
while agitating and maintaining the pH between 11 and 12 with sodium
hydroxide (Paragraph 5.2).
CAUTION; The Initial reaction product of alkaline chlorlnatlon 1s
th"e very toxic gas cyanogen chloride; therefore, 1t 1s
recommended that this reaction be performed In a hood. For
convenience, the sample may be agitated 1n a 1-Hter beaker by
means of a magnetic stirring device.
7.1.2 Test for residual chlorine with Kl-starch paper (Paragraph
4.4) and maintain this excess for 1 hr, continuing agitation. A distinct
blue color on the test paper Indicates a sufficient chlorine level. If
necessary, add additional hypochlorlte solution.
7.1.3 After 1 hr, add 0.5 g portions of ascorbic add until KI-
starch paper shows no residual chlorine. Add an additional 0.5 g of
ascorbic add to ensure the presence of excess reducing agent.
7.1.4 Test for total cyanide 1n both the chlorinated and
unchlorlnated allquots. (The difference of total cyanide 1n the
chlorinated and unchlorlnated allquots 1s the cyanide amenable to
chlorlnatlon.)
7.2 Distillation Procedure;
7.2.1 Place 500 ml of sample, or an aliquot diluted to 500 ml, 1n
the 1-Hter boiling flask. P1pet 50 ml of sodium hydroxide (Paragraph
5.2) Into the absorbing tube. If the apparatus 1n Figure 1 1s used, add
Type II water until the spiral 1s covered. Connect the boiling flask,
condenser, absorber, and trap 1n the train (Figure 1 or 2).
7.2.2 By adjusting the vacuum source, start a slow stream of air
entering the boiling flask so that approximately two bubbles of air per
second enter the flask through the air Inlet tube.
9012 - 6
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Date September 1986
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7.2.3 Use lead acetate paper to check the sample for the presence
of sulfide. A positive test 1s Indicated by a black color on the paper.
If positive, treat the sample by adding 50 mL of bismuth nitrate solution
(Paragraph 5.3) through the air inlet tube after the air rate 1s set.
Mix for 3 min prior to addition of
7.2.4 If samples are suspected to contain N03 and/or N02, add 50 mL
of sulfamic add solution (Paragraph 5.10) after the air rate is set
through the air inlet tube. Mix for 3 min prior to addition of ^$04.
7.2.5 Slowly add 50 mL 1:1 H2S04 (Paragraph 5.4) through the air
Inlet tube. Rinse the tube with Type II water and allow the airflow to
mix the flask contents for 3 min. Pour 20 ml of magnesium chloride
(Paragraph 5.9) into the air inlet and wash down with a stream of water.
7.2.6 Heat the solution to boiling. Reflux for 1 hr. Turn off
heat and continue the airflow for at least 15 min. After cooling the
boiling flask, disconnect absorber and close off the vacuum source.
7.2.7 Drain the solution from the absorber into a 250-mL volumetric
flask. Wash the absorber with Type II water and add the washings to the
flask. Dilute to the mark with Type II water.
7.3 Automated colorimetric determination;
7.3.1 Set up the manifold in a hood or a well -ventilated area as
shown in Figure 3.
7.3.2 Allow colorimeter and recorder to warm up for 30 min. Run a
baseline with all reagents, feeding Type II water through the sample
line.
7.3.3 Place appropriate standards in the sampler 1n order of
decreasing concentration. Complete loading of the sampler tray with
unknown samples.
7.3.4 When the baseline becomes steady, begin the analysis.
7.4 Standard curve for samples without sulfide;
7.4.1 Prepare a series of standards by pipetting suitable volumes
of standard solution (Paragraph 5.8) Into 250-mL volumetric flasks. To
each standard add 50 mL of 1.25 N sodium hydroxide and dilute to 250 mL
with Type II water. Prepare as follows:
9012 - 7
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Date September 1986
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ID
O
oo
J
TO SAMPLER WASH
_^ RECEPTACLE
f
<«
PROPORTIONING
PUMP
figure 3. Cyanide manifold A A11.
vo
oo
o>
-------
mL of Working Standard Solution Concentration
(1 mL = 10 ug CN) (ug CN/250 ml)
0 BLANK
1.0 10
2.0 20
5.0 50
10.0 100
15.0 150
20.0 200
7.4.2 It 1s not Imperative that all standards be distilled 1n the
same manner as the samples. It 1s recommended that at least two
standards (a high and a low) be distilled and compared with similar
values on the curve to ensure that the distillation technique 1s
reliable. If distilled standards do not agree within + 10X of the
undlstHled standards, the analyst should find the cause of the apparent
error before proceeding.
7.4.3 Prepare a standard curve by plotting absorbances of standards
vs. cyanide concentrations.
7.4.4 To check the efficiency of the sample distillation, add an
Increment of cyanide from either the Intermediate standard (Paragraph
5.7) or the working standard (Paragraph 5.8) to 500 ml of sample to
ensure a level of 20 ug/L. Proceed with the analysis as 1n Paragraph
7.2.1.
7.5 Standard curve for samples with sulflde:
7.5.1 All standards must be distilled 1n the same manner as the
samples. A minimum of 3 standards shall be distilled.
7.5.2 Prepare a standard curve by plotting absorbances of standards
vs. cyanide concentration.
7.6 Calculation; Prepare a standard curve by plotting peak heights of
standards against tfielr concentration values. Compute concentrations of
samples by comparing sample peak heights with the standard curve.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Employ a minimum of one blank per sample batch to determine 1f
contamination or any memory effects are occurring.
8.3 Verify calibration with an Independently prepared check standard
every 15 samples.
9012 - 9
Revision
Date September 1986
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8.4 Run one spike duplicate sample for every 10 samples. A duplicate
sample 1s a sample brought through the whole sample preparation process.
8.5 The method of standard additions shall be used for the analysis of
all samples that suffer from matrix Interferences.
9.0 METHOD PERFORMANCE
9.1 Precision and accuracy data are not available at this time.
10.0 REFERENCES
1. Annual Book of ASTM Standards, Part 31, "Water," Standard D2036-75,
Method B, p. 505 (1976).
2. Goulden, P.O., B.K. Afghan, and P. Brooksbank, Determination of Nanogram
Quantities of Simple and Complex Cyanides 1n Water, Anal. Chem., 44(11). pp.
1845-49 (1972).
3. Standard Methods for the Examination of Water and Wastewater, 14th ed.,
pp. 376 and 370, Method 413F and D (1975).
4. Technlcon AutoAnalyzer II Methodology, Industrial Method No. 315-74 WCUV
Digestion and Distillation, Technlcon Industrial Systems, Tarrytown, New York,
10591 (1974).
9012 - 10
Revision
Date September 1986
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METHOD 9012
TOTAL AND AMENABLE CYANIDE (COLORIMETP.IC. AUTOMATED UV)
7. 1
Pretreat
to determine
cyanides
amenable to
cnlorlnat ion
7.2.1
Place
••molt
in flasie:
plBCt BOdlum
hydroxide into
absorbing tube
7.3.*!
I AOO
lulf•nic
•CIO solution
tnrouo" •»*
inlet tuDe
rinse tuoe with
Type II w«ter;
moa •tignesulm
chloride
7.3.2
Introduce *lr
ctreem into
OOi1 ing
7.Z.3
7.3.6
Bol 1
• olut ion;
reflux; cool:
c lo»e off
vacuum cource
Treet
••mole by
•dding t>i«i*utn
nitrate
•olutlon
7.3.7
Drain solution
from aocorocr
into flack
7.3
Perform
baacline
colorimetric
analycl*
o
9012 - 11
Revision 0
Date September 1986
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METHOD SO12
TOTAL AND AMENABLE CYANIDE fCOLO«IMET«IC. AUTOMATED UV)
(Cortt Jnuco)
7.5.1
Distill
standards In
• •(*• «anner
as ••mole
7.4.1
Prepare •
•erics of
CN »t»na»ro»
•t«nq»ra curve
of •bsorbanco
7.4.a| oistui
two standards
to cneck
distillation
techniques
7.4.3
Preosre
stsnasra curve
of •Osorb»nce»
7.6
Conpute
concsntrstlons
7.4.41
Chvck
efficiency
of *«mol«
0lstin«tior
r stop )
9012 - 12
Revision 0
Date September 1986
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-------
METHOD 9013
(APPENDIX TO METHOD 9010)
CYANIDE EXTRACTION PROCEDURE FOR SOLIDS AND OILS
1.0 SCOPE AND APPLICATION
1.1 The extraction procedure described in this method is designed for
the extraction of soluble cyanides from solid and oil wastes. The method is
applicable to oil, solid, and multiphasic samples. This method is not applicable
to samples containing insoluble cyanide compounds.
2.0 SUMMARY OF METHOD
2.1 If the waste sample contains so much solid, or solids of such a
size as to interfere with agitation and homogenization of the sample mixture in
the distillation flask, or so much oil or grease as to interfere with the
formation of a homogeneous emulsion, the sample may be extracted with water at
pH 10 or greater, and the extract distilled and analyzed by Method 9010. Samples
that contain free water are filtered and separated into an aqueous component and
a combined oil and solid component. The nonaqueous component may then be
extracted, and an aliquot of the extract combined with an aliquot of the filtrate
in proportion to the composition of the sample. Alternatively, the components
may be analyzed separately, and cyanide levels reported for each component.
However, if the sample solids are known to contain sufficient levels of cyanide
(about 50 Mg/g) as to be well above the limit of detection, the extraction step
may be deleted and the solids analyzed directly by Method 9010. This can be
accomplished by diluting a small aliquot of the waste solid (1-10 g) in 500 mL
water in the distillation flask and suspending the slurry during distillation
with a magnetic stir-bar.
3.0 INTERFERENCES
3.1 Potential interferences that may be encountered during analysis are
discussed in Method 9010.
4.0 APPARATUS AND MATERIALS
4.1 Extractor - Any suitable device that sufficiently agitates a sealed
container of one liter volume or greater. For the purpose of this analysis,
agitation is sufficient when:
1. All sample surfaces are continuously brought into contact
with extraction fluid, and
2. The agitation prevents stratification of the sample and
fluid.
4.2 Buchner funnel apparatus
4.2.1 Buchner funnel - 500-mL capacity, with 1-liter vacuum
filtration flask.
9013 - 1 Revision 0
July 1992
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4.2.2 Glass wool - Suitable for fiHerirujj 0.8 m diameter such
as Corning Pyrex 3950.
4.2.3 Vacuum source - Preferably a water driven aspirator. A
valve or stopcock to release vacuum is required.
4.3 Top-loading balance - capable of weighing 0.1 g.
4.4 Separatory funnels - 500 ml.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless otherwise
indicated, it is intended that all reagents shall conform to the specifications
of the Committee on Analytical Reagents of the American Chemical Society, where
such specifications are available. Other grades may be used, provided it is
first ascertained that the reagent is of sufficiently high purity to permit its
use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Sodium hydroxide (50%w/v), NaOH. Commercially available.
5.4 n-Hexane, C6H14.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a plan that addresses the
considerations discussed in Chapter 4 of this manual. See Section 6.0 of Method
9010 for additional guidance.
7.0 PROCEDURE
7.1 If the waste does not contain any free aqueous phase, go to Step
7.5. If the sa.nple is a homogeneous fluid or slurry that does not separate or
settle in the distillation flask when using a Teflon coated magnetic stirring bar
but mixes so that the solids are entirely suspended, then the sample may be
analyzed by Method 9010 without an extraction step.
7.2 Assemble Buchner funnel apparatus. Unroll glass filtering fiber
and fold the fiber over itself-several times to make a pad about 1 cm thick when
lightly compressed. Cut the pad to fit the Buchner funnel. Weigh the pad, then
place it in the funnel. Turn the aspirator on and wet the pad with a known
amount of water.
7.3 Transfer the sample to the Buchner funnel in small aliquots, first
decanting the fluid. Rinse the sample container with known amounts of water and
add the rinses to the Buchner funnel. When no free water remains in the funnel,
slowly open the stopcock to allow air to enter the vacuum flask. A small amount
of sediment may have passed through the glass fiber pad. This will not interfere
with the analysis.
9013 - 2 Revision 0
July 1992
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7.4 Transfer the solid and the glass fiber pad to a tared weighing
dish. Since most greases and oils will not pass through the fiber pad, solids,
oils, and greases will be extracted together. If the filtrate includes an oil
phase, transfer the filtrate to a separatory funnel. Collect and measure the
volume of the aqueous phase. Transfer the oil phase to the weighing dish with
the solid.
7.5 Heigh the dish containing solid, oil (if any), and filter pad.
Subtract the weight of the dry filter pad. Calculate the net volume of water
present in the original sample by subtracting the total volume of rinses used
from the measured volume of the filtrate.
7.6 Place the following in a 1-liter wide-mouthed bottle:
500 ml water
5 mL 50% w/v NaOH
50 ml n-Hexane (if a heavy grease is present)
If the weight of the solids (Step 7.5) is greater than 25 g, weigh
out a representative aliquot of 25 g and add it to the bottle; otherwise
add all of the solids. Cap the bottle.
7.7 The pH of the extract must be maintained above 10 throughout the
extraction step and subsequent filtration. Since some samples may release acid,
the pH must be monitored as follows. Shake the extraction bottle and after one
minute, check the pH. If the pH is below 12, add 50% NaOH in 5 ml increments
until it is at least 12. Recap the bottle, and repeat the procedure until the
pH does not drop.
7.8 Place the bottle or bottles in the tumbler, making sure
there is enough foam insulation to cushion the bottle. Turn the tumbler on and
allow the extraction to run for about 16 hours.
7.9 Prepare a Buchner funnel apparatus as in Step 7.2 with a glass fiber
pad filter.
7.10 Decant the extract to the Buchner funnel. Full recovery of the
extract is not necessary.
7.11 If the extract contains an oil phase, separate the aqueous phase
using a separatory funnel. Neither the separation nor the filtration are
critical, but are necessary to be able to measure the volume of the aliquot of
the aqueous extract analyzed. Small amounts of suspended solids and oil
emulsions will not interfere.
7.12 At this point, an aliquot of the filtrate of the original sample may
be combined with an aliquot of the extract in a proportion representative of the
sample. Alternatively, they may be distilled and analyzed separately and
concentrations given for each phase. This is described by the following
equation:
Liquid Sample AliauotfrnU . Solid Extracted(q)a x Total Sample Fi1trate(ml)c
Extract Aliquot(mL) Total Solid(g)6 Total Extraction Fluid(mL)Q
9013 - 3 Revision 0
July 1992
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"From Step 7.6. Weight of solid sample used for extraction.
bFrom Step 7.5. Weight of solids and oil phase with the dry weight of
filter and tared dish subtracted.
Includes volume of all rinses added to the filtrate (Steps 7.2 and 7.3).
d500 ml water plus total volume of NaOH solution. Does not include hexane,
which is subsequently removed {Step 7.11).
Alternatively, the aliquots may be distilled and analyzed separately,
concentrations for each phase reported separately, and the amounts of each phase
present in the sample reported separately.
8.0 QUALITY CONTROL
8.1 Refer to Method 9010.
9.0 METHOD PERFORMANCE
9.1 In a single laboratory study, recoveries of 60 to 90% are reported
for solids and 88 to 92% for oils. The reported CVs are less than 13.
10.0 REFERENCES
10.1 Refer to Method 9010.
9013 - 4 Revision 0
July 1992
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METHOD 9013
(APPENDIX TO METHOD 9010)
CYANIDE EXTRACTION PROCEDURE FOR SOLIDS AND OILS
7 1 Analyze by
Metho'B-TOlO
7 4 S«parat« phaic*
in »«paratory
funnel; tranifar
oil pha»» to
weighing di>h
7 2 Ai»«mbU filter
apparatus, ••igh
filter pad. place
in funnel, wet pad
with known amount
of wa ta r
73 Pillar tampl*:
rins* lampl•
container with
tinown amount of
7 S W.igh >olid &
oil phaivt in tarad
w«ighinc| dith:
calculate amount of
water in sample
9013 - 5
Revision 0
July 1992
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METHOD 9013
(APPENDIX TO METHOD 9010)
CYANIDE EXTRACTION PROCEDURE FOR SOLIDS AND OILS (CONTINUED)
7 7 Shak.
• x t rJG11on
Dot 11•. ch«ek
pH
9013 - 6
Revision 0
July 1992
-------
\o
o
-------
METHOD 9020A
TOTAL ORGANIC HALIDES (TOX1
1.0 SCOPE AND APPLICATION
1.1 Method 9020 determines Total Organic Hal ides (TOX) as chloride in
drinking water and ground waters. The method uses carbon adsorption with a
microcoulometric-titration detector.
1.2 Method 9020 detects all organic halides containing chlorine,
bromine, and iodine that are adsorbed by granular activated carbon under the
conditions of the method. Fluorine-containing species are not determined by this
method.
1.3 Method 9020 is applicable to samples whose inorganic-halide concen-
tration does not exceed the organic-halide concentration by more than 20,000
times.
1.4 Method 9020 does not measure TOX of compounds adsorbed to
undissolved sol ids.
1.5 Method 9020 is restricted to use by, or under the supervision of,
analysts experienced in the operation of a pyrolysis/microcoulometer and in the
interpretation of the results.
1.6 This method is provided as a recommended procedure. It may be used
as a reference for comparing the suitability of other methods thought to be
appropriate for measurement of TOX (i .e., by comparison of sensitivity, accuracy,
and precision of data). There are three instruments that can be used to carry
out this method. They are the TOX-10 available from Cosa Instruments, and the
DX-20 and DX-20A available from Xertex-Dohrmann Instruments.
2.0 SUMMARY OF METHOD
2.1 A sample of water that has been protected against the loss of
volatiles by the elimination of headspace in the sampling container, and that is
free of undissolved solids, is passed through a column containing 40 mg of
activated carbon. The column is washed to remove any trapped inorganic halides
and is then combusted to convert the adsorbed organohalides to HX, which is
trapped and titrated electrolytically using a microcoulometric detector.
3.0 INTERFERENCES
3.1 Method interferences may be caused by contaminants, reagents,
glassware* and other sample-processing hardware. All these materials must be
routinely demonstrated to be free from interferences under the conditions of the
analysis by running method blanks.
3.1.1 Glassware must be scrupulously cleaned. Clean all
glassware as soon as possible after use by treating with chromate cleaning
solution. This should be followed by detergent washing in hot water.
Rinse with tap water and distilled water and drain dry; glassware which is
9020A - 1 Revision 1
July 1992
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not volumetric should, in addition, be heated in a muffle furnace at 400°C
for 15 to 30 min. (Volumetric ware should not be heated in a muffle
furnace.) Glassware should be sealed and stored in a clean environment
after drying and cooling to prevent any accumulation of dust or other
contaminants.
3.1.2 The use of high-purity reagents and gases helps to minimize
interference problems.
3.2 Purity of the activated carbon must be verified before use. Only
carbon samples that register less than 1,000 ng CT/40 mg should be used. The
stock of activated carbon should be stored in its granular form in a glass
container with a Teflon seal. Exposure to the air must be minimized, especially
during and after milling and sieving the activated carbon. No more than a 2-wk
supply should be prepared in advance. Protect carbon at all times from all
sources of halogenated organic vapors. Store prepared carbon and packed columns
in glass containers with Teflon seals.
3.3 Particulate matter will prevent the passage of the sample through
the adsorption column. Particulates must, therefore, be eliminated from the
sample. This must be done as gently as possible, with the least possible sample
manipulation, in order to minimize the loss of volatiles. It should also be
noted that the measured TOX will be biased by the exclusion of TOX from compounds
adsorbed onto the particulates. The following techniques may be used to remove
particulates; however, data users must be informed of the techniques used and
their possible effects on the data. These techniques are listed in order of
preference:
3.3.1 Allow the particulates to settle in the sample container
and decant the supernatant liquid into the adsorption system.
3.3.2 Centrifuge sample and decant the supernatant liquid into
the adsorption system.
3.3.3 Measure Purgeable Organic Hal ides (POX) of sample (see SW-
846 Method 9021) and Non-Purgeable Organic Hal ides (NPOX, that is, TOX of
sample that has been purged of volatiles) separately, where the NPOX
sample is centrifuged or filtered.
4.0 APPARATUS AND MATERIALS
4.1 Adsorption system (a schematic diagram of the adsorption system is
shown in Figure 1):
4,1.1 Adsorption module: Pressurized sample and nitrate-wash
reservoirs. (There are three instruments known to EPA at this time that
can be used to carry out this method. They are the TOX-10, available from
Cosa Instruments, and the DX-20 and DX-20A, available from Xertex-Dohrmann
Instruments.)
4.1.2 Adsorption columns: Pyrex, 5-cm-long x 6-mm-O.D. x 2-mm-
I.D.
9020A - 2 Revision 1
July 1992
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4.1.3 Granular activated carbon (GAC): Filtrasorb-400, Calgon-
APC or equivalent, ground or milled, and screened to a 100/200 mesh range.
Upon combustion of 40 mg of GAC, the apparent halide background should be
1,000 ng Cl" equivalent or less.
4.1.4 Cerafelt (available from Johns-Manville) or equivalent:
Form this material into plugs to fit the adsorption module and to hold 40
mg of GAC in the adsorption columns.
CAUTION: Do not touch this material with your fingers. Oily residue will
contaminate carbon.
4.1.5 Column holders.
4.1.6 Class A volumetric flasks: 100-mL and 50-mL.
4.2 Analytical system:
4.2.1 Microcoulometric-titration system: Containing the
following components (a flowchart of the analytical system is shown in
Figure 2):
4.2.1.1 Boat sampler: Muffled at 800'C for at least 2-4
min and cleaned of any residue by vacuuming after each run.
4.2.1.2 Pyrolysis furnace.
4.2.1.3 Microcoulometer with integrator.
4.2.1.4 Titration cell.
4.2.2 Strip-chart recorder.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Sodium sulfite (0.1 M), Na,S03: Dissolve 12.6 g ACS reagent grade
Na2S03 in reagent water and dilute to 1 L.
5.4 Concentrated nitric acid (HN03).
5.5 Nitrate-wash solution (5,000 mg NO,"/!.), KN03: Prepare a nitrate-
wash solution by transferring approximately 8.2 g of potassium nitrate (KN03)
into a 1-liter Class A volumetric flask and diluting to volume with reagent
water.
9020A - 3 Revision 1
July 1992
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5.6 Carbon dioxide (C02): Gas, 99.9% purity.
5.7 Oxygen (02) : 99.9% purity.
5.8 Nitrogen (N2): Prepurified.
5.9 Acetic acid in water (70%), CjH^: Dilute 7 volumes of glacial
acetic acid with 3 volumes of reagent water.
5.10 Trichlorophenol solution, stock (1 /nL = 10 /ig CT): Prepare a
stock solution by accurately weighing accurately 1.856 g of trichlorophenol into
a 100-mL Class A volumetric flask. Dilute to volume with methanol .
5.11 Trichlorophenol solution, calibration (1 pi = 500 ng Cl"), C6H,C130:
Dilute 5 ml of the trichlorophenol stock solution to 100 mL with methanol.
5.12 Trichlorophenol standard, instrument calibration: First, nitrate-
wash a single column packed with 40 mg of activated carbon, as instructed for
sample analysis, and then inject the column with 10 pi of the calibration
solution.
5.13 Trichlorophenol standard, adsorption efficiency (100 ^g cr/l
Prepare an adsorption-efficiency standard by injecting 10 pi of stock solution
into 1 liter of reagent water.
5.14 Blank standard: The methanol used to prepare the calibration
standard should be used as the blank standard.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed in Chapter Nine.
6.2 All samples should be collected in bottles with Teflon septa (e.g. .
Pierce #12722 or equivalent) and be protected from light. If this is not
possible, use amber glass 250-mL bottles fitted with Teflon-lined caps. Foil may
be substituted for Teflon if the sample is not corrosive. Samples must be
preserved by acidification to pH <2 with sulfuric acid, stored at 4"C, and
protected against loss of volatiles by eliminating headspace in the container.
Samples should be analyzed within 28 days. The container must be washed and
muffled at 400°C before use, to minimize contamination.
6.3 All glassware must be dried prior to use according to the method
discussed in Step 3.1.1.
7.0 PROCEDURE
7.1 Sample preparation:
7.1.1 Special care should be taken in handling the sample in
order to minimize the loss of volatile organohalides. The adsorption
procedure should be performed simultaneously on duplicates.
9020A - 4 Revision 1
July 1992
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7.1.2 Reduce residual chlorine by adding sulfite (5 mg sodium
sulfite crystals per liter of sample). Sulfite should be added at the
time of sampling if the analysis is meant to determine the TOX
concentration at the time of sampling. It should be recognized that TOX
may increase on storage of the sample. Samples should be stored at 4*C
without headspace.
7.2 Calibration:
7.2.1 Check the adsorption efficiency of each newly prepared
batch of carbon by analyzing 100 ml of the adsorption efficiency standard,
in duplicate, along with duplicates of the blank standard. The net
recovery should be within 5% of the standard value.
7.2.2 Nitrate-wash blanks (method blanks): Establish the
repeatability of the method background each day by first analyzing several
nitrate-wash blanks. Monitor this background by spacing nitrate-wash
blanks between each group of eight pyrolysis determinations. The nitrate-
wash blank values are obtained on single columns packed with
40 mg of activated carbon. Wash with the nitrate solution, as instructed
for sample analysis, and then pyrolyze the carbon.
7.2.3 Pyrolyze duplicate instrument-calibration standards and the
blank standard each day before beginning sample analysis. The net
response to the calibration standard should be within 3% of the
calibration-standard value. Repeat analysis of the instrument-calibration
standard after each group of eight pyrolysis determinations and before
resuming sample analysis, and after cleaning or reconditioning the
titration cell or pyrolysis system.
7.3 Adsorption procedure:
7.3.1 Connect two columns in series, each containing 40 mg of
100/200-mesh activated carbon.
7.3.2 Fill the sample reservoir and pass a metered amount of
sample through the activated-carbon columns at a rate of approximately 3
mL/mi n.
NOTE: 100 mL of sample is the preferred volume for concentrations of TOX
between 5 and 500 M9/L, 50 ml for 501 to 1000 M9/U and 25 ml for
1001 to 2000 /ig/L. If the anticipated TOX is greater than 2000
/ig/L, dilute the sample so that 100 ml will contain between 1 and
50 M9 TOX.
7.3.3 Wash the columns-in-series with 2 ml of the 5,000-mg/L
nitrate solution at a rate of approximately 2 mL/min to displace inorganic
chloride ions.
7.4 Pyrolysis procedure:
7.4.1 The contents of each column are pyrolyzed separately.
After being rinsed with the nitrate solution, the columns should be
9020A - 5 Revision 1
July 1992
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protected from the atmosphere and other sources of contamination until
ready for further analysis.
7.4.2 Pyrolysis of the sample .is accomplished in two stages. The
volatile components are pyrolyzed in a C02-rich atmosphere at a low
temperature to ensure the conversion of brominated trihalomethanes to a
titratable species. The less volatile components are then pyrolyzed at a
high temperature in an 02-rich atmosphere.
7.4.3 Transfer the contents of each column to the quartz boat for
individual analysis.
7.4.4 Adjust gas flow according to manufacturer's directions.
7.4.5 Position the sample for 2 min in the 200'C zone of the
pyrolysis tube.
7.4.6 After 2 min, advance the boat into the 800'C zone (center)
of the pyrolysis furnace. This second and final stage of pyrolysis may
require from 6 to 10 min to complete.
7.5 Detection: The effluent gases are directly analyzed in the micro-
coulometric-titration cell. Carefully follow manual instructions for optimizing
cell performance.
7.6 Breakthrough: The unpredictable nature of the background bias
makes it especially difficult to recognize the extent of breakthrough of
organohalides from one column to another. All second;column measurements for a
properly operating system should not exceed 10^'of the two-column total
measurement. If the 10% figure is exceeded, one of three events could have
happened: (1) the first column was overloaded and a legitimate measure of
breakthrough was obtained, in which case taking a smaller sample may be
necessary; (2) channeling or some other failure occurred, in which case the
sample may need to be rerun; or (3) a high random bias occurred, and the result
should be rejected and the sample rerun. Because it may not be possible to
determine which event occurred, a sample analysis should be repeated often enough
to gain confidence in results. As a general rule, any analysis that is rejected
should be repeated whenever a sample is available. In the event that repeated
analyses show that the second column consistently exceeds the 10% figure and the
total is too low for the first column to be saturated and the inorganic Cl is
less than 20,000 times the organic chlorine value, then the result should be
reported, but the data user should be informed of the problem. If the second-
column measurement is equal to or less than the nitrate-wash blank value, the
second-column value should be disregarded.
7.7 Calculations: TOX as Cl is calculated using the following formula:
(C, - C3) + (C2 - C3)
= M9/L Total Organic Halide
9020A - 6 Revision 1
July 1992
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where:
C., = M9 CT on the first column in series;
C2 - jig CT on the second column in series;
C3 = predetermined, daily, average, method-blank value
(nitrate-wash blank for a 40-mg carbon column); and
V = the sample volume in liters.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for
easy reference or inspection.
8.2 Employ a minimum of one blank per sample batch to determine if
contamination or any memory effects are occurring.
8.3 Verify calibration with an independently prepared check standard
every 15 samples.
8.4 Run one spike duplicate sample for every 10 samples. A duplicate
sample is a sample brought through the whole sample-preparation and analytical
process.
9.0 METHOD PERFORMANCE
9.1 Under conditions of duplicate analysis, the method detection limit
is 5 M9/L.
9,2 Analyses of distilled water, uncontaminated ground water, and
ground water from RCRA waste management facilities spiked with volatile
chlorinated organics generally gave recoveries between 75-100% over the
concentration range 10-500 ng/L. Relative standard deviations were generally
20% at concentrations greater than 25 M9/L. These data are shown in Tables 1
and 2.
10.0 REFERENCES
1. Gaskill, A., Compilation and Evaluation of RCRA Method Performance Data,
Work Assignment No. 2, EPA Contract No. 68-01-7075, September 1986.
2. Stevens, A.A., R.C. Dressman, R.K. Sorrel 1, and H.J. Brass, Organic Halogen
Measurements: Current Uses and Future Prospects, Journal of the American Water
Works Association, pp. 146-154, April 1985.
3. Tate, C., 8. Chow, et al., EPA Method Study 32, Method 450.1, Total Organic
Hal ides (TOX), EPA/600/S4-85/080, NTIS: PB 86 136538/AS.
9020A - 7 Revision 1
July 1992
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TABLE 1. METHOD PERFORMANCE DATA"
Spiked
Compound
Bromobenzene
Bromodi chl oromethane
Bromoform
Bromoform
Bromoform
Bromoform
Bromoform
Chloroform
Chloroform
Chloroform
Chloroform
Chloroform
Di bromodi chl oromethane
Di bromodi chloromethane
Tetrachloroethylene
Tetrachl oroethyl ene
Tetrachl oroethyl ene
trans -Oi chl oroethyl ene
trans -Oi chl oroethyl ene
trans -Di chl oroethyl ene
Matrix"
O.W.
D.W
D.W.
D.W.
G.W.
G.W.
G.W.
D.W.
D.W.
G.W.
G.W.
G.W.
D.W.
D.W.
G.W.
G.W.
G.W.
G.W.
G.W.
G.W.
TOX
Concentration
(M9/L)
443
160
160
238
10
31
100
98
112
10
30
100
155
374
10
30
101
10
30
98
Percent
Recovery
95
98
110
100
140
93
120
89
94
79
76
81
86
73
79
75
78
84
63
60
aResults from Reference 2.
bG.W. = Ground Water.
D.W. = Distilled Water.
9020A - 8
Revision 1
July 1992
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TABLE 2. METHOD PERFORMANCE DATA8
Sample Unspiked Spike Percent
Matrix TOX (ng/L) Level Recovery
Ground Water 68, 69 100 98, 99
Ground Water 5, 12 100 110, 110
Ground Water 5, 10 100 95, 105
Ground Water 54, 37 100 111, 106
Ground Water 17, 15 100 98, 89
Ground Water 11, 21 100 97, 89
"Results from Reference 3.
9020A - 9 Revision 1
July 1992
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Sample
Reservoir
(1 of 4)
Nitrate Wash
Reservoir
GAC Column 1
GAC Column 2
Figure 1. Schematic Diagram of Adsorption System
9020A - 10
Revision 1
July 1992
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Sparging
Device
Tltration
Cell
Pyrolysis
Furnace
Boat
Inlet
Microcoulometer
with Integrator
Strip Chart
Recorder
Adsorption
Module
Figure 2. Flowchart of Analytical System
9020A - 11
Revision 1
July 1992
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METHOD 9020A
TOTAL ORGANIC HALIDES (TOX)
START
711 Take special
ear* in handling
sample to minimize
vo 1 a 1 1 1 e loss
712 Add sulfite
to reduce residual
chlorine . s tore at
4 C without
headspace
1
7 2 1 Check
abs o r pi ion
efficiency for each
batch of carbon
1
7 I 2 Analyze
nitrate-wat*. hlanks
lo establish
background
723 Pyrolyie
dupl ica te
i ns t r umen t
calibration and
blank standards
each day
^
X^v'
' 3 L Connect in
containing
activated carbon
7 3 2 Fill sample
sample through
activated carbon
co 1 umns
733 Hash columns
with nitrate
so lution
741 Protect
columns from
con tamina t ion
1
742 Pyrolyze
volatile components
in C02 • r ich
atmosphere at Ion
temperature
•»
742 Pyrolyze less
volatile compounds
at high temperature
in 02-rich
a tmosphere
1
743 Transfer
contents of each
col umn to qua r t z
boat for analysis
1
744 Adjust gas
flow
,
7 4 5 Position
sample for 2
minutes in 200 C
zone of pyrolyfii
tube
1
746 Advance boat
into 800 C zone
9020A - 12
Revision 1
July 1992
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METHOD 9020A
(Continued)
7 S Analyze
eff Lu«nt gaie* in
microcouioro«lric-
11t r* 11on eel 1
7 S I> 2nd
col ucnn
iur*m«nt >10%
of Z column
total''
7 6 Diaregard
lecond-coLumn valu«
9020A - 13
Revision 1
July 1992
-------
O
Qd
-------
METHOD 9020B
TOTAL ORGANIC HALIDES (TOX)
1.0 SCOPE AND APPLICATION
1.1 Method 9020 determines Total Organic Hal ides (TOX) as chloride in
drinking water and ground waters. The method uses carbon adsorption with a
microcoulometric-titration detector.
1.2 Method 9020 detects all organic halides containing chlorine,
bromine, and iodine that are adsorbed by granular activated carbon under the
conditions of the method. Fluorine-containing species are not determined by this
method.
1.3 Method 9020 is applicable to samples whose inorganic-halide concen-
tration does not exceed the organic-halide concentration by more than 20,000
times.
1.4 Method 9020 does not measure TOX of compounds adsorbed to
undissolved solids.
1.5 Method 9020 is restricted to use by, or under the supervision of,
analysts experienced in the operation of a pyrolysis/microcoulometer and in the
interpretation of the results.
1.6 This method is provided as a recommended procedure. It may be used
as a reference for comparing the suitability of other methods thought to be
appropriate for measurement of TOX (i.e., by comparison of sensitivity, accuracy,
and precision of data).
2.0 SUMMARY OF METHOD
2.1 A sample of water that has been protected against the loss of
volatiles by the elimination of headspace in the sampling container, and that is
free of undissolved solids, is passed through a column containing 40 mg of
activated carbon. The column is washed to remove any trapped inorganic halides
and is then combusted to convert the adsorbed organohalides to HX, which is
trapped and titrated electrolytically using a microcoulometric detector.
3.0 INTERFERENCES
3.1 Method interferences may be caused by contaminants, reagents,
glassware, and other sample-processing hardware. All these materials must be
routinely demonstrated to be free from interferences under the conditions of the
analysis by running method blanks.
3.1.1 Glassware must be scrupulously cleaned. Clean all
glassware as soon as possible after use by treating with chromate cleaning
solution. This should be followed by detergent washing in hot water.
Rinse with tap water and distilled water and drain dry; glassware which is
not volumetric should, in addition, be heated in a muffle furnace at 400°C
for 15 to 30 min. (Volumetric ware should not be heated in a muffle
9020B - 1 Revision 2
September 1994
-------
furnace.) Glassware should be sealed and stored in a clean environment
after drying and cooling to prevent any accumulation of dust or other
contaminants.
3.1.2 The use of high-purity reagents and gases helps to minimize
interference problems.
3.2 Purity of the activated carbon must be verified before use. Only
carbon samples that register less than 1,000 ng CV/40 mg should be used. The
stock of activated carbon should be stored in its granular form in a glass
container with a Teflon seal. Exposure to the air must be minimized, especially
during and after milling and sieving the activated carbon. No more than a 2-wk
supply should be prepared in advance. Protect carbon at all times from all
sources of halogenated organic vapors. Store prepared carbon and packed columns
in glass containers with Teflon seals.
3.3 Particulate matter will prevent the passage of the sample through
the adsorption column. Particulates must, therefore, be eliminated from the
sample. This must be done as gently as possible, with the least possible sample
manipulation, in order to minimize the loss of volatiles. It should also be
noted that the measured TOX will be biased by the exclusion of TOX from compounds
adsorbed onto the particulates. The following techniques may be used to remove
particulates; however, data users must be informed of the techniques used and
their possible effects on the data. These techniques are listed in order of
preference:
3.3.1 Allow the particulates to settle in the sample container
and decant the supernatant liquid into the adsorption system.
3.3.2 Centrifuge sample and decant the supernatant liquid into
the adsorption system.
3.3.3 Measure Purgeable Organic Hal ides (POX) of sample (see SW-
846 Method 9021) and Non-Purgeable Organic Hal ides (NPOX, that is, TOX of
sample that has been purged of volatiles) separately, where the NPOX
sample is centrifuged or filtered.
4.0 APPARATUS AND MATERIALS
4.1 Adsorption system (a schematic diagram of the adsorption system is
shown in Figure 1):
4.1.1 Adsorption module: Pressurized sample and nitrate-wash
reservoirs.
4.1.2 Adsorption columns: Pyrex, 5-cm-long x 6-mm-O.D. x
2-mm-I.D.
4.1.3 Granular activated carbon (GAC): Filtrasorb-400, Calgon-
APC or equivalent, ground or milled, and screened to a 100/200 mesh range.
Upon combustion of 40 mg of GAC, the apparent halide background should be
1,000 ng CV equivalent or less.
9020B - 2 Revision 2
September 1994
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4.1.4 Cerafelt (available from Johns-Manville) or equivalent:
Form this material into plugs to fit the adsorption module and to hold
40 mg of GAC in the adsorption columns.
CAUTION: Do not touch this material with your fingers. Oily residue
will contaminate carbon.
4.1.5 Column holders.
4.1.6 Class A volumetric flasks: 100-mL and 50-mL.
4.2 Analytical system:
4.2.1 Microcoulometric-titration system: Containing the
following components (a flowchart of the analytical system is shown in
Figure 2):
4.2.1.1 Boat sampler: Muffled at 800°C for at least 2-
4 min and cleaned of any residue by vacuuming after each run.
4.2.1.2 Pyrolysis furnace.
4.2.1.3 Microcoulometer with integrator.
4.2.1.4 Titration cell.
4.2.2 Recording device.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Sodium sulfite (0.1 M), Na2S03: Dissolve 12.6 g ACS reagent grade
Na2S03 in reagent water and dilute to 1 L.
5.4 Concentrated nitric acid (HN03).'
5.5 Nitrate-wash solution {5,000 mg N03/L), KN03: Prepare a nitrate-
wash solution by transferring approximately 8.2 g of potassium nitrate (KN03)
into a 1-liter Class A volumetric flask and diluting to volume with reagent
water.
5.6 Carbon dioxide (C02): Gas, 99.9% purity.
5.7 Oxygen (02): 99.9% purity.
9020B - 3 Revision 2
September 1994
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5.8 Nitrogen (N2): Prepurified.
5.9 Acetic acid in water (70%), C2H402: Dilute 7 volumes of glacial
acetic acid with 3 volumes of reagent water.
5.10 Trichlorophenol solution, stock (1 pi = 10 M9 CV): Prepare a stock
solution by accurately weighing accurately 1.856 g of trichlorophenol into a 100-
ml Class A volumetric flask. Dilute to volume with methanol.
5.11 Trichlorophenol solution, calibration (1 jiL = 500 ng CV), C6H3C130:
Dilute 5 ml of the trichlorophenol stock solution to 100 ml with methanol.
5.12 Trichlorophenol standard, instrument calibration: First, nitrate-
wash a single column packed with 40 mg of activated carbon, as instructed for
sample analysis, and then inject the column with 10 p.1 of the calibration
solution.
5.13 Trichlorophenol standard, adsorption efficiency (100/zg CT/liter):
Prepare an adsorption-efficiency standard by injecting 10 /zl of stock solution
into 1 liter of reagent water.
5.14 Blank standard: The methanol used to prepare the calibration
standard should be used as the blank standard.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed in Chapter Nine.
6.2 All samples should be collected in bottles with Teflon septa (e.g..
Pierce #12722 or equivalent) and be protected from light. If this is not
possible, use amber glass 250-mL bottles fitted with Teflon-lined caps. Foil may
be substituted for Teflon if the sample is not corrosive. Samples must be
preserved by acidification to pH <2 with sulfuric acid, stored at 4°C, and
protected against loss of volatiles by eliminating headspace in the container.
Samples should be analyzed within 28 days. The container must be washed and
muffled at 400°C before use, to minimize contamination.
6.3 All glassware must be dried prior to use according to the method
discussed in Sec. 3.1.1.
7.0 PROCEDURE
7.1 Sample preparation:
7.1.1 Special care should be taken in handling the sample in
order to minimize the loss of volatile organohalides. The adsorption
procedure should be performed simultaneously on duplicates.
7.1.2 Reduce residual chlorine by adding sulfite (5 mg sodium
sulfite crystals per liter of sample). Sulfite should be added at the
time of sampling if the analysis is meant to determine the TOX
concentration at the time of sampling. It should be recognized that TOX
9020B - 4 Revision 2
September 1994
-------
may increase on storage of the sample. Samples should be stored at 4°C
without headspace.
7.2 Calibration:
7.2.1 Check the adsorption efficiency of each newly prepared
batch of carbon by analyzing 100 mL of the adsorption efficiency standard,
in duplicate, along with duplicates of the blank standard. The net
recovery should be within 10% of the standard value.
7.2.2 Nitrate-wash blanks (method blanks): Establish the
repeatability of the method background each day by first analyzing several
nitrate-wash blanks. Monitor this background by spacing nitrate-wash
blanks between each group of ten pyrolysis determinations. The nitrate-
wash blank values are obtained on s.ingle columns packed with40mgof
activated carbon. Wash with the nitrate solution, as instructed for
sample analysis, and then pyrolyze the carbon.
7.2.3 Pyrolyze duplicate instrument-calibration standards and the
blank standard each day before beginning sample analysis. The net
response to the calibration standard should be within 10% of the
calibration-standard value. Repeat analysis of the instrument-calibration
standard after each group of ten pyrolysis determinations and before
resuming sample analysis, and after cleaning or reconditioning the
titration cell or pyrolysis system.
7.3 Adsorption procedure:
7.3.1 Connect two columns in series, each containing 40 mg of
100/200-mesh activated carbon.
7.3.2 Fill the sample reservoir and pass a metered amount of
sample through the activated-carbon columns at a rate of approximately
3 mL/min.
NOTE: 100 ml of sample is the preferred volume for concentrations
of TOX between 5 and 500 /ig/L, 50 ml for 501 to 1000 /xg/L, and 25
ml for 1001 to 2000 M9/L. If the anticipated TOX is greater than
2000 M9/L, dilute the sample so that 100 ml will contain between
1 and 50 /xg TOX.
7.3.3 Wash the columns-in-series with 2 ml of the 5,000-mg/L
nitrate solution at a rate of approximately 2 mL/min to displace inorganic
chloride ions.
7.4 Pyrolysis procedure:
7.4.1 The contents of each column are pyrolyzed separately.
After being rinsed with the nitrate solution, the columns should be
protected from the atmosphere and other sources of contamination until
ready for further analysis.
9020B - 5 Revision 2
September 1994
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7.4.2 Pyrolysis of the sample is accomplished in two stages. The
volatile components are pyrolyzed in a C02-rich atmosphere at a low
temperature to ensure the conversion of brominated trihalomethanes to a
titratable species. The less volatile components are then pyrolyzed at a
high temperature in an 02-rich atmosphere.
7.4.3 Transfer the contents of each column to the quartz boat for
individual analysis.
7.4.4 Adjust gas flow according to manufacturer's directions.
7.4.5 Position the sample for 2 min in the 200eC zone of the
pyrolysis tube.
7.4.6 After 2 min, advance the boat into the 800°C zone (center)
of the pyrolysis furnace. This second and final stage of pyrolysis may
require from 6 to 10 min to complete.
7.5 Detection: The effluent gases are directly analyzed in the micro-
coulometric-titration cell. Carefully follow manual instructions for optimizing
cell performance.
7.6 Breakthrough: The unpredictable nature of the background bias
makes it especially difficult to recognize the extent of breakthrough of
organohalides from one column to another. ATI second-column measurements for a
properly operating system should not exceed 10% of the two-column total
measurement. If the 10% figure is exceeded, one of three events could have
happened: (1) the first column was overloaded and a legitimate measure of
breakthrough was obtained, in which case taking a smaller sample may be
necessary; (2) channeling or some other failure occurred, in which case the
sample may need to be rerun; or (3) a high random bias occurred, and the result
should be rejected and the sample rerun. Because it may not be possible to
determine which event occurred, a sample analysis should be repeated often enough
to gain confidence in results. As a general rule, any analysis that is rejected
should be repeated whenever a sample is available. In the event that repeated
analyses show that the second column consistently exceeds the 10% figure and the
total is too low for the first column to be saturated and the inorganic Cl is
less than 20,000 times the organic chlorine value, then the result should be
reported, but the data user should be informed of the problem. If the second-
column measurement is equal to or less than the nitrate-wash blank value, the
second-column value should be disregarded.
9020B - 6 Revision 2
September 1994
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7.7 Calculations: TOX as CT is calculated using the following formula:
(C, - C3) + (C2 - C3)
= /Lig/L Total Organic Halide
V
where:
CT = jug CV on the first column in series;
C2 = jug CV on the second column in series;
C3 = predetermined, daily, average, method-blank value
(nitrate-wash blank for a 40-mg carbon column); and
V = the sample volume in liters.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for specific quality control guidelines.
8.2 This method requires that all samples be run in duplicate.
8.3 Employ a minimum of two blanks to establish the repeatability of
the method background, and monitor the background by spacing method blanks
between each group of eight analytical determinations.
8.4 After calibration, verify it with an independently prepared check
standard.
8.5 Run matrix spike between every 10 samples and bring it through the
entire sample preparation and analytical process.
9.0 METHOD PERFORMANCE
9.1 Under conditions of duplicate analysis, the method detection limit
is 10 /zg/L.
9.2 Analyses of distilled water, uncontaminated ground water, and
ground water from RCRA waste management facilities spiked with volatile
chlorinated organics generally gave recoveries between 75-100% over the
concentration range 10-500 jug/L. Relative standard deviations were generally
20% at concentrations greater than 25 ^tg/L. These data are shown in Tables 1
and 2.
10.0 REFERENCES
1. Gaskill, A., Compilation and Evaluation of RCRA Method Performance Data,
Work Assignment No. 2, EPA Contract No. 68-01-7075, September 1986.
9020B - 7 Revision 2
September 1994
-------
2. Stevens, A.A., R.C. Dressman, R.K. Sorrel!, and H.J. Brass, Organic Halogen
Measurements: Current Uses and Future Prospects, Journal of the American Water
Works Association, pp. 146-154, April 1985.
3. Tate, C., B. Chow, et al., EPA Method Study 32, Method 450.1, Total Organic
Hal ides (TOX), EPA/600/S4-85/080, NTIS: PB 86 136538/AS.
9020B - 8 Revision 2
September 1994
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TABLE 1. METHOD PERFORMANCE DATAa
Spiked
Compound
Bromobenzene
Bromodichloromethane
Bromoform
Bromoform
Bromoform
Bromoform
Bromoform
Chloroform
Chloroform
Chloroform
Chloroform
Chloroform
Di bromodi chl oromethane
Di bromod i chl oromethane
Tetrachl oroethyl ene
Tetrachl oroethyl ene
Tetrachl oroethyl ene
trans -Di chl oroethyl ene
trans -Di chl oroethyl ene
trans-Dichl oroethyl ene
Matrix6
D.W.
D.W
D.W.
D.W.
G.W.
G.W.
G.W.
D.W.
D.W.
G.W.
G.W.
G.W.
D.W.
D.W.
G.W.
G.W.
G.W.
G.W.
G.W.
G.W.
TOX
Concentration
(M9/L)
443
160
160
238
10
31
100
98
112
10
30
100
155
374
10
30
101
10
30
98
Percent
Recovery
95
98
110
100
140
93
120
89
94
79
76
81
86
73
79
75
78
84
63
60
aResults from Reference 2.
hG.W. = Ground Water.
D.W. = Distilled Water.
9020B - 9
Revision 2
September 1994
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TABLE 2. METHOD PERFORMANCE DATA3
Sample Unspiked Spike Percent
Matrix TOX Levels Level Recoveries
(M9/L)
Ground Water 68, 69 100 98, 99
Ground Water 5, 12 100 110, 110
Ground Water 5, 10 100 95, 105
Ground Water 54, 37 100 111, 106
Ground Water 17, 15 100 98, 89
Ground Water 11, 21 100 97, 89
aResults from Reference 3.
9020B - 10 Revision 2
September 1994
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Fig. 1. Schematic Diagram of Adsorption System
Sample
Reservoir
(1 of 4)
Nitrate Wash
Reservoir
GAC Column 1
GAC Column 2
9020B - 11
Revision 2
September 1994
-------
Fig. 2. Flowchart of Analytical System
Sparging
Device
Titration
Cell
Pyrolysis
Furnace
Boat
Inlet
Adsorption
Module
Microcoulometer
with Integrator
Strip Chart
Recorder
9020B - 12
Revision 2
September 1994
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METHOD 9020B
TOTAL ORGANIC HALIDES (TOX)
START
7 1 1 TaUe specia 1
c*r« In handling
sample to minimize
volatile 1 oss
.,
7 1 2 Add luUite
chlorine; store at
4 C without
headapace
721 Check
abso r p 1 1 on
ba tch of carbon
7 2 2 Analyze
nitrate-wash blanks
to os tabl i sh
backgr ound
1
7.2 3 Py rolyze
dupl ica te
ins t r urRen t
cal ibra t i on and
blank 3 tandards
each day
?31 Connec t in
aeries two columns
containing
activated carbon
.*
732 Till sample
sampl a through
activated carbon
co 1 urons
7 3 3 Wash co 1 umns
with nitrate
solution
1
7 A 1 Protect
co lumns f r om
contamina tion
742 Pyrolyze
volatile components
in C02-rich
atmosphere at low
tempera lure
7 4 2 Py rolyze leas
volatile compounds
in 02 • r ich
a tmosphe re
?43 Transfer
contents of each
co Lumn to qua r t, z
boat for analysis
-»
? 4 4 Ad jus t gas
flow
745 Position
sample for 2
minutes in 200 C
zone of pyroly»is
tuba
i
746 Advance boat
into 800 C zone
7 5 Analyze
effluent gases in
microcoulometric •
titration celt
/I 6 Is 2ndN.
/ column X^
C measurement >10% y—
X- of 2 column S
\^ lolal' /
Yes
7 6 Reject and
r epea t
No
7 6 Disregard
second-column va L
9020B - 13
Revision 2
September 1994
-------
-------
METHOD 9021
PURGEABLE ORGANIC HALIDES (POX)
1.0 SCOPE AND APPLICATION
1.1 Method 9021 determines organically bound halides (chloride,
bromide, and iodide) purged from a sample of drinking water or ground water.
They are reported as chloride. This method is a quick screening procedure
requiring about 10 minutes. The method uses a sparging device, a pyrolysis
furnace, and a microcoulometric-titration detector.
1.2 Method 9021 detects purgeable organically bound chlorine, bromine,
and iodine. Fluorine containing species are not determined by this method.
Method 9021 measures POX concentrations ranging from 5 to 1,000 /^g/L.
1.3 Method 9021 is restricted to use by, or under the supervision of,
analysts experienced in the operation of a pyrolysis/microcoulometer and in the
interpretation of the results.
2.0 SUMMARY OF METHOD
2.1 A sample of water, protected against the loss of volatiles by the
elimination of headspace in the sampling container, is transferred to a purging
vessel. The volatile organic halides are purged into a pyrolysis furnace using
a stream of C02 and the hydrogen halide (HX) pyrolysis product is trapped and
titrated electrolytically using a microcoulometric detector.
3.0 INTERFERENCES
3.1 Contaminants, reagents, glassware, and other sample processing
hardware may cause interferences. Method blanks must be routinely run to
demonstrate freedom from interferences under the conditions of the analysis.
3.1.1 Glassware must be scrupulously clean. Clean all glassware
as soon as possible after use by treating with chromate cleaning solution.
This should be followed by detergent washing in hot water. Rinse with tap
water and reagent water and dry at 105°C for 1 hour or until dry.
Glassware which is not volumetric should, in addition, be heated in a
muffle furnace at 300"C for 15 to 30 minutes (Class A volumetric ware
should not be heated in a muffle furnace). Glassware should be sealed and
stored in a clean environment after drying and cooling to prevent any
accumulation of dust or other contaminants.
3.1.2 Use high purity reagents and gases to minimize interference
problems.
3.1.3 Avoid using non-PTFE (polytetrafluoroethylene) plastic
tubing, non-TFE thread sealants, or flow controllers with rubber
components in the purge gas stream.
9021 - 1 Revision 0
July 1992
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3.2 Samples can be contaminated by diffusion of volatile organics
(methylene chloride) through the septum seal into the sample during shipment and
especially during storage. A trip blank prepared from water and carried through
the sampling and handling protocol serves as a check on such contamination, A
trip blank should be run with each analytical batch.
3.3 Contamination by carry-over occurs whenever high level and low
level samples are sequentially analyzed. To-reduce carryover, the purging device
and sample "syringe must be rinsed with water between sample analyses. Whenever
an unusually concentrated sample is encountered, it should be followed by an
analysis of water to check for cross contamination. For samples containing large
amounts of water-soluble materials, suspended solids, high boiling compounds or
high organohalide levels, wash out the purging device with a detergent solution,
rinse it with water, and then dry it in a 1059C oven between analyses.
3.4 All operations should be carried out in an area where halogenated
solvents, such as methylene chloride, are not being used.
3.5 Residual free chlorine interferes in the method. Free chlorine
must be destroyed by adding sodium sulfite when the sample is collected.
4.0 APPARATUS AND MATERIALS
4.1 Sampling equipment (for discrete sampling)
4.1.1 Vial - 25-mL capacity or larger, equipped with a screw-cap
with hole in center (Pierce #13075 or equivalent).
4.1.2 Septum - Teflon lined silicone (Pierce #12722 or
equivalent). Detergent wash, rinse with tap and reagent water, and dry at
105'C for 1 hour before use.
4.2 Analytical system
4.2.1 Microcoulometric-titration system containing the following
components (a schematic diagram of the microcoulometric-titration system
is shown in Figure 1).
4.2.1.1 Purging device.
4.2.1.2 Pyrolysis furnace.
4.2.1.3 Titration cell.
4.2.2 Strip chart recorder (optional) - The recorder is
recommended to make sure the peak is down to baselines before stopping
integration.
4.2.3 Microsyringes - 10-^L and 25-juL with 0.006 in i.d. needle
(Hamilton 702N or equivalent).
4.2.4 Syringe valve - 2 way, with Luer ends.
9021 - 2 Revision 0
July 1992
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5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit .its use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Sodium sulfide, Na2S. Granular, anhydrous.
5.4 Acetic acid in water (70%), CH3COOH. Dilute 7 volumes of glacial
acetic acid with 3 volumes of water.
5.5 Sodium chloride calibration standard (1 fj.g ClV/nL). Dissolve
1.648 g NaCl in water and dilute to 1 liter.
5.6 Carbon dioxide.
5.7 Methanol, CH3OH. Store away from other solvents.
5.8 Chloroform, CHC13.
5.9 Chloroform (stock) solution (1 ML = H-2 M9 of CHC1, or 10 M9 CT).
Prepare a stock solution by delivering accurately 760 /iL (1120 mg) of chloroform
into a 100-mL Class A volumetric flask containing approximately 90 ml of
methanol. Dilute to volume with methanol (10,000 mg of chlorine/L).
5.10 Chloroform (calibration) solution (1 ^L = 0.1 ng Cl"). Dilute 1 ml
of the chloroform stock solution to 100 mL with methanol (100 mg of chlorine/L).
5.11 Chloroform Quality Control (QC) reference sample (100 M9/L).
Prepare an aqueous standard by injecting 100 pi of the chloroform calibration
standard (100 mg of Cl'/L) into a Class A volumetric flask containing 100 mL of
water. Mix and store in a bottle with zero headspace. Analyze within two hours
after preparation.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed in Chapter Nine of this manual.
6.2 All samples should be collected in bottles with Teflon lined
silicone septa (e.g., Pierce #12722 or equivalent) and be protected from light.
If this is not possible, use amber glass 250-mL bottles fitted with Teflon lined
caps.
6.3 AIT glassware must be cleaned prior to use according to the process
described in Step 3.1.1.
9021 - 3 Revision 0
July 1992
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6.4 Special care should be taken in handling the sample in order to
minimize the loss of volatile organohalides. This is accomplished through
elimination of headspace and by minimizing the number of transfers.
6.5 Reduce residual chlorine, if present, by adding sodium sulfite
(5 mg of sodium sulfite crystals per liter of sample). Sodium sulfite should be
added to empty sample bottles at the time of sampling. Shake vigorously for 1
minute after bottle has been filled with sample and properly sealed. Samples
should be stored at 4°C without headspace. POX may increase during storage of
the sample.
6.6 All samples must be analyzed within 14 days of collection.
7.0 PROCEDURE
7.1 Calibration.
7.1.1 Assemble thesparging/pyrolysis/microcoulometric-titration
apparatus shown in Figure 1 in accordance with the manufacturer's
specifications. Typically a C02 flow of 150 mL/min and a sparger
temperature of 45 ± 5eC are employed. The pyrolysis furnace should be set
at 800 ± 10'C. Attach the titration cell to the pyrolysis tube outlet and
fill with electrolyte (70% acetic acid). Flow rate and temperature
changes will affect the compounds that are purged and change the percent
recovery of marginal compounds. Therefore, these parameters should not be
varied. Adjust gas flow rate according to manufacturer's directions.
7.1.2 Turn on the instrument and allow the gas flow and
temperatures to stabilize. When the background current of the titration
cell has stabilized the instrument is ready for use.
7.1.3 Calibrate the microcoulometric-titration system for Cl"
equivalents by injecting various amounts (1 to 80 ;iL) of the sodium
chloride calibration standard directly into the titration cell and
integrating the response using the POX integration mode. If desired, the
analog output of the titration cell can be displayed on a strip chart
recorder. The range of sodium chloride amounts should cover the range of
expected ,sample concentrations and should always be less than 80 /ig of
CIV The integrated response should read within 2% or 0.05 jug of the
quantity injected (whichever is larger) over the range 1-80 /^g CT. If
this calibration requirement is not met, then the instrument sensitivity
parameters should be adjusted according to the manufacturer's
specifications to achieve an accurate response.
7.1.4 Check the performance of the analytical system daily by
analyzing three 5-mL aliquots of a freshly prepared 100 ^g/L chloroform
check standard. The mean of these three analyses should be between 0.4-
0.55 ng of CV and the percent relative standard deviation should be 5% or
less. If these criteria are not met, the system should be checked as
described in the instrument maintenance manual in order to isolate the
problem.
9021 - 4 Revision 0
July 1992
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NOTE: Low chloroform recovery can often be traced to a vitrified inlet
tube. The tube should be checked regularly and the analyst should
be able to determine, based on chloroform recoveries, when the tube
should be replaced.
7.1.5 Determine an instrument blank daily by running an analysis
with the purge vessel empty. The instrument blank should be 0.00 ± 0.05
Mg of CT. Analyze a calibration blank sample daily. The calibration
blank should be within 0.02 n9 of Cl~ of the reagent blank.
7.2 Sample analysis
7.2.1 Select a chloroform spike concentration representative of
the expected levels in the samples. Using the chloroform stock solution,
prepare a spiking solution in methanol which is 500 times more
concentrated than the selected spike concentration. Add 10 pi of the
spiking solution to 5-mL aliquots of the samples chosen for spiking (refer
to Section 8.0, Quality Control, for guidance in selecting the appropriate
number of samples to be spiked).
7.2.2 Allow sample to come to ambient temperature prior to
drawing it into the syringe. Remove the plunger from a 5-mL or 10-mL
syringe and attach a closed syringe valve. If maximum sensitivity is
desired and the sample does not foam excessively, a 10-mL sample aliquot
may be analyzed. Otherwise 5-mL aliquots should be used. Open the sample
bottle (or standard) and carefully pour the sample into the syringe barrel
to just short of overflowing. Replace the syringe plunger and compress
the sample. Open the syringe valve and vent any residual air while
adjusting the sample volume to 5 mL. Since this process of taking an
aliquot destroys the validity of the sample for future analysis, the
analyst should fill a second syringe at this time to protect against
possible loss of data (e.g., accidental spill), or for duplicate analysis.
7.2.3 Attach the syringe valve assembly to the syringe valve on
the purging device. Place the pyrolysis/microcoulometer system in the POX
integration mode to activate the integration system. Immediately open the
syringe valves and inject the sample into the purging chamber.
7.2.4 Close both valves and purge the sample for 10 minutes.
7.2.5 After integration is complete, open the syringe valves and
withdraw the purged sample. Flush the syringe and purging device with
water prior to analyzing other samples.
7.2.6 If the integrated response exceeds the working range of the
instrument, prepare a dilution of the sample from the aliquot in the
second syringe with water and reanalyze. The water must meet the criteria
of Step 7.1.5. It may be necessary to heat and purge dilution waters.
7.3 Pyrolysis procedure
7.3.1 Pyrolysis of the purged organic component of the sample is
accomplished by pyrolyzing in a C02-rich atmosphere at a low temperature
9021 - 5 Revision 0
July 1992
-------
to ensure the conversion of brominated trihalomethanes to a titratable
species.
7.4 Directly analyze the effluent gases in the microcoulometric-
titration cell. Carefully follow instrument manual instructions for optimizing
cell performance.
7.5 . Calculations - POX as CT is calculated using the following formula:
JL_ x 1000 = jug/L Purgeable Organic Halide
V
where:
Qs = Quantity of POX as ^g of Cl" in the sample aliquot.
V = Volume of sample aliquot in ml.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for
easy reference or inspection for 3 years. This method is restricted to use by
or under supervision of experienced analysts. Refer to the appropriate section
of Chapter One for additional quality control guidelines.
8.2 Analyze a minimum of one reagent blank every 20 samples or per
analytical batch, whichever is more frequent, to determine if contamination or
any memory effects are occurring.
8.3 In addition to the performance check mentioned in Step 7.1.4,
verify calibration with an independently prepared chloroform QC reference sample
every 15 samples.
8.4 Analyze matrix spiked samples for every 10 samples or analytical
batch, whichever is more frequent. The spiked sample is carried through the
whole sample preparation process and analytical process.
8.5 Analyze all samples in replicate,
9.0 METHOD PERFORMANCE
9.1 Under conditions of duplicate analysis, the reliable limit of
detection is 5 M9/L.
9.2 Analyses of distilled water, uncontaminated ground water, and
ground water from RCRA waste management facilities spiked with volatile
chlorinated organics generally give recoveries of 44-128% over the concentration
range of 29-4500 M9/L. Relative standard deviations are generally less than 20%
at concentrations greater than 25 Mg/L. These data are shown in Tables 1 and
2.
9021 - 6 Revision 0
July 1992
-------
10.0 REFERENCES
1. Takahashi, Y.; Moore, R.T.; Joyce, R.J. "Measurement of Total Organic
Hal ides (TOX) and Purgeable Organic Hal ides (POX) in Water Using Carbon
Adsorption and Microcoulometric Determination"; Proceedings from Division
of Environmental Chemistry, American Chemical Society Meeting, March 23-
28, 1980.
2. Methods for Chemical Analysis of Water and Wastes; U.S. Environmental
Protection Agency. Office of Research and Development. Environmental
Monitoring and Support Laboratory. ORD Publication Offices of Center for
Environmental Research Information: Cincinnati, OH, 1983; EPA-600/4-79-
020.
3. Fed. Regist. 1979, 45, 69468-69473; December 3.
4. Rohrbough, W.G.; et al. Reagent Chemicals. American Chemical Society
Specifications. 7th ed.; American Chemical Society: Washington, DC, 1986.
5. "Development and Evaluation of Methods for Total Organic Halide and
Purgeable Organic Halide in Wastewater"; U.S. Environmental Protection
Agency. Environmental Monitoring and Support Laboratory. Cincinnati, OH,
1984; EPA-600/4-84-008; NTIS-PB-84-134-337.
6. 1985 Annual Book of ASTM Standards. Vol. 11.01; "Standard Specification
for Reagent Water"; ATSM: Philadelphia, PA, 1985; D1193-77.
7. Dohrmann. Rosemount Analytical Division. Santa Clara, CA 95052-8007.
8. Cosa Instruments. Norwood, NJ 21942.
9021 - 7 Revision 0
July 1992
-------
TABLE 1.
PRECISION AND ACCURACY DATA FOR SELECTED PURGEABLE ORGANIC HALIDES
(Reference 5)
Compound
Chloroform
Trichloroethene
Tetrachloroethene
Chlorobenzene
Dose1
(M9/L
as CT)
11
10
10
8
Average
Recovery
(M9/L
as Cl")
11
6
5
3
Average
Percent
Recovery
100
60
50
38
Standard
Deviation
1.4
0.7
0.8
0.6
MDL2
(M9/L)
4.5
2.2
3.2
2.03
Number of
Replicates
7
7
7
7
1Ten milliliter aliquot of spiked reagent water analyzed.
2The method detection limit (MDL) is defined as the minimum concentration of a
substance that can be measured and reported with 99% confidence that the value
is above zero.
Practical MDL probably greater (approximately 5 to 6 jug/L) due to low recovery.
9021 - 8
Revision 0
July 1992
-------
TABLE 2.
PRECISION AND ACCURACY DATA FOR VARIOUS WATER SAMPLES'
(Reference 5)
Sample1
Tap Water
POTW Sewage
Chlorinated
Hydrocarbon
Plant
Wastewater
Chlorinated
Hydrocarbon
Plant
Wastewater
Chlorinated
Hydrocarbon
Plant
Wastewater
Solid Waste2
Leachate
Industrial
Wastewater
Aniline3
Wastewater
Aniline3
Wastewater
Background Level
Spike (M9/L
Component as CT)
... —
Chloroform 68
Chloroform 114
Chloroform 32
Chloroform 32
1,1-Dichloro- 171
ethane
Methyl ene 510
chloride
Chloroform 15,700
Chloroform 15,700
Spike Level
as'CT)
0
29
460
1,500
4,500
800
800
15,000
45,000
Average
Percent
Recovery
—
128
77
50
87
41
65
150
91
Standard
Deviation
2
5
36
32
470
17
120
58
400
Number
of
Replicates
3
3
3
3
3
3
3
3
3
Vive milliliter sample aliquots analyzed.
2Diluted 200:1 prior to analysis. Values for this sample are in mg/L for original
sample.
3Diluted 10:1 prior to analysis. Values are for undiluted sample.
9021 - 9
Revision 0
July 1992
-------
POX Determination Step
Microcoulometer
Integrator
Acidic
Silver
Titration
Cell
800 °C
250°C
Combustion Tube
POX Sparger
45°C
I
CO,
" c:
. 2
co
(/I
1O O
«0 3
f\>
-------
METHOD 9021
PURGEABLE ORGANIC HALIDES (POX)-
START
711 Assemble
appa ratai. seI
carbon di o*ida flo*
ra te. set spa rge r
and py rolys is
furnace temperature
712 Turn on
instrument, allow
gas flow and
temperalures to
»tabiLiz e, allow
background current
of 111ra tion ceM
to stabilize
7 I 2 Calibrate the
microcoulorret: t c •
I i I ra 11 on s y 3 t e1*
for Cl- equivalents
7 ! 3
i ns '» r unen t
aensivity
para.-eteri.
reca1ib ra te
7 1 4 Analyze 3
a 1 xquoIs of
chloroform check
s tandard
7 1 4 Check system.
reanalyze check
s tandard
7 1 S Analyx*
cal ibration blank;
determine
instrument blank
7 2 1 Select
spiking
concentration: add
spiking solution to
appropriate samples
722 Transfer
sample to syringe:
fill second syringe
9021 - 11
Revision 0
July 1992
-------
METHOD 9021
(Continued)
7 2 3 Attach syringe
va I ve a»sernbly to
purging devic*; plac*
pyroly»i«/
microcoul omett>r
system in POX
tntegration mod«,
inject sample into
purging chamber
724 Purge for 10
minutes
7 2 5 Milhdrax
purged samp Ie;
flush syringe and
purging nevice with
wa t er
726 Dilute simple
from second syringe
with wa ter
7 3 1 Pyrolyie
samole in a carbon
dioHide rich
atmosphere at a loo
temceralure
7 4 Analyze the
effluent gas>e» in
the
microcoulomatric-
titraI ion eel 1
75 Calculate POX
as Cl-
STOP
9021 - 12
Revision 0
July 1992
-------
vo
o
-------
METHOD 9022
TOTAL ORGANIC HALIDES (TOX) BY NEUTRON ACTIVATION ANALYSIS
1.0 SCOPE AND APPLICATION
1.1 Method 9022 determines Total Organic Halldes (TOX) 1n aqueous
samples. The method uses a carbon adsorption procedure Identical to that of
Method 9020 (TOX analysis using a m1crocoulometr1c-t1tration detector),
Irradiation by neutron bombardment, and then detection using a gamma-ray
detector.
1.2 Method 9022 detects all organic halldes containing chlorine,
bromine, and Iodine that are adsorbed by granular activated carbon under the
conditions of the method. Each halogen can be quantltated Independently.
1.3 Method 9022 1s restricted to use by, or under the supervision of,
analysts experienced 1n the operation of neutron activation analysis and
familiar with spectral Interferences.
1.4 This method, which may be used in place of Method 9020, has the
advantage of determining the Individual concentrations of the halogens
chlorine, bromine, and Iodine 1n addition to TOX.
2.0 SUMMARY OF METHOD
2.1 A sample of water that has been protected against the loss of
volatlles by the elimination of headspace 1n the sampling container, and that
Is free of undlssolved solids, 1s passed through a column containing 40 mg of
granular activated carbon (6AC). The column 1s washed to remove any trapped
Inorganic halldes. the GAC sample 1s exposed to thermal neutron bombardment,
creating a radioactive Isotope. Gamma-ray emission, which 1s unique to each
halogen, 1s counted. The areas of the resulting peaks are directly
proportional to the concentrations of the halogens.
3.0 INTERFERENCES
3.1 Method Interferences may be caused by contaminants, reagents,
glassware, and other sample processing hardware. All these materials must be
routinely demonstrated to be free from Interferences under the conditions of
the analysis by running method blanks.
3.1.1 Glassware must be scrupulously cleaned. Clean all glassware
as soon as possible after use by treating with chromatic cleaning
solution. This should be followed by detergent washing 1n hot water.
Rinse with tap water and distilled water and drain dry; glassware which
1s not volumetric should, 1n addition, be heated 1n a muffle furnace at
9022 - 1
Revision 0
Date September 1986
-------
400*C for 15 to 30 mln. Volumetric ware should not be heated 1n a muffle
furnace. Glassware should be sealed and stored 1n a clean environment
after drying and cooling to prevent any accumulation of dust or other
contaminants.
3,1.2 The use of high-purity reagents and gases helps to minimize
Interference problems.
3.2 Purity of the activated carbon must be verified before use. Only
carbon samples that register less than 2,000 ng Cl"/40 mg GAC should be used.
The stock of activated carbon should be stored In its granular form 1n a glass
container with a Teflon seal. Exposure to the air must be minimized,
especially during and after milling and sieving the activated carbon. No more
than a 2-wk supply should be prepared 1n advance. Protect carbon at all times
from all sources of halogenated organic vapors. Store prepared carbon and
packed columns 1n glass containers with Teflon seals.
4.0 APPARATUS AND MATERIALS
4.1 Adsorption system (a general schematic of the adsorption system 1s
shown 1n Figure 1):
4.1.1 Adsorption nodule with pressurized sample and nitrate-wash
reservoirs.
4.1.2 Adsorption columns: Pyrex, 5-cm long x 6-mm O.D. x 2-mm I.D.
4.1.3 Granular activated carbon (GAC): F1ltrasorb-400, Calgon-APC
or equivalent, ground or milled, and screened to a 100/200 mesh range.
Upon combustion of 40 mg of GAC, the apparent hallde background should be
1000 ng Cl* equivalent or less.
4.1.4 Cerafelt (available from Johns-Manvllle) or equivalent: Form
this material Into plugs using a 2-mni-I.D. stainless steel borer with
ejection rod to hold 40 mg of GAC 1n the adsorption columns.
CAUTION: Do not touch this material with your fingers. Oily
residue will contaminate carbon.
4.1.5 Column holders.
4.1.6 Volumetric flasks: 100-mL, 50-mL.
4.2 Containers suitable for containment of samples and standards during
Irradiation (e.g., l/5-dram polyethylene snap-cap vial).
4.3 Sample Introduction system and a reactor generating a thermal
neutron flux capableo?achieving enough halogen activity for counting
purposes (e.g., a reactor having a neutron flux of 5 x 1012 neutrons/cm2/sec).
4.4 A gamma-ray detector and data-handling system capable of resolving
the halogen peaks from potential Interferences and background.
9022 - 2
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Date September 1986
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to
o
ro
iv>
I
**>
Rewrvoif
GAC Column 1
o
L*~-D
Nitratr W«h
Reservoir
oo
GAC Column 2
O 73
at n
r> <
n> -^
o
n
§
n
Figure 1. Schematic diayam of adsorption system.
-------
5.0 REAGENTS
5.1 Prepur1f1ed nitrogen.
5.2 ASTM Type II water (ASTM D1193): Water should be monitored for
Impurities.
5.3 Nitrate-wash solution (5,000 mg N03~/L): Prepare a nitrate-wash
solution by transferring approximately 8.2 g of potassium nitrate (KNOs) Into
a 1-lUer volumetric flask and diluting to volume with Type II water.
5.4 Acetone and nanoqrade hexane (50% v/v mixture).
5.5 Sodium sulflte. 0.1 M (ACS reagent grade, 12.6 g/L).
5.6 Concentrated nitric add (HN03): Reagent grade.
5.7 Standards; 25-ug Cl, 2.5-ug Br, and 2.5-ug I.
5.8 Radioactive standards to be used for calibrating gamma-ray detection
systems.
5.9 Trlchlorophenol solution, stock (1 uL » 10 ug Cl~): Prepare a stock
solution by accurately weighing accurately 1.856 g of trlchlorophenol Into a
100-mL volumetric flask. Dilute to volume with methanol.
5.10 Trlchlorophenol standard, adsorption efficiency (100 ug Cl'/Hter):
Prepare an adsorption-efficiency standard by Injecting 10 uL of stock solution
Into 1 liter of Type II water.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed 1n Chapter Nine of this manual.
6.2 All samples should be collected 1n bottles with Teflon septa (e.g.,
Pierce #12722 or equivalent) and be protected from light. If this 1s not
possible, use amber glass, 250-mL, fitted with Teflon-Hned caps. Foil may be
substituted for Teflon If the sample 1s not corrosive. Samples must be
protected against loss of volatlles by eliminating headspace 1n the container.
Containers must be washed and muffled at 400'C before use, to minimize
contamination.
6.3 All glassware must be dried prior to use according to the method
discussed 1n Paragraph 3.1.1.
9022 - 4
Revision
Date September 1986
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7.0 PROCEDURE
7.1 Sample preparation:
7.1.1 Special care should be taken 1n handling the sample 1n order
to minimize the loss of volatile organohalldes. The adsorption procedure
should be performed simultaneously on the front and back columns.
7.1.2 Reduce residual chlorine by adding sulflte (1 ml of 0.1 M
sulfHe per liter of sample). Sulflte should be added at the time of
sampling 1f the analysis 1s meant to determine the TOX concentration at
the time of sampling. It should be recognized that TOX may Increase on
storage of the sample. Samples should be stored at 4*C without
headspace.
7.1.3 Samples containing undlssolved solids should be centrlfuged
and decanted.
7.1.4 Adjust the pH of the sample to approximately 2 with
concentrated HNOs just prior to adding the sample to the reservoir,
7.2 Calibration;
7.2.1 Check the adsorption efficiency of each newly prepared batch
of carbon by analyzing 100 ml of the adsorption efficiency standard, 1n
duplicate, along with duplicates of the blank standard. The net recovery
should be within 5% of the standard value.
7.2.2 Nitrate-wash blanks (method blanks): Establish the
repeatability of the method background each day by first analyzing
several nitrate-wash blanks. Monitor this background by spacing nitrate-
wash blanks between each group of eight analysis determinations. The
nitrate-wash blank values are obtained on single columns packed with 40
mg of activated carbon. Wash with the nitrate solution, as Instructed
for sample analysis, and then analyze the carbon.
7.2.3 Prior to each day's operation, calibrate the Instrument using
radioactive standards (e.g., cobalt-60 and rad1um-226 sources). The
Instrument 1s calibrated such that gamma rays from the standards fall
within one channel of their true energies. A 100-sec blank 1s then
counted to verify that no stray radioactive sources are within sensing
distance of the detector. As data are obtained throughout the day, peak
locations 1n the standards are monitored to ensure there 1s no electronic
drift of the Instrument. If drift 1s noted, the system must be recali-
brated.
7.3 Adsorption procedure;
7.3.1 Connect 1n series two columns, each containing 40 mg of
100/200-mesh activated carbon.
9022 - 5
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Date September 1986
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7.3.2 Fill the sample reservoir and pass a metered amount of sample
through the activated-carbon columns at a rate of approximately 3 mL/m1n.
NOTE: 100 mL of sample 1s the preferred volume for concentrations
of TOX between 5 and 500 ug/L, 50 ml for 501 to 1000 ug/L,
and 25 ml for 1,001 to 2,000 ug/L.
7.3.3 Wash the columns-1n-ser1es with at least 2 mL of the 5,000-
mg/L nitrate solution at a rate of approximately 2 mL/min to displace
Inorganic chloride Ions.
7.4 Activation;
7.4.1 After the quartz collection tube with the GAC 1s removed from
the extraction unit, the GAC and cerafelt pads are extruded, using the
packing rod, Into a prewashed plastic container (e.g., l/5-dram
polyethylene snap-cap vial). The vial has been prewashed to remove
Inorganic and organic chlorine by a soak in distilled water, followed by
storage 1n a glass jar containing 50% v/v acetone and hexane. After
extrusion, the vial 1s removed by forceps and air-dried to remove
residual water, acetone, and hexane. After extrusion, the vial 1s
snapped shut, the hinge removed with a scalpel blade, the cap heat-sealed
to the vial with an electric soldering gun reserved for that purpose, and
a single-digit number placed on the vial with a marker pen.
7.4.2 Samples plus a similar vial containing 25 ug CI, 2.5 ug Br,
and 2.5 ug I standards are then Introduced Into the reactor, generally by
placing them together 1n a 5-dram polyethylene vial and inserting them
Into a pneumatic-tube transfer "rabbit" for neutron Irradiation.
Irradiation 1s typically for a 15-min period at a thermal neutron
Irradiation flux of 5 x 10*2 neutrons/cm2/sec. After returning from the
reactor, the rabbit is allowed to "cool" for 20 min to allow short-lived
radlolsotopes (primarily Al) present 1n the GAC to decay.
7.5 Detection:
7.5.1 Analysis is performed using a lithium-drifted germanium
[Ge(L1)] gamma-ray detector with an amplifier and a 4096-channel memory
unit for data storage. The analyses can be performed either manually,
with the operator changing samples and transferring the data to magnetic
tape, or automatically, with both functions performed by an automatic
sample changer.
7.5.2 Analysis begins by counting the standard and samples for a
suitable time period (e.g., 200-sec "live" time for the standards and
samples). The operator records the time Intervals between samples and
the "dead" time of each sample 1n a logbook for later use 1n calculating
halogen concentrations in each sample.
7.5.3 Breakthrough; The unpredictable nature of the background
bias makes 1t especially difficult to recognize the extent of
breakthrough of organohalides from one column to another. All second-
9022 - 6
Revision 0
Date September 1986
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column measurements for a properly operating system should not exceed 10X
of the two-column total measurement. If the 10% ffgure is exceeded, one
of three events could have happened: (1) the first column was overloaded
and a legitimate measure of breakthrough was obtained, 1n which case
taking a smaller sample may be necessary; (2) channeling or some other
failure occurred, 1n which case the sample may need to be rerun; or (3) a
high random bias occurred, and the result should be rejected and the
sample rerun. Because 1t may not be possible to determine which event
occurred, a sample analysis should be repeated often enough to gain
confidence 1n results. As a general rule, any analysis that is rejected
should be repeated whenever a sample 1s available. In the event that
repeated analyses show that the second column consistently exceeds the
10% figure and the total Is too low for the first column to be saturated
and the Inorganic Cl 1s less than 20,000 times the organic chlorine
value, then the result should be reported, but the data user should be
Informed of the problem. If the second-column measurement 1s equal to or
less than the nitrate-wash blank value, the second-column value should be
disregarded.
7.6 Calculations;
7.6.1 Chlorine, bromine, and Iodine can be analyzed within a 200-
sec counting period taking place 20 to 40 mln after irradiation.
7.6.2 Chlorine 1s analyzed using the 1642-KeV gamma ray produced by
37.l-m1n 38C1. Bromine 1s analyzed using the 616-KeV gamma ray from
!7.7-m1n 80Br, and Iodine 1s analyzed using the 442-KeV gamma ray
produced by 25-m1n I28I.
7.6.3 The calculation used for quantltatlon 1s:
r,nm hai«ft«« - cts unk. „ counting time std. v uq 1n std. „ 0Xt
ppm halogen - cts std< x Count1n^ t1me Unk. x sample vol. x e
where:
cts unk. = the Integrated area of the appropriate gamma-ray peak 1n
the unknown with background subtracted and the total
multiplied by 1 + [(% dead time unknown - X dead time
std.)/200]. The latter correction 1s usually less than
4% and corrects for pile-up errors.
cts std. = the Integrated area of the appropriate gamma-ray peak 1n
the standard with background subtracted.
counting time std. = the "live" counting time 1n seconds of the
standard.
counting time unk. = the "live" counting time 1n seconds of the
unknown.
9022 - 7
Revision
Date September 1986
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ug 1n std. = the number of mlcrograms of the stable element 1n
question 1n the standard (25 for Cl, 2.5 for Br and I).
sample vol. - the volume of sample passed through the GAC column, 1n
ml.
e^t = the decay correction to bring all statistics back to
t ~ 0; X * 0.693/ti/2, where tj/2 « the half-life In
minutes.
t " the time Interval 1n minutes from the end of the count of the
standard until the end of the count of the sample.
7.6.4 No further calculations are necessary as long as the final
sample 1s counted within 40 m1n after the end of Irradiation.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Before performing any analyses, the analyst must demonstrate the
ability to generate acceptable accuracy and precision with this procedure by
analyzing appropriate quality-control check samples.
8.3 The laboratory must develop and maintain a statement of method
accuracy for their laboratory. The laboratory should update the accuracy
statement regularly as new recovery measurements are made.
8.4 Employ a minimum of one blank per sample batch to determine If
contamination 1s occurring.
8.5 Verify calibration with an Independently prepared check standard
every 15 samples.
8.6 Run one spike duplicate sample for every 10 samples. A duplicate
sample 1s a sample brought through the whole sample preparation and analytical
process.
8.7 It 1s recommended that the laboratory adopt additional quality-
assurance practices for use with this method. The specific practices that
would be most productive will depend upon the needs of the laboratory and the
precision of the sampling technique. Whenever possible, the laboratory should
perform analysis of standard reference materials and participate in relevant
performance-evaluation studies.
8.8 Quality control for the analysis phase is very straightforward in as
much as the Instrument 1s a noncontact analyzer. That 1s, only the radiation
emitted from the sample -- not the sample Itself -- should touch the analyzer.
9022 - 8
Revision
Date September 1986
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Because contamination of the system 1s not usually a problem (unless a sample
spills on 1t), the most serious quality-control Issues deal with uniform
neutron flux, counting geometry, and spectral Interpretation. The amount of
radioactivity Induced 1n a sample 1s directly proportional to the neutron flux
1t 1s exposed to. Because this flux can vary depending on how the sample 1s
positioned 1n relation to the reactor core during Irradiation, It Is essential
that a known standard be Irradiated with every sample batch to act as a flux
monitor. Care must also be taken to ensure that the standard and all samples
associated with the standard are counted at the same distance from the
detector.
9.0 METHOD PERFORMANCE
9.1 The following statistics are based on seven replicate analyses:
Combined Pooled
Chlorine Bromine Iodine average
River water 7
Well water 7 (ppb)
WWTP effluent
38.2
0.16
50.7
0.30
242
0.56
17
0.076
4.7
0.038
35.2
0.033
<1
—
-------
METHOD 9O22
TOTAL ORGANIC HAL I DCS (TOxl BY
NEUTRON ACTIVATION ANALYSIS
C
7.1.1
Take
special
cere handling
••mol* to
Minimize loss
of volatile*
7.2.2
Analyze nltratc-xaih
Blanks to establish
repeatability of
method background
•ach day
7.1.2
AOO SulMtC to
reduce residual
chlorine
7.2.31
JCalibrate
Instrument
each day using
radioactive
stenoergt
Centrifuge and
decant camples
with undls-
solvco aollas
7.3.1
Connect
in aeries
MO columns
containing
activated
carbon
7.1.3
Adjust
pM of sample
prior to adding
sample to
reservoir
7.3.2] Fill
• cample
reservior; oass
sample througn
activates
caroon columns
7.2.1
1 cneck
aosorpt Ion
efficiency for
e»cn datcn of
carbon
7.3.31 Olaplace
* inorganic
chloride ions
by Meaning
columns «ith
nitrate eolut.
7.4.1
Remove CAC
Ouarti
collection tube
7.4.1
Extrude
CAC ana
cerafelt
pads into a
pre«asnee
plastic vial
7.4.21 Introduce
' samples
and standards
into reactor
for neutron
Irradiation
7 .3. 1
Anaylze using
Ge (LI) gamma
ray detector
7.5.2
To
analyze.
count stanoaro
and samples
for a auitaele
time period
9022 - 10
Revision 0
Date September 1986
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TOTAL ORGANIC HAIIOES
MCTHOO 9022
(TOX) BY NEUTRON ACTIVATION ANALYSIS
(Continued)
9022 - 11
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Date September 1986
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o
w
o
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METHOD 9030A
ACID-SOLUBLE AND ACID-INSOLUBLE SULFIDES
1.0 SCOPE AND APPLICATION
1.1 The distillation procedure described in this method is designed for
the determination of sulfides in aqueous, solid waste materials, or effluents.
1.2 This method provides only a semi-quantitative determination of
sulfide compounds considered "acid-insoluble" (e.g.. CuS and SnS2) in solid
samples. Recovery has been shown to be 20 to 40% for CuS, one of the most stable
and insoluble compounds, and 40 to 60% for SnS2 which is slightly more soluble.
1.3 This method is not applicable to oil or multiphasic samples or
samples not amenable to the distillation procedure which can be analyzed by
Method 9031.
1.4 Method 9030 is suitable for measuring sulfide concentrations in
samples which contain between 0.2 and 50 mg/kg of sulfide.
1.5 This method is not applicable for distilling reactive sulfide,
however, Method 9030 is used to quantify the concentration of sulfide from the
reactivity test. Refer to Chapter Seven, Step 7.3.4.1 for the determination of
reactive sulfi.de.
1.6 This method measures total sulfide which is usually defined as the
acid-soluble fraction of a waste. If, however, one has previous knowledge of the
waste and is concerned about both soluble sulfides such as H»S, and metal
sulfides, such as CuS and SnS., then total sulfide is defined as tne combination
of both acid-soluble and acia-insoluble fractions. For wastes where only metal
sulfides are suspected to be present, only the acid-insoluble fraction needs to
be performed.
2.0 SUMMARY OF METHOD
2.1 For acid-soluble sulfide samples, separation of sulfide from the
sample matrix is accomplished by the addition of sulfuric acid to the sample.
The sample is heated to 70°C and the hydrogen sulfide (H2S) which is formed is
distilled under acidic conditions and carried by a nitrogen stream into zinc
acetate gas scrubbing bottles where it is precipitated as zinc sulfide.
2.2 For acid-insoluble sulfide samples, separation of sulfide from the
sample matrix is accomplished by suspending the sample in concentrated
hydrochloric acid by vigorous agitation. Tin(II) chloride is present to prevent
oxidation of sulfide to sulfur by the metal ion (as in copper(II)), by the
matrix, or by dissolved oxygen in the reagents. The prepared sample is distilled
under acidic conditions at 100'C under a stream of nitrogen. Hydrogen sulfide
gas is released from the sample and collected in gas scrubbing bottles containing
zinc(II) and a strong acetate buffer. Zinc sulfide precipitates.
9030A - 1 Revision 1
July 1992
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2.3 The sulfide in the zinc sulfide precipitate is oxidized to sulfur
with a known excess amount of iodine. Then the excess iodine is determined by
titration with a standard solution of phenyl arsine oxide (PAO) or sodium
thiosulfate until the blue iodine starch complex disappears. As the use of
standard sulfide solutions is not possible because of oxidative degradation,
quantitation is based on the PAO or sodium thiosulfate.
3.0 INTERFERENCES
3.1 Aqueous samples must be taken with a minimum of aeration to avoid
volatilization of sulfide or reaction with oxygen, which oxidizes sulfide to
sulfur compounds that are not detected.
3.2 Reduced sulfur compounds, such as sulfite and hydrosulfite,
decompose in acid, and may form sulfur dioxide. This gas may be carried over to
the zinc acetate gas scrubbing bottles and subsequently react with the iodine
solution yielding false high values. The addition of formaldehyde into the zinc
acetate gas scrubbing bottles removes this interference. Any sulfur dioxide
entering the scrubber will form an addition compound with the formaldehyde which
is unreactive towards the iodine in the acidified mixture. This method shows no
sensitivity to sulfite or hydrosulfite at concentrations up to 10 mg/kg of the
interferant.
3.3 Interferences for acid-insoluble sulfides have not been fully
investigated. However, sodium sulfite and sodium thiosulfate are known to
interfere in the procedure for soluble sulfides. Sulfur also interferes because
it may be reduced to sulfide by tin(II) chloride in this procedure.
3.4 The iodometric method suffers interference from reducing substances
that react with iodine, including thiosulfate, sulfite, and various organic
compounds.
3.5 The insoluble method should not be used for the determination of
soluble sulfides because it can reduce sulfur to sulfide, thus creating a
positive interference.
4.0 APPARATUS AND MATERIALS
4.1 Gas evolution apparatus as shown in Figure 1
4.1.1 Three neck flask - 500-mL, 24/40 standard taper joints.
4.1.2 Dropping funnel - 100-mL, 24/40 outlet joint.
4.1.3 Purge gas inlet tube - 24/40 joint, with coarse frit.
4.1.4 Purge gas outlet - 24/40 joint reduced to 1/4 in. tube.
4.1.5 Gas scrubbing bottles - 125-mL, with 1/4 in. o.d. inlet
and outlet tubes. Impinger tube must be fritted.
rubber.
4.1.6 Tubing - 1/4 in. o.d. Teflon or polypropylene. Oo not use
9030A - 2
Revision 1
July 1992
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NOTE: When analyzing for acid-insoluble sulfides, the distillation
apparatus is identical to that used in the distillation procedure
for acid-soluble sulfides except that the tubing and unions
downstream of the distillation flask must be all Teflon,
polypropylene or other material resistant to gaseous HC1. The
ground glass joints should be fitted with Teflon sleeves to prevent
seizing and to prevent gas leaks. Pinch clamps should also be used
on the joints to prevent leaks.
4.2 Hot plate stirrer.
4.3 pH meter.
4.4 Nitrogen regulator.
4.5 Flowmeter.
4.6 Top-loading balance - capable of weighing 0.1 g.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Zinc acetate solution for sample preservation (2N), Zn(CH,COO)2 •
2H20. Dissolve 220 g of zinc acetate dihydrate in 500 ml of reagent water.
5.4 Sodium hydroxide (IN), NaOH. Dissolve 40 g of NaOH in reagent
water and dilute to 1 liter.
5.5 Formaldehyde (37% solution), CH20. This solution is commercially
available.
5.6 Zinc acetate for the scrubber
5.6.1 For acid-soluble sulfides: Zinc acetate solution
(approximately 0.5M). Dissolve about 110 g zinc acetate dihydrate in
200 ml of reagent water. Add 1 ml hydrochloric acid (concentrated), HC1,
to prevent precipitation of zinc hydroxide. Dilute to 1 liter.
5.6.2 For acid-insoluble sulfides: Zinc acetate/sodium acetate
buffer. Dissolve 100 g sodium acetate, NaC2H302, and 11 g zinc acetate
dihydrate in 800 ml of reagent water. Add 1 ml concentrated hydrochloric
acid and dilute to 1 liter. The resulting pH should be 6.8.
5.7 Acid to acidify ,the sample
9030A - 3 Revision 1
July 1992
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H2S04.
5.7.1 For acid-soluble sulfides: Sulfuric acid (concentrated),
5.7.2 For acid-insoluble sulfides: Hydrochloric acid (9.8N),
HC1. Place 200 mL of reagent water in a 1-liter beaker. Slowly add
concentrated HC1 to bring the total volume to 1 liter.
5.8 Starch solution - Use either an aqueous solution or soluble starch
powder mixtures. Prepare an aqueous solution as follows. Dissolve 2 g soluble
starch and 2 g salicylic acid, C7H603, as a preservative, in 100 ml hot reagent
water.
5.9 Nitrogen.
5.10 Iodine solution (approximately 0.025N)
5.10.1 Dissolve 25 g potassium iodide, KI, in 700 mL of reagent
water in a 1-liter volumetric flask. Add 3.2 g iodine, I2. Allow to
dissolve. Add the type and amount of acid specified in Step 7.3.2.
Dilute to 1 liter and standardize as follows.
5.10.2 Dissolve approximately 2 g KI in 150 ml of reagent water.
Add exactly 20 mL of the iodine solution (Step 5.10.1) to be titrated and
dilute to 300 mL with reagent water.
5.10.3 Titrate with 0.025N standardized phenylarsine oxide or
0.025N sodium thiosulfate until the amber color fades to yellow. Add
starch indicator solution. Continue titration drop by drop until the blue
color disappears.
5.10.4- Run in replicate.
5.10.5 Calculate the normality as follows.
Normality (I2) = mL of titrant x normality of titrant
sample size in mL
5.11 Sodium sulfide nonanhydrate, Na2S • 9H20. For the preparation of
standard solutions to be used for calibration curves. Standards must be prepared
at pH > 9 and < 11. Protect standard from exposure to oxygen by preparing it
without headspace. These standards are unstable and should be prepared daily.
5.12 Tin(II) chloride, SnCl2, granular.
5.13 Titrant.
5.13.1 Standard phenylarsine oxide solution (PAD) (0.025N),
C6H5AsO. This solution is commercially available.
CAUTION: PAO is toxic.
9030A - 4 Revision 1
July 1992
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5.13.2 Standard sodium thiosulfate solution (0.025N), Na2S20, •
5H20. Dissolve 6.205 ± 0.005 g Na2S203 • 5H20 in 500 ml reagent water. Add
9 mi IN NaOH and dilute to 1 liter.
5.14 Sodium hydroxide (6N), NaOH. Dissolve 240 g of sodium hydroxide
in 1.liter of reagent water.
5.15 Hydrochloric acid (6N), HC1. Place 51 ml of reagent water in a 100
ml Class A volumetric flask. Slowly add concentrated HC1 to bring the total
volume to 100 ml.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed in Chapter Nine of this manual.
6.2 All aqueous samples and. effluents must be preserved with zinc
acetate and sodium hydroxide. Use four drops of 2N zinc acetate solution per 100
mL of sample. Adjust the pH to greater than 9 with 6N sodium hydroxide solution.
Fill the sample bottle completely and stopper with a minimum of aeration. The
treated sample is relatively stable and can be held for up to seven days. If
high concentrations of sulfide are expected to be in the sample, continue adding
zinc acetate until all the sulfide has precipitated. For solid samples, fill the
surface of the solid with 2N zinc acetate until moistened. Samples must be
cooled to 4°C and stored headspace free.
6.3 Sample Preparation
6.3.1 For an efficient distillation, the mixture in the
distillation flask must be of such a consistency that the motion of the
stirring bar is sufficient to keep the solids from settling. The mixture
must be free of solid objects that could disrupt the stirring bar.
Prepare the sample using one of the procedures in this section then
proceed with the distillation step (Section 7.0).
6.3.2 If the sample is aqueous, shake the sample container to
suspend any solids, then quickly decant the appropriate volume (up to
250 mL) of the sample to a graduated cylinder, weigh the cylinder,
transfer to the distillation flask and reweigh the cylinder to the nearest
milligram. Be sure that a representative aliquot is used, or use the
entire sample.
6.3.3 If the sample is aqueous but contains soft clumps of
solid, it may be possible to break the clumps and homogenize the sample by
placing the sample container on a jar mill and tumble or roll the sample
for a few hours. The slurry may then be aliquotted and weighed as above
to the nearest milligram then diluted with reagent water up to a total
volume of 250 mL to produce a mixture that is completely suspended by the
stirring bar.
6.3.4 If the sample is primarily aqueous, but contains a large
proportion of solid, the sample may be roughly separated by phase and the
amount of each phase measured and weighed to the nearest milligram into
9030A - 5 Revision 1
July 1992
-------
the distillation flask in proportion to their abundance in the sample.
Reagent water may be added up to a total volume of 250 ml. As a
guideline, no more than 25 g dry weight or 50 g of sludge can be
adequately suspended in the apparatus.
6.3.5 If the sample contains solids which absorb water and
swell, limit the sample size to 25 g dry weight. Otherwise, the solids
will restrict the fluid motion and lower the recovery.
6.3.6 If the sample contains solid objects that cannot be
reduced in size by tumbling, the solids must be broken manually. Clay-
like solids should be cut with a spatula or scalpel in a crystallizing
dish. If the solids can be reduced to a size that they can be suspended
by the stirring bar, the solid and liquid can be proportionately weighed.
6.3.7 Non-porous harder objects, for example stones or pieces
of metal, may be weighed and discarded. The percent weight of non-porous
objects should be reported and should be used in the calculation of
sulfide concentration if it has a significant effect on the reported
result.
7.0 PROCEDURE
For acid-soluble sulfide samples, go to 7.1
For acid-insoluble sulfide samples, go to 7.2
7.1 Acid-Soluble Sulfide
7.1.1 In a preliminary experiment, determine the approximate
amount of sulfuric acid required to adjust a measured amount of the sample to pH
less than or equal to 1. The sample size should be chosen so that it contains
between 0.2 and 50 mg of sulfide. Place a known amount of sample or sample
slurry in a beaker. Add reagent water until the total volume is 200 ml. Stir
the mixture and determine the pH. Slowly add sulfuric acid until the pH is less
than or equal to 1. Discard this preliminary sample.
CAUTION: Toxic hydrogen sulfide may be generated from the acidified sample.
This operation must be performed in the hood and the sample left
in the hood until the sample has been made alkaline or the sulfide
has been destroyed. From the amount of sulfuric acid required to
acidify the sample and the mass or volume of the sample acidified,
calculate the amount of acid required to acidify the sample to be
placed in the distillation flask.
7.1.2 Prepare the gas evolution apparatus as shown in Figure 1
in a fume hood.
7.1.2.1 Prepare a hot water bath at 70°C by filling a
crystallizing dish or other suitable container with water and place
it on a hot plate stirrer. Place a thermometer in the bath and
monitor the temperature to maintain the bath at 70°C.
9030A - 6 Revision 1
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7.1.2.2 Assemble the three neck 500-mL flask, fritted
gas inlet tube, and exhaust tube. Use Teflon sleeves to seal the
ground glass joints. Place a Teflon coated stirring bar into the
flask.
7.1.2.3 Place into each gas scrubbing bottle 10 ± 0.5
mL of the 0.5M zinc acetate solution, 5.0 ± 0.1 ml of 37%
formaldehyde and 100 ± 5.0 ml reagent water.
7.1.2.4 Connect the gas evolution flask and gas
scrubbing bottles as shown in Figure 1. Secure all fittings and
joints.
7.1.3 Carefully place an accurately weighed sample which
contains 0.2 to 50 mg of sulfide into the flask. If necessary, dilute to
approximately 200 ml with reagent water.
7.1.4 Place the dropping funnel onto the flask making sure its
stopcock is closed. Add the volume of sulfuric acid calculated in Step
7.1.1 plus an additional 50 ml into the dropping funnel. The bottom
stopcock must be closed.
7.1.5 Attach the nitrogen inlet to the top of the dropping
funnel gas shut-off valve. Turn on the nitrogen purge gas and adjust the
flow through the sample flask to 25 mL/min. The nitrogen in the gas
scrubbing bottles should bubble at about five bubbles per second.
Nitrogen pressure should be limited to approximately 10 psi to prevent
excess stress on the glass system and fittings. Verify that there are no
leaks in the system. Open the nitrogen shut-off valve leading to the
dropping funnel. Observe that the gas flow into the sample vessel will
stop for a short period while the pressure throughout the system
equalizes. If the gas flow through the sample flask does not return
within a minute, check for leaks around the dropping funnel. Once flow
has stabilized, turn on magnetic stirrer. Purge system for 15 minutes
with nitrogen to remove oxygen.
7.1.6 Heat sample to 70*C, Open dropping funnel to a position
that will allow a flow of sulfuric acid of approximately 5 mL/min.
Monitor the system until most of the sulfuric acid within the dropping
funnel has entered the sample flask. Solids which absorb water and swell
will restrict fluid motion and, therefore, lower recovery will be
obtained. Such samples should be limited to 25 g dry weight.
7.1.7 Purge, stir, and maintain a temperature of 70°C for a
total of 90 minutes from start to finish. Shut off nitrogen supply. Turn
off heat.
7.1.8 Proceed to Step 7.3 for the analysis of the zinc sulfide
by titration.
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7.2 Acid-Insoluble Sulfide
7.2.1 As the concentration of HC1 during distillation must be
within a narrow range for successful distillation of HjS, the water content
must be controlled. It is imperative that the final concentration of HC1
in the distillation flask be about 6.5N and that, the sample is mostly
suspended in the fluid by the action of the stirring bar. This is
achieved by adding 50 ml of reagent water, including water in the sample,
100 ml of 9.8N HC1, and the sample to the distillation flask. Solids
which absorb water and swell will restrict fluid motion and, therefore,
lower recovery will be obtained. Such samples should be limited to 25 g
dry weight. Other samples can range from 25 to 50 g.
7.2.2 If the matrix is a dry solid, weigh a portion of the
sample such that it contains 0.2 to 50 mg of sulfide. The solid should be
crushed to reduce particle size to 1 mm or less. Add 50 mL of reagent
water.
7.2.3 If the matrix is aqueous, then a maximum of 50 g of the
sample may be used. No additional water may be added. As none of the
target compounds are volatile, drying the sample may be preferable to
enhance the sensitivity by concentrating the sample. If less than 50 g of
the sample is required to achieve the 0.2 to 50 mg of sulfide range for
the test, then add reagent water to a total volume of 50 ml.
7.2.4 If the matrix is a moist solid, the water content of the
sample must be determined (Karl Fischer titration, loss on drying, or
other suitable means) and the water in the sample included in the total
50 ml of water needed for the correct HC1 concentration. For example, if
a 20 g sample weight is needed to achieve the desired sulfide level of
0.2 to 50 mg and the sample is 50% water then 40 mL rather than 50 ml of
reagent water is added along with the sample and 100 ml of 9.8N HC1 to the
distillation flask.
7.2.5 Weigh the sample and 5 g SnCK into the distillation
flask. Use up to 50 ml of reagent water, as calculated above, to rinse
any glassware.
7.2.6 Assemble the distillation apparatus as in Figure 1. Place
100 + 2.0 ml of zinc acetate/sodium acetate buffer solution and 5.0 ± 0.1
ml of 37% formaldehyde in each gas scrubbing bottle. Tighten the pinch
clamps on the distillation flask joints.
7.2.7 Make sure the stopcock is closed and then add 100 ± 1.0
ml of 9.8N HC1 to the dropping funnel. Connect the nitrogen line to the
top of the funnel and turn the nitrogen on to pressurize the dropping
funnel headspace.
7.2.8 Set the nitrogen flow at 25 mL/min. The nitrogen in the
gas scrubbing bottles should bubble at about five bubbles per second.
Purge the oxygen from the system for about 15 minutes.
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7.2.9 Turn on the magnetic stirrer. Set the stirring bar to
spin as fast as possible. The fluid should form a vortex. If not, the
distillation will exhibit poor recovery. Add all of the HCL from the
dropping funnel to the flask.
7.2.10 Heat the water bath to the boiling point (100"C), The
sample may or may not be boiling. Allow the purged distillation to
proceed for 90 minutes at 100'C. Shut off nitrogen supply. Turn off
heat.
7.2.11
by titration.
Proceed to Step 7.3 for the analysis of the zinc sulfide
7.3 Titration of Distillate
7.3.1 Pipet a known amount of standardized 0.025N iodine
solution (See Step 5.10.5) in a 500-mL flask, adding an amount in excess
of that needed to oxidize the sulfide. Add enough reagent water to bring
the volume to 100 ml. The volume of standardized iodine solution should
be about 65 ml for samples with 50 mg of sulfide.
7.3.2 If the distillation for acid-soluble sulfide is being
used, add 2 ml of 6N HC1. If the distillation for acid-insoluble sulfides
is performed, 10 ml of 6N HC1 should be added to the iodine.
7.3.3 Pipet both of the gas scrubbing bottle solutions to the
flask, keeping the end of the pipet below the surface of the iodine
solution. If at any point in transferring the zinc acetate solution or
rinsing the bottles, the amber color of the iodine disappears or fades to
yellow, more 0.025N iodine must be added. This additional amount must be
added to the amount from Step 7.3.1 for calculations. Record the total
volume of standardized 0.025N iodine solution used.
7.3.4 Prepare a rinse solution of a known amount of standardized
0.025N iodine solution, 1 ml of 6N HC1, and reagent water to rinse the
remaining white precipitate (zinc sulfide) from the gas scrubbing bottles
into the flask. There should be no visible traces of precipitate after
rinsing.
iodine from the
rinsate to
gas
the
7.3.5 Rinse any remaining traces of iodine
scrubbing bottles with reagent water, and transfer the
flask.
7.3.6 Titrate the solution in the flask with standard 0.025N
phenylarsine oxide or 0.025N sodium thiosulfate solution until the amber
color fades to yellow. Add enough starch indicator for the solution to
turn dark blue and titrate until the blue disappears. Record the volume
of titrant used.
9030A - 9
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7.3.7 Calculate the concentration of sulfide using the following
equation:
| 32.06 a
(ml I2 x N I2) - (mL titrant x N titrant) x 1 2 eq.
•. — > = sulfide (mg/kg) or
sample weight (kg) or sample volume .(L) (mg/L)
8.0 QUALITY CONTROL
8.1 All quality control data must be maintained and available for
reference or inspection for a period of three years. This method is restricted
to use by or under supervision of experienced analysts. Refer to the appropriate
section of Chapter One for additional quality control guidelines.
8.2 A reagent blank should be run once in twenty analyses or per
analytical batch, whichever is more frequent.
8.3 Check standards are prepared from water and a known amount of
sodium sulfide. A check standard should be run with each analytical batch of
samples, or once in twenty samples. Acceptable recovery will depend on the level
and matrix.
8.4 A matrix spiked sample should be run for each analytical batch or
twenty samples, whichever is more frequent, to determine matrix effects. If
recovery is low, acid-insoluble sulfides are indicated. A matrix spiked sample
is a sample brought through the whole sample preparation and analytical process.
9.0 METHOD PERFORMANCE
9.1 Accuracy - Accuracy for this method was determined by three
independent laboratories by measuring percent recoveries of spikes for both clean
matrices (water) and actual waste samples. The results are summarized below.
For Acid-Soluble Sulfide
Accuracy of titration step only
Lab A 84-100% recovery
Lab B 110-122% recovery
Accuracy for entire method for clean matrices (H20)
Lab C 94-106% recovery
Accuracy of entire method for actual waste samples
Lab C 77-92% recovery
Spiking levels ranged from 0.4 to 8 mg/L
For Acid-Insoluble Sulfide
The percent recovery was not as thoroughly studied for acid-insoluble
sulfide as it was for acid-soluble sulfide.
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Accuracy of entire method for synthetic waste samples
Lab C 21-81% recovery
Spiking levels ranged from 2.2 to 22 mg/kg
9.2 Precision
For Acid-Soluble Sulfide
Precision of titration step only
Lab A CV% 2.0 to 37
Lab B CV% 1.1 to 3.8
Precision of entire method for clean matrices (H20)
Lab C CV% 3.0 to 12
Precision of entire method for actual waste samples
Lab C CV% 0.86 to 45
For Acid-Insoluble Sulfide
Precision of entire method with synthetic wastes
Lab C CV 1.2 to 42
9.3 Detection Limit - The detection limit was determined by analyzing
seven replicates at 0.45 and 4.5 mg/L. The detection limit was calculated as the
standard deviation times the student's t-value for a one-tailed test with n-1
degrees of freedom at 99% confidence level. The detection limit for a clean
matrix (H20) was found to be between 0.2 and 0.4 mg/L.
10.0 REFERENCES
1. Test Methods for Evaluating Solid Waste, Physical/Chemical Methods, 2nd
ed.; U.S. Environmental Protection Agency. Office of Solid Waste and Emergency
Response. U.S. Government Printing Office: Washington, DC, 1982, revised 1984;
SW-846.
2. Methods for Chemical Analysis of Water and Wastes; U.S. Environmental
Protection Agency, Office of Research and Development. Environmental Monitoring
and Support Laboratory. ORD Publication Offices of Center for Environmental
Research Information: Cincinnati, OH, 1979; EPA-600/4-79-020.
3. CRC Handbook of Chemistry and Physics. 66th ed.; Weast, R., Ed.; CRC: Boca
Raton, FL, 1985.
4. Standard Methods for the Examination of Water and Wastewater, 16th ed.;
Greenberg, A.E.; Trussell, R.R.; Clesceri, L.S., Eds.; American Water Works
Association, Water Pollution Control Federation, American Public Health
Association: Washington, DC, 1985; Methods 427, 427A, 427B, and 427D.
5. Andreae, M.O.; Banard, W.R. Anal. Chem. 1983, 55, 608-612.
6. Barclay, H. Adv. Instrum. 1980, 35(2), 59-61.
9030A - 11 Revision 1
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7. Bateson, S.W.; Moody, G.J.; Thomas, O.P.R. Analyst 1986, 111. 3-9.
8. Berthage, P.O. Anal. Chim. Acta 1954, 10 310-311.
9. Craig, P.J.; Moreton, P.A. Environ. Technol. Lett. 1982, 3, 511-520.
10. Franklin, G.O.; Fitchett, A.M. Pulp & Paper Canada 1982, 83(10). 40-44.
11. Fuller, W. In Cyanide in the Environment; Van Zyl, D., Ed.; Proceedings of
Symposium; December, 1984.
12. Gottfried, G.J. "Precision, Accuracy, and MDL Statements for EPA Methods
9010, 9030, 9060, 7520, 7521, 7550, 7551, 7910, and 7911"; final report to the
U.S. Environmental Protection Agency (EMSL-CI); Biopheric.
13. Kilroy, W.P. Talanta 1983, 30(61. 419-422.
14. Kurtenacher, V.A.; Wallak, R. Z. Anora. U. Alia. Chem. 1927. 161 202-209.
15. Landers, D.H.; David. M.B.; Mitchell. M.J. Int. J. Anal. Chem 1983. 14,
245-256.
16. Opekar, F.; Brukenstein, S. Anal. Chem. 1984, 56, 1206-1209.
17. Ricklin, R.D.; Johnson, E.L. Anal. Chem. 1983, 55, 4.
18. Rohrbough, W.G.; et al. Reagent Chemicals. American Chemical Society
Specifications. 7th ed.; American Chemical Society: Washington, DC, 1986.
19. Snedecor, G.W.; Ghran, W.G. Statistical Methods; Iowa State University:
Ames, IA, 1980.
20. Umafia, M.; Beach, J.; Sheldon, L. "Revisions to Method 9010"; final report
to the U.S. Environmental Protection Agency on Contract No. 68-01-7266; Research
Triangle Institute: Research Triangle Park, NC, 1986; Work Assignment No. 1.
21. Umafia, M.; Sheldon, L. "Interim Report: Literature Review"; interim report
to the U.S. Environmental Protection Agency in Contract No. 68-01-7266; Research
Triangle Institute: Research Triangle Park, NC, 1986; Work Assignment No. 3.
22. Wang, W.; Barcelona, M.J. Environ. Inter. 1983, 9, 129-133.
23. Wronski, M. Talanta 1981, 28, 173-176.
24. Application Note 156; Princeton Applied Research Corp.: Princeton, NJ.
25. Guidelines for Assessing and Reporting Data Quality for Environmental
Measurements; U.S. Environmental Protection Agency. Office of Research and
Development. U.S. Government Printing Office: Washington, DC, 1983.
26. Fed. Regist. 1980, 45(98). 33122.
9030A - 12 Revision 1
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27. The Analytical Chemistry of Sulfur and Its Compounds. Part I; Karchmer,
J.H., Ed.; Wiley-Interscience: New York, 1970.
28. Methods for the Examination of Water and Associated Materials: Department
of the Environment: England, 1983.
29. "Development and Evaluation of a Test Procedure for Reactivity Criteria for
Hazardous Waste"; final report to the U.S. Environmental Protection Agency on
Contract 68-03-2961; EAL: Richmond, CA.
30. Test Method to Determine Hydrogen Sulfide Released from Wastes; U.S.
Environmental Protection Agency. Office of Solid Waste. Preliminary unpubl ished
protocol, 1985.
31. 1985 Annual Book of ASTH Standards. Vol. 11.01; "Standard Specification for
Reagent Water"; ASTM: Philadelphia, PA, 1985; D1193-77.
9030A - 13 Revision 1
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FIGURE 1.
GAS EVOLUTION APPARATUS
H2SO4 (HCl for Acid Insoluble Sulfides)
Hot Water Bath
with Magnetic Stirrer
Stirring Bar
Acetate
and
Formaldehyde
Scrubbing
Bottles
N20ut
9030A - 14
Revision 1
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METHOD 9030A
ACID-SOLUBLE AND ACID-INSOLUBLE SULFIDES
START
711 Choose >amp1«
size, a I ace Jam pie
in beaxer. add
water, measure pH.
add cone sulfucxc
acia to pH i.
discard sampl•
7 1 ! Calculate
ant t of sulfuric
acid needed to
acidify f resh
sample for purge.
fresh sample is to
be used for Step
1 1 4
? 1 2 Prepara gas
evolution apparatus
7 1 3 Place weighed
sacnpl e in flask.
dilute *i*,h water
v ( rveces sat y
7 1 4 Place
dropping funnel
onto flask; add
sulfuric acid (frorr
Step ? 1 1) to
d r a pping funnel
7.1 S Adjust
nit r ogen flow.
check for L ealcs .
turn on'slirrer.
purge 'y> tem of
oxygen for 15 mins
Acid-Solubl
Acid•Ins o1ubl<
7 i 6 Heat to 70 C.
add julfuric acid
Lo flask. cIosv
d r oppxng funnel
«h*n acid near»
depIetion
717 Purge, » Lir.
and heat for 90
mins . shut off
nitrogen. turn off
heat
718 Analyze by
tit ration
9030A - 15
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(Continued)
721 Hater content
of distillation
must be controlled;
cone of HC1 should
be 6 SN
721 Limit sample
size to 25 g dry
weigh t
721 Sample size
nay be 25 - 50 g
722 Weigh sample
crush if necessary.
add 50 ml »ater
723 Us* SO
samp 1•
Moist Solid
724 Determine
water content of
sample: include
total water nteded
for correct HC1
cone
723 Add oatir to
sample for a total
volume of 50 mL
9030A - 16
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(Continued)
'25 Place sample
in flask, add
slannoui chloride
7 2 6 Assemble
distillation
apparatus, place
zinc acetale/sodlum
acetate buffer and
formaldehyde in
scrubbing bottles
7 2 7 Add 100 rrL
9 8N HC1 lo
dropping funnel
7 2 8 Set nitrogen
flow, purge system
of oKygen for 15
mi ns
729 Turn on
stirrer: add HC1 to
distillation flask'
1 2 10 Heat .ater
bath to boi1.
distill for 90
mins at 100 C.
shut off nitrogen.
turn off heal
7 2 11 Analyj. by
titration
7 3 1 Pipel knovn
amount, of 0 02SN
iodine solution in
flask, bring lo
volume with valer
7 3 2 Add 10 mL &N
HC1
7 3 2 Add 2 ml.
KC1
9030A - 17
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MtiriOD 9030A
(Continued)
733 Pipet
scrubbing battle
solution into
Erlenmeyer flask
734 Prepare rin»«
solution of 0 02SN
iodine solution. 6N
HC1. and »ater
Ho
733 Add Bor«
iodin«; rvcord
total volume of
lodin* u««d
7 3 S Rin»« traoi
of iodin« from
scrubbing bottlvs;
transfer rin*a> to
flask
7 3 6 Titrata
solution until
amber color fad«s;
add starch
indicator, titrat*
until blu« color
disappears; record
volume of titrant
used
737 Calculate the
cone of sulfide in
the sample
STOP
9030A - 18
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METHOD 9031
EXTRACTABLE SULFIDES
1.0 SCOPE AND APPLICATION
1.1 The extraction procedure described in this method is designed for
the extraction of sulfides from matrices that are not directly amenable to the
distillation procedure Method 9030. Specifically, this method is designed for
the extraction of soluble sulfides. This method is applicable to oil, solid,
multiphasic, and all other matrices not amenable to analysis by Method 9030.
This method is not applicable for reactive sulfide. Refer to Chapter Seven for
the determination of reactive sulfide.
1.2 Method 9031 is suitable for measuring sulfide in solid samples at
concentrations above 1 mg/kg.
2.0 SUMMARY OF METHOD
2.1 If the sample contains solids that will interfere with agitation
and homogenization of the sample mixture, or so much oil or grease as to
interfere with the formation of a homogeneous emulsion in the distillation
procedure, the sample may be filtered and the solids extracted with water at pH
> 9 and < 11. The extract is then combined with the filtrate and analyzed by the
distillation procedure. Separation of sulfide from the sample matrix is
accomplished by the addition of sulfuric acid to the sample. The sample is
heated to 70°C and the hydrogen sulfide (H2S) which is formed is distilled under
acidic conditions and carried by a nitrogen stream into zinc acetate gas
scrubbing bottles where it is precipitated as zinc sulfide.
2.2 The sulfide in the zinc sulfide precipitate is oxidized to sulfur
with a known amount of excess iodine. Then the excess iodine is determined by
titration with a standard solution of phenylarsine oxide (PAO) or sodium
thiosulfate until the blue iodine starch complex disappears. The use of standard
sulfide solutions is not possible because of their instability to oxidative
degradation. Therefore quantitation is based on the PAO or sodium thiosulfate.
3.0 INTERFERENCES
3.1 Samples with aqueous layers must be taken with a minimum of
aeration to avoid volatilization of sulfide or reaction with oxygen which
oxidizes sulfide to sulfur compounds that are not detected.
3.2 Sulfur compounds such as sulfite and hydrosulfite decompose in acid
and may form sulfur dioxide. This gas may be carried over to the zinc acetate
gas scrubbing bottles and subsequently react with the iodine solution yielding
false high values. The addition of formaldehyde into the zinc acetate gas
scrubbing bottles removes this interference. Any sulfur dioxide entering the
scrubber will form an addition compound with the formaldehyde which is unreactive
towards the iodine in the acidified mixture. This method shows no sensitivity
to sulfite or hydrosulfite at concentrations up to 10 mg/kg of the interferant.
9031 - 1 Revision 0
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3.3 The iodometric method suffers interference from reducing substances
that react with iodine including thiosulfate, sulfite, and various organic
compounds.
3.4 Interferences have been observed when analyzing samples with high
metal content such as electroplating waste and chromium containing tannery waste.
4.0 APPARATUS AND MATERIALS
4.1 Extractor - Any suitable device that sufficiently agitates a sealed
container of one liter volume or greater. For the purpose of this analysis,
agitation is sufficient when:
1. All sample surfaces are continuously brought into contact
with extraction fluid, and
2. The agitation prevents stratification of the sample and
fluid.
Examples of suitable extractors are shown in Figures 2 and 3. The tumble-
extractors turn the extraction bottles end-over-end at a rate of about 30 rpm.
The apparatus in Figure 2 may be easily fabricated from plywood. The jar
compartments must be padded with polyurethane foaro to absorb shock. The drive
apparatus is a Norton jar mill.
4.2 Buchner funnel apparatus
4.2.1 Buchner funnel - 500-mL capacity, with 1-liter vacuum
filtration flask.
4.2.2 Glass wool - Suitable for filtering, 0.8 m diameter such
as Corning Pyrex 3950.
4.2.3 Vacuum source - Preferably a water driven aspirator. A
valve or stopcock to release vacuum is required.
4.3 Gas Evolution apparatus as shown in Figure 1
4.3.1 Three neck flask - 500-mL, 24/40 standard tapered joints.
4.3.2 Dropping funnel - 100-mL, 24/40 outlet joint.
4.3.3 Purge gas inlet tube - 24/40 joint with course frit.
4.3.4 Purge gas outlet - 24/40 joint reduced to 1/4 inch tube.
4.3.5 Gas scrubbing bottles - 125-mL, with 1/4 in. o.d. inlet and
outlet tubes. Impinger tube must not be fritted.
4.3.6 Tubing - 1/4 in. o.d. Teflon or polypropylene. Do not use
rubber.
4.4 Hot plate stirrer.
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4.5 pH meter.
4.6 Nitrogen regulator.
4.7 Flowmeter.
4.8 Separatory funnels - 500-mL.
4.9 Tumbler - See Figures 2 and 3.
4.10 Top-loading balance - capable of weighing 0.1 g.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Zinc acetate (for sample preservation) (2N}> Zn(CH3COO)2 • 2H20.
Dissolve 220 g of zinc acetate dihydrate in 500 ml of water.
5.4 Sodium hydroxide (50%w/v in water), NaOH. Commercially available.
5.5 Tin (II) chloride, SnCl2 • 2H20, granular.
5.6 n-Hexane, C6H,4.
5.7 Nitrogen, N2.
5.8 Sulfuric acid (concentrated), H2S04.
5.9 Zinc acetate for the scrubber (approximately 0.5M). Dissolve 110
g zinc acetate dihydrate in 200 ml of water. Add 1 mL concentrated hydrochloric
acid, HC1, to prevent precipitation of zinc hydroxide. Dilute to 1 liter.
5.10 Formaldehyde (37% solution), CH20. Commercially available.
5.11 Starch solution. Use either an aqueous solution or soluble starch
powder mixtures. Prepare an aqueous solution as follows. Dissolve 2 g soluble
starch and 2 g salicylic acid, C7H603, as a preservative, in 100 ml hot water.
5.12 Iodine solution (approximately 0.025N). Dissolve 25 g of potassium
iodide, KI, in 700 ml of water in a 1-liter volumetric flask. Add 3.2 g of
iodine, I2. Allow to dissolve. Dilute to 1 liter and standardize as follows.
Dissolve approximately 2 g KI in 150 mL of water. Pipet exactly 20 ml of the
iodine solution to be titrated and dilute to 300 mL with water. Titrate with
0.025N standard phenylarsine oxide, or 0.025N sodium thiosulfate, Na2S203, until
the amber color fades. Add starch indicator solution until the solution turns
9031 - 3 Revision 0
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deep blue. Continue titration drop by drop until the blue color disappears.
Run in replicate. Calculate the normality as follows:
Normality (I2) = ml of titrant x normality of titrant
Volume of sample (ml)
5.13 Sodium sulfide nonanhydrate Na2S • 9H20, for the preparation of
standard solutions to be used for calibration curves. Standards must be prepared
at pH > 9 and < 11.
5.14 Titrant.
5.14.1 Standard phenylarsine oxide (PAD) solution (0.025N),
C6H5AsO. This solution is commercially available.
CAUTION: PAO is toxic.
5.14.2 Standard sodium thiosulfate solution (0.025N), Na,S,0, •
5H20. Dissolve 6.205 ± 0.005 g Na2S20, • 5H20 in 500 ml of water. Add
9 ml IN NaOH and dilute to 1 liter.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed in Chapter Nine of this manual.
6.2 All samples must be preserved with zinc acetate and sodium
hydroxide. Use four drops of 2N zinc acetate solution per 100 mL of aqueous or
multiphasic sample. Adjust the pH to greater than 9.0 with 50% NaOH. Fill the
sample bottle completely and stopper with a minimum of aeration. For solid
samples, fill the surface of solid with 2N zinc acetate until moistened. Samples
must be cooled to 4"C during storage.
7.0 PROCEDURE
7.1 Assemble the Buchner funnel apparatus. Unroll the glass wool and
fold the fiber over itself several times to make a pad about 1 cm thick when
lightly compressed. Cut the pad to fit the Buchner funnel. Dry and weigh the
pad, then place it in the funnel. Turn on the aspirator and wet the pad with a
known amount of water.
7.2 Transfer a sample that contains between 1 and 50 mg of sulfide to
the Buchner funnel. Rinse the sample container with known amounts of water and
add the rinses to the Buchner funnel. When no free water remains in the funnel,
slowly open the stopcock to allow air to enter the vacuum flask. A small amount
of sediment may have passed through the glass fiber pad. This will not interfere
with the analysis.
7.3 Transfer the solid and the glass fiber pad to a dried tared
weighing dish. Since most greases and oils will not pass through the fiber pad,
solids, oils, and greases will be extracted together. If the filtrate includes
an oil phase, transfer the filtrate to a separatory funnel. Collect and measure
the volume of the aqueous phase. Transfer the oil phase to the weighing dish
with the solid and glass fiber pad.
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7.4 Weigh the dish containing solid, oil (if any), and glass fiber pad.
Subtract the weight of the dry glass fiber pad. Calculate the volume of water
present in the original sample by subtracting the total volume of rinses from the
measured volume of the filtrate.
7.5 Place the following in a 1-liter wide-mouth bottle:
500 ml water
5 ml 50% w/v NaOH
1 g SnCl, • 2H20
50 ml n-nexane (if an oil or grease is present).
Cap the bottle with a Teflon or polyethylene lined cap and shake vigorously to
saturate the solution with stannous chloride. Direct a stream of nitrogen gas
at about 10 mL/min into the bottle for about 1 minute to purge the headspace of
oxygen. If the weight of the solids (Step 7.4) is greater than 25 g, weigh out
a representative aliquot of 25 g and add it to the bottle while still purging
with nitrogen. Otherwise, add all of the solids. Cap the bottle; avoid the
influx of air.
7.6 The pH of the extract must be maintained at > 9 or < 11 throughout
the extraction step and subsequent filtration. Since some samples may release
acid, the pH must be monitored as follows. Shake the extraction bottle and wait
1 minute. Open the bottle under a stream of nitrogen and check the pH. If the
pH is below 9, add 50% NaOH in 5 ml increments until it is at least 9. Recap the
bottle, and repeat the procedure until the pH does not drop. The bottle must be
thoroughly purged of oxygen before each recapping. Oxygen will oxidize sulfide
to elemental sulfur or other sulfur containing compounds that will not be
detected.
7.7 Place the bottle in the tumbler, making sure there is enough foam
insulation to cushion the bottle. Turn the tumbler on and allow the extraction
to run for about 18 hours.
7.8 Prepare a Buchner funnel apparatus as in Step 7.1 with a glass
fiber pad filter.
7.9 Decant the extract to the Buchner funnel.
7.10 If the extract contains an oil phase, separate the aqueous phase
using a separatory funnel. Neither the separation nor the filtration are
critical, but are necessary to be able to measure the volume of the aqueous
extract analyzed. Small amounts of suspended solids and oil emulsions will not
interfere with the extraction.
7.11 At this point, an aliquot of the filtrate of the original sample
may be combined with an aliquot of the extract in a proportion representative cf
the sample. Calculate the proportions as follows:
Aliquot of the Filtrate(mL) _ Solid Extracted(q)8 x Total Sample Filtrate(mL)c
Aliquot of the Extract(mL) " Total Solid(g)d Total Extraction Fluid(mL)d
9031 - 5 Revision 0
July 1992
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"From Step 7.5. Weight of solid sample used for extraction.
bFrom Step 7.4. Weight of solids and oil phase with the dry weight of filter and
tared dish subtracted.
clncludes volume of all rinses added to the filtrate (Steps 7.1 and 7.2).
d500 ml water plus total volume of NaOH solution. Does not include hexane, which
is subsequently removed (Step 7.10).
Alternatively, the samples may be distilled and analyzed separately,
concentrations for each phase reported separately, and the amounts of each phase
present in the sample reported separately.
7.12 Distillation of Sulfide
7.12.1 In a preliminary experiment, determine the approximate
amount of sulfuric acid required to adjust a measured amount of the sample
to pH less than or equal to 1. The sample size should be chosen so that
it contains between 1.0 and 50 mg of sulfide. Place a known amount of
sample or sample slurry in a beaker. Add water until the total volume is
200 ml. Stir the mixture and determine the pH. Slowly add sulfuric acid
until the pH is less than or equal to 1.
CAUTION: Toxic hydrogen sulfide may be generated from the acidified sample.
This operation must be performed in the hood and the sample left
in the hood until the sample has been made alkaline or the sulfide
has been destroyed.
From the amount of sulfuric acid required to acidify the sample and the
mass or volume of the sample acidified, calculate the amount of acid
required to acidify the sample to be placed in the distillation flask.
7.12.2 Prepare the gas evolution apparatus as shown in Figure 1
in a fume hood.
7.12.2.1 Prepare a hot water bath at 70°C by filling a
crystallizing dish or other suitable container with water and place
it on a hotplate stirrer. Place a thermometer in the bath and
monitor the temperature to maintain the bath at 70*C.
7.12.2.2 Assemble the three neck 500-mL flask, fritted
gas inlet tube, and exhaust tube. Use Teflon sleeves to seal the
ground glass joints. Place a Teflon coated stirring bar into the
flask.
7.12.2.3 Place into each gas scrubbing bottle 10 + 0.5
ml of the 0.5M zinc acetate solution, 5.0 + 0.1 ml of 37%
formaldehyde and 100 ± 5.0 ml water.
7.12.2.4 Connect the gas evolution flask and gas
scrubbing bottles as shown in Figure 1. Secure all fittings and
joints.
9031 - 6 Revision 0
July 1992
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7.12.3 Carefully place an accurately weighed sample which contains
1.0 to 50 mg of sulfide into the flask. If necessary, dilute to
approximately 200 ml with water.
7.12.4 Place the dropping funnel onto the flask making sure its
stopcock is closed. Add the volume of sulfuric acid calculated in Step
7.1.1 plus an additional 50 ml into the dropping funnel. The bottom
stopcock must be closed.
7.12.5 Attach the nitrogen inlet to the top of the dropping funnel
gas shut-off valve. Turn on the nitrogen purge gas and adjust the flow
through the sample flask to 25 mL/min. The nitrogen in the gas scrubbing
bottles should bubble at a rate of about five bubbTes per second.
Nitrogen pressure should be limited to approximately 10 psi to prevent
excess stress on the glass system and fittings. Verify that there are no
leaks in the system. Open the nitrogen shut-off valve leading to the
dropping funnel. Observe that the gas flow into the sample vessel will
stop for a short period while the pressure throughout the system
equalizes. If the gas flow through the sample flask does not return
within a minute, check for leaks around the dropping funnel. Once flow
has stabilized, turn on the magnetic stirrer. Purge the system for 15
minutes with nitrogen to remove oxygen.
7.12.6 Heat sample to 70°C. Open dropping funnel to a position
that will allow a flow of sulfuric acid of approximately 5 mL/min. Monitor
the system until most of the sulfuric acid contained within the dropping
funnel has entered the sample flask. Close the dropping funnel while a
small amount of acid remains. Immediately close the gas shut-off valve to
the dropping funnel.
7.12.7 Purge, stir, and maintain a temperature of 70'C for a total
of 90 minutes from start to finish. Shut off nitrogen supply. Turn off
heat.
7.13 Titration of Distillate
7.13.1 Pipet a known amount of standardized 0.025N iodine solution
(see Step 5.12). in a 500-mL flask, adding an amount in excess of that
needed to oxidize the sulfide. Add enough water to bring the volume to
100 ml. The volume of standardized iodine solution should be about 65 ml
for samples with 50 mg of sulfide.
7.13.2 Add 2 mL of 6N HC1 to the iodine.
7.13.3 Pipet both of the gas scrubbing bottle solutions into the
flask, keeping the end of the pipet below the surface of the iodine
solution. If at any point in transferring the zinc acetate solution or
rinsing the bottles, the amber color of the iodine disappears or fades to
yellow, more 0.025N iodine must be added. This additional amount must be
added to the amount from Step 7.13.1 for calculations. Record the total
volume of standardized 0.025N iodine solution used.
7.13.4 Prepare a rinse solution of a known amount of standardized
0.025N iodine solution, 1 ml of 6N HC1, and water to rinse the remaining
9031 - 7 Revision 0
July 1992
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white precipitate (zinc sulfide) from the gas scrubbing bottles into the
flask. There should be no visible traces of precipitate after rinsing.
7.13.5 Rinse any remaining traces of iodine from the gas scrubbing
bottles with water, and transfer the rinses to the flask.
7.13.6 Titrate the solution in the flask with standard 0.025N
phenylarsine oxide or 0.025N sodium thiosulfate solution until the ambe>"
color fades to yellow. Add enough starch indicator for the solution to
turn dark blue and titrate until the blue disappears. Record the volume
of titrant used.
7.13.7 Calculate the concentration of sulfide in the sample as
follows:
[(ml of I2 x N of 12) - (ml of titrant x N of titrant)](16.03)
. = sulfide(mg/kg)
sample weight (kg)
8.0 QUALITY CONTROL
8.1 All quality control data must be maintained and available for
reference or inspection for a period of three years. This method is restricted
to use by or under supervision of experienced analysts. Refer to the appropriate
section of Chapter One for additional quality control requirements.
8.2 A reagent blank should be run every twenty analyses or per
analytical batch, whichever is more frequent.
8.3 Check standards are prepared from water and a known amount of
sodium sulfide. A check standard should be run with each analytical batch of
samples or once every twenty samples. Acceptable recovery will depend on the
level and matrix.
8.4 A matrix spiked sample should be run for each analytical batch or
twenty samples, whichever is more frequent, to determine matrix effects. If
recovery is low, acid-insoluble sulfides are indicated. A matrix spiked sample
is a sample brought through the whole sample preparation and analytical process.
8.5 Verify the calibration with an independently prepared QC reference
sample every twenty samples or once per analytical batch, whichever is more
frequent.
9.0 METHOD PERFORMANCE
9.1 Accuracy - Accuracy for this method was determined by three
independent laboratories by measuring percent recoveries of spikes for waste
samples. The results are summarized below.
Accuracy for the entire method for four synthetic waste samples 70-104%
recovery
9031 - 8 Revision 0
Ouly 1992
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9.2 Precision
Precision of entire method for four synthetic waste samples
Percent coefficient of variation 1.0-34
10.0 REFERENCES
1. Test Methods for Evaluating Solid Waste. Physical/Chemical Methods. 3rd ed.;
U.S. Environmental Protection Agency. Office of Solid Waste and Emergency
Response. U.S. Government Printing Office: Washington, DC,1987; SW-846; 955-001-
00000-1.
2. Methods for Chemical Analysis of Water and Wastes; U.S. Environmental
Protection Agency. Office of Research and Development. Environmental Monitoring
and Support Laboratory. ORD Publications Office. Center for Environmental
Research Information: Cincinnati, OH, 1979; EPA-600/4-79-020, Method 376.1.
3. CRC Handbook of Chemistry and Physics. 66th ed.; Weast, R., Ed.; CRC: Boca
Raton, FL, 1985.
4. Standard Methods for the Examination of Water and Wastewater. 16th ed.;
Greenberg, A.E.; Trussell, R.R.; Clesceri, L.S., Eds.; American Water Works
Association, Water Pollution Control Federation, American Public Health
Association: Washington, DC, 1985; Methods 427, 427A, 427B, and 427D.
5. Andreae, M.O.; Bernard, W.R. Anal. Chem. 1983, 5_5, 608-612.
6. Barclay, H. Adv. Instrum. 1980, 35(2). 59-61.
7. Bateson, S.W.; Moody, G.J.; Thomas, J.P.R. Analyst 1986, 111. 3-9.
8. Berthage, P.O. Anal. Chim. Acta 1954, 10, 310-311.
9. Craig, P.J.; Moreton, P.A. Environ. Technol. Lett. 1982, 3, 511-520.
10. Franklin, G.O.; Fitchett, A.W. Pulp & Paper Canada 1982, 83(101. 40-44.
11. Fuller, W. Cyanide in the Environment; Van Zyl, D., Ed.; Proceedings of
Symposium; December 1984.
12. Gottfried, G.J. "Precision, Accuracy, and MDL Statements for EPA Methods
9010, 9030, 9060, 7520, 7521, 7550, 7551, 7910, and 7911"; final report to the
U.S. Environmental Protection Agency (EMSL-CI); Biopheric.
13. Kilroy, W.P. Talanta 1983, 30(6), 419-422.
14. Kurtenacher, V.A.; Wallak, R. Z. Anorg. U. Chem. 1927, 161. 202-209.
15. Landers, D.H.; David, M.B.; Mitchell, M.J. Int. J. Anal. Chem. 1983, 14,
245-256.
16. Opekar, F.; Brukenstein, S. Anal. Chem. 1984, 56> 1206-1209.
17. Ricklin, R.D.; Johnson, E.L. Anal. Chem. 1983, 55, 4.
9031 - 9 Revision 0
July 1992
-------
18. Rohrbough, W.G.; et al. Reaoent Chemicals. American Chemical Society
Specifications, 7th ed.; American Chemical Society: Washington, DC, 1986.
19. Snedecor, G.W.; Ghran, W.G. Statistical Methods; Iowa State University
Press: Ames, IA, 1980.
20. Umafia, M.; Beach, J.; Sheldon, L. "Revisions to Method 9010"; final report
to the Environmental Protection Agency on Contract No. 68-01-7266; Research
Triangle Institute: Research Triangle Park, NC, 1986; Work Assignment No. 1.
21. Umafia, M.; Sheldon, L. "Interim Report: Literature Review"; interim report
to the U.S. Environmental Protection Agency in Contract No. 68-01-7266; Research
Triangle Institute: Research Triangle Park, NC, 1986; Work Assignment No. 3.
22. Wang, W.; Barcelona, M.J. Environ. Inter. 1983, 9, 129-133.
23. Wronksi, M. Talanta 1981, 28, 173-176.
24. Application Note 156; Princeton Applied Research Corp.: Princeton, NJ.
25. Guidelines for Assessing and Reporting Data Quality for Environmental
Measurements; U.S. Environmental Protection Agency Office of Research and
Development: Washington, DC, 1983.
26. Fed. Regist. 1980, 45(98). 33122.
27. The Analytical Chemistry of Sulfur and Its Compounds. Part I; Karchmer,
J.H., Ed.; Wiley-Interscience: New York, 1970.
28. Methods for the Examination of Water and Associated Materials; Department
of the Environment: England, 1983.
29. "Development and Evaluation of a Test Procedure for Reactivity Criteria for
Hazardous Waste"; final report to the U.S. Environmental Protection Agency on
Contract 68-03-2961; EAL: Richmond, CA.
30. 1985 Annual Book of ASTM Standards. Vol. 11.01; "Standard Specification for
Reagent Water"; ATSM: Philadelphia, PA, 1985; 01193-77.
9031 - 10 Revision 0
July 1992
-------
FIGURE 1.
GAS EVOLUTION APPARATUS
H2SO4 (HCI for Acid Insoluble Sulfides)
Hot Water Bath
with Magnetic Stirrer
N2 Out
Zinc Acetate
and
Formaldehyde
Scrubbing
Bottles
Stirring Bar
9031 - 11
Revision 0
July 1992
-------
FIGURE 2.
TUMBLER-EXTRACTOR
Foam-Inner Uner
1-L Bottle
with Cap
Jar Mill Drive
Box Wheels Plywood Construction
9031 - 12
Revision 0
July 1992
-------
FIGURE 3.
EXTRACTOR
L-Gailoo PUttic
or Glass Book
Foam Bonded to Cover
Box Assembly
Plywood Construction
Totally Endoted
F» Cooted Motor
30 rpm, 1/8 HP
9031 - 13
Revision 0
July 1992
-------
METHOD 9031
SULFIDES
START
7 3 Transfer
filtrate lo
separator/ funnel.
collect aqueous
phase and measure
volume, transfer
oil phase lo
weighing dish
7 1 Assemble
Buchner funnel
apparatus
7 2 Transfer sample
to funnel; cinse
sample container w/
known afflt of
valer. add ciniei
t o funnel. filter
until no free water
remains in funnel
7 3 Transfer solid
and fiber pad Lo
dried la red
weighing dish
7 4 Meigh dish and
contents, subtract
glass fiber pad (if
any). subt ract
total volume of
rinses from volume
of filtrate
7 5 Place «aler.
NaOH. itannou*
chloride, and
n-h«»ane (if oil or
grease is present)
in 1 t bottle
7 S Cap bottle Kith
Teflon 1ined cap
and shake, direct
nitrogen into
bottle for 1 minute
to purge oxygen
7 S Add all solids:
cap botI1e
Yes
7 5 Weigh out 25 g.
add to bottle .hilt
purging
9031 - 14
Revision 0
July 1992
-------
METHOD 9031
(Continued)
7 & pH of
extraction must, be
> 9 and < 11. shake
bo t.11 B I mm . open
under nit r ogen.
check pH -
7 6 Add 5 mL
aliquot of MaOH
No
? 7 Place bottle in
tumbier. turn on
and extract for 18
hour a
? 8 Prvpare Buchnvc
funnel apparatus as
in Step 7 I
7 9 Decani extract
into funnel
7 11 Combine
aqu«ous extract and
original sample
fit Irate in
a Iiquo ts
proportional to the
sample, calculat*
proportion*
7 12 1 Choo*e
sample size, place
known ami of
sample in beaker;
add water, measure
pH. add cone
sulfunc acid to a
pH • 1
7 12 2 CaLculaL*
amount of sulfuric
acid needed to
acidify aample
7 10 Place extract
in separatory
funnel, collect and
measure volume of
aqueous phase
9031 - 15
Revision 0
July 1992
-------
METHOD 9031
(Continued)
7 12 2 Prepare gas
evol u I ion
7 12 3
apparatus
Place
weighed sample in
flask di
1
7 12 4
Place
dropping funnel
sulfuric acid from
Step 7 12 1 to
dr opping funnel
1
7 12 5
ni 1 r ogen
check for
lu r n on *
Ad;us I
flow.
Leaks .
tir r«r .
pu rge sys I em o I
oxygen
for IS
minutes
1
7 12 6 Hea
t to 70C.
add sulfuric acid
to flask
. close
funnel when acid
nears depletion
-J
•-•
7 12 7 Purge, stir.
and hea
for 90
mm . shut off
nitrogen.
hej
turn off
it
7 13 Analyze by
i
7 13 1 Pipol knovn
amount of 0 02SN
an Crlenmeyer
wa ter
J
7 13 2 Ade
HC1 to
J
7 13 3
scrubber
2 ml 6N
flask
Pipet
solution
into flask
— J
/ 7 13 3 N.
7 13 3 Add more
jr Does amber ^vYes iodine solution
C iodine color \' > record total volume
X. disappear' >X of iodine solution
>v / used
No
7 13 A Prepare
rinse solution of
I
7 13 S Rinse trace*
scrubbing bottle:
flask »ilh pipet
1
7 13 6 Titrate
flask solution
until amber color
fades, add starch
indicator: titrate •
un t i 1 blue color
disappears; record
volume of titranl
used
1
7 13 7 Calculate
the concentration
of sulfide in the
sample
.
STOP
9031 - 16
Revision 0
July 1992
-------
o
w
en
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METHOD 9035
SULFATE (COLORIMETRIC. AUTOMATED. CHLORANILATE)
1.0 SCOPE AND APPLICATION
1.1 This automated method 1s applicable to ground water, drinking and
surface waters, and domestic and Industrial wastes containing 10 to 400 mg
$04-2/11ter.
2.0 SUMMARY OF METHOD
2.1 When solid barium chloranllate 1s added to a solution containing
sulfate, barium sulfate 1s precipitated, releasing the highly colored add
chloranllate 1on. The color Intensity 1n the resulting chloranillc acid
solution 1s proportional to the amount of sulfate present.
3.0 INTERFERENCES
3.1 Cations such as calcium, aluminum, and Iron Interfere by precipi-
tating the chloranllate. These Ions are removed by passage through an ion-
exchange column.
3.2 Samples should be centrlfuged or filtered before analysis.
4.0 APPARATUS AND MATERIALS
4.1 Automated continuous-flow analytical Instrument, with:
4.1.1 Sampler I.
4.1.2 Continuous filter.
4.1.3 Manifold.
4.1.4 Proportioning pump.
4.1.5 Colorimeter: Equipped with 15 mm tubular flowcell and 520 nm
filters.
4.1.6 Recorder.
4.1.7 Heating bath, 45'C.
4.2 Magnetic stlrrer.
9035 - 1
Revision
Date September 1986
-------
5.0 REAGENTS
5.1 ASTM Type II water (ASTM D1193): Water should be monitored for
Impurities.
5.2 Barium chloranllate; Add 9 g of barium chloranllate (BaCgCl204) to
333 mL of spectrophotometrlc grade ethyl alcohol and dilute to 1 liter with
Type II water.
5.3 Acetate buffer. pH 4.63: Dissolve 13.6 g of sodium acetate 1n Type
II water. Add 6.4 mL of acetic add and dilute to 1 liter with Type II water.
Make.fresh weekly.
5.4 NaOH-EDTA solution; Dissolve 65 g of NaOH and 6 g EDTA 1n Type II
water and dilute to 1 liter. This solution 1s also used to clean out the
manifold system at end of sampling run.
5.5 Ion exchange resin; Dowex-50 W-X8, 1on1c form-H"1". The column 1s
prepared by sucking a slurry of the resin Into 12 1n. of 3/l6-1n O.D. tubing.
This may be conveniently done by using a plpet and a loose-fitting glass wool
plug 1n the tube. The column, upon exhaustion, turns red. Ensure that air
does not enter the column.
5.6 Stock solution! Dissolve 1.4790 g of oven-dried (105*C) Na2$04 1n
Type II water and dilute to 1 liter 1n a volumetric flask (1.0 mL = 1.0 mg).
5.7 Standards; Prepare a series of standards by diluting suitable
volumes of stock solution to 100.0 mL with Type II water. The following
dilutions are suggested.
Stock Solution (mL) Concentration (mg/L)
1.0 10
2.0 20
4.0 40
6.0 60
8.0 80
10.0 100
15.0 150
20.0 200
30.0 300
40.0 400
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed 1n Chapter Nine of this manual.
6.2 Refrigerate at 4*C.
9035 - 2
Revision
Date September 1986
-------
7.0 PROCEDURE
7.1 Set up manifold as shown 1n Figure 1. (Note that any precipitated
BaS04 and the unused barium chloranilate are removed by filtration. If any
BaS04 should come through the filter, It 1s complexed by the NaOH-EDTA
reagent.)
7.2 Allow both colorimeter and recorder to warm up for 30 min. Run a
baseline with all reagents, feeding Type II water through the sample line.
Adjust dark current and operative opening on colorimeter to obtain suitable
baseline.
7.3 Place Type II water wash tubes 1n alternate openings in sampler and
set sample timing at 2.0 m1n.
7.4 Place working standards 1n sampler in order of decreasing concen-
tration. Complete filling of sampler tray with unknown samples.
7.5 Switch sample line from Type II water to sampler and begin analysis.
7.6 Calculation;
7.6.1 Prepare a standard curve by plotting peak heights of
processed standards against known concentrations. Compute concentration
of samples by comparing sample peak heights with standard curve.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Calibration curves must be composed of a minimum of a blank and
three standards. A linear calibration curve should be made for every hour of
continuous sample analysis.
8.3 Dilute samples 1f they are more concentrated than the highest
standard or 1f they fall on the plateau of a calibration curve.
8.4 Employ a minimum of cne blank per sample batch to determine 1f
contamination has occurred.
8.5 Verify calibration with an Independently prepared check standard
every 15 samples.
8.6 Run one spike duplicate sample for every 10 samples. A spike
duplicate sample is a sample brought through the whole sample preparation and
analytical process.
9035 - 3
Revision
Date September 1986
-------
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FICORE 1 - SUIFATE MANIFOLD AA I
-------
9.0 METHOD PERFORMANCE
9.1 Precision and accuracy data are available 1n Method 375.1 of Methods
for Chemical Analysis of Water and Wastes.
10.0 REFERENCES
1. Bertolacinl, R.J., and J.E. Barney, II, Colorlmetric Determination of
Sulfate with Barium Chloranllate, Anal. Chem., 29(2), pp. 281-283 (1957).
2. Gales, M.E., Jr., W.H. Kaylor, and J.E. Longbottom, Determination of
Sulphate by Automatic Colorlraetric Analysis, Analyst, 93, 97 (1968).
9035 - 5
Revision
Date September 1986
-------
METHOD 9035
SULFATE (COUORIMETRIC. AUTOMATED. CMUOHANILATE)
7. 1
Set up manifold
7.2
Place
working
standards In
sampler; fill
sampler tray
Warm up
colorimeter,
recprd«r;
obtain suitable
baseline
7.5
Switch cample
line to toile
and analyze
7.3
Place water
M«*h tuba* In
•ampler
7.5.1
Compute
concentration
Of samples
o
StOP
9035 - 6
Revision 0
Date September 1986
-------
o
w
ON
-------
METHOD 9036
SULFATE (COLORIMETRIC. AUTOMATED. METHYLTHYMOL BLUE. AA IlV
1.0 SCOPE AND APPLICATION
1.1 This automated method 1s applicable to ground water, drinking and
surface waters, and domestic and Industrial wastes.
1.2 Samples 1n the range of 0.5 to 300 mg S04~2/I1ter can be analyzed,
2.0 SUMMARY OF METHOD
2.1 The sample 1s first passed through a sodium-form cation-exchange
column to remove multlvalent metal Ions. The sample containing sulfate 1s
then reacted with an alcohol solution of barium chloride and methylthymol blue
(MTB) at a pH of 2.5-3.0 to form barium sulfate. The combined solution Is
raised to a pH of 12.5-13.0 so that excess barium reacts with MTB. The
uncomplexed MTB color 1s gray; 1f 1t 1s all chelated with barium, the color 1s
blue. Initially, the barium and MTB are equlmolar and equivalent to 30 mg
$04-2/1 Her; thus the amount of uncomplexed MTB 1s equal to the sulfate
present.
3.0 INTERFERENCES
3.1 The Ion-exchange column eliminates Interferences from multlvalent
cations. A mid-scale sulfate standard containing Ca"*"1" should be analyzed
periodically to ensure that the column 1s functioning properly.
3.2 Samples with pH below 2 should be neutralized because high add
concentrations elute cations from the Ion-exchange resin.
3.3 Turbid samples should be filtered or centrlfuged.
4.0 APPARATUS AND MATERIALS
4.1 Automated continuous-flow analytical Instrument;
4.1.1 Sampler.
4.1.2 Manifold: High- or low-level (Figure 1).
4.1.3 Proportioning pump.
4.1.4 Heating bath: Operable at the temperature specified.
4.1.5 Colorimeter: Equipped with 15 mm flowcell and 460 nm
Interference filters.
9036 - 1
Revision
Date September 1986
-------
TO WASTE
-------
4.1.6 Filters: Of specified transmlttance.
4.1.7 Recorder.
5.0 REAGENTS
5.1 ASTM Type II water (ASTM D1193): Water should be monitored for
Impurities.
5.2 Barium chloride; Dissolve 1.526 g of barium chloride dlhydrate
(BaCl2'2H20) 1n 500 ml of Type II water and dilute to 1 liter.
5.3 Methyl thymol blue; Dissolve 0.1182 g of methyl thymol blue
(3'3"-b1s-N,N-b1s carboxymethyl-amino roethyHhymolsulfone-phthale1n
pentasodlum salt) 1n 25 ml of barium chloride solution (Paragraph 5.2). Add
4 ml of 1.0.N hydrochloric add, which changes the color to bright orange.
Add 71 ml of water and dilute to 500 ml with ethanol. The pH of this solution
1s 2.6. This reagent should be prepared the day before and stored 1n a brown
plastic bottle 1n the freezer.
5.4 Buffer, pH 10.5 + 0.5: Dissolve 6.75 g of ammonium chloride 1n
500 ml of Type II water. Add 57 ml of concentrated ammonium hydroxide and
dilute to 1 liter with Type II water.
5.5 Buffered EDTA; Dissolve 40 g of tetrasodlum EDTA 1n pH 10.5 buffer
(Paragraph 5.4) and dilute to 1 liter with buffer.
5.6 Sodium hydroxide solution (50%); Dissolve 500 g NaOH 1n 600 ml of
Type II water, cool, and dilute to 1 liter.
5.7 Sodium hydroxide, 0.18 N: Dilute 14.4 ml of sodium hydroxide
solution (Paragraph 5.6) to 1 liter.
5.8 Ion-exchange resin; B1o-Rex 70, 20-50 mesh, sodium form, B1o-Rad
Laboratories, Richmond, California. Free from fines by stirring with several
portions of Type II water and decant the supernate before settling 1s
complete.
5.9 Dilution, water: Add 0.75 ml of sulfate stock solution (Paragraph
5.10) and 3 drops of Br1j-35 (available from Technlcon) to 2 liters of Type II
water.
5.10 Sulfate stock solution. 1 ml = 1 mg S04'2: Dissolve 1.479 g of
dried Na2S04 (105*0) 1n Type II water and dilute to 1 liter.
5.11 Dilute sulfate solution, 1 ml = 0.1 mg SO^2: Dilute 100 ml of
sulfate stock solution (Paragraph 5.10) to 1 liter.
9036 - 3
Revision
Date September 1986
-------
5.12 High-level working standards, 10-300 mg/L: Prepare high-level
working standardsbydilutingthefollowing volumes of stock standard
(Paragraph 5.10) to 100 ml:
Stock Solution (ml) Concentration (mq/L)
1 10
5 50
10 100
15 150
25 250
30 300
5.13 Low-level working standards, 0.5-30 mg/L: Prepare low-level
working standards by diluting the following volumes of dilute sulfate solution
(Paragraph 5.11) to 100 ml:
Stock Solution (ml) Concentration (mg/L)
0.5 0.5
1 1.0
5 5.0
10 10.0
15 15.0
25 25.0
30 30.0
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed in Chapter Nine of this manual.
6.2 Refrigerate at 4*C.
7.0 PROCEDURE
7.1 Set up manifold for high- (10-300 mg/L SO^2) or low- (0.5-30 mg/L
S04"2) level samples as described 1n Figure 1.
7.2 The Ion-exchange column 1s prepared by pulling a slurry of the resin
into a piece of glass tubing 7.5-1n. long, 2.0-mm I.D., and 3.6-mm O.D. This
is conveniently done by using a pipet and a loose-fitting glass wool plug in
the tubing. Care should be taken to avoid allowing air bubbles to enter the
column. If air bubbles become trapped, the column should be prepared again.
The column can exchange the equivalent of 35 mg of calcium. For the high-
level manifold, this corresponds to about 900 samples with 200 mg/L Ca. The
column should be prepared as often as necessary to ensure that no more than
50% of its capacity 1s used.
9036 - 4
Revision
Date September 1986
-------
7.3 Allow the colorimeter, recorder, and printer to warm up for 30 min.
Pump all reagents until a stable baseline 1s achieved.
7.4 Analyze all working standards 1n duplicate at the beginning of a run
to develop a standard curve. The A and B control standards must be analyzed
every hour to ensure that the system remains properly calibrated. Because the
chemistry 1s nonlinear, the 180-mg/L standard 1s set at 50% on the recorder
using the standard calibration control on the colorimeter.
7.5 At the end of each day, the system should be washed with the
buffered EDTA solution (Paragraph 5.5). This is done by placing the
methyl thymol blue line and the sodium hydroxide line in water for a few
minutes and then 1n the buffered EDTA solution for 10 m1n. Wash the system
with water for 15 min before shutting down.
7.6 Prepare a standard curve by plotting peak heights of five processed
standards against known concentrations. Compute concentration of samples by
comparing sample peak heights with the standard curve. Note that this is not
a linear curve but a third order curve.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Calibration curves must be composed of a minimum of a blank and
three standards. A calibration curve should be made for every hour of
continuous sample analysis.
8.3 Dilute samples 1f they are more concentrated than the highest
standard or 1f they fall on the plateau of a calibration curve.
8.4 Employ a minimum of one blank per sample batch to determine if
contamination has occurred.
8.5 Verify calibration with an Independently prepared check standard
every 15 samples.
8.6 Run one spike duplicate sample for every 10 samples. A duplicate
sample is a sample brought through the whole sample preparation and analytical
process.
9.0 METHOD PERFORMANCE
9.1 Precision and accuracy data are available in Method 375.2 of Methods
for Chemical Analysis of Water and Wastes.
9036 - 5
Revision 0
Date September 1986
-------
10.0 REFERENCES
1. Coloros, E., M.R. Panesar, and P.P. Parry, "Linearizing the Calibration
Curve in Determination of Sulfate by the Methylthymol Blue Method," Anal.
Chem. 48, 1693 (1976).
2. Lazrus, A.L., K.C. Hill, and J.P. Lodge, "Automation in Analytical
Chemistry," Technicon Symposia, 1965.
9036 - 6
Revision
Date September 1986
-------
METHOD 9036
SUI.FATE (COLORIMETRIC. AUTOMATED. METHYLTHVKOL SLUE. A* II)
c
St»rt
7. 1
o
Set up ••njfolo
7.Z
7 .4
Develop
• »t»nd»ro
curve; check
Cillbrat ion
•very hour
Prepare ion
••change column
7.3
7.5
Ooxn «t «na of
em-,
M»r» up
colorl»«ter.
recorder «na
printer. Get
•(•bit
7.6
Corvc*ntr*t ion
Of ••moles
O
Stop
9036 - 7
Revision 0
Date September 1986
-------
o
OJ
00
-------
METHOD 9038
SULFATE (TURBIDIMETRIC)
1.0 SCOPE AND APPLICATION
1.1 This method 1s applicable to ground water, drinking and surface
waters, and domestic and Industrial wastes.
1.2 This method 1s suitable for all concentration ranges of sulfate
(S04~2); however, 1n order to obtain reliable readings, use a sample aliquot
containing not more than 40 mg/L of S04~2.
1.3 The minimum detectable limit 1s approximately 1 mg/L of S04~2.
2.0 SUMMARY OF METHOD
2.1 Sulfate 1on 1s converted to a barium sulfate suspension under
controlled conditions. The resulting turbidity 1s determined by a nephelo-
meter, filter photometer, or spectrophotometer and compared with a curve
prepared from standard sulfate solution.
3.0 INTERFERENCES
3.1 Color and turbidity due to the sample matrix can cause positive
Interferences which must be accounted for by use of blanks.
3.2 Silica 1n concentrations over 500 mg/L will Interfere.
4.0 APPARATUS AND MATERIALS
4.1 Magnetic stlrrer; Variable speed so that 1t can be held constant
just below splashing.Use Identical shapes and sizes of magnetic stirring
bars.
4.2 Photometer (one of the following, given 1n order of preference):
4.2.1 Nephelometer.
4.2.2 Spectrophotometer: For use at 420 nm with light path of
4 to 5 cm.
4.2.3 Filter photometer: With a violet filter having a maximum
near 420 nm and a light path of 4 to 5 cm.
4.3 Stopwatch; If the magnetic stlrrer 1s not equipped with an accurate
timer.
9038 - 1
Revision
Date September 1986
-------
4.4 Measuring spoon; Capacity 0.2 to 0.3 ml.
5.0 REAGENTS
5.1 ASTH Type II water (ASTM D1193): Water should be monitored for
Impurities.
5.2 Conditioning reagent; Slowly add 30 ml concentrated HC1 to 300 ml
Type II water, 100 ml 95% ethanol or Isopropanol, and 75 g NaCl 1n solution in
a container. Add 50 ml glycerol and mix.b
5.3 Barium chloride (BaCl2): Crystals, 20 to 30 mesh.
5.4 Sodium carbonate solution; (approximately 0.05 N) : Dry 3 to 5 g
primary standard N32C03 at 250*C for 4 hr and cool 1n a desiccator. Weigh
2.5 + 0.2 g (to the nearest mg), transfer to a 1-Hter volumetric flask, and
fill to the mark with Type II water.
5.5 Proprietary reagents; Such as Hach Sulfaver or equivalent, are
acceptable.
5.6 Standard sulfate solution (1.00 ml = 100 ug S04"2); Prepare by
Paragraph 5.6.1 or 5.6.2.
5.6.1 Standard sulfate solution from
5.6.1.1 Standard sulfurlc add, 0.1 N; Dilute 3.0 ml
concentrated ^SOi to 1 liter with Type II water. Standardize
against 40.0 ml of 0.05 N Na2COa solution (Paragraph 5.4) with about
60 ml Type II water by titrating potent1ometr1cally to a pH of about
5. Lift electrodes and rinse Into beaker. Boil gently for 3 to 5
min under a watch glass cover. Cool to room temperature. Rinse
cover glass Into beaker. Continue tltration to the pH Inflection
point. Calculate the normality of H2S04 using;
N
53.00 x C
where:
A = g Na2COs weighed Into 1 liter flask (Paragraph 5.4);
B = mL Na2C03 solution used in the standardization;
C = mL acid used 1n tltration;
5.6.1.2 Standard add, 0.02 N; Dilute appropriate amount of
standard acid, 0.1 N (Paragraph 5.6.1.1) to 1 liter (use 200.00 ml
standard acid if normality 1s 0.1000 N) . Check by standardization
against 15 ml of 0.05 N Na2C03 solution (Paragraph 5.4).
9038 - 2
Revision
Date September 1986
-------
5.6.1.3 Place 10 ml standard sulfurlc add, 0.02 N (Paragraph
5.6.1.2) in a 100-tnL volumetric flask and dilute to the mark.
5.6.2 Standard sulfate solution from NapSO^ Dissolve 147.9 mg
anhydrous Na2S04 in Type II water in a 1-Titer volumetric flask and
dilute to the mark with Type II water.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed In Chapter Nine of this manual.
6.2 Preserve by refrigerating at 4*C.
7.0 PROCEDURE
7.1 Formation of barium sulfate turbidity;
7.1.1 Place a 100-mL sample, or a suitable portion diluted to
100 mL, into a 250-mL Erlenmeyer flask.
7.1.2 Add exactly 5.0 mL conditioning reagent (Paragraph 5.2).
7.1.3 Mix 1n the stirring apparatus.
7.1.4 While the solution 1s being stirred, add a measured spoonful
of BaCl2 crystals (Paragraph 5.3) and begin timing Immediately.
7.1.5 Stir exactly 1.0 min at constant speed.
7.2 Measurement of barium sulfate turbidity;
7.2.1 Immediately after the stirring period has ended, pour
solution Into absorbance cell.
7.2.2 Measure turbidity at 30-sec intervals for 4 min.
7.2.3 Record the maximum reading obtained in the 4-min period.
7.3 Preparation of calibration curve;
7.3.1 Prepare calibration curve using standard sulfate solution
(Paragraph 5.6).
7.3.2 Space standards at 5-mg/L increments in the 0-40 mg/L sulfate
range.
7.3.3 Above 50 mg/L the accuracy decreases and the suspensions lose
stability.
9038 - 3
Revision 0
Date September 1986
-------
7.3.4 Check reliability of calibration curve by running a standard
with every three or four samples.
7.4 Correction for sample color and turbidity;
7.4.1 Run a sample blank using steps 7.1 and 7.2, without the
addition of barium chloride (Paragraph 7.1.4).
7.5 Calculation;
7.5.1 Read mg SC>4~2 from linear calibration curve:
? mg SO/2 x 1,000
"B S04~ /L = ml sample
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Calibration curves must be composed of a minimum of a blank and
three standards. A calibration curve should be made for every hour of
continuous sample analysis.
8.3 Dilute samples 1f they are more concentrated than the highest
standard or 1f they fall on the plateau of a calibration curve.
8.4 Employ a minimum of one blank per sample batch to determine 1f
contamination has occurred.
8.5 Verify calibration with an Independently prepared check standard
every 15 samples.
8.6 Run one spike duplicate sample for every 10 samples. A spike
duplicate sample 1s a sample brought through the whole sample preparation and
analytical process.
9.0 METHOD PERFORMANCE
9.1 Thirty-four analysts 1n 16 laboratories analyzed six synthetic water
samples containing exact Increments of Inorganic sulfate with the following
results:
9038 - 4
Revision
Date September 1986
-------
Increment as
Sulfate
(mg/L)
8.6
9.2
110
122
188
199
Precision as
Standard Deviation
(mg/L)
2.30
1.78
7.86
7.50
9.58
11.8
Accuracy
Bias
(X)
-3.72
-8.26
-3.01
-3.37
+0.04
-1.70
as
Bias
(mg/L)
-0.3
-0.8
-3.3
-4.1
+0.1
-3.4
(Data from: FWPCA Method Study 1, Mineral and Physical Analyses.)
9.2 A synthetic unknown sample containing 259 mg/L sulfate, 108 mg/L Ca,
82 mg/L Mg, 3.1 mg/L K, 19.9 mg/L Na, 241 mg/L chloride, 0.250 mg/L nitrite N,
1.1 mg/L nitrate N, and 42.5 mg/L total alkalinity (contributed by NaHC03),
was analyzed 1n 19 laboratories by the turb1d1metr1c method, with a relative
standard deviation of 9.IX and a relative error of 1.2X.
10.0 REFERENCES
1. Annual Book of ASTM Standards, Part 31, "Water," Standard D516-68,
Method B, p. 430 (1976).
2. Standard Methods for the Examination of Water and Wastewater, 14th ed.,
p. 496, Method 427C, (1975).
9038 - 5
Revision
Date September 1986
-------
METHOD 9038
(TuftaXOIMCTRIC)
7. J.I
o
Place
in fle*k tar
formation of
beriun sulfete
turbidity
7.1.2
7.2.2
turbidity:
record *>mx .
reading
Add
conditioning
reagent ana mix
7.1.4
7.3
Prepare
calibration
Add BaCli
crystals: stir
for l nlnute
7.4
Correct for
•anole color
and turbidity
7.2.1
Pour solution
into abaorbance
ctll
7.5
-2
Calculate SOX
f Stop J
9038 - 6
Revision 0
Date September 1986
-------
o
*».
O
-------
METHOD 9040A
PH ELECTRQMETRIC MEASUREMENT
1.0 SCOPE AND APPLICATION
1.1 Method 9040 is used to measure the pH of aqueous wastes and those
multiphase wastes where the aqueous phase constitutes at least 20% of the total
volume of the waste.
1.2 The corrosivity of concentrated acids and bases, or of concentrated
acids and bases mixed with inert substances, cannot be measured. The pH
measurement requires some water content.
2.0 SUMMARY
2.1 The pH of the sample is determined electrometrically using either
a glass electrode in combination with a reference potential or a combination
electrode. The measuring device is calibrated using a series of standard
solutions of known pH.
3.0 INTERFERENCES
3.1 The glass electrode, in general, is not subject to solution
interferences from color, turbidity, colloidal matter, oxidants, reductants, or
moderate (<0.1 molar solution) salinity.
3.2 Sodium error at pH levels >10 can be reduced or eliminated by using
a low-sodium-error electrode.
3.3 Coatings of oily material or particulate matter can impair
electrode response. These coatings can usually be removed by gentle wiping or
detergent washing, followed by rinsing with distilled water. An additional
treatment with hydrochloric acid (1:10) may be necessary to remove any remaining
film.
3.4 Temperature effects on the electrometric determination of pH arise
from two sources. The first is caused by the change in electrode output at
various temperatures. This interference should be controlled with instruments
having temperature compensation or by calibrating the electrode-instrument system
at the temperature of the samples. The second source of temperature effects is
the change of pH due to changes in the sample as the temperature changes. This
error is sample-dependent and cannot be controlled. It should, therefore, be
noted by reporting both the pH and temperature at the time of analysis.
4.0 APPARATUS AND MATERIALS
4.1 pH meter: Laboratory or field model. Many instruments are commer-
cially available with various specifications and optional equipment.
4.2 Glass electrode.
9040A - 1 Revision 1
September 1994
-------
4.3 Reference electrode: A silver-silver chloride or other reference
electrode of constant potential may be used.
NOTE: Combination electrodes incorporating both measuring and
referenced functions are convenient to use and are available with
solid, gel-type filling materials that require minimal maintenance.
4.4 Magnetic stirrer and Teflon-coated stirring bar.
4.5 Thermometer and/or temperature sensor for automatic compensation.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Primary standard buffer salts are available from the National
Institute of Standards and Technology (NIST) and should be used in situations
where extreme accuracy is necessary. Preparation of reference solutions from
these salts requires some special precautions and handling, such as low-
conductivity dilution water, drying ovens, and carbon-dioxide-free purge gas.
These solutions should be replaced at least once each month.
5.3 Secondary standard buffers may be prepared from NIST salts or
purchased as solutions from commercial vendors. These commercially available
solutions have been validated by comparison with NIST standards and are
recommended for routine use.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed in Chapter Nine of this manual.
6.2 Samples should be analyzed as soon as possible.
7.0 PROCEDURE
7.1 Calibration:
7.1.1 Because of the wide variety of pH meters and accessories,
detailed operating procedures cannot be incorporated into this method.
Each analyst must be acquainted with the operation of each system and
familiar with all instrument functions. Special attention to care of the
electrodes is recommended.
7.1.2 Each instrument/electrode system must be calibrated at a
minimum of two points that bracket the expected pH of the samples and are
approximately three pH units or more apart. (For corrosivity characteri-
zation, the calibration of the pH meter should include a buffer of pH 2
for acidic wastes and a pH 12 buffer for caustic wastes.) Various
9040A - 2 Revision 1
September 1994
-------
"balance" or
manufacturer's
instrument designs may involve use of a dial (to
"standardize") or a slope adjustment, as outlined in the .. .
instructions. Repeat adjustments on successive portions of the two buffer
solutions until readings are within 0.05 pH units of the buffer solution
value.
7.2 Place the sample or buffer solution in a clean glass beaker using
a sufficient volume to cover the sensing elements of the electrodes and to give
adequate clearance for the magnetic stirring bar. If field measurements are
being made, the electrodes may be immersed directly into the sample stream to an
adequate depth and moved in a manner to ensure sufficient sample movement across
the electrode-sensing element as indicated by drift-free readings (<0.1 pH).
7.3 If the sample temperature differs by more than 2CC from the buffer
solution, the measured pH values must be corrected. Instruments are equipped
with automatic or manual compensators that electronically adjust for temperature
differences. Refer to manufacturer's instructions.
7.4 Thoroughly rinse and gently wipe the electrodes prior to measuring
pH of samples. Immerse the electrodes into the sample beaker or sample stream
and gently stir at a constant rate to provide homogeneity and suspension of
solids. Note and record sample pH and temperature. Repeat measurement on
successive aliquots of sample until values differ by <0.1 pH units. Two or three
volume changes are usually sufficient.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for the appropriate QC protocols.
8.2 Electrodes must be thoroughly rinsed between samples.
9.0 METHOD PERFORMANCE
9.1 Forty-four analysts in twenty laboratories analyzed six synthetic
water samples containing exact increments of hydrogen-hydroxyl ions, with the
following results:
Accuracy as
Standard Deviation Bias Bias
pH Units pH Units % pH Units
3.5
3.5
7.1
7.2
8.0
8.0
0.10
0.11
0.20
0.18
0.13
0.12
-0.29
-0.00
+ 1.01
-0.03
-0.12
+0.16
-0.01
+0.07
-0.002
-0.01
+0.01
10.0 REFERENCES
1. National Bureau of Standards, Standard Reference Material Catalog 1986-87,
Special Publication 260.
9040A - 3
Revision 1
September 1994
-------
METHOD 9040A
pH ELECTROMETRIC MEASUREMENT
( Start J
7.1 Calibrate pH
meter.
7.2 Place sample
or buffer solution
in glass beaker.
7.3 Does
temperature
differ by more
than 2C from
buffer?
7.3 Correct
measured pH
values.
7.4 Immerce
electrodes and
measure pH of
sample.
7.4 Note and record
pH and temperature;
repeat 2 or 3 times
with different
aliquots.
I
( Stop j
9040A - 4
Revision 1
September 1994
-------
o
*>.
-------
METHOD 9041A
DH PAPER METHOD
1.0 SCOPE AND APPLICATION
1.1 Method 9041 may be used to measure pH as an alternative to Method
9040 (except as noted in Step 1.3) or in cases where pH measurements by Method
9040 are not possible.
1.2 Method 9041 is not applicable to wastes that contain components
that may mask or alter the pH paper color change.
1.3 pH paper is not considered to be as accurate a form of pH
measurement as pH meters. For this reason, pH measurements taken with Method
9041 cannot be used to define a waste as corrosive or noncorrosive (see RCRA
regulations 40 CFR §261.22(a)(l).
2.0 SUMMARY OF METHOD
2.1 The approximate pH of the waste is determined with wide-range pH
paper. Then a more accurate pH determination is made using "narrow-range" pH
paper whose accuracy has been determined (1) using a series of buffers or (2) by
comparison with a calibrated pH meter.
3.0 INTERFERENCES
3.1 Certain wastes may inhibit or mask changes in the pH paper. This
interference can be determined by adding small amounts of acid or base to a small
aliquot of the waste and observing whether the pH paper undergoes the appropriate
changes.
CAUTION: THE ADDITION OF ACID OR BASE TO WASTES MAY RESULT IN VIOLENT
REACTIONS OR THE GENERATION OF TOXIC FUMES (e.g.. hydrogen
cyanide). Thus, a decision to take this step requires some
knowledge of the waste. See Step 7.3.3 for additional precautions.
4.0 APPARATUS AND MATERIALS
4.1 Wide-range pH paper.
4.2 Narrow-range pH paper: With,a distinct color change for every 0.5
pH unit (e.g., Alkaacid Full-Range pH Kit, Fisher Scientific or equivalent).
Each batch of narrow-range pH paper must be calibrated versus certified pH
buffers or by comparison with a pH meter which has been calibrated with certified
pH buffers. If the incremental reading of the narrow-range pH paper is within
0.5 pH units, then the agreement between the buffer or the calibrated pH meter
with the paper must be within 0.5 pH units.
4.3 pH Meter (optional).
9041A - 1 Revision 1
July 1992
-------
5.0 REAGENTS
5.1 Certified pH buffers: To be used for calibrating the pH paper or
for calibrating the pH meter that will be used subsequently to calibrate the pH
paper.
5.2 Dilute acid (e.g.. 1:4 HC1).
5.3 Dilute base (e.g.. 0.1 N NaOH).
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a sampling plan which addresses
the considerations discussed in Chapter Nine of this manual.
7.0 PROCEDURE
7.1 A representative aliquot of the waste must be tested with wide-
range pH paper to determine the approximate pH.
7.2 The appropriate narrow-range pH paper is chosen and the pH of a
second aliquot of the waste is determined. This measurement should be performed
in duplicate.
7.3 Identification of interference:
7.3.1 Take a third aliquot of the waste, approximately 2 mL in
volume, and add acid dropwise until a pH change is observed. Note the
color change.
7.3.2 Add base dropwise to a fourth aliquot and note the color
change. (Wastes that have a buffering capacity may require additional
acid or base to result in a measurable pH change.)
7.33 The observation of the appropriate color change is a strong
indication that no interferences have occurred.
CAUTION ADDITION OF ACID OR BASE TO SAMPLES MAY RESULT IN VIOLENT REACTIONS
OR THE GENERATION OF TOXIC FUMES. PRECAUTIONS MUST BE TAKEN. THE
ANALYST SHOULD PERFORM THESE TESTS IN A WELL-VENTILATED HOOD WHEN
DEALING WITH UNKNOWN SAMPLES.
8.0 QUALITY CONTROL
8.1 All quality control data must be maintained and available for easy
reference or inspection.
8,2 All pH determinations must be performed in duplicate.
8.3 Each batch of pH paper must be calibrated versus certified pH
buffers or a pH meter which has been calibrated with certified pH buffers.
9041A - 2 Revision 1
July 1992
-------
9.0 METHOD PERFORMANCE
9.1 No data provided.
10.0 REFERENCES
10.1 None required.
9041A - 3 Revision 1
July 1992
-------
METHOD 9041A
pH PAPER METHOD
START
7 1 D«l«rmin*
approitifliat*; pH with
wid*-rang* pH pap*r
7 2 S*l*ct
appropriate
narrow-rang* pH
pap*r. d*t*rmin« pH
in duplicate on 2nd
a 1iquo1
731 Ujing 3rd
aliquot, add acid
to wast* until pH
chang««; not* color
changa
732 Add bas« to
4th aliquot, not*
color chang*
733 D*t*rmin* if
int*rf*r*nc«s hav*
occur r*d
STOP
9041A - 4
Revision 1
July 1992
-------
o
«t
in
-------
METHOD 9045A
SOIL oH
1.0 SCOPE AND APPLICATION
1.1 Method 9045 is an electrometric procedure which has been approved
for measuring pH in calcareous and noncalcareous soils.
2.0 SUMMARY OF METHOD
2.1 The soil sample is mixed either with reagent water or with a
calcium chloride solution (see Section 5.0), depending on whether the soil is
considered calcareous or non-calcareous. The pH of the solution is then measured
with a pH meter.
3.0 INTERFERENCES
3.1 Samples with very low or very high pH may give incorrect readings
on the meter. For samples with a true pH of >10, the measured pH may be
incorrectly low. This error can be minimized by using a low-sodium-error
electrode. Strong acid solutions, with a true pH of <1, may give incorrectly
high pH measurements.
3.2 Temperature fluctuations will cause measurement errors.
3.3 Errors will occur when the electrodes become coated. If an
electrode becomes coated with an oily material that will not rinse free, the
electrode can either (1) be cleaned with an ultrasonic bath, or (2) be washed
with detergent, rinsed several times with water, placed in 1:10 HC1 so that the
lower third of the electrode is submerged, and then thoroughly rinsed with water.
4.0 APPARATUS AND MATERIALS
4.1 pH Meter with means for temperature compensation.
4.2 Electrodes:
4.2.1 Calomel electrode.
4.2.2 Glass electrode.
4.2.3 A combination electrode can be employed instead of calomel
or glass.
4.3 Beaker: 50-mL.
4.4 Class A volumetric flasks: 1 L and 2 L.
4.5 Analytical balance: capable of weighing 0.1 g.
4.6 Aluminum foil.
9045A - 1 Revision 1
July 1992
-------
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently purity to
permit its use without lessening the accuracy of the determination.
5.2 Reagent water. All reference to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Primary standard buffer salts are available from the National
Institute of Standards and Technology (NIST) and should be used in situations
where extreme accuracy is necessary. Preparation of reference solutions from
these salts requires some special precautions and handling, such as low-
conductivity dilution water, drying ovens, and carbon-dioxide-free purge gas.
These solutions should be replaced at least once each month.
5.4 Secondary standard buffers may be prepared from NIST salts or
purchased as solutions from commercial vendors. These commercially available
solutions, which have been validated by comparison with NIST standards, are
recommended for routine use.
5.5 Stock calcium chloride solution (CaCK), 3.6 M: Dissolve 1059 g of
CaCl, • 2H20 in reagent water in a 2-liter volumetric flask. Cool the solution,
dilute it to volume with reagent water, and mix it well. Dilute 20 ml of this
solution to 1 liter with reagent water in a volumetric flask and standardize it
by titrating a 25-mL aliquot of the diluted solution with standard 0.1 N AgN03,
using 1 ml of 5% K2Cr04 as the indicator.
5.6 Calcium chloride (CaCl,), 0.01 M: Dilute 5 mL of stock 3.6 M CaCl,
to 1.8 liters with reagent water. If the pH of this solution is not between 5
and 6.5, adjust the pH by adding a little Ca(OH)2 or HC1. As a check on the
preparation of this solution, measure its electrical conductivity. The specific
conductivity should be 2.32 ± 0.08 mmho per cm at 25"C.
5.7 Hydrochloric acid (HC1): 1:3 mixture with reagent water.
6.0 SAMPLE PRESERVATION AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed in Chapter Nine of this manual.
6.2 Samples should be analyzed as soon as possible.
7.0 PROCEDURE
7.1 Calibration:
7.1.1 Because of the wide variety of pH meters and accessories,
detailed operating procedures cannot be incorporated into this method.
Each analyst must be acquainted with the operation of each system and
9045A - 2 Revision 1
July 1992
-------
familiar with all instrument functions. Special attention to care of the
electrodes is recommended.
7.1.2 Each instrument/electrode system must be calibrated at a
minimum of two points that bracket the expected pH of the samples and are
approximately three pH units or more apart. Repeat adjustments on
successive portions of the two buffer, solutions until readings are within
0.05 pH units of the buffer solution value.
7.2 Determination of calcareous vs. non-calcareous soils:
7.2.1 Place approximately 0.5 g of sample (less than 60 mesh) on
a piece of aluminum foil.
7.2.2 Add one or two drops of 1:3 HC1 to the sample. The
presence of CaC03 is indicated by a bubbling or audible fizz.
7.2.3 If the sample produces bubbling or fizzing, it is a
calcareous soil. If no bubbling or fizzing occurs, the sample is a non-
calcareous soil.
7.3 Sample preparation and pH measurement of non-calcareous soils:
7.3.1 To 20 g of soil in a 50-mL beaker, add 20 ml of reagent
water and stir the suspension several times during the next 30 minutes.
7.3.2 Let the soil suspension stand for about 1 hour to allow
most of the suspended clay to settle out from the suspension.
7.3.3 Adjust the electrodes in the clamps of the electrode holder
so that, upon lowering the electrodes into the beaker, the glass electrode
will be immersed just deep enough into the clear supernatant solution to
establish a good electrical contact through the ground-glass joint or the
fiber-capillary hole. Insert the electrodes into the sample solution in
this manner. For combination electrodes, immerse just below the
suspension.
7.3.4 If the sample temperature differs by more than 2°C from the
buffer solution, the measured pH values must be corrected.
7.3.5 Report the results as "soil pH measured in water."
7.4 Sample preparation and pH measurement of calcareous soils:
7.4.1 To 10 g of soil in a 50-mL beaker, add 20 mL of 0.01 M
CaCl2 (Step 5.6) solution and stir the suspension several times during the
next 30 minutes.
7.4.2 Let the soil suspension stand for about 30 minutes to allow
most of the suspended clay to settle out from the suspension.
7.4.3 Adjust the electrodes in the clamps of the electrode holder
so that, upon lowering the electrodes into the beaker, the glass electrode
will be immersed well into the partly settled suspension and the calomel
9045A - 3 Revision 1
July 1992
-------
electrode will be immersed just deep enough into the clear supernatant
solution to establish a good electrical contact through the ground-glass
joint or the fiber-capillary hole. Insert the electrode into the sample
solution in this manner.
7.4.4 If the sample temperature differs by more than 2*C from the
buffer solution, the measured pH values must be corrected.
7.4.5 Report the results as "soil pH measured in 0.01 M CaCl2."
8.0 QUALITY CONTROL
8.1 Duplicate samples and check standards should be analyzed routinely.
8.2 Electrodes must be thoroughly rinsed between samples.
9.0 METHOD PERFORMANCE
9.1 No data provided.
10.0 REFERENCES
10.1 Black, Charles Allen; Methods of Soil Analysis; American Society
of Agronomy: Madison, WI. 1973
9045A - 4 Revision 1
July 1992
-------
METHOD 9045A
SOIL pH
7 3 1 Add
•attr to 20 g
soiL: atir
7 3 2 Let
soil
luspcniion
stand for 1
hour
7 2 1 Place
0 5 g tample
on aluminum
foil
4 1 Add
calcium
chloride
solution to lOg
soi 1 , itir
Correct
7 4 2 Let
1011
iu*p«niion
jtand for 30
minu t«i
Ir.iat I
• l«c t rodes
into sampli
*o1ution
pH
Report
raiulti
STOP
9045A - 5
Revision 1
July 1992
-------
©
«t
in
-------
METHOD 9045B
SOIL AND WASTE pH
1.0 SCOPE AND APPLICATION
1.1 Method 9045 is an electrometric procedure for measuring pH in
soils and waste samples. Wastes may be solids, sludges, or non-aqueous
liquids. If water is present, it must constitute less than 20% of the total
volume of the sample.
2.0 SUMMARY OF METHOD
2.1 The sample is mixed with reagent water, and the pH of the
resulting aqueous solution is measured.
3.0 INTERFERENCES
3.1 Samples with very low or very high pH may give incorrect
readings on the meter. For samples with a true pH of >10, the measured pH may
be incorrectly low. This error can be minimized by using a low-sodium-error
electrode. Strong acid solutions, with a true pH of <1, may give incorrectly
high pH measurements.
3.2 Temperature fluctuations will cause measurement errors.
3.3 Errors will occur when the electrodes become coated. If an
electrode becomes coated with an oily material that will not rinse free, the
electrode can (1) be cleaned with an ultrasonic bath, or (2) be washed with
detergent, rinsed several times with water, placed in 1:10 HC1 so that the
lower third of the electrode is submerged, and then thoroughly rinsed with
water, or (3) be cleaned per the manufacturer's instructions.
4.0 APPARATUS AND MATERIALS
4.1 pH Meter with means for temperature compensation.
4.2 Glass Electrode.
4.3 Reference electrode: A silver-silver chloride or other
reference electrode of constant potential may be used.
NOTE: Combination electrodes incorporating both measuring and
referenced functions are convenient to use and are available
with solid, gel-type filling materials that require minimal
maintenance.
4.4 Beaker: 50-mL.
4.5 Thermometer and/or temperature sensor for automatic
compensation.
4.6 Analytical balance: capable of weighing 0.1 g.
9045B - 1 Revision 2
September 1994
-------
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American
Chemical Society, where such specifications are available. Other grades may
be used, provided it is first ascertained that the reagent is of sufficiently
high purity to permit its use without lessening the accuracy of the
determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Primary standard buffer salts are available from the National
Institute of Standards and Technology (NIST) and should be used in situations
where extreme accuracy is necessary. Preparation of reference solutions from
these salts requires some special precautions and handling, such as low-
conductivity dilution water, drying ovens, and carbon-dioxide-free purge gas.
These solutions should be replaced at least once each month.
5.4 Secondary standard buffers may be prepared from NIST salts or
purchased as solutions from commercial vendors. These commercially available
solutions, which have been validated by comparison with NIST standards, are
recommended for routine use.
6.0 SAMPLE PRESERVATION AND HANDLING
6.1 All samples must be collected using a sampling plan that
addresses the considerations discussed in Chapter Nine of this manual.
6.2 Samples should be analyzed as soon as possible.
7.0 PROCEDURE
7.1 Calibration:
7.1.1 Because of the wide variety of pH meters and
accessories, detailed operating procedures cannot be incorporated into
this method. Each analyst must be acquainted with the operation of each
system and familiar with all instrument functions. Special attention to
care of the electrodes is recommended.
7.1.2 Each instrument/electrode system must be calibrated at a
minimum of two points that bracket the expected pH of the samples and
are approximately three pH units or more apart. Repeat adjustments on
successive portions of the two buffer solutions until readings are
within 0.05 pH units of the buffer solution value.
7.2 Sample preparation and pH measurement of soils:
7.2.1 To 20 g of soil in a 50-mL beaker, add 20 mL of reagent
water, cover, and continuously stir the suspension for 5 minutes.
9045B - 2 Revision 2
September 1994
-------
Additional dilutions are allowed if working with hygroscopic soils and
salts or other problematic matrices.
7.2.2 Let the soil suspension stand for about 1 hour to allow
most of the suspended clay to settle out from the suspension or filter
or centrifuge off the aqueous phase for pH measurement.
7.2.3 Adjust the electrodes in the clamps of the electrode
holder so that, upon lowering the electrodes into the beaker, the glass
electrode will be immersed just deep enough into the clear supernatant
solution to establish a good electrical contact through the ground-glass
joint or the fiber-capillary hole. Insert the electrodes into the
sample solution in this manner. For combination electrodes, immerse
just below the suspension.
7.2.4 If the sample temperature differs by more than 2°C from
the buffer solution, the measured pH values must be corrected.
7.2.5 Report the results as "soil pH measured in water at
°C" where " °C" is the temperature at which the test was conducted.
7.3 Sample preparation and pH measurement of waste materials:
7.3.1 To 20 g of waste sample in a 50-mL beaker, add 20 ml of
reagent water, cover, and continuously stir the suspension for 5
minutes. . Additional dilutions are allowed if working with hygroscopic
wastes and salts or other problematic matrices.
7.3.2 Let the waste suspension stand for about 15 minutes to
allow most of the suspended waste to settle out from the suspension or
filter or centrifuge off aqueous phase for pH measurement.
NOTE: If the waste is hygroscopic and absorbs all the reagent
water, begin the experiment again using 20 g of waste and 40 mL
of reagent water.
NOTE: If the supernatant is multiphasic, decant the oily phase
and measure the pH of the aqueous phase. The electrode may need
to be cleaned (Step 3.3) if it becomes coated with an oily
material.
7.3.3 Adjust the electrodes in the clamps of the electrode
holder so that, upon lowering the electrodes into the beaker, the glass
electrode will be immersed just deep enough into the clear supernatant
to establish good electrical contact through the ground-glass joint or
the fiber-capillary hole. Insert the electrode into the sample solution
in this manner. For combination electrodes, immerse just below the
suspension.
7.3.4 If the sample temperature differs by more than 2°C from
the buffer solution, the measured pH values must be corrected.
7.3.5 Report the results as "waste pH measured in water at
°C" where " °C" is the temperature at which the test was conducted.
9045B - 3 Revision 2
September 1994
-------
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for the appropriate QC protocols.
8.2 Electrodes must be thoroughly rinsed between samples.
9.0 METHOD PERFORMANCE
9.1 No data provided.
10.0 REFERENCES
1. Black, Charles Allen; Methods of Soil Analysis; American Society of
Agronomy: Madison, WI, 1973.
2. National Bureau of Standards, Standard Reference Material Catalog, 1986-
87, Special Publication 260.
9045B - 4 Revision 2
September 1994
-------
METHOD 90458
SOIL AND WASTE pH
f Start J
7.1 Calibrate
each instrument/
electrode
system.
7.2.1 Add 20 mL
water to 20 g soil;
stir continuously
for 5 minutes.
7.3.1 Add 20 mL
water to 20 g waste;
stir continuously
for 5 minutes.
7.2.2 Let soil
suspension
stand for 1
hour or filter.
7.3.2 Let waste
suspension
stand for 15
minutes or filter.
Insert
electrodes
into sample
solution.
Do
sample
and buffer
sol'n temps
vary by
2C?
Correct
measured pH
values.
Report
results and
temperature
Is
supernatant
multiphasic?
Repeat experiment
with 20 g waste
and 40 mL water.
Decant oily
phase;
measure pH of
aqueous phase.
Aqueous
Phase
9045B - 5
Revision 2
September 1994
-------
o
tn
o
-------
METHOD 9050
SPECIFIC CONDUCTANCE
1.0 SCOPE AND APPLICATION
1.1 Method 9050 1s used to measure the specific conductance of drinking,
ground, surface, and saline waters and domestic and Industrial aqueous wastes.
Method 9050 1s not applicable to solid samples.
2.0 SUMMARY OF METHOD
2.1 The specific conductance of a sample 1s measured using a self-
contained conductivity meter (Wheatstone bridge-type or equivalent).
2.2 Whenever possible, samples are analyzed at 25*C. If samples are
analyzed at different temperatures, temperature corrections must be made and
results reported at 25*C.
3.0 INTERFERENCES
3.1 Platinum electrodes can degrade and cause erratic results. When
this happens, as evidenced by erratic results or flaking off of the platinum
black, the electrode should be replatlnlzed.
3.2 The specific conductance cell can become coated with oil and other
materials. It 1s essential that the cell be thoroughly rinsed and, 1f
necessary, cleaned between samples.
4.0 APPARATUS AND MATERIALS
4.1 Self-contained conductivity Instruments; an Instrument consisting
of a source of alternating current, a Wheatstone bridge, null Indicator, and a
conductivity cell or other Instrument measuring the ratio of alternating
current through the cell to voltage across 1t. The latter has the advantage
of a linear reading of conductivity. Choose an Instrument capable of
measuring conductivity with an error not exceeding IX or 1 umho/cm, whichever
1s greater.
4.2 Platinum-electrode or non-piatlnum-electrode specific conductance
cell.
4.3 Water bath.
4.4 Thermometer; capable of being read to the nearest 0.1*C and
covering the range 23* to 27*C. An electrical thermometer having a small
thermistor sensing element 1s convenient because of Its rapid response.
9050 - 1
Revision 0
Date September 1986
-------
5.0 REAGENTS
5.1 Conductivity water; Pass distilled water through a mixed-bed
delonlzer and discard first1,000 ml. Conductivity should be less than 1
umho/cm.
5.2 Standard potassium chloride (0.0100 M): Dissolve 0.7456 g anhydrous
KC1 1n conductivity water and make up to 1,000 ml at 25*C. This solution will
have a specific conductance of 1,413 umho/cm at 25*C.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed 1n Chapter Nine of this manual.
6.2 All sample containers must be prewashed and thoroughly rinsed. Both
plastic and glass containers are suitable.
6.3 Aqueous samples should be stored at 4'C and analyzed within 24 hr.
7.0 PROCEDURE
7.1 Determination of cell constant; Rinse conductivity cell with at
least three portions of 0.01 NKClsolution. Adjust temperature of a fourth
portion to 25.0 + 0.1*C. Measure resistance of this portion and note
temperature. Compute cell constant, C:
c = (o.001413)(RKCI) 1 + 0.0191 (t - 25)
where;
RKC1 = measured resistance, ohms; and
t = observed temperature, *C.
7.2 Conductivity measurement; Rinse cell with one or more portions of
sample. Adjust temperatureofa final portion to 25.0 + 0.1*C. Measure
sample resistance or conductivity and note temperature.
7.3 Calculation: The temperature coefficient of most waters is only
approximately thesame as that of standard KCl solution; the more the
temperature of measurement deviates from 25.0*C, the greater the uncertainty
in applying the temperature correction. Report all conductivities at 25.0*C.
9050 - 2
Revision
Date September 1986
-------
7.3.1 When sample resistance is measured, conductivity at 25*C 1s:
v fl.OOO.OOOUC)
* " Rm 1 + 0.0191 (t - 25)
where:
K = conductivity, umho/cm;
C = cell constant, cm-L;
Rm = measured resistance of sample, ohms; and
t - temperature of measurement.
7.3.2 When sample conductivity is measured, conductivity at 25*C
is:
(iga. ooo.ooo) (c)
K = 1 + 0.0191 (t - 25)
where:
Km = measured conductivity, umho at t*C, and other units
are defined as above.
NOTE: If conductivity readout is in umho/cm, delete the factor 1,000,000
in the numerator.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or inspection.
8.2 Analyze check standards after approximately every 15 samples.
8.3 Run 1 duplicate sample for every 10 samples.
9.0 METHOD PERFORMANCE
9.1 Three synthetic samples were tested with the following results:
Conduc-
tivity
umhos/cm
147.0
303.0
228.0
No. of
Results
117
120
120
Relative
Standard
Deviation
%
8.6
7.8
8.4
Relative
Error
%
9.4
1.9
3.0
9050 - 3
Revision
Date September 1986
-------
10.0 REFERENCES
1. Standard Methods for the Examination of Water and Wastewater, 16th ed.
(1985), Method 205.
9050 - 4
Revision
Date September 1986
-------
SPECIFIC CONDUCTANCE
7. 1
r
• no te
tolutl
call
Measure
es istance
mp of KC1
on: calc.
constant
7.2
red
cpr
1
ten
Measure
•ample
•tanca or
lOuct J vlty
ind note
iipcrature
7.3
Calculate
•ample
conductivity
at ZS *C
{ Stop J
9050 - 5
Revision 0
Date September 1986
-------
so
o
(J\
OS
-------
METHOD 9056
DETERMINATION OF INORGANIC ANIONS BY ION CHROMATOGRAPHY
1.0 SCOPE AND APPLICATION
1.1 This method addresses the sequential determination of the anions
chloride, fluoride, bromide, nitrate, nitrite, phosphate, and sulfate in the
collection solutions from the bomb combustion of solid waste samples, as well as
all water samples.
1.2 The method detection limit (MDL), the minimum concentration of a
substance that can be measured and reported with 99% confidence that the value
is above zero, varies for anions as a function of sample size and the
conductivity scale used. Generally, minimum detectable concentrations are in the
range of 0.05 mg/L for F' and 0.1 mg/L for Br', CV, N03", N02", P043', and S042' with
a 100-/nL sample loop and a 10-^mho full-scale setting on the conductivity
detector. Similar values may be achieved by using a higher scale setting and an
electronic integrator. Idealized detection limits of an order of magnitude lower
have been determined in reagent water by using a l-/imho/cm full-scale setting
(Table 1). The upper limit of the method is dependent on total anion
concentration and may be determined experimentally. These limits may be extended
by appropriate dilution.
2.0 SUMMARY OF METHOD
2.1 A small volume of combustate collection solution or other water
sample, typically 2 to 3 ml, is injected into an ion chromatograph to flush and
fill a constant volume sample loop. The sample is then injected into a stream
of carbonate-bicarbonate eluent of the same strength as the collection solution
or water sample.
2.2 The sample is pumped through three different ion exchange columns and
into a conductivity detector. The first two columns, a precolumn or guard column
and a separator column, are packed with low-capacity, strongly basic anion
exchanger. Ions are separated into discrete bands based on their affinity for
the exchange sites of the resin. The last column is a suppressor column that
reduces the background conductivity of the eluent to a low or negligible level
and converts the anions in the sample to their corresponding acids. The
separated anions in their acid form are measured using an electrical-conductivity
cell. Anions are identified based on their retention times compared to known
standards. Quantitation is accomplished by measuring the peak height or area and
comparing it to a calibration curve generated from known standards.
3.0 INTERFERENCES
3.1 Any species with a retention time similar to that of the desired ion
will interfere. Large quantities of ions eluting close to the ion of interest
will also result in an interference. Separation can be improved by adjusting the
eluent concentration and/or flow rate. Sample dilution and/or the use of the
method of standard additions can also be used. For example, high levels of
organic acids may be present in industrial wastes, which may interfere with
9056 - 1 Revision 0
September 1994
-------
inorganic anion analysis. Two common species, formate and acetate, elute between
fluoride and chloride.
3.2 Because bromide and nitrate elute very close together, they are
potential interferences for each other. It is advisable not to have Br"/N03
ratios higher than 1:10 or 10:1 if both anions are to be quantified. If nitrate
is observed to be an interference with bromide, use of an alternate detector
(e.g., electrochemical detector) is recommended.
3.3 Method interferences may be caused by contaminants in the reagent
water, reagents, glassware, and other sample processing apparatus that lead to
discrete artifacts or elevated baseline in ion chromatograms.
3.4 Samples that contain particles larger than 0.45 urn and reagent
solutions that contain particles larger than 0.20 fj.m require filtration to
prevent damage to instrument columns and flow systems.
3.5 If a packed bed suppressor column is used, it will be slowly consumed
during analysis and, therefore, will need to be regenerated. Use of either an
anion fiber suppressor or an anion micromembrane suppressor eliminates the time-
consuming regeneration step through the use of a continuous flow of regenerant.
4.0 APPARATUS AND MATERIALS
4.1 Ion chromatograph, capable of delivering 2 to 5 ml of eluent per
minute at a pressure of 200 to 700 psi (1.3 to 4.8 MPa). The chromatograph shall
be equipped with an injection valve, a 100-jitL sample loop, and set up with the
following components, as schematically illustrated in Figure 1.
4.1.1 Precolumn, a guard column placed before the separator column
to protect the separator column from being fouled by particulates or
certain organic constituents (4 x 50 mm, Dionex P/N 030825 [normal run],
or P/N 030830 [fast run], or equivalent).
4.1.2 Separator column, a column packed with low-capacity
pellicular anion exchange resin that is styrene divinylbenzene-based has
been found to be suitable for resolving F", Cl", N02', P04"3, Br", N03", and
S04'2 (see Figure 2) (4 x 250 mm, Dionex P/N 03827 [normal run], or P/N
030831 [fast run], or equivalent).
4.1.3 Suppressor column, a column that is capable of converting
the eluent and separated anions to their respective acid forms (fiber,
Dionex P/N 35350, micromembrane, Dionex P/N 38019 or equivalent).
4.1.4 Detector, a low-volume, flowthrough, temperature-
compensated, electrical conductivity cell (approximately 6 /zL volume,
Dionex, or equivalent) equipped with a meter capable of reading from 0 to
1,000 /^seconds/cm on a linear scale.
4.1.5 Pump, capable of delivering a constant flow of approximately
2 to 5 mL/min throughout the test and tolerating a pressure of 200 to
700 psi (1.3 to 4.8 MPa).
9056 - 2 Revision 0
September 1994
-------
4.2 Recorder, compatible with the detector output with a full-scale
response time in 2 seconds or less.
4.3 Syringe, minimum capacity of 2 mL and equipped with a male pressure
fitting.
4.4 Eluent and regenerant reservoirs, suitable containers for storing
eluents and regenerant. For example, 4 L collapsible bags can be used.
4.5 Integrator, to integrate the area under the chromatogram. Different
integrators can perform this task when compatible with the electronics of the
detector meter or recorder. If an integrator is used, the maximum area
measurement must be within the linear range of the integrator.
4.6 Analytical balance, capable of weighing to the nearest 0.0001 g.
4.7 Pipets, Class A volumetric flasks, beakers: assorted sizes.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless otherwise
indicated, it is intended that all reagents shall conform to the specifications
of the Committee on Analytical Reagents of the American Chemical Society, where
such specifications are available. Other grades may be used, provided it is
first ascertained that the reagent is of sufficiently high purity to permit its
use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One. Column life may be extended by passing
reagent water through a 0.22-jim filter prior to use.
5.3 Eluent, 0.003M NaHC03/0.0024M Na2C03. Dissolve 1.0080 g of sodium
bicarbonate (0.003M NaHC03) and 1.0176 g of sodium carbonate (0.0024M Na2C03) in
reagent water and dilute to 4 L with reagent water.
5.4 Suppressor regenerant solution. Add 100 ml of IN H2S04 to 3 L of
reagent water in a collapsible bag and dilute to 4 L with reagent water.
5.5 Stock solutions (1,000 mg/L).
5.5.1 Bromide stock solution (1.00 ml = 1.00 mg Br"). Dry
approximately 2 g of sodium bromide (NaBr) for 6 hours at 150°C, and cool
in a desiccator. Dissolve 1.2877 g of the dried salt in reagent water,
and dilute to 1 L with reagent water.
5.5.2 Chloride stock solution (1.00 ml = 1.00 mg CT). Dry sodium
chloride (NaCl) for 1 hour at 600°C, and cool in a desiccator. Dissolve
1.6484 g of the dry salt in reagent water, and dilute to 1 L with reagent
water.
5.5.3 Fluoride stock solution (1.00 ml = 1.00 mg F"). Dissolve
2.2100 g of sodium fluoride (NaF) in reagent water, and dilute to 1 L with
reagent water. Store in chemical-resistant glass or polyethylene.
9056 - 3 Revision 0
September 1994
-------
5.5.4 Nitrate stock solution (1.00 ml = 1.00 mg N03"). Dry
approximately 2 g of sodium nitrate (NaN03) at 105°C for 24 hours.
Dissolve exactly 1.3707 g of the dried salt in reagent water, and dilute
to 1 L with reagent water.
5.5.5 Nitrite stock solution (1.00 mL = 1.00 mg N02"). Place
approximately 2 g of sodium nitrate (NaN02) in a 125 ml beaker and dry to
constant weight (about 24 hours) in a desiccator containing concentrated
H2S04. Dissolve 1.4998 g of the dried salt in reagent water, and dilute
to 1 L with reagent water. Store in a sterilized glass bottle.
Refrigerate and prepare monthly.
NOTE: Nitrite is easily oxidized, especially in the presence of
moisture, and only fresh reagents are to be used.
NOTE: Prepare sterile bottles for storing nitrite solutions by
heating for 1 hour at 170°C in an air oven.
5.5.6 Phosphate stock solution (1.00 mL = 1.00 mg P043'). Dissolve
1.4330 g of potassium dihydrogen phosphate (KH2P04) in reagent water, and
dilute to 1 L with reagent water. Dry sodium sulfate (Na2S04) for 1 hour
at 105°C and cool in a desiccator.
5.5.7 Sulfate stock solution (1.00 mL = 1.00 mg S042'). Dissolve
1.4790 g of the dried salt in reagent water, and dilute to 1 L with
reagent water.
5.6 Anion working solutions. Prepare a blank and at least three
different working solutions containing the following combinations of anions. The
combination anion solutions must be prepared in Class A volumetric flasks. See
Table 2.
5.6.1 Prepare a high-range standard solution by diluting the
volumes of each anion specified in Table 2 together to 1 L with reagent
water.
5.6.2 Prepare the intermediate-range standard solution by diluting
10.0 mL of the high-range standard solution (see Table 2) to 100 mL with
reagent water.
5.6.3 Prepare the low-range standard solution by diluting 20.0 mL
of the intermediate-range standard solution (see Table 2) to 100 mL with
reagent water.
5.7 Stability of standards. Stock standards are stable for at least 1
month when stored at 4°C. Dilute working standards should be prepared weekly,
except those that contain nitrite and phosphate, which should be prepared fresh
daily.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed in Chapter Nine of this manual.
9056 - 4 Revision 0
September 1994
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6.2 Analyze the samples as soon as possible after collection. Preserve
by refrigeration at 4°C.
7.0 PROCEDURE
7.1 Calibration
7.1.1 Establish ion chromatographic operating parameters
equivalent to those indicated in Table 1.
7.1.2 For each analyte of interest, prepare calibration standards
at a minimum of three concentration levels and a blank by adding
accurately measured volumes of one or more stock standards to a Class A
volumetric flask and diluting to volume with reagent water. If the
working range exceeds the linear range of the system, a sufficient number
of standards must be analyzed to allow an accurate calibration curve to be
established. One of the standards should be representative of a concen-
tration near, but above, the method detection limit if the system is
operated on an applicable attenuator range. The other standards should
correspond to the range of concentrations expected in the sample or should
define the working range of the detector. Unless the attenuator range
settings are proven to be linear, each setting must be calibrated
individually.
7.1.3 Using injections of 0.1 to 1.0 ml (determined by injection
loop volume) of each calibration standard, tabulate peak height or area
responses against the concentration. The results are used to prepare a
calibration curve for each analyte. During this procedure, retention
times must be recorded.
7.1.4 The working calibration curve must be verified on each
working day, or whenever the anion eluent strength is changed, and for
every batch of samples. If the response or retention time for any analyte
varies from the expected values by more than + 10%, the test must be
repeated, using fresh calibration standards. If the results are still
more than + 10%, an entirely new calibration curve must be prepared for
that analyte.
7.1.5 Nonlinear response can result when the separator column
capacity is exceeded (overloading). Maximum column loading (all anions)
should not exceed about 400 ppm.
7.2 Analyses
7.2.1 Sample preparation. When aqueous samples are injected, the
water passes rapidly through the columns, and a negative "water dip" is
observed that may interfere with the early-eluting fluoride and/or
chloride ions. The water dip should not be observed in the combustate
samples; the collecting solution is a concentrated eluent solution that
will "match" the eluent strength when diluted to 100-mL with reagent water
according to the bomb combustion procedure. Any dilutions required in
analyzing other water samples should be made with the eluent solution.
The water dip, if present, may be removed by adding concentrated eluent to
9056 - 5 Revision 0
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all samples and standards. When a manual system is used, it is necessary
to micropipet concentrated buffer into each sample. The recommended
procedures follow:
(1) Prepare a 100-mL stock of eluent 100 times normal concentration by
dissolving 2.5202 g NaHC03 and 2.5438 g Na2C03 in 100-mL reagent
water. Protect the volumetric flask from air.
(2) Pipet 5 ml of each sample into a clean polystyrene micro-beaker.
Micropipet 50 juL of the concentrated buffer into the beaker and stir
well.
Dilute the samples with eluent, if necessary, to concentrations within the
linear range of the calibration.
7.2.2 Sample analysis.
7.2.2.1 Start the flow of regenerant through the
suppressor column.
7.2.2.2 Set up the recorder range for maximum sensitivity
and any additional ranges needed.
7.2.2.3 Begin to pump the eluent through the columns.
After a stable baseline is obtained, inject a midrange standard. If
the peak height deviates by more than 10% from that of the previous
run, prepare fresh standards.
7.2.2.4 Begin to inject standards starting with the
highest concentration standard and decreasing in concentration. The
first sample should be a quality control reference sample to check
the calibration.
7.2.2.5 Using the procedures described in Step 7.2.1,
calculate the regression parameters for the initial standard curve.
Compare these values with those obtained in the past. If they
exceed the control limits, stop the analysis and look for the
problem.
7.2.2.6 Inject a quality control reference sample. A
spiked sample or a sample of known content must be analyzed with
each batch of samples. Calculate the concentration from the
calibration curve and compare the known value. If the control
limits are exceeded, stop the analysis until the problem is found.
Recalibration is necessary.
7.2.2.7 When an acceptable value has been obtained for
the quality control sample, begin to inject the samples.
7.2.2.8 Load and inject a fixed amount of well-mixed
sample. Flush injection loop thoroughly, using each new sample.
Use the same size loop for standards and samples. Record the
9056 - 6 Revision 0
September 1994
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resulting peak size in area or peak height units. An automated
constant volume injection system may also be used.
7.2.2.9 The width of the retention time window used to
make identifications should be based on measurements of actual
retention time variations of standards over the course of a day.
Three times the standard deviation of a retention time can be used
to calculate a suggested window size for a compound. However, the
experience of the analyst should weigh heavily in the interpretation
of chromatograms.
7.2.2.10 If the response for the peak exceeds the working
range of the system, dilute the sample with an appropriate amount of
reagent water and reanalyze.
7.2.2.11 If the resulting chromatogram fails to produce
adequate resolution, or if identification of specific anions is
questionable, spike the sample with an appropriate amount of
standard and reanalyze.
NOTE: Nitrate and sulfate exhibit the greatest amount of change,
although all anions are affected to some degree. In some cases,
this peak migration can produce poor resolution or
mis identification.
7.3 Calculation
7.3.1 Prepare separate calibration curves for each anion of
interest by plotting peak size in area, or peak height units of standards
against concentration values. Compute sample concentration by comparing
sample peak response with the standard curve.
7.3.2 Enter the calibration standard concentrations and peak
heights from the integrator or recorder into a calculator with linear
least squares capabilities.
7.3.3 Calculate the following parameters: slope (s), intercept
(I), and correlation coefficient (r). The slope and intercept define a
relationship between the concentration and instrument response of the
form:
Yi = s, xs + I (1)
where:
y; = predicted instrument response
Sj = response slope
Xj = concentration of standard i
I = intercept
9056 - 7 Revision 0
September 1994
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Rearrangement of the above equation yields the concentration corresponding
to an instrumental measurement:
X; - (y, - D/s, (2)
where:
Xj = calculated concentration for a sample
YJ = actual instrument response for a sample
Sj and I are calculated slope and intercept from calibration above.
7.3.4 Enter the sample peak height into the calculator, and
calculate the sample concentration in milligrams per liter.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference and inspection. Refer to Chapter One for additional quality control
guidelines.
8.2 After every 10 injections, analyze a midrange calibration standard.
If the instrument response has changed by more than 5%, recalibrate.
8.3 Analyze one in every ten samples in duplicate. Take the duplicate
sample through the entire sample preparation and analytical process.
8.4 A matrix spiked sample should be run for each analytical batch or
twenty samples, whatever is more frequent, to determine matrix effects.
9.0 METHOD PERFORMANCE
9.1 Single-operator accuracy and precision for reagent, drinking and
surface water, and mixed domestic and industrial wastewater are listed in Table
3.
9.2 Combustate samples. These data are based on 41 data points obtained
by six laboratories who each analyzed four used crankcase oils and three fuel oil
blends with crankcase in duplicate. The oil samples were combusted using Method
5050. A data point represents one duplicate analysis of a sample. One data
point was judged to be an outlier and was not included in the results.
9.2.1 Precision. The precision of the method as determined by the
statistical examination of interlaboratory test results is as follows:
Repeatability - The difference between successive results obtained
by the sample operator with the same apparatus under constant operating
conditions on identical test material would exceed, in the long run, in
the normal and correct operation of the test method, the following values
only in 1 case in 20 (see Table 4):
*where x is the average of two results in /ig/g.
9056 - 8 Revision 0
September 1994
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Repeatability =20.9
Reproducibilitv - The difference between two single and independent
results obtained by different operators working in different laboratories
on identical test material would exceed, in the long run, the following
values only in 1 case in 20:
Reproducibility =42.1
*where x is the average value of two results in /ug/g.
9.2.2 Bias. The bias of this method varies with concentration,
as shown in Table 5:
Bias = Amount found - Amount expected
10.0 REFERENCES
1. Environmental Protection Agency. Test Method for the Determination of
Inorganic Anions in Water by Ion Chromatography. EPA Method 300.0. EPA-600/4-
84-017. 1984.
2. Annual Book of ASTM Standards, Volume 11.01 Water D4327, Standard Test
Method for Anions in Water by Ion Chromatography, pp. 696-703. 1988.
3. Standard Methods for the Examination of Water and Wastewater, Method 429,
"Determination of Anions by Ion Chromatography with Conductivity Measurement,"
16th Edition of Standard Methods.
4. Dionex, 1C 16 Operation and Maintenance Manual, PN 30579, Dionex Corp.,
Sunnyvale, CA 94086.
5. Method detection limit (MDL) as described in "Trace Analyses for
Wastewater," J. Glaser, D. Foerst, G. McKee, S. Quave, W. Budde, Environmental
Science and Technology, Vol. 15, Number 12, p. 1426, December 1981.
6. Gaskill, A.; Estes, E. D.; Hardison, D. L.; and Myers, L. E. Validation
of Methods for Determining Chlorine in Used Oils and Oil Fuels. Prepared for
U.S. Environmental Protection Agency Office of Solid Waste. EPA Contract No. 68-
01-7075, WA 80. July 1988.
9056 - 9 Revision 0
September 1994
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TABLE 1.
CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION
LIMITS IN REAGENT WATER
Analyte
Fluoride
Chlorine
Nitrite-N
o-Phosphate-P
Nitrate-N
Sulfate
Retention8
time
min
1.2
3.4
4.5
9.0
11.3
21.4
Relative
retention
time
1.0
2.8
3.8
7.5
9.4
17.8
Method6
detection limit,
mg/L
0.005
0.015
0.004
0.061
0.013
0.206
Standard conditions:
Columns - As specified in 4.1.1-4.1.3
Detector - As specified in 4.1.4
Eluent - As specified in 5.3
Concentrations of mixed standard (mg/L)
Fluoride 3.0
Chloride 4.0
Nitrite-N 10.0
Sample loop - 100 /xL
Pump volume - 2.30 mL/min
o-Phosphate-P 9.0
Nitrate-N 30.0
Sulfate 50.0
"The retention time given for each anion is based on the equipment and analytical
conditions described in the method. Use of other analytical columns or different
elutant concentrations will effect retention times accordingly.
bMDL calculated from data obtained using an attentuator setting of 1-jumho/ctn full
scale. Other settings would produce an MDL proportional to their value.
9056 - 10
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September 1994
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TABLE 2.
PREPARATION OF STANDARD SOLUTIONS FOR INSTRUMENT CALIBRATION
High
Range
Standard1
Fluoride (F")
Chloride (CV)
Nitrite (N02')
Phosphate (P043-)
Bromide (Br')
Nitrate (N03-J
Sulfate (S042')
10
10
20
50
10
30
100
An ion
concentration
mg/L
10
10
20
50
10
30
100
Intermediate-
range standard,
mg/L
(see 5.6.2)
1.0
1.0
2.0
5.0
1.0
3.0
10.0
Low-range
standard,
mg/L (see
5.6.3)
0.2
0.2
0.4
1.0
0.2
0.6
2.0
1Milliliters of each stock solution (1.00 mL = 1.00 mg) diluted to 1 L (see sec.
5.6.1).
9056 - 11
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September 1994
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TABLE 3.
SINGLE-OPERATOR ACCURACY AND PRECISION
Sample
Analyte type
Chloride
Fluoride
Nitrate-N
Nitrite-N
o-Phosphate-P
Sulfate
RW
DM
SW
WW
RW
DW
SW
WW
RW
DW
SW
WW
RW
DW
SW
WW
RW
DE
SW
WW
RW
DW
SW
WW
Spike
mg/L
0.050
10.0
1.0
7.5
0.24
9.3
0.50
1.0
0.10
31.0
0.50
4.0
0.10
19.6
0.51
0.52
0.50
45.7
0.51
4.0
1.02
98.5
10.0
12.5
Number
of
replicates
7
7
7
7
7
7
7
7
7
7
7.
7
7
7
7
7
7
7
7
7
7
7
7
7
Mean
recovery,
%
97.7
98.2
105.0
82.7
103.1
87.7
74.0
92.0
100.9
100.7
100.0
94.3
97.7
103.3
88.2
100.0
100.4
102.5
94.1
97.3
102.1
104.3
111.6
134.9
Standard
deviation,
mg/L
0.0047
0.289
0.139
0.445
0.0009
0.075
0.0038
0.011
0.0041
0.356
0.0058
0.058
0.0014
0.150
0.0053
0.018
0.019
0.386
0.020
0.04
0.066
1.475
0.709
0.466
RW = Reagent water.
DW = Drinking water.
SW = Surface water.
WW = Wastewater.
9056 - 12
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September 1994
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TABLE 4.
REPEATABILITY AND REPRODUCIBILITY FOR CHLORINE IN
USED OILS BY BOMB OXIDATION AND ION CHROMATOGRAPHY
Average value, Repeatability, Reproducibility,
M9/9 M9/9
500
1,000
1,500
2,000
2,500
3,000
467
661
809
935
1,045
1,145
941
1,331
1,631
1,883
2,105
2,306
TABLE 5.
RECOVERY AND BIAS DATA FOR CHLORINE IN USED OILS BY
BOMB OXIDATION AND ION CHROMATOGRAPHY
Amount
Expected
Atg/9
Amount
found
M9/9
Bias,
M9/9
Percent,
bias
320 567 247 +77
480 773 293 +61
920 1,050 130 +14
1,498 1,694 196 +13
1,527 1,772 245 +16
3,029 3,026 -3 0
3,045 2,745 -300 -10
9056 - 13 Revision 0
Septent)er 1994
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FIGURE 1
SCHEMATIC OF ION CHROMATOGRAPH
WASTE
(1) Eluent reservoir
(2) Pump
(3) Precolumn
(4) Separator column
(5) Suppressor column
(6) Detector
(7) Recorder or integrator, or both
9056 - 14
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September 1994
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FIGURE 2
TYPICAL ANION PROFILE
so;'
u
MINUTES
9056 - 15
Revision 0
Septenter 1994
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[ Start j
METHOD 9056
DETERMINATION OF INORGANIC ANIONS BY ION CHROMATOGRAPHY
1
7.1.1 Establish ion
chromatographic
operating
parameters.
v
7.1.2 Prepare
calibration
standards at a
minimum of three
concentration
levels and a blank.
V
7.1 .3 Prepare
calibration curve.
V
7.1.4 Verify the
calibration curves
each working day or
whenever the anion
eluent is changed,
and for every batch
of samples.
|
/ ^^ 7.2.1 If a dilution
/7.2.1 Are\Aqueous '* "e^"""l,rv '£*
/samples aqueou>S_ w, dilution should
X of extracts?/ ^ be made with
N. / eluent solution.
[Extracts
7.2.2 Analyze
standards beginning
with the highest
concentration and
decreasing in
concentration.
V
7.2.1 Add
concentrated
^_ «lu»nt to all
"^ samples and
standards to
remove water dip.
>,
7.2.2.5 Compare
results to
calibration curve;
if results exceed
control limits,
identify problem
before proceeding.
V
7.2.2.6 Inject a
spiked sample of
known cone.;
calculate the cone.
from the calibration
curve; if result
exceeds control
limits, find problem
before proceeding.
4
7.2.2.7 Begin
sample analysis.
\r
7.2.2.8 Analyze all
samples in same
manner.
A
/7.2.2.10\
/ Does responseV
( for peak exceed
>v working /
^^ range? /
|No
V
7.3.1 Prepare
sample calibration
curves for each
anion of interest
and compute sample
concentration.
•^
7.2.2.10 Dilute
i es w sample with
reagent water.
7.3.3 Calculate
w concentrations
* from mstrumenial
response.
1
( Stop J
9056 - 16
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Septaiter 1994
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o
O\
O
-------
METHOD 9060
TOTAL ORGANIC CARBON
1.0 SCOPE AND APPLICATION
1.1 Method 9060 1s used to determine the concentration of organic carbon
1n ground water, surface and saline waters, and domestic and Industrial
wastes. Some restrictions are noted 1n Sections 2.0 and 3.0.
1.2 Method 9060 1s most applicable to measurement of organic carbon
above 1 mg/L.
2.0 SUMMARY OF METHOD
2.1 Organic carbon 1s measured using a carbonaceous analyzer. This
Instrument converts the organic carbon 1n a sample to carbon dioxide (C02) by
either catalytic combustion or wet chemical oxidation. The C02 formed 1s then
either measured directly by an Infrared detector or converted to methane (CH4)
and measured by a flame 1on1zat1on detector. The amount of C0£ or Cfy in a
sample 1s directly proportional to the concentration of carbonaceous material
1n the sample.
2.2 Carbonaceous analyzers are capable of measuring all forms of carbon
1n a sample. However, because of various properties of carbon-containing
compounds 1n liquid samples, the manner of preliminary sample treatment as
well as the Instrument settings will determine which forms of carbon are
actually measured. The forms of carbon that can be measured by Method 9060
are:
1. Soluble, nonvolatile organic carbon: e.g., natural sugars.
2. Soluble, volatile organic carbon: e.g., mercaptans, alkanes, low
molecular weight alcohols.
3. Insoluble, partially volatile carbon: e.g., low molecular weight
oils.
4. Insoluble, partlculate carbonaceous materials: e.g., cellulose
fibers.
5. Soluble or Insoluble carbonaceous materials adsorbed or entrapped
on Insoluble Inorganic suspended matter: e.g., oily matter adsorbed
on silt particles.
2.3 Carbonate and bicarbonate are Inorganic forms of carbon and must be
separated from the total organic carbon value. Depending on the Instrument
manufacturer's Instructions, this separation can be accomplished by either a
simple mathematical subtraction, or by removing the carbonate and bicarbonate
by converting them to C02 with degassing prior to analysis.
9060 - 1
Revision 0
Date September 1986
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3.0 INTERFERENCES
3.1 Carbonate and bicarbonate carbon represent an Interference under the
terms of this test and must be removed or accounted for 1n the final calcula-
tion.
3.2 This procedure 1s applicable only to homogeneous samples which can
be Injected Into the apparatus reprodudbly by means of a mlcrolHer-type
syringe or plpet. The openings of the syringe or plpet limit the maximum size
of particle which may be Included 1n the sample.
3.3 Removal of carbonate and bicarbonate by acidification and purging
with nitrogen, or other Inert gas, can result In the loss of volatile organic
substances.
4.0 APPARATUS AND MATERIALS
4.1 Apparatus for blending or homogenizing samples; Generally, a
War1ng-type blender 1s satisfactory.
4.2 Apparatus for total and dissolved organic carbon;
4.2.1 Several companies manufacture analyzers for measuring
carbonaceous material 1n liquid samples. The most appropriate system
should be selected based on consideration of the types of samples to be
analyzed, the expected concentration range, and the forms of carbon to be
measured.
4.2.2 No specific analyzer 1s recommended as superior. If the
technique of chemical oxidation 1s used, the laboratory must be certain
that the Instrument 1s capable of achieving good carbon recoveries 1n
samples containing particulates.
5.0 REAGENTS
5.1 ASTM Type II water (ASTM D1193): Water should be monitored for
Impurities, and should be boiled and cooled to remove C02.
5.2 Potassium hydrogen phthalate. stock solution. 1,000 mg/L carbon;
Dissolve 0.2128 g of potassium hydrogen phthalate (primary standard grade) 1n
Type II water and dilute to 100.0 tnL.
NOTE; Sodium oxalate and acetic add are not recommended as stock
solutions.
5.3 Potassium hydrogen phthalate, standard solutions; Prepare standard
solutions from the stock solution by dilution with Type II water.
9060 - 2
Revision
Date September 1986
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5.4 Carbonate-bicarbonate, stock solution. 1,000 mg/L carbon: Weigh
0.3500 g of sodium bicarbonate and 0.4418 g ot sodium carbonate and.transfer
both to the same 100-mL volumetric flask. Dissolve with Type II water.
5.5 Carbonate-bicarbonate, standard solution; Prepare a series of
standards similar to Step 5.3.
NOTE; This standard 1s not required by some Instruments.
5.6 Blank solution; Use the same Type II water as was used to prepare
the standard solutions.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed 1n Chapter Nine of this manual.
6.2 Sampling and storage of samples 1n glass bottles 1s preferable.
Sampling and storage 1n plastic bottles such as conventional polyethylene and
cubltalners 1s permissible If 1t Is established that the containers do not
contribute contaminating organlcs to the samples.
NOTE; A brief study performed 1n the EPA Laboratory Indicated that Type
II water stored 1n new, 1-qt cubltalners did not show any Increase
1n organic carbon after 2 weeks' exposure.
6.3 Because of the possibility of oxidation or bacterial decomposition
of some components of aqueous samples, the time between sample collection and
the start of analysis should be minimized. Also, samples should be kept cool
(4*C) and protected from sunlight and atmospheric oxygen.
6.4 In Instances where analysis cannot be performed within 2 hr from
time of sampling, the sample 1s acidified (pH £ 2) with HC1 or ^$04.
7.0 PROCEDURE
7.1 Homogenize the sample 1n a blender.
NOTE; To avoid erroneously high results, Inorganic carbon must be
accounted for. The preferred method 1s to measure total carbon and
Inorganic carbon and to obtain the organic carbon by subtraction.
If this 1s not possible, follow Steps 7.2 and 7.3 prior to analysis;
however, volatile organic carbon may be lost.
7.2 Lower the pH of the sample to 2.
7.3 Purge the sample with nitrogen for 10 m1n.
7.4 Follow Instrument manufacturer's Instructions for calibration,
procedure, and calculations.
7.5 For calibration of the Instrument, a series of standards should be
used that encompasses the expected concentration range of the samples.
9060 - 3
Revision 0
Date September 1986
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7.6 Quadruplicate analysis 1s required. Report both the average and the
range.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Employ a minimum of one blank per sample batch to determine 1f
contamination or any memory effects are occurring.
8.3 Verify calibration with an Independently prepared check standard
every 15 samples.
8.4 Run one spike duplicate sample for every 10 samples. A duplicate
sample 1s a sample brought through the whole sample preparation and analytical
process.
9.0 METHOD PERFORMANCE
9.1 Precision and accuracy data are available 1n Method 415.1 of Methods
for Chemical Analysis of Water and Wastes.
10.0 REFERENCES
1. Annual Book of ASTM Standards, Part 31, "Water," Standard 0 2574-79,
p. 469 (1976).
2. Standard Methods for the Examination of Water and Wastewater, 14th ed.(
p. 532, Method 505 (1975).
9060 - 4
Revision
Date September 1986
-------
M£TMCC 906
TOTAL ORGANIC
c
Start
7.1
Homogenize
the temple in
• blenoer
7.2
7.4
Follow manufacturer'
Instructions for
eel Jor»tion.
proceoure. »no
calculations using
cerDoneceous *
Lower tn«
••mple OH
7.3
7.5
Use series of
standards for
c*l lt-rr*t ion
Pure* the
••mple with
nJtrooen
7.6
OueOr-uo 1 lc» te
• nclys is
o
Stoo
9060 - 5
Revision 0
Date September 1986
-------
o
ON
Ui
-------
METHOD 9065
PHENOLICS (SPECTROPHOTOMETRIC. MANUAL 4-AAP WITH DISTILLATION)
1.0 SCOPE AND APPLICATION
1.1 This method 1s applicable to the analysis of ground water, drinking,
surface, and saline waters, and domestic and Industrial wastes.
1.2 The method 1s capable of measuring phenolic materials at the 5 ug/L
level when the colored end product 1s extracted and concentrated 1n a solvent
phase using phenol as a standard.
1.3 The method 1s capable of measuring phenolic materials that contain
more than 50 ug/L 1n the aqueous phase (without solvent extraction) using
phenol as a standard.
1.4 It 1s not possible to use this method to differentiate'between
different kinds of phenols.
2.0 SUMMARY OF METHOD
2.1 Phenolic materials react with 4-am1noant1pyr1ne 1n the presence of
potassium ferrlcyanlde at a pH of 10 to form a stable reddish-brown antlpyrlne
dye. The amount of color produced 1s a function of the concentration of
phenolic material.
3.0 INTERFERENCES
3.1 For most samples a preliminary distillation 1s required to remove
Interfering materials.
3.2 Color response of phenolic materials with 4-am1noant1pyr1ne 1s not
the same for all compounds. Because phenol1c-type wastes usually contain a
variety of phenols, 1t 1s not possible to duplicate a mixture of phenols to be
used as a standard. For this reason phenol has been selected as a standard
and any color produced by the reaction of other phenolic compounds 1s reported
as phenol. This value will represent the minimum concentration of phenolic
compounds present In the sample.
3.3 Interferences from sulfur compounds are eliminated by acidifying the
sample to a pH of <4 with H2$04 and aerating briefly by stirring.
3.4 Oxidizing agents such as chlorine, detected by the liberation of
Iodine upon acidification 1n the presence of potassium Iodide, are removed
Immediately after sampling by the addition of an excess of ferrous ammonium
sulfate. If chlorine 1s not removed, the phenolic compounds may be partially
oxidized and the results may be low.
9065 - 1
Revision 0
Date September 1986
-------
4.0 APPARATUS AND MATERIALS
4.1 Distillation apparatus: All glass, consisting of a 1-liter Pyrex
distilling apparatus with Graham condenser.
4.2 pH meter.
4.3 Spectrophotometer; For use at 460 or 510 nm.
4.4 Funnels.
4.5 Filter paper.
4.6 Membrane filters.
4.7 Separatory funnels; 500- or 1,000-mL.
4.8 Nessler tubes: Short or long form.
5.0 REAGENTS
5.1 ASTM Type II water (ASTM D1193): Water should be monitored for
Impurities.
5.2 Sulfurlc add solution. ^$04: Concentrated.
5.3 Buffer solution; Dissolve 16.9 g NfyCl 1n 143 ml concentrated NfyOH
and dilute to 250 ml with Type II water. Two ml of buffer should adjust
100 ml of distillate to pH 10.
5.4 Aro1noant1pyr1ne solution; Dissolve 2 g of 4-am1noant1pyr1ne (4-AAP)
1n Type II water and dilute to 100 ml.
5.5 Potassium ferrlcyanlde solution; Dissolve 8 g of K3Fe(CN)s 1n Type
II water and dilute to 100ml.
5.6 Stock phenol solution; Dissolve l.o g phenol 1n freshly boiled and
cooled Type II water and dilute to 1 liter (1 ml « 1 mg phenol).
NOTE; This solution 1s hydroscoplc and toxic.
5.7 Working solution A; Dilute 10 ml stock phenol solution to 1 liter
with Type II water (1 ml » 10 ug phenol).
5.8 Working solution B; Dilute 100 ml of working solution A to 1,000 ml
with Type II water (1 ml » 1 ug phenol).
5.9 Chloroform.
9065 - 2
Revision
Date September 1986
-------
5.10 Ferrous ammonium sulfate; Dissolve 1.1 g 1n 500 ml Type II water
containing 1 mL concentrated H2S04 and dilute to 1 liter with freshly boiled
and cooled Type II water.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed 1n Chapter Nine of this manual.
6.2 Biological degradation 1s Inhibited by the addition of ^$04 to
pH <4. Store at 4*C. The sample should be stable for 28 days.
7.0 PROCEDURE
7.1 Distillation;
7.1.1 Measure 500 mL of sample Into a beaker. Lower the pH to
approximately 4 with concentrated ^$04 (1 mL/L), and transfer to the
distillation apparatus.
7.1.2 Distill 450 mL of sample, stop the distillation, and when
boiling ceases, add 50 mL of warm Type II water to the flask and resume
distillation until 500 mL have been collected.
7.1.3 If the distillate 1s turbid, filter through a prewashed
•enbrane filter.
7.2 Direct photometric method;
7.2.1 Using working solution A (5.6), prepare the following
standards In 100-mL volumetric flasks:
Working Solution A (mL) Concentration (ug/L)
0.0 0.0
0.5 50.0
1.0 100.0
2.0 200.0
5.0 500.0
8.0 800.0
10.0 1000.0
7.2.2 To 100 mL of distillate or to an aliquot diluted to 100 mL
and/or standards, add 2 mL of buffer solution (5.2) and mix. The pH of
the sample and standards should be 10 + 0.2.
7.2.3 Add 2.0 mL amlnoantlpyrine solution (5.3) and mix.
7.2.4 Add 2.0 mL potassium ferricyanide solution (5.4) and mix.
7.2.5 After 15 min read absorbance at 510 nm.
9065 - 3
Revision 0
Date September 1986
-------
7.3 Chloroform extraction method:
CAUTION:This method should be performed 1n a hood; chloroform.
1s toxic.
7.3.1 Using working solution B (5.7), prepare the following
standards. Standards may be prepared by pipetting the required volumes
Into the separatory funnels and diluting to 500 ml with Type II water:
Working Solution B (ml) Concentration (ug/L)
0.0 0.0
3.0 6.0
5.0 10.0
10.0 20.0
20.0 40.0
25.0 50.0
7.3.2 Place 500 ml of distillate or an aliquot diluted to 500 ml 1n
a separatory funnel. The sample should not contain more than 50 ug/L
phenol.
7.3.3 To sample and standards add 10 ml of buffer solution (5.2)
and mix. The pH should be 10 + 0.2.
7.3.4 Add 3.0 mL amlnoantlpyrlne solution (5.3) and mix.
7.3.5 Add 3.0 ml potassium ferrlcyanide solution (5.4) and mix.
7.3.6 After 3 m1n, extract with 25 ml of chloroform (5.9). Shake
the separatory funnel at least 10 tiroes, let CHCla settle, shake again 10
times, and let chloroform settle again.
7.3.7 Filter chloroform extract through filter paper. Do not add
more chloroform.
7.3.8 Read the absorbance of the samples and standards against the
blank at 460 nm.
7.4 Calculation;
7.4.1 Prepare a standard curve by plotting the absorbance values of
standards versus the corresponding phenol concentrations.
7.4.2 Obtain concentration value of sample directly from standard
curve.
9065 - 4
Revision
Date September 1986
-------
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Calibration curves must be composed of a minimum of a blank and
three standards. A calibration curve should be made for every hour of
continuous sample analysis.
8.3 Dilute samples 1f they are more concentrated than the highest
standard or 1f they fall on the plateau of a calibration curve.
8.4 Employ a minimum of one blank per sample batch to determine 1f
contamination has occurred.
8.5 Verify calibration with an Independently prepared check standard
every 15 samples.
8.6 Run one spike duplicate sample for every 10 samples. A duplicate
sample 1s a sample brought through the whole sample preparation and analytical
process.
9.0 METHOD PERFORMANCE
9.1 In a single laboratory using sewage samples at concentrations of
3.8, 15, 43, and and 89 ug/L, the standard deviations were +0.5, +0.6, +0.6,
and +1.0 ug/L, respectively. At concentrations of 73, 146, 299, an3 447 ug/L,
the standard deviations were +1.0, +1.8, +4.2, and +5.3 ug/L, respectively.
9.2 In a single laboratory using sewage samples at concentrations of 5.3
and 82 ug/L, the recoveries were 78X and 98X,respectively. At concentrations
of 168 and 489 ug/L, the recoveries were 97* and 98%, respectively.
10.0 REFERENCES
1. Annual Book of ASTM Standards. Part 31, "Water," Standard D1783-70,
p. 553 (1976).
2. Standard Methods for the Examination of Water and Wastewater, 14th ed.,
pp. 574-581, Method 510 through 510C (1975).
9065 - 5
Revision 0
Date September 1986
-------
METHOD 9O6S
PHENOLICS (SPeCTROPHOTOMCTRIC. MANUAL 4-AAP WITH DISTILLATION)
f Start J
O
7.1.1
Measure
sample
into beaker;
lower pH with
concentrated
Is distillate
turcio?
Prepare
•tandarus
uslno
•oiutlon A
7.3.2
Ada Duffer
aolutlon; nl*
7.2.3
Add
aminaantpyrlna
uolutlon
7.2.4
Add potasslu»
ferrlcyanlda
solution; «lx
7.2.5
Read absorbance
7.3.1}
Prepare
standards
uslno working
solution 8
O
9065 - 6
Revision 0
Date September 1986
-------
METHOD 9065
PHENOLICS tSP£CTHO«>HOTOMET«IC. MANUAL 4-AAP WITH OrSTlLUATION)
(Cont inutd)
7.3.2
0
Piece
OKtlllete or
dilutee aliauot
in ieo«r«tory
tunnel
7.3.3
7.3.6
Extrect with
cnloroforin
A £30
uffer colutlon
to eemole mna
»t«nO»ro»: mix
7.3.4
7.3.7
Filter
chloroform
•xtrect*
ABO
•mlnoentlpyrlne
•olutlon; mix
7.3.5
«oeoroence
Ada ooteteium
ferricyenioe
•olutlon: mix
Calculate
coocentretion
ot «
( Stop J
9065 - 7
Revision 0
Date September 1986
-------
vo
o
o\
o\
-------
METHOD 9066
PHENOLICS (COLORIMETRIC, AUTOMATED 4-AAP WITH DISTILLATION)
1.0 SCOPE AND APPLICATION
1.1 This method 1s applicable to the analysis of ground water and of
drinking, surface, and saline waters.
1.2 The method 1s capable of measuring phenolic materials from 2 to
500 ug/L 1n the aqueous phase using phenol as a standard.
2.0 SUMMARY OF METHOD
2.1 This automated method 1s based on the distillation of phenol and
subsequent reaction of the distillate with alkaline ferrlcyanlde (K3Fe(CN)e)
and 4-am1no-ant1pyr1ne (4-AAP) to form a red complex which 1s measured at 505
or 520 nm.
3.0 INTERFERENCES
3.1 Interferences from sulfur compounds are eliminated by acidifying the
sample to a pH of <4.0 with H2S04 and aerating briefly by stirring.
3.2 Oxidizing agents such as chlorine, detected by the liberation of
Iodine upon acidification 1n the presence of potassium Iodide, are removed
Immediately after sampling by the addition of an excess of ferrous ammonium
sulfate (5.5). If chlorine 1s not removed, the phenolic compounds may be
partially oxidized and the results may be low.
3.3 Background contamination from plastic tubing and sample containers
1s eliminated by filling the wash receptacle by siphon (using Kel-F tubing)
and using glass tubes for the samples and standards.
4.0 APPARATUS AND MATERIALS
4.1 Automated continuous-flow analytical Instrument;
4.1.1 Sadler: Equipped with continuous mixer.
4.1.2 Manifold.
4.1.3 Proportioning pump II or III.
4.1.4 Heating bath with distillation coll.
4.1.5 Distillation head.
9066 - 1
Revision
Date September 1986
-------
4.1.6 Colorlneter: Equipped with a 50 mm flowcell and 505 or
520 nm filter.
4.1.7 Recorder.
5.0 REAGENTS
5.1 ASTM Type II water (ASTM D1193): Water should be monitored for
Impurities.
5.2 Distillation reagent; Add 100 ml of concentrated phosphoric add
(85X H3P04) to 800 ml of Type II water, cool and dilute to 1 liter.
5.3 Buffered potassium_ ferrlcyanlde; Dissolve 2.0 g potassium
ferrlcyanide, 3.1 g boric add, and"3.75 g potassium chloride 1n 800 ml of
Type II water. Adjust to pH of 10.3 with 1 N sodium hydroxide (5.3) and
dilute to lllter. Add 0.5mL of Brlj-35 (available from Technlcon).
(Brlj-35 1s a wetting agent and 1s a proprietary Technlcon product.) Prepare
fresh weekly.
5.4 Sodium hydroxide (1 N): Dissolve 40 g NaOH 1n 500 ml of Type II
water, cool and dilute to 1 liter.
5.5 4-Am1noantlpvr1ne; Dissolve 0.65 g of 4-am1noant1pyr1ne in 800 ml
of Type II water and dilute to 1 liter. Prepare fresh each day.
5.6 Ferrous ammonium sulfate; Dissolve 1.1 g ferrous ammonium sulfate
1n 500 ml Type IIwatercontaining 1 ml ^$04 and dilute to 1 liter with
freshly boiled and cooled Type II water.
5.7 Stock phenol; Dissolve 1.00 g phenol 1n 500 ml of Type II water and
dilute to 1,000 ml.Add 0.5 ml concentrated H2S04 as preservative (1.0 mL =
1.0 mg phenol).
CAUTION: This solution 1s toxic.
5.8 Standard phenol solution A; Dilute 10.0 ml of stock phenol solution
(5.6) to 1,000 ml (1.0 ml - 0.01 mg phenol).
5.9 Standard phenol solution B: Dilute 100.0 ml of standard phenol
solution A (5.8) to 1,000 ml with Type II water (1.0 ml * 0.001 mg phenol).
5.10 Standard phenol solution C; Dilute 100.0 ml of standard phenol
solution B (5.9) to 1,000 ml with Type II water (1.0 ml = 0.0001 mg phenol).
5.11 Using standard solution A, B, or C, prepare the following standards
in 100-mL volumetric flasks. Each standard should be preserved by adding 2
drops of concentrated H2S04 to 100.0 ml:
9066 - 2
Revision
Date September 1986
-------
Standard Solution (ml) Concentration (ug/L)
Solution C
1.0 1.0
2.0 2.0
3.0 3.0
5.0 5.0
Solution B
1.0 10.0
2.0 20.0
5.0 50.0
10.0 100.0
Solution A
2.0 200.0
3.0 300.0
5.0 500.0
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed In Chapter Nine of this manual.
6.2 Biological degradation 1s Inhibited by the acidification to a pH <4
with H£S04. The sample should be kept at 4*C and analyzed within 28 days of
collection.
7.0 PROCEDURE
7.1 Set up the manifold as shown 1n Figure 1.
7.2 Fill the wash receptacle by siphon. Use Kel-F tubing with a fast
flow (1 I1ter/hr).
7.3 Allow colorimeter and recorder to warm up for 30 m1n. Run a
baseline with all reagents, feeding Type II water through the sample line.
Use polyethylene tubing for sample line. When new tubing Is used, about 2 hr
may be required to obtain a stable baseline. This 2-hr time period may be
necessary to remove the residual phenol from the tubing.
7.4 Place appropriate phenol standards 1n sampler 1n order of decreasing
concentration. Complete loading of sampler tray with unknown samples, using
glass tubes. If samples have not been preserved as Instructed 1n Paragraph
6.2, add concentrated H2S04 to 100 mL of sample. Run with sensitivity setting
at full scale or 500.
9066 - 3
Revision
Date September 1986
-------
To Wast*
M »
tO
o
CT>
I
J*
O X>
a> a>
r+ <
(D —>.
V)
— J.
t/) O
n>
cr
O)
VD
00
Ml /min
SAMPLER
X"
RESAMPLE
\ WP <
M
' SM
BATH WITH
TION COIL
4
1
157-8089
nnnn
i
i
1
^
5O5yn filters
50 mm Tubulor f/c
«J
*3
'
f
i
y
GRAY
0 32 AIR
2 OO SAMPLE
0.42 DISTILLING SOL.
0.42 WASTE FROM
STILL
1.0 RESAMPLE WASTE
0.32 AIR n
1.2 RESAMPLE
O 23 4 AAP
A-2
023 BUFFERED POTASSIUM
FERRI CYANIDE
J.O WASTE FROM F/C
PROPORTIONING
PUMP
SAMPLE RATE 20/hr. 1:2
» K«l-f
• •IOO ACIDFLEX
• •» POLYETHYLENE
COLORIMETER RECORDER
Figure 1. Phenol Autoanalyzer II
-------
7.5 Switch sample from Type II water to sampler and begin analysis.
7.6 Calculation;
7.6.1 Prepare a linear standard curve by plotting peak heights of
standards against concentration values. Compute concentration of samples
by comparing sample peak heights with standards.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for
easy reference or Inspection.
8.2 Calibration curves must be composed of a minimum of a blank and
three standards. A calibration curve should be made for every hour of
continuous sample analysis.
8.3 Dilute samples 1f they are more concentrated than the highest
standard or 1f they fall on the plateau of a calibration curve.
8.4 Employ a minimum of one blank per sample batch to determine 1f
contamination has occurred.
8.5 Verify calibration with an Independently prepared check standard
every 15 samples.
8.6 Run one spike duplicate sample for every 10 samples. A duplicate
sample 1s a sample brought through the whole sample preparation and analytical
process.
9.0 METHOD PERFORMANCE
9.1 In a single laboratory using sewage samples at concentrations of
3.8, 15, 43, and 89 ug/L, the standard deviations were +0.5, +.0.6, +.0.6, and
+1.0 ug/L, respectively. At concentrations of 73, 146, 2997 and 447 ug/L,
the standard deviations were +1.0, +1.8, +4.2, and +5.3 ug/L, respectively.
9.2 In a single laboratory using sewage samples at concentrations of 5.3
and 82 ug/L, the recoveries were 78% and 98%, respectively. At concentrations
of 168 and 489 ug/L, the recoveries were 97% and 98%, respectively.
9066 - 5
Revision
Date September 1986
-------
10.0 REFERENCES
1. Gales, M.E. and R.L. Booth, "Automated 4AAP Phenolic Method" AWWA 68.
540 (1976). ~~
2. Standard Methods for the Examination of Water and Wastewater, 14th ed.,
p. 574, Method 510, (1975).
3. Technlcon AutoAnalyzer II Methodology, Industrial Method N0.127-71W,
AA II.
9066 - 6
Revision
Date September 1986
-------
(COLOftlMeTRXC. AUTOMATED 4-AAP WITH DISTILLATION)
7. J
Set up ••nlfold
7.2
Fill w«»h
r«c«pt«cl«
7.3
W»r» up
co lor lm«t.«r >nd
r«cord»r
7.3
Run •
O
O
7.4
Lead pftvnol
•t«nd»rd« «nd
7.S
Switch •••pl«
to ••«pl«r;
•nalyz*
7.6
7.4
Add cone.
Compute
concentration
of
( stop J
9066 - 7
Revision 0
Date September 1986
-------
-------
METHOD 9067
PHENOLICS (SPECTROPHOTOMETRIC. HBTH WITH DISTILLATION)
1.0 SCOPE AND APPLICATION
1.1 This method 1s applicable to the analysis of ground water, drinking,
surface, and saline waters, and domestic and Industrial wastes.
1.2 The method 1s capable of measuring phenolic materials at the 2 ug/L
level when the colored end product 1s extracted and concentrated 1n a solvent
phase using phenol as a standard.
1.3 The method 1s capable of measuring phenolic materials that contain
from 50 to 1,000 ug/L 1n the aqueous phase (without solvent extraction) using
different kinds of phenols.
1.4 It 1s not possible to use this method to differentiate between
different kinds of phenols.
2.0 SUMMARY OF METHOD
2.1 This method 1s based on the coupling of phenol with MBTH 1n an add
medium using eerie ammonium sulfate as an oxldant. The coupling takes place
1n the p-pos1t1on; 1f this position 1s occupied, the MBTH reagent will react
at a free o-pos1t1on. The colors obtained have maximum absorbance from 460 to
595 nm. For phenol and most phenolic mixtures, the absorbance 1s 520 and
490 nm.
3.0 INTERFERENCES
3.1 For most samples a preliminary distillation 1s required to remove
Interfering materials.
3.2 Color response of phenolic materials with MBTH 1s not the same for
all compounds. Because phenol1c-type wastes usually contain a variety of
phenols, 1t 1s not possible to duplicate a mixture of phenols to be used as a
standard. For this reason, phenol has been selected as a standard and any
color produced by the reaction of other phenolic compounds 1s reported as
phenol. This value will represent the minimum concentration of phenolic
compounds present 1n the sample.
3.3 Interferences from sulfur compounds are eliminated by acidifying the
sample to a pH of less than 4.0 with H2S04 and aerating briefly by stirring.
3.4 Oxidizing agents such as chlorine, detected by the liberation of
Iodine upon acidification 1n the presence of potassium Iodide, are removed
Immediately after sampling by the addition of an excess of ferrous ammonium
9067 - 1
Revision 0
Date September 1986
-------
sulfate (see Paragraph 5.11). If chlorine 1s not removed, the phenolic
compounds may be partially oxidized and the results may be low.
3.5 Phosphate causes a precipitate to form; therefore, phosphoric add
should not be used for preservation. All glassware should be phosphate free.
3.5 High concentrations of aldehydes may cause Interferences.
4.0 APPARATUS AND MATERIALS
4.1 Distillation apparatus; All glass, consisting of a 1-11ter Pyrex
distilling apparatus with Graham condenser.
4.2 pH Meter.
4.3 Spectrophotometer.
4.4 Funnels.
4.5 Filter paper.
4.6 Membrane filters.
4.7 Separatory funnels.
5.0 REAGENTS
5.1 ASTM Type II water (ASTM D1193): Water should be monitored for
Impurities.
5.2 SulfuHc add. IN: Add 28 mL of concentrated H2S04 to 900 mL of
Type II water, mix, and dilute to 1 liter.
5.3 MBTH solution. 0.05X: Dissolve 0.1 g of 3-methyl-2-benzo-
thlazollnone hydrazone hydrochlorlde 1n 200 ml of Type II water.
5.4 Cerlc ammonium sulfate solution; Add 2.0 g of
and 2.0 ml of concentrated H2S04 to 150 mL of Type
solid has dissolved, dilute to 200 mL with Type II water.
2H70 and 2.0 mL of concentrated H2S04 to 150 mL of Type II water. After the
5.5 Buffer solution: Dissolve, 1n the following order: 8 g of sodium
hydroxide, 2 g EDTA (d1 sodium salt), and 8 g boric add 1n 200 ml of Type II
water. Dilute to 250 ml with Type II water.
5.6 Working buffer solution; Make a working solution by mixing an
appropriate volume of buffer solution (5.5) with an equal volume of ethanol.
5.7 Chloroform.
9067 - 2
Revision
Date September 1986
-------
5.8 Stock phenol; Dissolve 1.00 g phenol 1n 500 ml of Type II water and
dilute to 1,000 ml. Add 1 g CuS04 and 0.5 ml concentrated H2S04 as
preservative (1.0 ml * 1.0 mg phenol).
5.9 Standard phenol solution A; Dilute 10.0 ml of stock phenol solution
(5.8) to 1,000 ml (1.0 ml = 0.01 mg phenol).
5.10 Standard phenol solution B; Dilute 100.0 ml of standard phenol
solution A (5.9) to 1,000 ml with Type II water (1.0 ml « 0.001 mg phenol).
5.11 Ferrous ammonium sulfate: Dissolve 1.1 g ferrous ammonium sulfate
1n 500 ml Type II watercontaining 1 ml concentrated ^$04 and dilute to 1
liter with freshly sorted and cooled Type II water.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed 1n Chapter Nine of this manual.
6.2 Biological degradation 1s Inhibited by acidification to a pH of <4
with H2S04. The sample should be kept at 4*C and analyzed within 28 days of
collection.
7.0 PROCEDURE
7.1 Distillation;
7.1.1 To 500 mL of sample, adjust the pH to approximately 4 with
1 N sulfurlc add solution (5.2).
7.1.2 Distill over 450 mL of sample, add 50 mL of warm Type II
water to flask, and resume distillation until 500 mL has been collected.
7.1.3 If the distillate 1s turbid, filter through a prewashed
membrane filter.
7.2 Concentration above 50 uq/L;
7.2.1 To 100 mL of distillate or an aliquot diluted to 100 mL, add
4 mL of MBTH solution (5.3).
7.2.2 After 5 m1n, add 2.5 mL of eerie ammonium sulfate solution
(5.4).
7.2.3 Walt another 5 m1n and add 7 mL of working buffer solution
(5.6).
7.2.4 After 15 win, read the absorbance at 520 nm against a reagent
blank. The color 1s stable for 4 hr.
9067 - 3
Revision
Date September 1986
-------
7.3 Concentration below 50 ug/L;
7.3.1 To 500 ml of distillate 1n a separatory funnel, add 4 ml of
MBTH solution (5.3).
7.3.2 After 5 m1n, add 2.5 ml of eerie ammonium sulfate solution
(5.4).
7.3.3 After an additional 5 m1n, add 7 ml of working buffer
solution (5.6).
7.3.4 After 15 m1n, add 25 ml of chloroform. Shake the separatory
funnel at least 20 times. Allow the layer to separate and pass the
chloroform layer through filter paper.
7.3.5 Read the absorbance at 490 nm against a reagent blank.
7.4 Calculation;
7.4.1 Prepare a standard curve by plotting absorbances against
concentration values.
7.4.2 Obtain concentration value of sample directly from prepared
standard curve.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Calibration curves must be composed of a minimum of a blank and
three standards. A calibration curve should be made for every hour of
continuous sample analysis.
8.3 Dilute samples 1f they are more concentrated than the highest
standard or 1f they fall on the plateau of a calibration curve.
8.4 Employ a .minimum of one blank per sample batch to determine 1f
contamination has occurred.
8.5 Verify calibration with an Independently prepared check standard
every 15 samples.
8.6 Run one spike duplicate sample for every 10 samples. A duplicate
sample 1s a sample brought through the whole sample preparation and analytical
process.
9067 - 4
Revision
Date September 1986
-------
9.0 METHOD PERFORMANCE
9.1 Precision and accuracy data are not available at this time.
10.0 REFERENCES
1. FHestad, H.O., E.E. Ott, and F.A. Gunther, "Automated Colorometrlc Micro
Determination of Phenol by Ox1dat1ve Coupling with 3-Methyl-benzoth1azol1none
Hydrazone," Technlcon International Congress, 1969.
2. Gales, M.E., "An Evaluation of the 3-Methyl-benzoth1azol1none Hydrazone
Method for the Determination of Phenols 1n Water and Wastewater," Analyst,
100, No. 1197, 841 (1975).
9067 - 5
Revision
Date September 1986
-------
PHENOLICS fSPECTRO*»HOTOMgTRIC. MBTM KITH
7.1.1
AOO
copp«r >ulf«te
solution
to «»mol« to
•ajutt pH
I* dlctlllate
turbja?
9067
Revision 0
Date September 1986
-------
M£Tr>O^ 9O6 .
PMENOLICS (SPECTROPHOTOMETRIC. M8TM WITH DISTILLATION!
(Continued)
AOC MBTH
SOlut
AOd MBTH
CO lut ion
of
concentrat 1O«
to olst11 late
or dilutee
• 1iguot
to a 1st11 late
In «eo»'~«tory
foone 1
AOO
cerlc ammoniu
Ado cerjc
ammonir ;^;
COlut ion
A<30 oorlclng
buffer
en] oro t ofw.
•naxe. ' 1 1 ter
chlo^o form
layer
Re«0 •ocoroance
Calculate
conc«ntratton
value of ••mole
9067 - 7
Revision 0
Date September 1986
-------
O
-4
-------
METHOD 9071
OIL AND GREASE EXTRACTION METHOD FOR SLUDGE SAMPLES
1.0 SCOPE AND APPLICATION
1.1 Method 9071 1s used to recover low levels of oil and grease
(10 mg/L) by chemically drying a wet sludge sample and then extracting via the
Soxhlet apparatus.
1.2 Method 9071 1s used when relatively polar, heavy petroleum fractions
are present, or when the levels of nonvolatile greases challenge the solu-
bility limit of the solvent.
1.3 Specifically, Method 9071 1s suitable for biological Uplds, mineral
hydrocarbons, and some Industrial wastewaters.
1.4 Method 9071 1s not recommended for measurement of low-boll1ng
fractions that volatilize at temperatures below 70*C.
2.0 SUMMARY OF METHOD
2.1 A 20-g sample of wet sludge with a known dry-sol Ids content 1s
acidified to pH 2.0 with 0.3 mL concentrated HC1.
2.2 Magnesium sulfate monohydrate will combine with 75% of Its own
weight 1n water 1n forming MgS04*7H20 and 1s used to dry the acidified sludge
sample.
2.3 After drying, the oil and grease are extracted with trlchlorotrl-
fluoroethane (Fluorocarbon 113) using the Soxhlet apparatus.
3.0 INTERFERENCES
3.1 The method 1s entirely empirical, and duplicate results can be
obtained only by strict adherence to all details of the processes.
3.2 The rate and time of extraction 1n the Soxhlet apparatus must be
exactly as directed because of varying solubilities of the different greases.
3.3 The length of time required for drying and cooling extracted
material must be constant.
3.4 A gradual Increase 1n weight may result due to the absorption of
oxygen; a gradual loss of weight may result due to volatilization.
9071 - 1
Revision
Date September 1986
-------
4.0 APPARATUS AND MATERIALS
4.1 Extraction apparatus: Soxhlet.
4.2 Analytical balance.
4.3 Vacuum pump or some other vacuum source.
4.4 Extraction thimble; Filter paper.
4.5 Glass wool or small glass beads to fill thimble.
4.6 Grease-free cotton: Extract nonabsorbent cotton with solvent.
4.7 Beaker; 150-mL.
4.8 pH Indicator to determine acidity.
4.9 Porcelain mortar.
4.10 Extraction flask; 150-mL.
4.11 Distilling apparatus; Waterbath at 70*C.
4.12 Desiccator.
5.0 REAGENTS
5.1 Concentrated hydrochloric add (HC1).
5.2 Magnesium sulfate monohydrate; Prepare MgSO^^O by spreading a
thin layer in a dish and drying in an oven at 150'C overnight.
5.3 Trichlorotrifluoroethane (I,l,2-tr1ch1oro-l,2,2,-trifluoroethane):
Boiling point, 47*C.Thesolvent should leave no measurable residue on
evaporation; distill 1f necessary.
5.4 ASTM Type II water (ASTM D1193): Water should be monitored for
impurities.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 Transfers of the solvent trichlorotrlfluoroethane should not Involve
any plastic tubing in the assembly.
6.2 Sample transfer implements; Implements are required to transfer
portions of solid, semi sol id, and liquid wastes from sample containers to
laboratory glassware. Liquids may be transferred using a glass hypodermic
syringe. Solids may be transferred using a spatula, spoon, or coring device.
9071 - 2
Revision 0
Date September 1986
-------
6.3 Any turbidity or suspended sol Ids 1n the extraction flask should be
removed by filtering through grease-free cotton or glass wool.
7.0 PROCEDURE
7.1 Weigh out 20 + 0.5 g of wet sludge with a known dry-solid content.
Place 1n a 150-ml beaker.
7.2 Acidify to a pH of 2 with approximately 0.3 ml concentrated HC1 .
7.3 Add 25 g prepared t^SO/p^O and stir to a smooth paste.
7.4 Spread paste on sides of beaker to facilitate evaporation. Let
stand about 15-30 m1n or until substance 1s solidified.
7.5 Remove sol Ids and grind to fine powder 1n a mortar.
7.6 Add the powder to the paper extraction thimble.
7,7 Wipe beaker and mortar with pieces of filter paper moistened with
solvent and add to thimble.
7.8 Fill thimble with glass wool (or glass beads).
7.9 Extract 1n Soxhlet apparatus using tr1chlorotr1fluoroethane at a
rate of 20 cycles/hr for 4 hr.
7.10 Using grease-free cotton, filter the extract Into a pre-we1ghed
250-mL boiling flask. Use gloves to avoid adding fingerprints to the flask.
7.11 Rinse flask and cotton with solvent.
7.12 Connect the boiling flask to the distilling head and evaporate the
solvent by Immersing the lower half of the flask 1n water at 70*C. Collect
the solvent for reuse. A solvent blank should accompany each set of samples.
7.13 When the temperature 1n the distilling head reaches 50*C or the
flask appears dry, remove the distilling head. To remove solvent vapor, sweep
out the flask for 15 sec with air by Inserting a glass tube that 1s connected
to a vacuum source. Immediately remove the flask from the heat source and
wipe the outside to remove excess moisture and fingerprints.
7.14 Cool the boiling flask in a desiccator for 30 m1n and weigh.
7.15 Calculate oil and grease as a percentage of the total dry sol Ids.
Generally:
* n< « of wet solids, g x dry solids fraction
9071 - 3
Revision
Date September 1986
-------
8.0 QUALITY CONTROL
8.1 Before processing any samples, the analyst should demonstrate
through the analysis of a Type II water method blank that all glassware 1s
free of organic contamination; 1f there 1s a change 1n reagents, a method
blank should be processed as a safeguard against reagent contamination. The
blank sample should be carried through all stages of the sample preparation
and measurement.
8.2 Standard quality assurance practices should be used with this
method. Laboratory replicates should be analyzed to validate the precision of
the analysis. Fortified samples should be carried through all stages of
sample preparation and measurement; they should be analyzed to validate the
sensitivity and accuracy of the analysis.
8.3 Comprehensive quality control procedures are specified for each
target compound 1n the referring analytical method.
8.4 All quality control data should be maintained and available for easy
reference or Inspection.
8.5 Employ a minimum of one blank per sample batch to determine 1f
contamination has occurred.
8.6 Verify calibration with an Independently prepared check standard
every 15 samples.
8.7 Run one spike duplicate sample for every 10 samples. A duplicate
sample 1s a sample brought through the whole sample preparation and analytical
process.
9.0 METHOD PERFORMANCE
9.1 No data provided.
10.0 REFERENCES
1. Blum, K.A. and M.J. Taras, "Determination of Emulsifying 011 1n
Industrial Wastewater," JWPCF Research Suppl., 40, R404 (1968).
2. Standard Methods for the Examination of Water and Wastewater, 14th ed.,
p. 515, Method 502A (1975).
9071 - 4
Revision 0
Date September 1986
-------
METHOD 9071
OIL ANO BMCACE EXTRACTION METHOD FOR SLUDGE SAMPLES
CrD O
O
7. 1
welgrt
•nd place In
beaker •••ele
of w«t sludge
7.6 I
Add
powder to paper
extraction
7.2
Acidify to
pH 2.0
7.3
7. 11
Rinse fleck
with solvent
7.7
Wipe beaker ena
•Mjrter; add to
thlMle
Ada end stir
7.4
7.8
7. 12
Evaporate and
collect eolvent
for reuse
Fill thJ»el«
wltn glaaa wool
L«t aubitenct
eolioify
7.a
7.9
7. 13
Remove solvent
vapor
Extract in
SoxMet
apparatue
Re«ove and
grind solid* to
• fln« powder
7. 14
Cool and weigh
boiling flat*
7.101
antract Into
bo Ulna fleck
O
7. 15
Calculate X of
oil and greaae
O GED
9071 - 5
Revision 0
Date September 1986
-------
o
•-1
H*
V
-------
METHOD 9071A
OIL AND GREASE EXTRACTION METHOD FOR SLUDGE AND SEDIMENT SAMPLES
1.0 SCOPE AND APPLICATION
1.1 Method 9071 is used to quantify low concentrations of oil and
grease (10 mg/L) by chemically drying a wet sludge sample and then extracting via
the Soxhlet apparatus. It is also used to recover oil and grease levels in
sediment and soil samples.
1.2 Method 9071 is used when relatively polar, heavy petroleum
fractions are present, or when the levels of nonvolatile greases challenge the
solubility limit of the solvent.
1.3 Specifically, Method 9071 is suitable for biological lipids,
mineral hydrocarbons, and some industrial wastewaters.
1.4 Method 9071 is not recommended for measurement of low-boiling
fractions that volatilize at temperatures below 70°C.
2.0 SUMMARY OF METHOD
2.1 A 20-g sample of wet sludge with a known dry-solids content is
acidified to pH 2.0 with 0.3 mL concentrated HC1.
2.2 Magnesium sulfate monohydrate will combine with 75% of its own
weight in water in forming MgS04 • 7H20 and is used to dry the acidified sludge
sample.
2.3 Anhydrous sodium sulfate is used to dry samples of soil and
sediment.
2.4 After drying, the oil and grease are extracted with
trichlorotrifluoroethane (Fluorocarbon-113)1 using the Soxhlet apparatus.
3.0 INTERFERENCES
3.1 The method is entirely empirical, and duplicate results can be
obtained only by strict adherence to all details of the processes.
3.2 The rate and time of extraction in the Soxhlet apparatus must be
exactly as directed because of varying solubilities of the different greases.
3.3 The length of time required for drying and cooling extracted
material must be constant.
3.4 A gradual increase in weight may result due to the absorption of
oxygen; a gradual loss of weight may result due to volatilization.
Replacement solvent will be specified in a forthcoming regulation.
9071A - 1 Revision 1
September 1994
-------
4.0 APPARATUS AND MATERIALS
4.1 Soxhlet extraction apparatus.
4.2 Analytical balance.
4.3 Vacuum pump or some other vacuum source.
4.4 Extraction thimble: Filter paper.
4.5 Glass wool or small glass beads to fill thimble.
4.6 Grease-free cotton: Extract nonabsorbent cotton with solvent.
4.7 Beaker: 150-mL.
4.8 pH Indicator to determine acidity.
4.9 Porcelain mortar.
4.10 Extraction flask: 150-mL.
4.11 Distilling apparatus: Waterbath at 70°C.
4.12 Desiccator.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Concentrated hydrochloric acid (HC1).
5.4 Magnesium sulfate monohydrate: Prepare MgS04 • H20 by spreading a
thin layer in a dish and drying in an oven at 150°C overnight.
5.5 Sodium sulfate, granular, anhydrous (Na2S04): Purify by heating at
400°C for 4 hours in a shallow tray, or by precleaning the sodium sulfate with
methylene chloride. If the sodium sulfate is precleaned with methylene chloride,
a method blank must be analyzed, demonstrating that there is no interference from
the sodium sulfate.
9071A - 2 Revision 1
September 1994
-------
5.6 Trichlorotrifluoroethane (l,l,2-trichloro-l,2,2-trifluoroethane):
Boiling point, 47°C. The solvent should leave no measurable residue on
evaporation; distill if necessary.2
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 Transfers of the solvent trichlorotrifluoroethane should not
involve any plastic tubing in the assembly.
6.2 Sample transfer implements: Implements are required to transfer
portions of solid, semisolid, and liquid wastes from sample containers to
laboratory glassware. Liquids may be transferred using a glass hypodermic
syringe. Solids may be transferred using a spatula, spoon, or coring device.
6.3 Any turbidity or suspended solids in the extraction flask should
be removed by filtering through grease-free cotton or glass wool.
7.0 PROCEDURE
7.1 Determination of Sample Dry Weight Fraction
Weigh 5-10 g of the sample into a tared crucible. Determine the dry weight
fraction of the sample by drying overnight at 105°C.
NOTE: The drying oven should be contained in a hood or vented.
Significant laboratory contamination may result from a heavily
contaminated hazardous waste sample.
Allow to cool in a desiccator before weighing:
dry weight fraction = q of dry sample
g of sample
7.2 Sample Handling
7.2.1 Sludge Samples
7.2.1.1 Weigh out 20 + 0.5 g of wet sludge with a known
dry-weight fraction (Section 7.1). Place in a 150-mL beaker.
7.2.1.2 Acidify to a pH of 2 with approximately 0.3 mL
concentrated HC1.
7.2.1.3 Add 25 g prepared Mg2S04 • H20 and stir to a
smooth paste.
7.2.1.4 Spread paste on sides of beaker to facilitate
evaporation. Let stand about 15-30 min or until substance is
solidified.
Replacement solvent will be specified in a forthcoming regulation.
9071A - 3 Revision 1
September 1994
-------
7.2.1.5 Remove solids and grind to fine powder in a
mortar.
7.2.1.6 Add the powder to the paper extraction thimble.
7.2.1.7 Wipe beaker and mortar with pieces of filter
paper moistened with solvent and add to thimble.
7.2.1.8 Fill thimble with glass wool (or glass beads).
7.2.2 Sediment/Soil Samples
7.2.2.1 Decant and discard any water layer on a sediment
sample. Mix sample thoroughly, especially composited samples.
Discard any foreign objects such as sticks, leaves, and rocks.
7.2.2.2 Blend 10 g of the solid sample of known dry
weight fraction with 10 g of anhydrous sodium sulfate, and place
1n an extraction thimble. The extraction thimble must drain freely
for the duration of the extraction period.
7.3 Extraction
7.3.1 Extract in Soxhlet apparatus using trichlorotrifluorocarbon
at a rate of 20 cycles/hr for 4 hr.
7.3.2 Using grease-free cotton, filter the extract into a pre-
weighed 250-mL boiling flask. Use gloves to avoid adding fingerprints to
the flask.
7.3.3 Rinse flask and cotton with solvent.
7.3.4 Connect the boiling flask to the distilling head and
evaporate the solvent by immersing the lower half of the flask in water at
70°C. Collect the solvent for reuse. A solvent blank should accompany
each analytical batch of samples.
7.3.5 When the temperature in the distilling head reaches 50°C
or the flask appears dry, remove the distilling head. To remove solvent
vapor, sweep out the flask for 15 sec with air by inserting a glass tube
that is connected to a vacuum source. Immediately remove the flask from
the heat source and wipe the outside to remove excess moisture and
fingerprints.
7.3.6 Cool the boiling flask in a desiccator for 30 min and
weigh.
7.3.7 Calculate oil and grease as a percentage of the total dry
solids. Generally:
% of oil and grease = gain in weight of flask (g) x 100
wt. of wet solids (g) x dry weight fraction
9071A - 4 Revision 1
September 1994
-------
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for
easy reference and inspection. Refer to Chapter One for additional quality
control guidelines.
8.2 Employ a minimum of one blank per analytical batch or twenty
samples, whichever is more frequent, to determine if contamination has occurred.
8.3 Run one matrix duplicate and matrix spike sample every twenty
samples or analytical batch, whichever is more frequent. Matrix duplicates and
spikes are brought through the whole sample preparation and analytical process.
8.4 The use of corn oil is recommended as a reference sample solution.
9.0 METHOD PERFORMANCE
9.1 Two oil and grease methods (Methods 9070 and 9071) were tested on
sewage by a single laboratory. When 1-liter portions of the sewage were dosed
with 14.0 mg of a mixture of No. 2 fuel oil and Wesson oil, the recovery was 93%,
with a standard deviation of + 0.9 mg/L.
10.0 REFERENCES
1. Blum, K.A. and M.J. Taras, "Determination of Emulsifying Oil in Industrial
Wastewater," JWPCF Research Suppl., 40, R404 (1968).
2. Standard Methods for the Examination of Water and Wastewater, 14th ed.,
p. 515, Method 502A (1975).
9071A - 5 Revision 1
September 1994
-------
METHOD 9071A
OIL AND GREASE EXTRACTION METHOD FOR SLUDGE AND SEDIMENT SAMPLES
f Start J
7.1 Determine
dry weight fraction
of sample.
7.2.1.1 Weigh
a sample of
wet sludge
and place in
beaker.
Sludge
Soil/Sediment
7.2.2.1 Decant
water; mix
sample; discard
foreign objects.
7.2.1.2
Acidify to
pH 2.
7.2.2.2 Blend
with sodium
sulfata; add
to extraction
thimble.
7.2.1.3 Add
and stir
magnesium sulfate
monohydrate.
7.2.1.6
Remove and
grind solids
to a fine
powder.
9071A - 6
Revision 1
Septenter 1994
-------
METHOD 9071A
OIL AND GREASE EXTRACTION METHOD FOR SLUDGE AND SEDIMENT SAMPLES
(Continued)
7.2.1.6 Add
powder to
paper
extraction
thimble.
7.2.1.7 Wipe
beaker and
mortar; add
to thimble.
7.2.1.8 Fill
thimble with
glass wool.
7.3.1 Extract
in Soxhlet
apparatus for
4 hours.
7.3.2 Filter
extract into
boiling flask.
7.3.3 Rinse
flask with
solvent.
7.3.4
Evaporate and
collect
solvent for reuse.
7.3.6 Remove
solvent vapor.
7.3.6 Cool
and weigh
boiling flask.
7.3.7
Calculate %
oil end
grease.
( Stop j
9071A - 7
Revision 1
Septenter 1994
-------
Cft
-------
METHOD 9075
TEST METHOD FOR TOTAL CHLORINE IN NEW AND USED
PETROLEUM PRODUCTS BY X-RAY FLUORESCENCE SPECTROMETRY (XRF)
1.0 SCOPE AND APPLICATION
1.1 This test method covers the determination of total chlorine in new
and used oils, fuels, and related materials, including crankcase, hydraulic,
diesel, lubricating and fuel oils, and kerosene. The chlorine content of
petroleum products is often required prior to their use as a fuel.
1.2 The applicable range of this method is from 200 jug/g to percent
levels.
1.3 Method 9075 is restricted to use by, or under the supervision of,
analysts experienced in the operation of an X-ray fluorescence spectrometer and
in the interpretation of the results.
2.0 SUMMARY OF METHOD
2.1 A well-mixed sample, contained in a disposable plastic sample cup,
is loaded into an X-ray fluorescence (XRF) spectrometer. The intensities of the
chlorine Ka and sulfur Ka lines are measured, as are the intensities of
appropriate background lines. After background correction, the net intensities
are used with a calibration equation to determine the chlorine content. The
sulfur intensity is used to correct for absorption by sulfur.
3.0 INTERFERENCES
3.1 Possible interferences include metals, water, and sediment in the
oil. Results of spike recovery measurements and measurements on diluted samples
can be used to check for interferences.
Each sample, or one sample from a group of closely related samples, should
be spiked to confirm that matrix effects are not significant. Dilution of
samples that may contain water or sediment can produce incorrect results, so
dilution should be undertaken with caution and checked by spiking. Sulfur
interferes with the chlorine determination, but a correction is made.
Spike recovery measurements of used crankcase oil showed that diluting
samples five to one allowed accurate measurements on approximately 80% of the
samples. The other 20% of the samples were' not accurately analyzed by XRF.
3.2 Water in samples absorbs X-rays emmitted by chlorine. For this
inter-ference, use of as short an X-ray counting time as possible is beneficial.
This appears to be related to stratification of samples into aqueous and
nonaqueous layers while in the analyzer.
Although a correction for water may be possible, none is currently
available. In general, the presence of any free water as a separate phase or a
9075 - 1 Revision 0
September 1994
-------
water content greater than 25% will reduce the chlorine signal by 50 to 90%. See
Sec. 6.4.
4.0 APPARATUS AND MATERIALS
4.1 XRF spectrometer, either energy dispersive or wavelength dispersive.
The instrument must be able to accurately resolve and measure the intensity of
the chlorine and sulfur lines with acceptable precision.
4.2 Disposable sample cups with suitable plastic film such as Mylar*.
5.0 REAGENTS
5.1 Purity of reagents. Reagent-grade chemicals shall be used in all
tests. Unless otherwise indicated, it is intended that all reagents shall
conform to the specifications of the Committee on Analytical Reagents of the
American Chemical Society, where such specifications are available. Other grades
may be used, provided it is first ascertained that the reagent is of sufficiently
high purity to permit its use without lessening the accuracy of the
determination.
5.2 Mineral oil, mineral spirits or paraffin oil (sulfur- and chlorine-
free), for preparing standards and dilutions.
5.3 1-Chlorodecane (Aldrich Chemical Co.), 20.1% chlorine, or similar
chlorine compound.
5.4 Di-n-butyl sulfide (Aldrich Chemical Co.), 21.9% sulfur by weight.
5.5 Quality control standards such as the standard reference materials
NBS 1620, 1621, 1622, 1623, and 1624 for sulfur in oil standards; and NBS 1818
for chlorine in oil standards.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed in Chapter Nine.
6.2 The collected sample should be kept headspace free prior to prepara-
tion and analysis to minimize volatilization losses of organic halogens. Because
waste oils may contain toxic and/or carcinogenic substances, appropriate field
and laboratory safety procedures should be followed.
6.3 Laboratory sampling of the sample should be performed on a well-mixed
sample of oil. The mixing should be kept to a minimum and carried out as nearly
headspace free as possible to minimize volatilization losses of organic halogens.
6.4 Free water, as a separate phase, should be removed and cannot be
analyzed by this method.
9075 - 2 Revision 0
September 1994
-------
7.0 PROCEDURE
7.1 Calibration and standardization.
7.1.1 Prepare primary calibration standards by diluting the
chlorodecane and n-butyl sulfide with mineral spirits or similar material.
7.1.2 Prepare working calibration standards that contain sulfur,
chlorine, or both according to the following table:
Cl:
S:
1.
2.
3.
4.
500, 1,000, 2,000, 4,000, and 6,000
0.5, 1.0, and 1.5% sulfur
0.5% s, 1,000 /xg/g ci
0.5% S, 4,000 pg/g Cl
1.0% S, 500 pg/g Cl
1.0% S, 2,000 ng/g Cl
5.
6.
7.
8.
1.0% S, 6,000 ng/g Cl
1.5% s, 1,000 /zg/g ci
1.5% S, 4,000 pg/g Cl
1.5% S, 6,000 pg/g Cl
Once the correction factor for sulfur interference
determined, fewer standards may be required.
with chlorine is
7.1.3 Measure the intensity of the chlorine Ka line and the
sulfur Ka line as well as the intensity of a suitably chosen background.
Based on counting statistics, the relative standard deviation of each peak
measurement should be 1% or less.
7.1.4 Determine the net chlorine and sulfur intensities by
correcting each peak for background. Do this for all of the calibration
standards as well as for a paraffin blank.
7.1.5 Obtain a linear calibration curve for sulfur by performing
a least squares fit of the net sulfur intensity to the standard concentra-
tions, including the blank. The chlorine content of a standard should
have little effect on the net sulfur intensity.
7.1.6 The calibration equation for chlorine must include a
correction term for the sulfur concentration. A suitable equation
follows:
where:
I
m,
S
b,
Cl = (ml + b) (1 + k*S)
= net chlorine intensity
= adjustable parameters
= sulfer concentration
(1)
Using a least squares procedure, the above equation or a suitable
substitute should be fitted to the data. Many XRF instruments are
equipped with suitable computer programs to perform this fit. In any
case, the resulting equation should be shown to be accurate by analysis of
suitable standard materials.
9075 - 3
Revision 0
September 1994
-------
7.2 Analysis.
7.2.1 Prepare a calibration curve as described in Sec. 7.1. By
periodically measuring a very stable sample containing both sulfur and
chlorine, it may be possible to use the calibration equations for more
than 1 day. During each day, the suitability of the calibration curve
should be checked by analyzing standards.
7.2.2 Determine the net chlorine and sulfur intensities for a
sample in the same manner as done for the standards.
7.2.3 Determine the chlorine and sulfur concentrations of the
samples from the calibration equations. If the sample concentration for
either element is beyond the range of the standards, the sample should be
diluted with mineral oil and reanalyzed.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for specific quality control procedures.
8.2 One sample in ten should be analyzed in triplicate and the relative
standard deviation reported. For each triplicate, a separate preparation should
be made, starting from the original sample.
8.3 Each sample, or one sample in ten from a group of similar samples,
should be spiked with the elements of interest by adding a known amount of
chlorine or sulfur to the sample. The spiked amount should be between 50% and
200% of the sample concentration, but the minimum addition should be at least
five times the limit of detection. The percent recovery should be reported and
should be between 80% and 120%. Any sample suspected of containing >25% water
should also be spiked with organic chlorine.
8.4 Quality control standard check samples should be analyzed every day
and should agree within 10% of the expected value of the standard.
9.0 METHOD PERFORMANCE
9.1 These data are based on 47 data points obtained by seven laboratories
who each analyzed four used crankcase oils and three fuel oil blends with
crankcase in duplicate. A data point represents one duplicate analysis of a
sample. Two data points were determined to be outliers and are not included in
these results.
9.2 Precision. The precision of the method as determined by the
statistical examination of interlaboratory test results is as follows:
Repeatability - The difference between successive results obtained
by the same operator with the same apparatus under constant operating
conditions on identical test material would exceed, in the long run, in
the normal and correct operation of the test method, the following values
only in 1 case in 20 (see Table 1):
9075 - 4 Revision 0
September 1994
-------
Repeatability = 5.72
*where x is the average of two results in
Reproducibility - The difference between two single and independent
results obtained by different operators working in different laboratories
on identical test material would exceed, in the long run, the following
values only in 1 case in 20:
Reproducibility =9.83
*where x is the average value of two results in
9.3 Bias. The bias of this test method varies with concentration, as
shown in Table 2:
Bias = Amount found - Amount expected.
10.0 REFERENCE
1. Gaskill, A.; Estes, E.D.; Hardison, D.L.; and Myers, I.E. Validation of
Methods for Determining Chlorine in Used Oils and Oil Fuels. Prepared for
U.S. Environmental Protection Agency, Office of Solid Waste. EPA Contract
No. 68-01-7075, WA 80. July 1988.
9075 - 5 Revision 0
September 1994
-------
TABLE 1. REPEATABILITY AND REPRODUCIBILITY
FOR CHLORINE IN USED OILS BY
X-RAY FLUORESCENCE SPECTROMETRY
Average value, Repeatability, Reproducibility,
M9/9 M9/9 M9/9
500
1,000
1,500
2,000
2,500
3,000
128
181
222
256
286
313
220
311
381
440
492
538
TABLE 2. RECOVERY AND BIAS DATA FOR CHLORINE IN
USED OILS BY X-RAY FLUORESCENCE SPECTROMETRY
Amount
expected,
M9/9
320
480
920
1,498
1,527
3,029
3,045
Amount
found,
M9/9
278
461
879
1,414
1,299
2,806
2,811
Bias,
M9/9
-42
-19
-41
-84
-228
-223
-234
Percent
bias
-13
-4
-4
-6
-15
-7
-8
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METHOD 9075
TEST METHOD FOR TOTAL CHLORINE IN NEW AND USED
PETROLEUM PRODUCTS BY X-RAY FLUORESCENCE SPECTROMETRY (XRF)
START
7 1 1 - 7 1. 2
Prepare calibration
standa rds
7 1 3 Measure
intensity of
standard! and
backgr ound
714 Determine ni
intensity for
standard! and a
paraffin blank
715-716
Cont t ruet
calibration curves
for sulfur and
chlorine
721 Check
calibration curvai
periodica11y
throughout the day
722 Determine net
chlorine and sulfur
intensities for
•ample
723 Determine
chlorine and sulfur
concentrations from
calibration curves
7 2 3
la sample
concent ration
beyond range of
standards'7
723 Dilute sampli
•ith mineral oil
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METHOD 9076
TEST METHOD FOR TOTAL CHLORINE IN NEU AND USED PETROLEUM
PRODUCTS BY OXIDATIVE COMBUSTION AND HICROCOULOMETRY
1.0 SCOPE AND APPLICATION
1.1 This test method covers the determination of total chlorine in new
and used oils, fuels and related materials, including crankcase, hydraulic,
diesel, lubricating and fuel oils, and kerosene by oxidative combustion and
microcoulometry. The chlorine content of petroleum products is often required
prior to their use as a fuel.
1.2 The applicable range of this method is from 10 to 10,000 /ug/g
chlorine.
2.0 SUMMARY OF METHOD
2.1 The sample is placed in a quartz boat at the inlet of a high-
temperature quartz combustion tube. An inert carrier gas such as argon, carbon
dioxide, or nitrogen sweeps across the inlet while oxygen flows into the center
of the combustion tube. The boat and sample are advanced into a vaporization
zone of approximately 300°C to volatilize the light ends. Then the boat is
advanced to the center of the combustion tube, which is at 1,000°C. The oxygen
is diverted to pass directly over the sample to oxidize any remaining refractory
material. All during this complete combustion cycle, the chlorine is converted
to chloride and oxychlorides, which then flow into an attached titration cell
where they quantitatively react with silver ions. The silver ions thus consumed
are coulometrically replaced. The total current required to replace the silver
ions is a measure of the chlorine present in the injected samples.
2.2 The reaction occurring in the titration cell as chloride enters is:
CT + Ag+ > AgCl (1)
The silver ion consumed in the above reaction is generated coulometrically
thus:
Ag° > Ag+ + Q (2)
2.3 These microequivalents of silver are equal to the number of micro-
equivalents of titratable sample ion entering the titration cell.
3.0 INTERFERENCES
3.1 Other titratable halides will also give a positive response. These
titratable halides include HBr and HI (HOBr + HOI do not precipitate silver).
Because these oxyhalides do not react in the titration cell, approximately 50%
microequivalent response is detected from bromine and iodine.
3.2 Fluorine as fluoride does not precipitate silver, so it is not an
interferant nor is it detected.
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3.3 This test method is applicable in the presence of total sulfur
concentrations of up to 10,000 times the chlorine level.
4.0 APPARATUS AND MATERIALS1
4.1 Combustion furnace. The sample should be oxidized in an electric
furnace capable of maintaining a temperature of 1,000°C to oxidize the organic
matrix.
4.2 Combustion tube, fabricated from quartz and constructed so that a
sample, which is vaporized completely in the inlet section, is swept into the
oxidation zone by an inert gas where it mixes with oxygen and is burned. The
inlet end of the tube connects to a boat insertion device where the sample can
be placed on a quartz boat by syringe, micropipet, or by being weighed
externally. Two gas ports are provided, one for an inert gas to flow across the
boat and one for oxygen to enter the combustion tube.
4.3 Microcoulometer, Stroehlein Coulomat 702 CL or equivalent, having
variable gain and bias control, and capable of measuring the potential of the
sensing-reference electrode pair, and comparing this potential with a bias
potential, and applying the amplified difference to the working-auxiliary
electrode pair so as to generate a titrant. The microcoulometer output signal
shall be proportional to the generating current. The microcoulometer may have
a digital meter and circuitry to convert this output signal directly to a mass
of chlorine (e.g., nanograms) or to a concentration of chlorine (e.g., micrograms
of chlorine or micrograms per gram).
4.4 Titration cell. Two different configurations have been applied to
coulometrically titrate chlorine for this method.
4.4.1 Type I uses a sensor-reference pair of electrodes to detect
changes in silver ion concentration and a generator anode-cathode pair of
electrodes to maintain constant silver ion concentration and an inlet for
a gaseous sample from the pyrolysis tube. The sensor, reference, and
anode electrodes are silver electrodes. The cathode electrode is a
platinum wire. The reference electrode resides in a saturated silver
acetate half-cell. The electrolyte contains 70% acetic acid in water.
4.4.2 Type II uses a sensor-reference pair of electrodes to
detect changes in silver ion concentration and a generator anode-cathode
pair of electrodes to maintain constant silver ion concentration, an inlet
for a gaseous sample that passes through a 95% sulfuric acid dehydrating
tube from the pyrolysis tube, and a sealed two-piece titration cell with
an exhaust tube to vent fumes to an external exhaust. All electrodes can
be removed and replaced independently without reconstructing the cell
assembly. The anode electrode Is constructed of silver. The cathode
electrode is constructed of platinum. The anode is separated from the
'Any apparatus that meets the performance criteria of this section may be
used to conduct analyses by this methodology. Three commercial analyzers that
fulfill the requirements for apparatus Steps 4.1 through 4.4 are: Dohrmann
Models DX-20B and MCTS-20 and Mitsubishi Model TSX-10 available from Cosa
Instrument.
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cathode by a 10% KN03 agar bridge, and continuity is maintained through an
aqueous 10% KN03 salt bridge. The sensor electrode is constructed of
silver. The reference electrode is a silver/silver chloride ground glass
sleeve, double-junction electrode with aqueous 1M KN03 in the outer chamber
and aqueous 1M KC1 in the inner chamber.
4.5 Sampling syringe, a microliter syringe of 10 /iL capacity capable of
accurately delivering 2 to 5 /uL of a viscous sample into the sample boat.
4.6 Micropipet, a positive displacement micropipet capable of accurately
delivering 2 to 5 n\. of a viscous sample into the sample boat.
4.7 Analytical balance. When used to weigh a sample of 2 to 5 mg onto
the boat, the balance shall be accurate to + 0.01 mg. When used to determine the
density of the sample, typically 8 g per 10 ml, the balance shall be accurate to
± o-i g.
4.8 Class A volumetric flasks: 100 ml.
5.0 REAGENTS
5.1 Purity of Reagents, Reagent-grade chemicals shall be used in all
tests. Unless otherwise indicated, it is intended that all reagents shall
conform to the specifications of the Committee on Analytical Reagents of the
American Chemical Society, where such specifications are available. Other grades
may be used, provided it is first ascertained that the reagent is of sufficiently
high purity to permit its use without lessening the accuracy of the
determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Acetic acid, CH3C02H. Glacial.
5.4 Isooctane, (CH3)2CHCH2C(CH3)3 (2,2,4-Trimethylpentane).
5.5 Chlorobenzene, C6H5C1.
5.6 Chlorine, standard stock solution - 10,000 ng Cl//jL, weigh
accurately 3.174 g of chlorobenzene into 100-mL Class A volumetric flask. Dilute
to the mark with isooctane.
5.7 Chlorine, standard solution. -1,000 ng Cl/^L, pipet 10.0 mL of
chlorine stock solution (Sec. 5.6) into a 100-mL volumetric flask and dilute to
volume with isooctane.
5.8 Argon, helium, nitrogen, or carbon dioxide, high-purity grade (HP)
used as the carrier gas. High-purity grade gas has a minimum purity of 99.995%.
5.9 Oxygen, high-purity grade (HP), used as the reactant gas.
5.10 Gas regulators. Two-stage regulator must be used on the reactant and
carrier gas.
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5.11 Cell Type 1.
5.11.1 Cell electrolyte solution. 70% acetic acid: combine 300
ml reagent water with 700 ml acetic acid (Sec. 5.3) and mix well.
5.11,2 Silver acetate, CH3C02Ag. Powder purified for saturated
reference electrode.
5.12 Cell Type 2.
5.12.1 Sodium acetate, CH3C02Na.
5.12.2 Potassium nitrate, KN03.
5.12.3 Potassium chloride, KC1.
5.12.4 Sulfuric acid (concentrated), H2S04.
5.12.5 Agar, (jelly strength 450 to 600 g/cm2).
5.12.6 Cell electrolyte solution - 85% acetic acid: combine 150
mL reagent water with 1.35 g sodium acetate (Sec. 5.12.1) and mix well;
add 850 ml acetic acid (Sec. 5.3) and mix well.
5.12.7 Dehydrating solution - Combine 95 ml sulfuric acid (Sec.
5.12.4) with 5 ml reagent water and mix well.
CAUTION: This is an exothermic reaction and may proceed with bumping
unless controlled by the addition of sulfuric acid. Slowly add
sulfuric acid to reagent water. Do not add water to sulfuric acid.
5.12.8 Potassium nitrate (10%), KN03. Add 10 g potassium nitrate
(Sec. 5.12.2) to 100 ml reagent water and mix well.
5.12.9 Potassium nitrate (1M), KN03. Add 10.11 g potassium
nitrate (Sec. 5.12.2) to 100 ml reagent water and mix well.
5.12.10 Potassium chloride (1M), KC1. Add 7.46 g potassium
chloride (Sec. 5.12.3) to 100 mL reagent water and mix well.
5.12.11 Agar bridge solution - Mix 0.7 g agar (Sec. 5.12.5), 2.5g
potassium nitrate (Sec. 5.12.2), and 25 ml reagent water and heat to
boiling.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed in Chapter Nine.
6.2 Because the collected sample will be analyzed for total halogens, it
should be kept headspace free and refrigerated prior to preparation and analysis
to minimize volatilization losses of organic halogens. Because waste oils may
9076 - 4 Revision 0
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contain toxic and/or carcinogenic substances, appropriate field and laboratory
safety procedures should be followed.
6.3 Laboratory subsampling of the sample should be performed on a well-
mixed sample of oil.
7.0 PROCEDURES
7.1 Preparation of apparatus.
Set up the analyzer as per the equipment manufacturer's
7.1.1
instructions.
7.1.2 Typical operating conditions: Type 1.
Furnace temperature ............... 1,000°C
Carrier gas flow .................. 43 cm3/min
Oxygen gas flow ................... 160 cm3/min
Coulometer
Bias ............................ 250 mV
Gain ............................ 25%
7.1.3 Typical operating conditions: Type 2.
Furnace temperature ............... H-l 850*C
H-2 1,000'C
Carrier gas flow .................. 250 cm3/roin
Oxygen gas flow ................... 250 cm3/roin
Coulometer
End point potential (bias) ...... 300 mV
Gain G-l .......................... 1.5 coulombs/A mV
G-2 .......................... 3.0 coulombs/A mV
G-3 .......................... 3.0 coul ombs/A mV
ES-1 (range 1) .................... 25 mV
ES-2 (range 2) .................... 30 mV
NOTE: Other conditions may be appropriate.
instrumentation manual.
7.2 Sample introduction.
7.2.1 Carefully fill a 10-/iL syringe with 2 to 5
depending on the expected concentration of total chlorine.
sample through the septum onto the cool boat, being certain
boat with the needle tip to displace the last droplet.
Refer to the
xL of sample
Inject the
to touch the
7.2.2 For viscous samples that cannot be drawn into the syringe
barrel, a positive displacement micropipet may be used. Here, the 2-5 juL
of sample is placed on the boat from the micropipet through the opened
hatch port. The same technique as with the syringe is used to displace
the last droplet into the boat. A tuft of quartz wool in the boat can aid
in completely transferring the sample from the micropipet into the boat.
9076 - 5
Revision 0
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NOTE: Dilution of samples to reduce viscosity is not recommended due
to uncertainty about the solubility of the sample and its
chlorinated constituents. If a positive displacement micropipet is
not available, dilution may be attempted to enable injection of
viscous samples.
7.2.3 Alternatively, the sample boat may be removed from the
instrument and tared on an analytical balance. A sample of 2-5 mg is
accurately weighed directly into the boat and the boat and sample returned
to the inlet of the instrument.
2-5 /iiL = 2-5 mg
NOTE: Sample dilution may be required to ensure that the titration
system is not overloaded with chlorine. This will be somewhat
system dependent and should be determined before analysis is
attempted. For example, the MCTS-20 can titrate up to 10,000 ng
chlorine in a single injection or weighed sample, while the DX-20B
has an upper limit of 50,000 ng chlorine. For 2 to 5 juL sample
sizes, these correspond to nominal concentrations in the sample of
800 to 2,000 jug/g and 4,000 to 10,000 jiig/g, respectively. If the
system is overloaded, especially with inorganic chloride, residual
chloride may persist in the system and affect results of subsequent
samples. In general, the analyst should ensure that the baseline
returns to normal before running the next sample. To speed baseline
recovery, the electrolyte can be drained from the cell and replaced
with fresh electrolyte.
NOTE: To determine total chlorine, do not extract the sample either
with reagent water or with an organic solvent such as toluene or
isooctane. This may lower the inorganic chlorine content as well as
result in losses of volatile solvents.
7.2.4 Follow the manufacturer's recommended procedure for moving
the sample and boat into the combustion tube.
7.3 Calibration and standardization.
7.3.1 System recovery - The fraction of chlorine in a standard
that is titrated should be verified every 4 hours by analyzing the
standard solution (Sec. 5.7). System recovery is typically 85% or better.
The pyrolysis tube should be replaced whenever system recovery drops below
75%.
NOTE: The 1,000 ^9/9 system recovery sample is suitable for all
systems except the MCTS-20 for which a 100 ng/g sample should be
used.
7.3.2 Repeat the measurement of this standard at least three
times.
9076 - 6 Revision 0
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7.3.3 System blank - The blank should be checked daily with
isooctane. It is typically less than 1 fj.g/g chlorine. The system blank
should be subtracted from both samples and standards.
7.4 Calculations.
7.4.1 For systems that read directly in mass units of chloride,
the following equations apply:
Chlorine, Mg/g (wt/wt) . ' B
or
Chlorine, M9/9 (wt/wt) = (H)P(Rh) ' B
where:
Display = Integrated value in nanograms (when the integrated values are
displayed in micrograms, they must be multiplied by 103)
DisplayB = blank measurement Displays = sample measurement
V = Volume of sample injected in micro!iters
VB = blank volume Vs = sample volume
D = Density of sample, grams per cubic centimeters
0B = blank density Ds = sample density
RF = Recovery factor = ratio of chlorine = Found - Blank
determined in standard minus the system Known
blank, divided by known standard content
B = System blank, ^g/g chlorine = DisplayB
(VBJ (UB)
M = Mass of sample, mg
7.4.2 Other systems internally compensate for recovery factor,
volume, density, or mass and blank, and thus read out directly in parts
per million chlorine units. Refer to instrumentation manual.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for specific quality control procedures.
8.2 Each sample should be analyzed twice. If the results do not agree
to within 10%, expressed as the relative percent difference of the results,
repeat the analysis.
8.3 Analyze matrix spike and matrix spike duplicates - spike samples with
a chlorinated organic at a level of total chlorine commensurate with the levels
being determined. The spike recovery should be reported and should be between
9076 - 7 Revision 0
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80 and 120% of the expected value. Any sample suspected of containing >25% water
should also be spiked with organic chlorine.
9.0 METHOD PERFORMANCE
9.1 These data are based on 66 data points obtained by 10 laboratories
who each analyzed four used crankcase oils and three fuel oil blends with
crankcase in duplicate. A data point represents one duplicate analysis of a
sample. One laboratory and four additional data points were determined to be
outliers and are not included in these results.
9.2 Precision. The precision of the method as determined by the
statistical examination of interlaboratory test results is as follows:
Repeatability - The difference between successive results obtained by the
same operator with the same apparatus under constant operating conditions on
identical test material would exceed, in the long run, in the normal and correct
operation of the test method the following values only in 1 case in 20 (see Table
1):
Repeatability = 0.137 x*
*where x is the average of two results in
Reproducibility - The difference between two single and independent
results obtained by different operators working in different laboratories
on identical test material would exceed, in the long run, the following
values only in 1 case in 20:
Reproducibility - 0.455 x*
*where x is the average value of two results in jug/g.
9.3 Bias. The bias of this test method varies with concentration, as
shown in Table 2:
Bias = Amount found - Amount expected
10.0 REFERENCE
1. Gaskill, A.; Estes, E.D.; Hardison, D.L.; and Myers, I.E. "Validation of
Methods for Determining Chlorine in Used Oils and Oil Fuels." Prepared
for U.S. Environmental Protection Agency, Office of Solid Waste. EPA
Contract No. 68-01-7075, WA80. July 1988.
2. Rohrobough, W.G.; et al . Reagent Chemicals, American Chemical Society
Specifications, 7th ed.; American Chemical Society: Washington, DC, 1986.
3. Standard Instrumentation, 3322 Pennsylvania Avenue, Charleston, WV 25302.
9076 - 8 Revision 0
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TABLE 1.
REPEATABILITY AND REPRODUCIBILITY FOR CHLORINE IN
USED OILS BY MICROCOULOMETRIC TITRATION
Average value Repeatability, Reproducibility,
M9/9 M9/9 M9/9
500
1,000
1,500
2,000
2,500
3,000
69
137
206
274
343
411
228
455
683
910
1,138
1,365
TABLE 2.
RECOVERY AND BIAS DATA FOR CHLORINE IN USED OILS
BY MICROCOULOMETRIC TITRATION
Amount
expected,
M9/9
320
480
920
1,498
1,527
3,029
3,045
Amount
found
M9/9
312
443
841
1,483
1,446
3,016
2,916
Bias,
M9/9
-8
-37
-79
-15
-81
-13
-129
Percent
bias
-3
-8
-9
-1
-5
0
-4
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METHOD 9076
TEST METHOD FOR TOTAL CHLORINE IN NEW AND USED PETROLEUM
PRODUCTS BY OXIDATIVE COMBUSTION AND MICROCOULOMETRY
722 In)«et
• ample- inio
cool boat
with
micropipat
724 Mov.
•ample and
boat into
combu* tion
tuba
7 3 1 Verify
lystam
racovary
avary 4 houri
7 2.1 Inject
sample into
cool boat
«ith syringe
732 Repeal
»tandard
naaauramant
at laait
thraa tma»
733 Chack
lyatam blank
daily »ith
i*ooctana
7 4 Calculate
chlorina
concantrat ion
c
STOP
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o
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METHOD 9077
TEST METHODS FOR TOTAL CHLORINE IN NEW AND USED PETROLEUM
PRODUCTS (FIELD TEST KIT METHODS)
1.0 SCOPE AND APPLICATION
1.1 The method may be used to determine if a new or used petroleum
product meets or exceeds requirements for total halogen measured as chloride.
An analysis of the chlorine content of petroleum products is often required prior
to their use as a fuel. The method is specifically designed for used oils
permitting onsite testing at remote locations by nontechnical personnel to avoid
the delays for laboratory testing.
1.2 In these field test methods, the entire analytical sequence,
including sampling, sample pretreatment, chemical reactions, extraction, and
quantification, are combined in a single kit using predispensed and encapsulated
reagents. The overall objective is to provide a simple, easy to use procedure,
permitting nontechnical personnel to perform a test with analytical accuracy
outside of a laboratory environment in under 10 minutes. One of the kits is
preset at 1,000 ng/g total chlorine to meet regulatory requirements for used
oils. The other kits provide quantitative results over a range of 750 to
7,000 /zg/g and 300 to 4,000
2.0 SUMMARY OF METHOD
2.1 The oil sample (around 0.4 g by volume) is dispersed in a solvent
and reacted with a mixture of metallic sodium catalyzed with naphthalene and
diglyme at ambient temperature. This process converts all organic halogens to
their respective sodium halides. All halides in the treated mixture, including
those present prior to the reaction, are then extracted into an aqueous buffer,
which is then titrated with mercuric nitrate using diphenyl carbazone as the
indicator. The end point of the titration is the formation of the blue-violet
mercury diphenyl carbazone complex. Bromide and iodide are titrated and reported
as chloride.
2.2 Reagent quantities are preset in the fixed end point kit (Method
A) so that the color of the solution at the end of the titration indicates
whether the sample is above 1,000 jug/g chlorine (yellow) or below 1,000 jug/g
chlorine (blue).
2.3 The first quantitative kit (Method B) involves a reverse titration
of a fixed volume of mercuric nitrate with the extracted sample such that the end
point is denoted by a change from blue to yellow in the titration vessel over the
range of the kit (750 to 7,000 Mg/g)- The final calculation is based on the
assumption that the oil has a specific gravity of 0.9 g/cm3.
2.4 The second quantitative kit (Method C) involves a titration of the
extracted sample with mercuric nitrate by means of a 1-mL microburette such that
the end point is denoted by a change from pale yellow to red-violet over the
range of the kit (300 to 4,000 iig/g). The concentration of chlorine in the
original oil is then read from a scale on the microburette.
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NOTE; Warning—All reagents are encapsulated or contained within
ampoules. Strict adherence to the operational procedures included
with the kits as well as accepted safety procedures (safety glasses
and gloves) should be observed.
NOTE; Warning—When crushing the glass ampoules, press firmly in
the center of the ampoule once. Never attempt to recrush broken
glass because the glass may come through the plastic and cut
fingers.
NOTE; Warning—In case of accidental breakage onto skin or
clothing, wash with large amounts of water. All the ampoules are
poisonous and should not be taken internally.
Warn ing --The gray ampoules contain metallic sodium. Metallic
sodium is a flammable water-reactive solid.
NOTE; Warning—Do not ship kits on passenger aircraft. Dispose of
used kits properly.
NOTE: Caution—When the sodium ampoule in either kit is crushed,
oils that contain more than 25% water will cause the sample to turn
clear to light gray. Under these circumstances, the results may
be biased excessively low and should be disregarded.
3.0 INTERFERENCES
3.1 Free water, as a second phase, should be removed. However, this
second phase can be analyzed separately for chloride content if desired.
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METHOD A
FIXED END POINT TEST KIT METHOD
4.0A APPARATUS AND MATERIALS
4.1A The CLOR-D-TECT 10001 is a complete self-contained kit. It
includes: a sampling tube to withdraw a fixed sample volume for analysis; a
polyethylene test tube #1 into which the sample is introduced for dilution and
reaction with metallic sodium; and a polyethylene tube #2 containing a buffered
aqueous extractant, the mercuric nitrate titrant, and diphenyl carbazone
indicator. Included are instructions to conduct the test and a color chart to
aid in determining the end point.
5.0A REAGENTS
5.1A Purity of reagents. Reagent-grade chemicals shall be used in all
tests. Unless otherwise indicated, it is intended that all reagents shall
conform to the specifications of the Committee on Analytical Reagents of the
American Chemical Society, where such specifications are available. Other grades
may be used, provided it is first ascertained that the reagent is of sufficiently
high purity to permit its use without lessening the accuracy of the determina-
tion.
5.2A All necessary reagents are contained within the kit.
5.3A The kit should be examined upon opening to see that all of the
components are present and that all the ampoules (4) are in place and not
leaking. The liquid in Tube #2 (yellow cap) should be approximately 1/2 in.
above the 5-mL line and the tube should not be leaking. The ampoules are not
supposed to be completely full.
6.0A SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1A All samples must be collected using a sampling plan that addresses
the considerations discussed in Chapter Nine.
6.2A Because the collected sample will be analyzed for total halogens,
it should be kept headspace free and refrigerated prior to preparation and
analysis to minimize volatilization losses of organic halogens. Because waste
oils may contain toxic and/or carcinogenic substances, appropriate field and
laboratory safety procedures should be followed.
7.0A PROCEDURE
7.1A Preparation. Open analysis carton, remove contents, mount plastic
test tubes in the provided holder. Remove syringe and glass sampling capillary
from foil pouch.
Available from Dexsil Corporation, One Hamden Park Drive, Hamden, CT 06517.
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NOTE: Perform the test in a warm, dry area with adequate light.
In cold weather, a truck cab is sufficient. If a warm area is not
available, Step 7.3A should be performed while warming Tube #1 in
palm of hand.
7.2A Sample introduction. Remove white cap from Tube #1. Using the
plastic syringe, slowly draw the oil up the capillary tube until it reaches the
flexible adapter tube. Wipe excess oil from the tube with the provided tissue,
keeping capillary vertical. Position capillary tube into Tube #1, and detach
adapter tubing, allowing capillary to drop to the bottom of the tube. Replace
white cap on tube. Crush the capillary by squeezing the test tube several times,
being careful not to break the glass reagent ampoules.
7.3A Reaction. Break the lower (colorless) capsule containing the clear
diluent solvent by squeezing the sides of the test tube. Mix thoroughly by
shaking the tube vigorously for 30 seconds. Crush the upper grey ampoule
containing metallic sodium, again by squeezing the sides of the test tube. Shake
vigorously for 20 seconds. Allow reaction to proceed for 60 seconds, shaking
intermittently several times while timing with a watch.
NOTE: Caution—Always crush the clear ampoule in each tube first.
Otherwise, stop the test and start over using another complete kit.
False (low) results may occur and allow a contaminated sample to
pass without detection if clear ampoule is not crushed first.
7.4A Extraction. Remove caps from both tubes. Pour the clear buffered
extraction solution from Tube #2 into Tube #1. Replace the white cap on Tube #1,
and shake vigorously for 10 seconds. Vent tube by partially unscrewing the
dispenser cap. Close cap securely, and shake for an additional 10 seconds. Vent
again, tighten cap, and stand tube upside down on white cap. Allow phases to
separate for 2 minutes.
7.5A Analysis. Put filtration funnel into Tube #2. Position Tube #1
over funnel and open nozzle on dispenser cap. Squeeze the sides of Tube #1 to
dispense the clear aqueous lower phase through the filter into Tube #2 to the
5 ml line on Tube #2. Remove the filter funnel. Replace the yellow cap on Tube
#2 and close the nozzle on the dispenser cap. Break the colorless lower capsule
containing mercuric nitrate solution by squeezing the sides of the tube, and
shake for 10 seconds. Then break the upper colored ampoule containing the
diphenylcarbazone indicator, and shake for 10 seconds. Observe color
immediately.
7.6A Interpretation of results
7.6.1A Because all reagent levels are preset, calculations are not
required. A blue solution in Tube #2 indicates a chlorine content in the
original oil of less than 1,000 jug/g, and a yellow color indicates that
the chlorine concentration is greater than 1,000 ^9/9- Refer to the color
chart enclosed with the kit in interpreting the titration end point.
7.6.2A Report the results as < or > 1,000 jug/g chlorine in the oil
sample.
9077 - 4 Revision 0
September 1994
-------
8.0A QUALITY CONTROL
8.1A Refer to Chapter One for specific quality control procedures.
8.2A Each sample should be tested two times. If the results do not
agree, then a third test must be performed. Report the results of the two that
agree.
9.0A METHOD PERFORMANCE
9.1A No formal statement is made about either the precision or bias of
the overall test kit method for determining chlorine in used oil because the
result merely states whether there is conformance to the criteria for success
specified in the procedure, (i .e.. a blue or yellow color in the final solution).
In a collaborative study, eight laboratories analyzed four used crankcase oils
and three fuel oil blends with crankcase in duplicate using the test kit. Of the
resulting 56 data points, 3 resulted in incorrect classification of the oil's
chlorine content (Table 1). A data point represents one duplicate analysis of
a sample. There were no disagreements within a laboratory on any duplicate
determinations.
9077 - 5 Revision 0
September 1994
-------
TABLE 1.
PRECISION AND BIAS INFORMATION FOR METHOD A-
FIXED END POINT TEST KIT METHOD
Expected
concentration,
M9/9
Percent agreement
Expected results, Percent
/ig/g correct4 Within Between
320
480
920
1,498
1,527
3,029
3,045
< 1,000
< 1,000
< 1,000
> 1,000
> 1,000
> 1,000
> 1,000
100
100
100
87
75
100
100
100
100
100
100
100
100
100
100
100
100
87
75
100
100
"Percent correct --percent correctly identified as above or below
1,000
bPercent agreement—percent agreement within or between laboratories.
9077 - 6
Revision 0
September 1994
-------
START
METHOD 9077, METHOD A
FIXED END POINT TEST KIT METHOD
7 1A Open test kit
7 2A Dra* oil into
capillary tube;
remove excess oil.
drop capillary tube
into Tube #1 and
cap Tube tl: ctush
capillary tube
7.3A Break
colorless capsule;
nix; crush grey
capsule; mix; allow
reaction to proceed
for E>0 sec
7.4A Pour Tube f2
solution into Tube
tl; mix; vent;
allot* phases to
separate
7.SA filter aqueous
lover phase in Tub*
#1 into Tube f2,
remove filter
funnel; break
colorless capsule;
mix; break upper
colored capsule;
• ix; observe color
7 6.1 Chlorine
content is > 1000
"9/9
7.6 1 Chi or me
content is < 1000
"9/9
762 Report
results
STOP
9077 - 7
Revision 0
September 1994
-------
METHOD B
REVERSE TITRATION QUANTITATIVE END POINT TEST KIT METHOD
4.OB APPARATUS AND MATERIALS
4.IB QuantiClor2 kit components (see Figure 1).
4.1.IB Plastic reaction bottle: 1 oz, with flip-top dropper cap
and a crushable glass ampoule containing sodium.
4.1.2B Plastic buffer bottle: contains 9.5 mL of aqueous buffer
solution.
4.1.3B Titration vial: contains buffer bottle and indicator-
impregnated paper.
4.1.4B Glass vial: contains 2.0 mL of solvents.
4.1.5B Micropipet and plunger, 0.25 mL.
4.1.6B Activated carbon filtering column.
4.1.7B Titret and valve assembly.
4.2B The reagents needed for the test are packaged in disposable
containers.
4.3B The procedure utilizes a Titret. Titrets are hand-held,
disposable cells for titrimetric analysis. A Titret is an evacuated glass
ampoule (13 mm diameter) that contains an exact amount of a standardized liquid
titrant^ A flexible valve assembly is attached to the tip of the ampoule.
Titrets employ the principle of reverse titration; that is, small doses of
sample are added to the titrant to the appearance of the end point color. The
color change indicates that the equivalency point has been reached. The flow of
the sample into the Titret may be controlled by using an accessory called a
Titrettor* .
5. OB REAGENTS
5.IB The crushable glass ampoule, which is inside the reaction bottle,
contains 85 mg of metallic sodium in a light oil dispersion.
5.2B The buffer bottle contains 0.44 g of NaH?PO, • 2H?0 and 0.32 mL of
HN03 in distilled water.
5.38 The glass vial contains 770 mg Stoddard Solvent (CAS No. 8052-
41-3), 260 mg toluene, 260 mg butyl ether, 260 mg diglyme, 130 mg naphthalene,
and 70 mg demulsifier.
?
Quanti-Chlor Kit, Titrets , and Titrettor*1 are manufactured by Chemetrics,
Inc., Calverton, VA 22016. U.S. Patent No. 4,332,769.
9077 - 8 Revision 0
September 1994
-------
5.4B The Titret contains 1.12 mg mercuric nitrate in distilled water.
5.58 The indicator-impregnated paper contains approximately 0.3 mg of
diphenylcarbazone and 0.2 mg of brilliant yellow.
6.OB SAMPLE COLLECTION, PRESERVATION, AND HANDLING
See Section 6.0A of Method A.
7.OB PROCEDURE
7.IB Shake the glass vial and pour its contents into the reaction
bottle.
7.2B Fill the micropipet with a well-shaken oil sample by pulling the
plunger until its top edge is even with the top edge of the micropipet. Wipe off
the excess oil and transfer the sample into the reaction bottle (see Figure 2.1).
7.3B Gently squeeze most of the air out of the reaction bottle (see
Figure 2.2). Cap the bottle securely, and shake vigorously for 30 seconds.
7.4B Crush the sodium ampoule by pressing against the outside wall of
the reaction bottle (see Figure 2.3).
CAUTION: Samples containing a high percentage of water will
generate heat and gas, causing the reaction bottle walls to
expand. To release the gas, briefly loosen the cap.
7.5B Shake the reaction bottle vigorously for 30 seconds.
7.6B Wait 1 minute. Shake the reaction bottle occasionally during this
time.
7.7B Remove the buffer bottle from the titration vial, and slowly pour
its contents into the reaction bottle (see Figure 2.4).
7.8B Cap the reaction bottle and shake gently for a few seconds. As
soon as the foam subsides, release the gas by loosening the cap. Tighten the
cap, and shake vigorously for 30 seconds. As before, release any gas that has
formed, then turn the reaction bottle upside down (see Figure 2.5).
7.9B Wait 1 minute.
7.10B While holding the filtering column in a vertical position, remove
the plug. Gently tap the column to settle the carbon particles.
7.11B Keeping the reaction bottle upside down, insert the flip top into
the end of the filtering column and position the column over the titration vial
(see Figure 2.6). Slowly squeeze the lower aqueous layer out of the reaction
bottle and into the filtering column. Keep squeezing until the first drop of oil
is squeezed out.
9077 - 9 Revision 0
September 1994
-------
NOTE: Caution--The aqueous layer should flow through the filtering
column into the titration vial in about 1 minute. In rare cases,
it may be necessary to gently tap the column to begin the flow.
The indicator paper should remain in the titration vial.
7.12B Cap the titration vial and shake it vigorously for 10 seconds.
7.13B Slide the flexible end of the valve assembly over the tapered tip
of the Titret so that it fits snugly (see Figure 3.1).
7.14B Lift (see Figure 3.2) the control bar and insert the assembled
Titret into the Titrettor* .
7.15B Hold the Titrettor*1 with the sample pipe in the sample, and press
the control bar to snap the pre-scored tip of the Titret (see Figure 3.3).
NOTE: Caution—Because the Titret is sealed under vacuum, the
fluid inside may be agitated when the tip snaps.
7.16B With the tip of the sample pipe in the sample, briefly press the
control bar to pull in a SMALL amount of sample (see Figure 3.3). The contents
of the Titret will turn purple.
CAUTION: During the titration, there will be some undissolved
powder inside the Titret. This does not interfere with the
accuracy of the test.
7.17B Wait 30 seconds.
7.18B Gently press the control bar again to allow another SMALL amount
of the sample to be drawn into the Titret.
CAUTION: Do not press the control bar unless the sample pipe is
immersed in the sample. This prevents air from being drawn into
the Titret.
7.198 After each addition, rock the entire assembly to mix the contents
of the Titret. Watch for a color change from purple to very pale yellow.
7.208 Repeat Steps 7.18B and 7.19B until the color change occurs.
CAUTION: The end point color change (from purple to pale yellow)
actually goes through an intermediate gray color. During this
intermediate stage, extra caution should be taken to bring in
SMALL amounts of sample and to mix the Titret contents well.
7.21B When the color of the liquid in the Titret changes to PALE YELLOW,
remove the Titret from the Titrettor* . Hold the Titret in a vertical position
and carefully read the test result on the scale opposite the liquid level.
7.22B Calculation
7.22.IB To obtain results in micrograms per gram total chlorine,
multiply scale units on the Titret by 1.3 and then subtract 200.
9077 - 10 Revision 0
September 1994
-------
8.OB QUALITY CONTROL
8.IB Refer to Chapter One for specific quality control procedures.
8.2B Each sample should be tested two times. If the results do not
agree to within 10%, expressed as the relative percent difference of the results,
a third test must be performed. Report the results of the two that agree.
9.OB METHOD PERFORMANCE
9.IB These data are based on 49 data points obtained by seven
laboratories who each analyzed four used crankcase oils and three fuel oil blends
with crankcase in duplicate. A data point represents one duplicate analysis of
a sample. There were no outlier data points or laboratories.
9.2B Precision. The precision of the method as determined by the
statistical examination of interlaboratory test results is as follows:
Repeatabil ity - The difference between successive results obtained
by the same operator with the same apparatus under constant
operating conditions on identical test material would exceed, in
the long run, in the normal and correct operation of the test
method, the following values only in 1 case in 20 (see Table 2):
Repeatability = 0.31 x*
*where x is the average of two results in M9/9«
Reproducibility - The difference between two single and
independent results obtained by different operators working in
different laboratories on identical test material would exceed,
in the long run, the following values only in 1 case in 20:
Reproducibility = 0.60 x*
*where x is the average value of two results in M9/9-
9.3B Bias. The bias of this test method varies with concentration, as
shown in Table 3:
Bias = Amount found - Amount expected
9077 - 11 Revision 0
September 1994
-------
TABLE 2.
REPEATABILITY AND REPRODUCIBILITY FOR CHLORINE IN USED
OILS BY THE QUANTITATIVE END POINT TEST KIT METHOD
Average value, Repeatability, Reproducibility,
M9/9 M9/9 M9/9
1,000
1,500
2,000
2,500
3,000
310
465
620
775
930
600
900
1,200
1,500
1,800
TABLE 3.
RECOVERY AND BIAS DATA FOR CHLORINE IN USED OILS BY THE
QUANTITATIVE END POINT TEST KIT METHOD
Amount
expected,
Mg/g
320 (< 750)a
480 {< 750)a
920
1,498
1,527
3,029
3,045
Amount
found,
/*g/g
776
782
1,020
1,129
1,434
1,853
2,380
Bias,
Mg/g
+16
+32
+100
-369
-93
-1,176
-665
Percent
bias
+3
+4
+11
-25
-6
-39
-22
The lower limit of the kit is 750
9077 - 12 Revision 0
September 1994
-------
Reaction boltle
Titration via
1 Glass vial
Filtering
Column
•—^ x
Buffer
bottle
assembly
Micro pipet
Figure 1. Components of CHEMetrics Total Chlorine in Waste Oil Test Kit
(Cat. No. K2610).
9077 - 13
Revision 0
September 1994
-------
Push plunger
down to
transfer
sample
Figure 2.1
Figure 2.2
«• Crush
Figure 2.3
Buffer Bottle
Figure 2.4
Reaction bottle
upsidedown in
component tray
Figure 2.5
Aqueous
Layer
Filtering Column
Figure 2.6
Titration Vial
Figure 2. Reaction-Extraction Procedure.
9077 - 14
Revision 0
September 1994
-------
Attaching
the Valve
Assembly
Figure 3.1
Valve
Assembly
/ \
Titret
Lift control bar
Snapping
the Tip
Figure 3.2
Performing the
Analysis
Figure 3.3
Watch for
color change
here
Press control bar
Sample pipe
Sample —
Readihg
the Result
Figure 3.4
Read
scale units
when color
changes
permanently
\
Figure 3. Titration Procedure
9077 - 15
Revision 0
September 1994
-------
METHOD 9077, METHOD B
REVERSE TITRATION QUANTITATIVE END POINT TEST KIT METHOD
STMJT
i
7 IB Shake glass
react j. on bot t le
i
7 2B Fill
micropi pe t uti th
oil , transfer 01 1
to reaction bottle
1 3B Squeeze ai r
bottle, cap; mix
7 IB Crush sodium
ampoul e
7 SB - ? 6B Shake
reaction bottle for
30 second* ; wai t
one minute
7 7B Pour buf/er
into reaction —
bottle
7 8B • 7 9B Shake
gently; release
upside down; wait
one minute
1
7 10B Prepare
filtering col umn
1
7 1JB Fil ter lower
aqueous layer
through filtering
column into
titration vial
i
7 12B Shake vial
1
7 13B Assemble
valve assembly over
Titret
i
7 14B In'sert Titret
into TitreHor
7 1SB Snap tip of
Titret
7.16B - 7 20B Pull
snal1 amount of
•ample into Titret.
mi*; »ait 30
seconds; repeat
process until color
changes from purple
to pale yel1ow
7 21B When color
changes to pale
yel1ow. remove
Titrel; record test
result from Titret
7 22B Calculate
concentration of
chlorine in ug/g
STOP
9077 - 16
Revision 0
Septarber 1994
-------
METHOD C
DIRECT TITRATION QUANTITAVE END POINT TEST KIT METHOD
4.0C APPARATUS AND MATERIALS
4.1C The CHLOR-D-TECT Q40003 is a complete self-contained kit. It
includes: a sampling syringe to withdraw a fixed sample volume for analysis; a
polyethylene test tube #1 into which the sample is introduced for dilution and
reaction with metallic sodium; a polyethylene tube #2 containing a buffered
aqueous extractant and the diphenylcarbazone indicator; a microburette containing
the mercuric nitrate titrant; and a plastic filtration funnel. Also included are
instructions to conduct the test.
5.0C REAGENTS
5.1C All necessary reagents are contained within the kit. The diluent
solvent containing the catalyst, the metallic sodium, and the diphenylcarbazone
are separately glass-encapsulated in the precise quantity required for analysis.
A predispensed volume of buffer is contained in the second polyethylene tube.
Mercuric nitrate titrant is also supplied in a sealed titration burette.
5.2C The kit should be examined upon opening to see that all of the
components are present and that all ampoules (3) are in place and not leaking.
The liquid in Tube #2 (clear cap) should be approximately 1/2 in. above the 5-mL
line and the tube should not be leaking. The ampoules are not supposed to be
completely full.
6.0C SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1C See Section 6.0A of Method A.
7.0C PROCEDURE
7.1C Preparation. Open analysis carton, remove contents, mount plastic
test tubes in the provided holder.
NOTE: Perform the test in a warm, dry area with adequate light.
In cold weather, a truck cab is sufficient. If a warm area is not
available, Step 7.3C should be performed while warming Tube #1 in
palm of hand.
7.2C Sample introduction. Unscrew the white dispenser cap from Tube #1.
Slide the plunger in the empty syringe a few times to make certain that it slides
easily. Place the top of the syringe in the oil sample to be tested, and pull
back on the plunger until it reaches the stop and cannot be pulled further.
Remove the syringe from the sample container, and wipe any excess oil from the
outside of the syringe with the enclosed tissue. Place the tip of the syringe
in Tube #1, and dispense the oil sample by depressing the plunger. Replace the
white cap on the tube.
3
'Available from Dexsil Corporation, One Hamden Park Drive, Hamden, CT 06517.
9077 - 17 Revision 0
September 1994
-------
7.3C Reaction. Break the lower (colorless) capsule containing the clear
diluent solvent by squeezing the sides of the test tube. Mix thoroughly by
shaking the tube vigorously for 30 seconds. Crush the upper grey ampoule
containing metallic sodium, again by squeezing the sides of the test tube. Shake
vigorously for 20 seconds. Allow reaction to proceed for 60 seconds, shaking
intermittently several times while timing with a watch.
CAUTION: Always crush the clear ampoule in each tube first.
Otherwise, stop the test and start over using another complete kit.
False (low) results may occur and allow a contaminated sample to
pass without detection if clear ampoule is not crushed first.
7.4C Extraction. Remove caps from both tubes. Pour the clear buffered
extraction solution from Tube #2 into Tube #1. Replace the white cap on Tube #1,
and shake vigorously for 10 seconds. Vent tube by partially unscrewing the
dispenser cap. Close cap securely, and shake for an additional 10 seconds. Vent
again, tighten cap, and stand tube upside down on white cap. Allow phases to
separate for 2 minutes.
NOTE: Tip Tube #2 to an angle of only about 45°. This will prevent
the holder from sliding out.
7.5C Analysis. Put filtration funnel into Tube #2. Position Tube #1
over funnel and open nozzle on dispenser cap. Squeeze the sides of Tube #1 to
dispense the clear aqueous lower phase through the filter into Tube #2 to the 5-
mL line on Tube #2. Remove the filter funnel, and close the nozzle on the
dispenser cap. Place the plunger rod in the titration burette and press until
it clicks into place. Break off (do not pull off) the tip on the titration
burette. Insert the burette into Tube #2, and tighten the cap. Break the
colored ampoule, and shake gently for 10 seconds. Dispense titrant dropwise by
pushing down on burette rod in small increments. Shake the tube gently to mix
titrant with solution in Tube #2 after each increment. Continue adding titrant
until solution turns from yellow to red-violet. An intermediate pink color may
develop in the solution, but should be disregarded. Continue titrating until a
true red-violet color is realized. The chlorine concentration of the original
oil sample is read directly off the titrating burette at the tip of the black
plunger. Record this result imtnediatley as the red-violet color will fade with
time.
8.0C QUALITY CONTROL
8.1C Refer to Chapter One for specific quality control procedures.
8.2C Each sample should be tested two times. If the results do not
agree to within 10%, expressed as the relative percent difference of the results,
a third test must be performed. Report the results of the two that agree.
9.0C METHOD PERFORMANCE
9.1C These data are based on 96 data points obtained by 12 laboratories
who each analyzed six used crankcase oils and two fuel oil blends with crankcase
in duplicate. A data point represents one duplicate analysis of a sample.
9077 - 18 Revision 0
September 1994
-------
9.2C Precision. The precision of the method as determined by the
statistical examination of inter!aboratory test results is as follows:
Repeatability - The difference between successive results obtained
by the same operator with the same apparatus under constant
operating conditions on identical test material would exceed, in
the long run, in the normal and correct operation of the test
method, the following values only in 1 case in 20 (see Table 4):
Repea tabi 1 i ty = 0 .17 5 x*
*where x is the average of two results in M9/9'
Reproducibility - The difference between two single and independent
results obtained by different operators working in different
laboratories on identical test material would exceed, in the long
run, the following values only in 1 case in 20:
Reproducibility = 0.331 x*
*where x is the average value of two results in jug/g.
9.3C Bias. The bias of this test method varies with concentration, as
shown in Table 5:
Bias = Amount found - Amount expected
10.0 REFERENCE
1. Gaskill, A.; Estes, E.D.; Hardison, D.L.; and Myers, I.E. Validation of
Methods for Determining Chlorine in Used Oils and Oil Fuels. Prepared for
U.S. Environmental Protection Agency, Office of Solid Waste. EPA Contract
No. 68-01-7075, wA 80. July 1988.
9077 - 19 Revision 0
September 1994
-------
TABLE 4.
REPEATABILITY AND REPRODUCIBILITY FOR CHLORINE IN USED
OILS BY THE QUANTITATIVE END POINT TEST KIT METHOD
Average value, Repeatability, Reproducibility,
M9/9
500
1,000
1,500
2,000
2,500
3,000
4,000
88
175
263
350
438
525
700
166
331
497
662
828
993
1,324
TABLE 5.
RECOVERY AND BIAS DATA FOR CHLORINE IN USED OILS BY THE
QUANTITATIVE END POINT TEST KIT METHOD
Amount
expected,
M9/9
664
964
1,230
1,445
2,014
2,913
3,812
4,190
Amount
found,
M9/9
695
906
1,116
1,255
1,618
2,119
2,776
3,211
Bias,
M9/9
31
-58
-114
-190
-396
-794
-1,036
-979
Percent
bias
+5
-6
-9
-13
-20
-27
-27
-23
9077 - 20 Revision 0
September 1994
-------
METHOD 9077, METHOD C
DIRECT TITRATION QUANTITAVE END POINT TEST KIT METHOD
START
7 1C Open test kit
7 2C Draw oil into
ay ringe: remove
excess 011.
dispense oil into
Tube j*l
7 3C Brtalc
colorless capsule.
miK. crush grey
capsule; mix; allow
reaction to proceed
for 60 seconds
7 4C Pour Tube #2
solution into Tube
/I; mix; vont;
allow phases to
separate
7 SC Filter aqueous
1ower pha*e in Tube
/I into Tube #2;
remove filter
funnel
7 SC Place plunger
in tiIra ton
burette; pre»:
break off burette
tip; intert burette
in Tube /2: break
colored ampoule;
• hake
7 SC Diapense
titrant. ihake.
repeat process
until solution
turns from yellow
to red-violet
^ SC Record level
from titrating
bure11e
STOP
9077 - 21
Revision 0
September 1994
-------
o
oo
o
-------
METHOD 9080
CATION-EXCHANGE CAPACITY OF SOILS (AMMONIUM ACETATE)
1.0 SCOPE AND APPLICATION
1.1 Method 9080 1s used to determine the cation-exchange capacity of
soils. The method is not applicable to soils containing appreciable amounts
of vermlculite clays, kaolin, halloysite, or other l:l-type clay minerals.
They should be analyzed by the sodium acetate method (Method 9081). That
method (9081) 1s also generally the preferred method for very calcareous
soils. For distinctly acid soils, the cation-exchange capacity by summation
method (Chapman, p. 900; see Paragraph 10.1) should be employed.
2.0 SUMMARY
2.1 The soil is mixed with an excess of 1 N ammonium acetate solution.
This results In an exchange of the ammonium cations for exchangeable cations
present 1n the soil. The excess ammonium 1s removed, and the amount of
exchangeable ammonium is determined.
3.0 INTERFERENCES
3.1 Soils containing appreciable vermlculite clays, kaolin, halloysite,
or other l:l-type clay minerals will often give lower values for exchange
capacity. See Paragraph 1.1 above.
3.2 With calcareous soils, the release of calcium carbonate from the
soil into the ammonium acetate solution limits the saturation of exchange
sites by the ammonium 1on. This results in artificially low cation-exchange
capacities.
4.0 APPARATUS AND MATERIALS
4.1 Erlenmeyer flask; 500-mL.
4.2 Buchner funnel or equivalent; 55-mm.
4.3 Sieve; 2-mm.
4.4 Aeration apparatus (assembled as in Figure 1):
4.4.1 KJeldahl flask: 800-mL.
4.4.2 Erlenmeyer flask: 800-mL.
4.4.3 Glass wool filter.
9080 - 1
Revision
Date September 1986
-------
to Ntxt Unit
From Air Scrubbers
Soil Sample
Plus 150 ml
5%
and
FBW Drops
PariHm Oil
Suction
(Aeration
450 to 500 Liters
Per Hour)
500-ml
Wide Mouth
Erlenmeyer
Flask
N/10H2S04
in 100 ml
Water
Figure 1. Diagram of aeration unit for determination of absorbed ammonia. Six to twelve
such units is a convenient number for routine work; they can be mounted on a portable rack.
(Apparatus as modified by Dr. A. P. Vanselow, Dept. of Soils & Plant Nutrition, Univerity of
California, Riverside, Calif.).
9080 - 2
Revision Q
Date September 1986
-------
4.4.4 Glass tubing.
4.4.5 Flow meter.
5.0 REAGENTS
5.1 Ammonium acetate (NfyOAc), 1 N: Dilute 114 mL of glacial acetic
acid (99.5%) with water to a volume of approximately 1 liter. Then add 138 ml
of concentrated ammonium hydroxide (NfyOH) and add water to obtain a volume of
about 1,980 ml. Check the pH of the resulting solution, add more NH40H, as
needed, to obtain a pH of 7, and dilute the solution to a volume of 2 liters
with water.
5.2 Isopropyl alcohol: 99%.
5.3 Ammonium chloride (NlfyCl), 1 N: Dissolve 53.49 g of NfyCl in Type
II water, adjust the pH to 7.0 with NH^OH, and dilute to 1 L.
5.4 Ammonium chloride (NH4C1), 0.25 N: Dissolve 13.37 g of NH4C1 in
Type II water, adjust the pH to 7.0 with NH40H, and dilute to 1 L.
5.5 Ammonium oxalate ((NH4)?C204'H20), 10%: Add 90 ml of Type II water
to 10 g of ammonium oxalate ((NH4)2C204'H20) and mix well.
5.6 Dilute ammonium hydroxide (NffyOH): Add 1 volume of concentrated
NlfyOH to an equal volume of water.
5.7 Silver nitrate (AgNOs), 0.10 N: Dissolve 15.39 g of NgNOs 1n Type
II water, mix well, and dilute to 1 L.
5,8 Reagents for aeration option:
5.8.1 Sodium carbonate solution (Nag^), 5%: Add 95 ml of Type II
water to 5 g of N32C03 and mix well.
5.8.2 Paraffin oil.
5.8.3 Sulfurfc acid (^04), 0.1 N standard: Add 2.8 ml
concentrated ^$04 to Type II water and dilute to 1 L. Standardize
against a base of known concentration.
5.8.4 Sodium hydroxide (NaOH), 0.1 N standard: Dissolve 4.0 g NaOH
in Type II water and dilute to 1 L.' Standardize against an add of known
concentration.
5.8.5 Methyl red Indicator, 0.1%: Dissolve 0.1 g in 99.9 ml of 95%
ethanol and mix well.
9080 - 3
Revision
Date September 1986
-------
5.9 Reagents for distillation option:
5.9.1 Sodium chloride, NaCl (acidified), 1035: Dissolve 100 g of
NaCI (ammonium-free) 1n 900 mL of Type II water; mix well. Add
approximately 0.42 mL of concentrated HC1 to make the solution
approximately 0.005 N.
5.9.2 Sodium hydroxide (NaOH), 1 N: Dissolve 40 g of NaOH 1n Type
II water and dilute to 1 L.
5.9.3 Boric acid (H3BCh), 2% solution: Dissolve 20 g H3B03 1n 980
ml Type II water and mix well.
5.9.4 Standard sulfurlc add (H2S04), 0.1 N: See Step 5.8.3.
5.9.5 Bromocresol green-methyl
0.1 g of bromocresol green with 2
add 95% ethyl alcohol to obtain
0.1 g of methyl red with a few
mortar. Add 3 ml of 0.1 N NaOH
100 ml with 95% ethyl alcohol.
solution with 25 ml of the methyl
200 ml with 95% ethyl alcohol.
red mixed Indicator: Triturate
ml 0.1 N NaOH 1n an agate mortar and
a total volume of 100 ml. Triturate
ml of 95% ethyl alcohol 1n an agate
and dilute the solution to a volume of
Mix 75 ml of the bromocresol green
red solution and dilute the mixture to
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed 1n Chapter Nine of this manual.
7.0 PROCEDURE
7.1 Sieve a sample aliquot of the soil through a 2-mm screen and allow
the sieved soil to air dry (at a temperature of <60*C). Place 10 g of the
a1r-dr1ed soil 1n a 500-mL Erlenmeyer flask and add 250 mL of neutral, 1 N
NfyOAc. (Use 25 g of soil 1f the exchange capacity 1s very low, e.g., 3-5 meq
per 100 g.) Shake the flask thoroughly and allow 1t to stand overnight.
7.2 Filter the soil with light suction using a 55-mm Buchner funnel or
equivalent. Do not allow the soil to become dry and cracked.
7.3 Leach the soil with the neutral NfyOAc reagent until no test for
calcium can be obtained 1n the effluent solution. (For the calcium test, add
a few drops each of 1 N NH4C1 and 10% ammonium oxalate, dilute NH40H to 10 mL
of the leachate in a test tube, and heat the solution to near the boiling
point. The presence of calcium is indicated by a white precipitate or
turbidity.)
7.4 Then leach the soil four times with
0.25 N NH4C1.
neutral 1 N NH4C1 and once with
9080 - 4
Revision 0
Date September 1986
-------
7,5 Wash out the electrolyte with 150 to 200 ml of 99% isopropyl
alcohol. When the test for chloride 1n the leachate (use 0.10 AgNOs) becomes
negligible, allow the soil to drain thoroughly.
7.6 Determine the adsorbed NH4 either by the aeration method (Paragraph
7.7) or by the acid-Nad method (Paragraph 7.8).
7.7 Aeration method:
7.7.1 Place an excess of 0.1 N standard ^04 in the 500-mL
Erlenmeyer flask on the aeration apparatus (50 ml is an ample quantity
for most soils) and add 10 drops of methyl red indicator and enough
distilled water to make the total volume about 100 ml.
7.7.2 Attach the flask to the apparatus. Then transfer the
ammonium-saturated sample of soil (from Paragraph 7.5) quantitatively to
the 800-ml Kjeldahl flask located 1n the flow line just before the
Erlenmeyer flask with the standard acid. Use a rubber policeman and a
stream of distilled water from a wash bottle, as needed, to complete the
transfer.
7.7.3 Add 150 ml Na2C(>3 solution and a few drops of paraffin oil
and attach the flask to the apparatus.
7.7.4 Apply suction to the outflow end of the apparatus and adjust
the rate of flow to 450 to 500 liters of air per hr. Continue the
aeration for 17 hr.
7.7.5 Shut off the suction and remove the flask. Titrate the
residual acid 1n the absorption solutions with standard 0.1 N NaOH from
the original red color through orange to yellow at the end point. From
the tltration values obtained with the soil and blank solutions,
calculate the content of adsorbed ammonium 1n milligram equivalents per
100 g soil.
7.8 Acid-NaCl method;
7.8.1 Leach the ammonium-saturated soil from Paragraph 7.5 with 10%
acidified NaCl until 225 ml have passed through the sample. Add small
portions at a time, allowing each portion to pass through the sample
before adding the next portion.
7.8.2 Transfer the leachate quantitatively to an 800-mL Kjeldahl
flask, add 25 ml of 1 N NaOH, and distill 60 ml of the solution into
50 ml of 2% H3B03.
7.8.3 Add 10 drops of bromocresol green-methyl red mixed Indicator
and titrate the boric acid solution with standard 0.1 N ^$04. The color
change is from bluish green through bluish purple to pink at the end
point. Run blanks on the reagents. Correct the titration figure for the
blanks and calculate the milliequivalents of ammonium in 100 g of soil.
9080 - 5
Revision 0
Date September 1986
-------
7.8.4 Results should be reported as "determined with ammonium
acetate" at pH 7.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Employ a minimum of one blank per sample batch to determine if
contamination or any memory effects are occurring.
8.3 Material of known cation-exchange capacity must be routinely
analyzed.
9.0 METHOD PERFORMANCE
9.1 No data provided.
10.0 REFERENCES
1. This method 1s based on Chapman, H.D., "Cation-exchange Capacity,"
pp. 891-900, 1n C.A. Black (ed.), Method of Soil Analysis, Part 2: Chemical
and Microbiological Properties, Am. Soc. Agron., Madison, Wisconsin (1965).
9080 - 6
Revision
Date September 1986
-------
CATJON-eXCHAMGC CAPACITY (AMMONIUM ACETATE)
7.1 I
Sl«ve •
••nplB of coll
througn 2-mm
•cre«n:
NM OAC: l«t
•tend dvernlgnt
Filter «oU
wltn lignt
•uetlon
7.3
L«ach toll -1th
o«utr«l NH^OAC
7.3
T««t for
cilclum
L«»cn coll
Kith NH,C1
9080 - 7
Revision 0
Date September 1986
-------
METHOD 908O
CATION-EXCHANGE CAPACITY (AMMONIUM ACETATE!
(Continued)
o
7.S
ell
• lit
Wash
out th,e
tctr-olyte:
>w soli to
dr«lo
7.6
7.7.1
Aeration nethod^Xwhlch method !»>
osea to determine.
edeoroeo
Aeld-NeCl method
Pl»ct HLSO 4 In
••rctton »op«r«tu«
fl»«k; «dd m«triyl
red lnaic»tor «no
aj«t»ll«0 Miter
7.7.e
7.6.1
Li«cM (Oil frgwi
Step 7.S with
d NaCl
Attech
to
transfer soil
• •mole (7.S) to
Klelaahl fleck
7.8.2 Transfer
Irachate
to KjelOahl
flasK: add
N»OH. distill
Into
9080 - 8
Revision 0
Date September 1986
-------
CATION-EXCHANGE CAPACITY (AMMONIUM ACETATE)
(Continued)
7.7.3
Add
NajCOj
solution and
paraffin oil:
attach Meek
to aoearatus
7.7.4
A«rat« for 17
Hour*
Titrate H.BO
solution
7.8.3
blanks: correct
titratlon
'tour* for
blanks:
7.7.5}
Shut aft
suction: rsMow*
flask; titrate
residual aclo
7.8.31
Calculate
aMKonlu*
in soil
7.7.51
Calculate
content of
absareed
9080 - 9
Revision 0
Date September 1986
-------
VO
o
00
-------
METHOD 9081
CATION-EXCHANGE CAPACITY OF SOILS (SODIUM ACETATE)
1.0 SCOPE AND APPLICATION
1.1 Method 9081 1s applicable to most soils, Including calcareous and
noncalcareous soils. The method of cation-exchange capacity by summation
(Chapman, 1965, p. 900; see Paragraph 10.1) should be employed for distinctly
add soils.
2.0 SUMMARY OF METHOD
2.1 The soil sample 1s mixed with an excess of sodium acetate solution,
resulting in an exchange of the added sodium cations for the matrix cations.
Subsequently, the sample 1s washed with Isopropyl alcohol. An ammonium
acetate solution 1s then added, which replaces the adsorbed sodium with
ammonium. The concentration of displaced sodium Is then determined by atomic
absorption, emission spectroscopy, or an equivalent means.
3.0 INTERFERENCES
3.1 Interferences can occur during analysis of the extract for sodlun
content. Thoroughly Investigate the chosen analytical method for potential
Interferences.
4.0 APPARATUS AND MATERIALS
4.1 Centrifuge tube and stopper; 50-mL, round-bottom, narrow neck.
4.2 Mechanical shaker.
4.3 Volumetric flask; 100-mL.
5.0 REAGENTS
5.1 Sodium acetate (NaOAc), 1.0 N: Dissolve 136 g of NaC2H202'3H20 1n
water and dilute 1t to 1,000 mL. The pH of this solution should be 8.2. If
needed, add a few drops of acetic add or NaOH solution to bring the reaction
of the solution to pH 8.2.
5.2 Ammonium acetate (NH^Ac), 1 N: Dilute 114 mL of glacial acetic
add (99.5%) with water to a volume of approximately 1 liter. Then add 138 mL
of concentrated ammonium hydroxide (NH40H) and add water to obtain a volume of
about 1,980 mL. Check the pH of the resulting solution, add more WtyOH, as
needed, to obtain a pH of 7, and dilute the solution to a volume of 2 liters
with water.
9081 - 1
Revision 0
Date September 1986
-------
5.3 Isopropyl alcohol; 99%.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must be collected using a sampling plan that addresses
the considerations discussed 1n Chapter Nine of this manual.
7.0 PROCEDURE
7,1 Weigh 4 g of medium- or fine-textured soil or 6 g of coarse-textured
soil and transfer the sample to a 50-mL, round-bottom, narrow-neck centrifuge
tube. (A fine soil has >50% of the particles <0.074 mm, medium soil has >50X
>0.425 mm, while a coarse soil has more than 50% of Its particles }2 mm.
7.2 Add 33 mL of 1.0 N NaOAc solution, stopper the tube, shake 1t 1n a
mechanical shaker for 5 m1n, and centrifuge 1t until the supernatant liquid Is
clear.
7.3 Decant the liquid, and repeat Paragraph 7.2 three more times.
7.4 Add 33 mL of 99% Isopropyl alcohol, stopper the tube, shake It In a
mechanical shaker for 5 m1n, and centrifuge 1t until the supernatant liquid 1s
clear.
7,5 Repeat the procedure described In Paragraph 7.4 two more times.
7.6 Add 33 mL of NH40Ac solution, stopper the tube, shake 1t 1n a
mechanical shaker for 5 m1n, and centrifuge 1t until the supernatant liquid 1s
clear. Decant the washing Into a 100-mL volumetric flask.
7.7 Repeat the procedure described 1n Paragraph 7.6 two more times.
7.8 Dilute the combined washing to the 100-mL mark with ammonium acetate
solution and determine the sodium concentration by atomic absorption, emission
spectroscopy, or an equivalent method.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Employ a minimum of one blank per sample batch to determine 1f
contamination or any memory effects are occurring.
8.3 Materials of known cation-exchange capacity must be routinely
analyzed.
9081 - 2
Revision
Date September 1986
-------
9.0 METHOD PERFORMANCE
9.1 No data provided.
10.0 REFERENCES
10.1 This method 1s based on Chapman, H.D., "Cation-exchange Capacity,"
pp. 891-900, 1n C.A. Black (ed.), Method of Soil Analysis, Part 2: Chemical
and Microbiological Properties, Am. Soc. Agron., Madison, Wisconsin (1965).
9081 - 3
Revision
Date September 1986
-------
§0e)l
CAPACITY or soxus CSOOIUM
7.1
weigh
out •••ola.
centrifuge tub*
Add
H«OAC •olutton;
ccntrlfug*
7.3
O«e«nt liquid;
»or«
7.4
Add icoprooyl
• Iconol; •heiie:
centrifuge
7.S
Mepeet t M
tlewa
O
o
7.6
Add
solution:
centra fug«:
Decent w»«hi
into ri*>k
7.7
procedure
Z
r.» I Dilute
• I combined
«««hlng
witn eiMonlua
•cetete
•olution
Oeter«irte
•odlu*
concentret ion
9081 - 4
Revision 0
Date September 1986
-------
vo
o
-------
METHOD 9090A
COMPATIBILITY TEST FOR WASTES AND MEMBRANE LINERS
1.0 SCOPE AND APPLICATION
1.1 Method 9090 is intended for use in determining the effects of
chemicals i'n a surface impoundment, waste pile, or landfill on the physical
properties of flexible membrane liner (FML) materials intended to contain them.
Data from these tests will assist in deciding whether a given liner material is
acceptable for the intended application.
2.0 SUMMARY OF METHOD
2.1 In order to estimate waste/liner compatibility, the liner material
is immersed in the chemical environment for minimum periods of 120 days at room
temperature (23 ± 2°C) and at 50 + 2"C. In cases where the FML will be used in
a chemical environment at elevated temperatures, the immersion testing shall be
run at the elevated temperatures if they are expected to be higher than 50*C.
Whenever possible, the use of longer exposure times is recommended. Comparison
of measurements of the membrane's physical properties, taken periodically before
and after contact with the waste fluid, is used to estimate the compatibility of
the liner with the waste over time.
3.0 INTERFERENCES (Not Applicable)
4.0 APPARATUS AND MATERIALS
NOTE: In general, the following definitions will be used in this method:
1. Sample - a representative piece of the liner material proposed for
use that is of sufficient size to allow for the removal of
all necessary specimens.
2. Specimen - a piece of material, cut from a sample, appropriately
shaped and prepared so that it is ready to use for a test.
4.1 Exposure tank - Of a size sufficient to contain the samples, with
provisions for supporting the samples so that they do not touch the bottom or
sides of the tank or each other, and for stirring the liquid in the tank. The
tank should be compatible with the waste fluid and impermeable to any of the
constituents they are intended to contain. The tank shall be equipped with a
means for maintaining the solution at room temperature (23 ± 2"C) and 50 ± 2°C
and for preventing evaporation of the solution (e.g., use a cover equipped with
a reflux condenser, or seal the tank with a Teflon gasket and use an airtight
cover). Both sides of the liner material shall be exposed to the chemical
environment. The pressure inside the tank must be the same as that outside the
tank. If the liner has a side that (1) is not exposed to the waste in actual use
and (2) is not designed to withstand exposure to the chemical environment, then
such a liner may be treated with only the barrier surface exposed.
9090A - 1 Revision 1
July 1992
-------
4.2 Stress-strain machine suitable for measuring elongation, tensile
strength, tear resistance, puncture resistance, modulus of elasticity, and ply
adhesion.
4.3 Jig for testing puncture resistance for use with FTMS 101C, Method
2065.
4.4 Liner sample labels and holders made of materials known to be
resistant to the specific wastes.
4.5 Oven at 105 ± 2°C.
4.6 Dial micrometer.
4.7 Analytical balance.
4.8 Apparatus for determining extractable content of liner materials.
NOTE: A minimum quantity of representative waste fluid necessary to conduct
this test has not been specified in this method because the amount will
vary depending upon the waste composition and the type of liner material.
For example, certain organic waste constituents, if present in the
representative waste fluid, can be absorbed by the liner material,
thereby changing the concentration of the chemicals in the waste. This
change in waste composition may require the waste fluid to be replaced
at least monthly in order to maintain representative conditions in the
waste fluid. The amount of waste fluid necessary to maintain
representative waste conditions will depend on factors such as the volume
of constituents absorbed by the specific liner material and the
concentration of the chemical constituents in the waste.
5.0 REAGENTS (Not Applicable)
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 For information on what constitutes a representative sample of the
waste fluid, refer to the following guidance document:
Permit Applicants' Guidance Manual for Hazardous Waste Land Treatment,
Storage, and Disposal Facilities; Final Draft; Chap. 5, pp. 15-17;
Chap. 6, pp. 18-21; and Chap. 8, pp. 13-16, May 1984.
7.0 PROCEDURE
7.1 Obtain a representative sample of the waste fluid. If a waste
sample is received in more than one container, blend thoroughly. Note any signs
of stratification. If stratification exists, liner samples must be placed in
each of the phases. In cases where the waste fluid is expected to stratify and
the phases cannot be separated, the number of immersed samples per exposure
period can be increased (e.g^, if the waste fluid has two phases, then 2 samples
per exposure period are needed) so that test samples exposed at each level of the
waste can be tested. If the waste to be contained in the land disposal unit is
in solid form, generate a synthetic leachate (see Step 7.9.1).
9090A - 2 Revision 1
July 1992
-------
7.2 Perform the following tests on unexoosed samples of the polymeric
membrane liner material at 23 ± 2*C (see Steps 7.9.2 and 7.9.3 below for
additional tests suggested for specific circumstances). Tests for tear
resistance and tensile properties are to be performed according to the protocols
referenced in Table 1. See Figure 1 for cutting patterns for nonreinforced
liners, Figure 2 for cutting patterns for reinforced liners, and Figure 3 for
cutting patterns for semicrystalline liners. (Table 2, at the end of this method,
gives characteristics of various polymeric liner materials.)
1. Tear resistance, machine and transverse directions, three specimens
each direction for nonreinforced liner- materials only. See Table
1 for appropriate test method, the recommended test speed, and the
values to be reported.
2. Puncture resistance, two specimens, FTMS 101C, Method 2065. See
Figure 1, 2, or 3, as applicable, for sample cutting patterns.
3. Tensile properties, machine and transverse directions, three
tensile specimens in each direction. See Table 1 for appropriate
test method, the recommended test speed, and the values to be
reported. See Figure 4 for tensile dumbbell cutting pattern
dimensions for nonreinforced liner samples.
4. Hardness, three specimens, Duro A (Duro D if Duro A reading is
greater than 80), ASTM D2240. The hardness specimen thickness for
Duro A is 1/4 in., and for Duro D it is 1/8 in. The specimen
dimensions are 1 in. by 1 in.
5. Elongation at break. This test is to be performed only on membrane
materials that do not have a fabric or other nonelastomeric support
as part of the liner.
6. Modulus of elasticity, machine and transverse directions, two
specimens each direction for semicrystalline liner materials only,
ASTM D882 modified Method A (see Table 1).
7. Volatiles content, SW 870, Appendix III-D.
8. Extractables content, SW 870, Appendix III-E.
9. Specific gravity, three specimens, ASTM D792 Method A.
10. Ply adhesion, machine and transverse directions, two specimens each
direction for fabric reinforced liner materials only, ASTM D413
Machine Method, Type A -- 180 degree peel.
11. Hydrostatic resistance test, ASTM D751 Method A, Procedure 1.
7.3 For each test condition, cut five pieces of the lining material of
a size to fit the sample holder, or at least 8 in. by 10 in. The fifth sample
is an extra sample. Inspect all samples for flaws and discard unsatisfactory
ones. Liner materials with fabric reinforcement require close inspection to
ensure that threads of the samples are evenly spaced and straight at 90*.
Samples containing a fiber scrim support may be flood-coated along the exposed
9090A - 3 Revision 1
July 1992
-------
edges with a solution recommended by the liner manufacturer, or another procedure
should be used to prevent the scrim from being directly exposed. The flood-
coating solution will typically contain 5-15% solids dissolved in a solvent. The
solids content can be the liner formula or the base polymer.
Measure the following:
1. Gauge thickness, in. -- average of the four corners.
2. Mass, Ib. -- to one-hundredth of a Ib.
3. Length, in. -- average of the lengths of the two sides plus the
length measured £hrough the liner center.
4. Width, in. -- average of the widths of the two ends plus the width
measured through the liner center.
NOTE; Do not cut these liner samples into the test specimen shapes shown in
Figure 1, 2, or 3 at this time. Test specimens will be cut as specified
in Step 7.7, after exposure to the waste fluid.
7.4 Label the liner samples (e.q^ notch or use metal staples to
identify the sample) and hang in the waste fluid by a wire hanger or a weight.
Different liner materials should be immersed in separate tanks to avoid exchange
of plasticizers and soluble constituents .when plasticized membranes are being
tested. Expose the liner samples to the stirred waste fluid held at room
temperature and at 50 + 2'C.
7.5 At the end of 30, 60, 90, and 120 days of exposure, remove one
liner sample from each test condition to determine the membrane's physical
properties (see Steps 7.6 and 7.7). Allow the liner sample to cool in the waste
fluid until the waste fluid has a stable room temperature. Wipe off as much
waste as possible and rinse briefly with water. Place wet sample in a labeled
polyethylene bag or aluminum foil to prevent the sample from drying out. The
liner sample should be tested as soon as possible after removal from the waste
fluid at room temperature, but in no case later than 24 hours after removal.
7.6 To test the immersed sample, wipe off any remaining waste and rinse
with deionized water. Blot sample dry and measure the following as in Step 7.3:
1. Gauge thickness, in.
2. Mass, Ib.
3. Length, in.
4. Width., in.
7.7 Perform the following tests on the exposed samples (see Steps 7.9.2
and 7.9.3 below for additional tests suggested for specific circumstances).
Tests for tear resistance and tensile properties are to be performed according
to the protocols referenced in Table 1. Die-cut test specimens following
suggested cutting patterns. See Figure 1 for cutting patterns for nonreinforced
9090A - 4 Revision 1
July 1992
-------
liners, Figure 2 for cutting patterns for reinforced liners, and Figure 3 for
semi crystal!ine liners.
1. Tear resistance, machine and transverse directions, three specimens
each direction for materials without fabric.reinforcement. See
Table 1 for appropriate test method, the recommended test specimen
and speed of test, and the values to be reported.
2. Puncture resistance, two specimens, FTMS 101C, Method 2065. See
Figure 1, 2, or 3, as applicable, for sample cutting patterns.
3. Tensile properties, machine and transverse directions, three
specimens each direction. See Table 1 for appropriate test method,
the recommended test specimen and speed of test, and the values to
be reported. See Figure 4 for tensile dumbbell cutting pattern
dimensions for nonreinforced liner samples.
4. Hardness, three specimens, Duro A (Duro D if Duro A reading is
greater than 80), ASTM 2240. The hardness specimen thickness for
Duro A is 1/4 in., and for Ouro D is 1/8 in. The specimen
dimensions are 1 in. by 1 in.
5. Elongation at break. This test is to be performed only on membrane
materials that do not have a fabric or other nonelastomeric support
as part of the 1iner.
6. Modulus of elasticity, machine and transverse directions, two
specimens each direction for semi crystal line liner materials only,
ASTM D882 modified Method A (see Table 1).
7. Volatiles content, SW 870, Appendix III-D.
8. Extractables content, SW 870, Appendix III-E.
9. Ply adhesion, machine and transverse directions, two specimens each
direction for fabric reinforced liner materials only, ASTM D413
Machine Method, Type A -- 180 degree peel.
10. Hydrostatic resistance test, ASTM D751 Method A, Procedure 1.
7.8 Results and reporting
7.8.1 Plot the curve for each property over the time period 0 to
120 days and display the spread in data points.
7.8.2 Report all raw, tabulated, and plotted data. Recommended
methods for collecting and presenting information are described in the
documents listed under Step 6.1 and in related agency guidance manuals.
7.8.3 Summarize the raw test results as follows:
1. Percent change in thickness.
2. Percent change in mass.
9090A - 5 Revision 1
July 1992
-------
3. Percent change in area (provide length an'd width
dimensions).
4. Percent retention of physical properties.
5. Change, in points, of hardness reading.
6. The modulus of elasticity calculated in pounds-force per
square inch.
7. Percent volatiles of unexposed and exposed liner material.
8. Percent extractables of unexposed and exposed liner
material.
9. The adhesion value, determined in accordance with ASTM
D413, Step 12.2.
10. The pressure and time elapsed at the first appearance of
water through the flexible membrane liner for the
hydrostatic resistance test.
7.9 The following additional procedures are suggested in specific
situations:
7.9.1 For the generation of a synthetic leachate, the Agency
suggests the use of the Toxicity Characteristic Leaching Procedure (TCLP)
that was finalized in the Federal Register on June 29, 1990, Vol. 55,
No. 126, p. 26986.
7.9.2 For semi crystal line membrane liners, the Agency suggests
the determination of the potential for environmental stress cracking. The
test that can be used to make this determination is either ASTM D1693 or
the National Institute of Standards and Technology Constant Tensile Load.
The evaluation of the results should be provided by an expert in this
field.
7.9.3 For field seams, the Agency suggests the determination of
seam strength in shear and peel modes. To determine seam strength in peel
mode, the test ASTM D413 can be used. To determine seam strength in shear
mode for nonreinforced FMLs, the test ASTM D3083 can be used, and for
reinforced FMLs, the test ASTM D751, Grab Method, can be used at a speed
of 12 in. per minute. The evaluation of the results should be provided by
an expert in this field.
8.0 QUALITY CONTROL
8.1 Determine the mechanical properties of identical nonimmersed and
immersed liner samples in accordance with the standard methods for the specific
physical property test. Conduct mechanical property tests on nonimmersed and
immersed liner samples prepared from the same sample or lot of material in the
same manner and run under identical conditions. Test liner samples immediately
after they are removed from the room temperature test solution.
9090A - 6 Revision 1
July 1992
-------
9.0 METHOD PERFORMANCE
9.1 No data provided.
10.0 REFERENCES
1. None required.
9090A - 7 Revision 1
July 1992
-------
Table 1. Physical tasting of a*x»sed
s In liner-waste Mould
ttblltty Ust
Type of ooepound and
construction
Tensile properties avthod
Type of yaclaan
Meter of *Mcta*ns
iteed of test
Values to to reported
Crossl Inked or vulcanized
ASM 04 12
Ou*tollb
3 In each direction
20 H»
Temtle slrength. psl
Elongation at Irak. 1
Tensile set after break. X
Stress at WO end 2001
elongation, psl
Therenplastlc
ASM DUB
Ou*tel)b
3 In each direction
20 tpe
Tensile strength, psl
Elongation at break. 1
Tensile sat after break. 1
Stress at 100 and 2001
elongation, pst
SeelcrysUlllne
ASM DUB
OU*tellb
3 tn each direction
2 tf?e>
Tensile strength at yield, pst
Elongation at yield. I
Tensile set at break, psl
Elongation at break, pst
Tensile set after break. I
Stress at 100 and 2001
elongation, pst
Fitor Ic -reinforced*
ASM 0751. MethodB
1-ln wide strip and 2- In. J«
separation
3 tn each direction
12 tpe
Tensile at fabric break, ppl
Elongation at fabric break. 1
Tensile at ultUute break, ppl
Elongation at ultleate break, ppl
Tensile set after break. 1
Stress at 100 and 2001
elongation, pst
vo
o
to
o
00
Mtdilus of elasticity
T]|»0f
Mater of **c\mm
Spwdof Ust
Valun reported
Taar rcststanc* Mtnod
ASIM 0624
ASM 1004
ASM 0882. Mrtfnd A
Strip: 0.5 In. wtdt *nd 6. In long
at a 2 In. Jav separation
2 In eadi direction
0.2 lp>
Greatest slope of Initial stress -
strata curve, pst
ASM 01004
VO O
VO 3
ro
Type of XMcleen
Mater of ipec leans
Steed of test
Values reported
Puncture resistance Method
Type of jMcteien
Meter of jpectacftt
Speed of test
Values reported
OteC
3 In each direction
20 \fm
Stress, ppl
FTMS 101C. Method 2066
2 In. sqave
2
20 Ip*
Gage, ertl
Stress. lb
Elongation. In.
e
3 In each direction
20 tp*
Stress, ppt
FTMS 101C. Method 2066
2 tn. souar*
2
20 toe
Cage, artl
Stress. lb
Elongation. In.
e
2 tn each direction
2 tp*
Mix to* stress, ppl
FTMS 101C. Method 2066
2 In. sojiare
2
20 Ip*
Gage, ertl
Stress. lb
Elongation. In.
—
_
—
—
me 101C. Method 2066
2 1n. so^iare
2
20 IP*
G*9e. .11
Stress. lb
Elongation. In.
Jc*n be therBoplastlc. cross)Inked, or vulcanized i
"See Flswre 4.
%t perfoneBd on this eetarUl.
°»to tear resistance test Is iene»ol>l for faVtc-relnforced sheetings In the teeerslon study.
as ASM 0624. Die C.
-------
TABLE 2.
POLYMERS USED IN FLEXIBLE MEMBRANE LINERS
Thermoplastic Materials (TPj
CPE (Chlorinated polyethylene)8
A family of polymers produced by a chemical reaction of chlorine on
polyethylene. The resulting thermoplastic elastomers contain 25 to 45%
chlorine by weight and 0 to 25% crystallinity.
CSPE (Chlorosulfonated polyethylene)8
A family of polymers that are produced by the reaction of polyethylene
with chlorine and sulfur dioxide, usually containing 25 to 43% chlorine
and 1.0 to 1.4% sulfur. Chlorosulfonated polyethylene is also known as
hypalon.
EIA (Ethylene interpolymer alloy)8
A blend of EVA and polyvinyl chloride resulting in a thermoplastic
elastomer.
PVC (Polyvinyl chloride)8
A synthetic thermoplastic polymer made by polymerizing vinyl chloride
monomer or vinyl chloride/vinyl acetate monomers. Normally rigid and
containing 50% of plasticizers.
PVC-CPE (Polyvinyl chloride - chlorinated polyethylene alloy)"
A blend of polyvinyl chloride and chlorinated polyethylene.
TN-PVC (Thermoplastic nitrile-polyvinyl chloride)8
An -alloy of thermoplastic unvulcanized nitrile rubber and polyvinyl
chloride.
Vulcanized Materials (XL)
Butyl rubber8
A synthetic rubber based on isobutylene and a small amount of isoprene to
provide sites for vulcanization.
aAlso supplied reinforced with fabric.
9090A - 9 Revision 1
July 1992
-------
TABLE 2. (Continued)
EPOM (Ethylene propylene diene monomer)3'
A synthetic elastomer based on ethylene, propylene, and a small amount of
nonconjugated diene to provide sites for vulcanization.
CM (Cross-linked chlorinated polyethylene)
No definition available by EPA.
CO, ECO (Epichlorohydrin polymers)8
Synthetic rubber, including two epichlorohydrin-based elastomers that are
saturated, high-molecular-weight aliphatic polyethers with chloromethyl
side chains. The two types include homopolymer (CO) and a copolymer of
epichlorohydrin and ethylene oxide (ECO).
CR (Polychloroprene)8
Generic name for a synthetic rubber based primarily on chlorobutadiene.
Polychloroprene is also known as neoprene.
Semicrvstalline Materials (CX)
HOPE - (High-density polyethylene)
A polymer prepared by the low-pressure polymerization of ethylene as the
principal monomer.
HOPE - ^ (High-density polyethylene/rubber alloy)
A biend of high-density polyethylene and rubber.
LLDPE (Liner low-density polyethylene)
A low-density polyethylene produced by the copolymerization of ethylene
with various alpha olefins in the presence of suitable catalysts.
PEL (Polyester elastomer)
A segmented thermoplastic copolyester elastomer containing recurring long-
chain ester units derived from dicarboxylic acids and long-chain glycols
and short-chain ester units derived from dicarboxylic acids and low-
molecular-weight diols.
"Also supplied reinforced with fabric.
bAlso supplied as a thermoplastic.
9090A - 10 Revision 1
July 1992
-------
TABLE 2. (Continued)
PE-EP-A (Polyethylene ethylene/propylene alloy)
A blend of polyethylene and ethylene and propylene polymer resulting in a
thermoplastic elastomer.
T-EPDM (Thermoplastic EPDM)
An ethylene-propylene diene monomer blend resulting in a thermoplastic
elastomer.
9090A - 11 Revision 1
July 1992
-------
FIGURE 1. SUGGESTED PATTERN FOR CUTTING TEST SPECIMENS FROM
NONREINFORCED CROSSLINKED OR THERMOPLASTIC IMMERSED LINER SAMPLES.
A
10'
Puncturt ttit jptcjmtnt
Tftr ttst iptdmtns
Volatllts ttst spfdmtn
Ttnsllt ttst spodmns
Not to teal*
9090A - 12
Revision 1
July 1992
-------
FIGURE 2. SUGGESTED PATTERN FOR CUTTING TEST SPECIMENS FROM
FABRIC REINFORCED IMMERSED LINER SAMPLES.
NOTE: TO AVOID EDGE EFFECTS, CUT SPECIMENS
1/8 - 1/4 INCH IN FROM EDGE OF IMMERSED SAMPLE.
Volatnt* ttst sptdmtn
Puncturt ttst sptcintns
~ M i^J f^J^ti ti i A iJ^'W 'M • •A'fc^^Tr** -*• t ^^ < • i
3"" Ttnsllt t«t tptcimtns^ - "•"''"
_—,, ~^r |tt * JUH*1.'.* Wl T\ <•'-•' . C".'.' '
:5*tf-r*.-' *•_.-*:**."- •.'**•! *.>~ V *~1+ ^; '• -'.-'*,««
Not to K*it
9090A - 13
Revision 1
July 1992
-------
FIGURE 3. SUGGESTED PATTERN FOR CUTTING TEST SPECIMENS FROM
SEMICRYSTALLINE IMMERSED LINER SAMPLES.
NOTE: TO AVOID EDGE EFFECTS, CUT SPECIMENS
1/8 TO 1/4 INCH IN FROM EDGE OF IMMERSED SAMPLE.
Modulus of tl*st1c1ty
ttst sptclmtn*
Ttnsllt ttst sptelMns
Volttllts ttst sptdMn
Puneturt ttst sptclntns
Tor ttst sptctatns
Mot to
9090A - 14
Revision 1
July 1992
-------
FIGURE 4. DIE FOR TENSILE DUMBBELL (NONREINFORCED LINERS)
HAVING THE FOLLOWING DIMENSIONS:
t
1
wo
1
1
"^x,
y
\
W
t
1
s
V
W - Width of narrow section
L - Length of narrow section
WO - Width overall
LO - Length overall
G - Gage length
D - Distance between gaps
0.25 inches
1.25 inches
0.625 inches
3.50 inches
1.00 inches
2.00 inches
9090A - 15
Revision 1
July 1992
-------
METHOD 9090A
COMPATIBILITY TEST FOR WASTES AND MEMBRANE LINERS
START
1 1 Obtain sampl*
of was la fluid
7 2 Perform tests
on uneMposed
• arnpl 99 of I iner
ma tec la 1
7 3 Cut pieces of
lining material for
each teat condition
7 4 Label test.
spec irn«ns and
expose to waste
fluid
7 5 Determine
membrane physical
properties at 30
day intervals (30.
60, 90. 120 days]
7 6 To test exposed
specimens. measure
gauge thickness.
mass , 1 eng th. and
width
7 7 Perform '.ests
on exposed samples
7 3 Reoo r t and
eva1ua te da la
STOP
9090A - 16
Revision 1
July 1992
-------
-------
METHOD 9096
LIQUID RELEASE TEST (LRT) PROCEDURE
1.0 SCOPE AND APPLICATION
1.1 The Liquid Release Test (LRT) is a laboratory test designed to
determine whether or not liquids will be released from sorbents when they are
subjected to overburden pressures in a landfill.
1.2 Any liquid-loaded sorbent that fails the EPA Paint Filter Free
Liquids Test (PFT) (SW-846 Method 9095), may be assumed to release liquids in
this test. Analysts should ensure that the material in question will pass the
PFT before performing the LRT.
2.0 SUMMARY OF METHOD
2.1 A representative sample of the liquid-loaded sorbent, standing 10
cm high in the device, is placed between twin stainless steel screens and two
stainless-steel grids, in a device capable of simulating landfill overburden
pressures. An absorptive filter paper is placed on the side of each stainless-
steel grid opposite the sample (i.e.. the stainless-steel screen separates the
sample and the filter paper, while the stainless-steel grid provides a small air
gap to prevent wicking of liquid from the sample onto the filter paper). A
compressive force of 50 psi is applied to the top of the sample. Release of
liquid is indicated when a visible wet spot is observed on either filter paper.
3.0 INTERFERENCES
3.1 When testing sorbents are loaded with volatile liquids (e.q±,
solvents), any released liquid migrating to the filter paper may rapidly
evaporate. For this reason, filter papers should be examined immediately after
the test has been conducted.
3.2 It is necessary to thoroughly clean and dry the stainless-steel
screens prior to testing to prevent false positive or false negative results.
Material caught in screen holes may impede liquid transmission through the screen
causing false negative results. A stiff bristled brush, like those used to clean
testing sieves, may be used to dislodge material from holes in the screens. The
screens should be ultrasonically cleaned with a laboratory detergent, rinsed with
deionized water, rinsed with acetone, and thoroughly dried.
When sorbents containing oily substances are tested, it may be necessary
to use solvents (e.g., methanol or methylene chloride) to remove any oily residue
from the screens and from the sample holder surfaces.
3.3 When placing the 76 mm screen on top of the loaded sample it is
important to ensure that no sorbent is present on top of the screen to contact
the filter paper and cause false positive results. In addition, some sorbent
residue may adhere to container sidewalls and contact the filter as the sample
9096 - 1 Revision 0
September 1994
-------
compresses under load, causing wet spots on the edges of the filter. This type
of false positive may be avoided by carefully centering the 76 mm filter paper
in the device prior to initiating the test.
3.4 Visual examination of the sample may indicate that a release is
certain (e.g.. free standing liquid or a sample that flows like a liquid),
raising concern over unnecessary clean-up of the LRT device. An optional 5
minute Pre-Test, described in Appendix A of this procedure, may be used to
determine whether or not an LRT must be performed.
4.0 APPARATUS AND MATERIALS
4.1 LRT Device (LRTD): A device capable of applying 50 psi of pressure
continuously to the top of a confined, cylindrical sample (see Figure 1). The
pressure is applied by a piston on the top of the sample. All device components
contacting the sample (i .e_._, sample-holder, screens, and piston) should be
resistant to attack by substances being tested. The LRTD consists of two basic
components, described below.
4.1.1 Sample holder: A rigid-wall cylinder, with a bottom plate,
capable of holding a 10 cm high by 76 mm diameter sample.
4.1.2 Pressure Application Device: In the LRTD (Figure 1),
pressure is applied to the sample by a pressure rod pushing against a
piston that lies directly over the sample. The rod may be pushed against
the piston at a set pressure using pneumatic, mechanical, or hydraulic
pressure. Pneumatic pressure application devices should be equipped with
a pressure gauge accurate to within +1 psi, to indicate when the desired
pressure has been attained and whether or not it is adequately maintained
during the test. Other types of pressure application devices (e.g.,
mechanical or hydraulic) may be used if they can apply the specified
pressure continuously over the ten minute testing time. The pressure
application device must be calibrated by the manufacturer, using a load
cell or similar device placed under the piston, to ensure that 50+1 psi
is applied to the top of the sample. The pressure application device
should be sufficiently rugged to deliver consistent pressure to the sample
with repeated use.
4.2 Stainless-Steel Screens: To separate the sample from the filter,
thereby preventing false positive results from particles falling on the filter
paper. The screens are made of stainless steel and have hole diameters of 0.012
inches with 2025 holes per square inch. Two diameters of screens are used: a
larger (90 mm) screen beneath the sample and a smaller (76 mm) screen that is
placed on top of the sample in the sample-holding cylinder.
4.3 Stainless-Steel Grids: To provide an air gap between the
stainless-steel screen and filter paper, preventing false positive results from
capillary action. The grids are made of 1/32" diameter, woven, stainless steel
wire cut to two diameters, 90 mm and 76 mm.
9096 - 2 Revision 0
September 1994
-------
4.4 Filter Papers: To detect released liquid. Two sizes, one 90 mm
and one 76 mm, are placed on the side of the screen opposite the sample. The
76 mm diameter filter paper has the outer 6 mm cut away except 3 conical points
used for centering the paper (see Figure 2). Blue, seed-germination filter paper
manufactured by Schleicher and Schuell (Catalog Number 33900) is suitable. Other
colored, absorptive papers may be used as long as they provide sufficient wet/dry
contrast for the operator to clearly see a wet spot.
4.5 Spatula: To assist in loading and removing the sample.
4.6 Rubber or wooden mallet: To tap the sides of the device to settle
and level the sample.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Acetone.
6.0 SAMPLE COLLECTION, PRESERVATION AND HANDLING
6.1 All samples should be collected using a sampling plan that
addresses the considerations discussed in "Test Methods for Evaluating Solid
Wastes (SW-846)." The sampling plan should be designed to detect and sample any
pockets of liquids that may be present in a container (i.e., in the bottom or top
of the container).
6.2 Preservatives should not be added to samples.
6.3 Samples should be tested as soon as possible after collection, but
in no case after more than three days after collection. If samples must be
stored, they can be stored in sealed containers and maintained under dark, cool
conditions (temperature ranging between 35* and 72° F). Samples should not be
frozen.
7.0 PROCEDURE
The procedure below was developed for the original LRTD, manufactured by
Associated Design and Manufacturing Company (ADM). Procedures for other LRTDs,
along with evidence for equivalency to the ADM device, should be supplied by the
manufacturer.
9096 - 3 Revision 0
September 1994
-------
7.1 Disassemble the LRTD and make sure that all parts are clean and
dry.
7.2 Invert the sample-holding cylinder and place the large stainless-
steel screen, the large stainless-steel grid, then a 90 mm filter paper on the
cylinder base (bottom-plate side).
7.3 Secure the bottom plate (plate with a hole in the center and four
holes located on the outer circumference) to the flange on the bottom of the
sample-holding cylinder using four knob screws.
7.4 Turn the sample holder assembly to the right-side-up position
(bottom-plate-side down). Fill the sample holder with a representative sample
until the sample height measures 10 cm (up to the etched line in the cylinder).
7.5 Tap the sides of the sample holder with a rubber or wooden mallet
to remove air pockets and to settle and level the sample.
7.6 Repeat filling, and tapping until a sample height of 10 cm is
maintained after tapping.
7.7 Smooth the top of the sample.with a spatula to create a horizontal
surface.
7.8 Place the small stainless-steel screen, then the small stainless-
steel grid on top of the sample.
NOTE: Prior to placing the stainless-steel grid on top of the
screen, make sure that no sorbent material is on the grid side of
the stainless-steel screen.
7.9 Place the 76 mm filter paper on top of the small stainless-steel
grid, making sure the filter paper is centered in the device.
7.10 Using the piston handle (screwed into the top of the piston) lower
the piston into the sample holder until it sits on top of the filter paper.
Unscrew and remove the handle.
7.11 Place the loaded sample holder into position on the baseplate and
lock into place with two toggle clamps.
7.12 Place the pressure application device on top of the sample-holder.
Rotate the device to lock it into place and insert the safety key.
7.13 Connect air 1ines.
7.14 Initiate rod movement and pressure application by pulling the air-
valve lever toward the operator and note time on data sheet. The pressure gauge
at the top of the pressure application device should read as specified in the
factory calibration record for the particular device. If not, adjust regulator
to attain the specified pressure.
9096 - 4 Revision 0
September 1994
-------
NOTE: After pressure application, the air lines can be disconnected, the
toggle clamps can be released, and the LRTD can be set aside for 10
minutes while other LRTDs are pressurized. LRTD pressures should be
checked every 3 minutes to ensure that the specified pressure is being
maintained. If the specified pressure is not being maintained to within
± 5 psi, the LRTD must be reconnected to the air lines and pressure
applied throughout the 10 minute test.
7.15 After 10 minutes place the LRTD on the baseplate, reconnect air
lines and toggle clamps, and turn off pressure (retract the rod) by pushing the
air-valve lever away from the operator. Note time on data sheet.
7.16 When the air gauge reaches 0 psi, disconnect the air lines and
remove the pressure-application device by removing the safety key, rotating the
device, and lifting it away from the sample holder.
7.17 Screw the piston handle into the top of the piston.
7.18 Lift out the piston.
7.19 Remove the filter paper and immediately examine it for wet spots
(wet area on the filter paper). The presence of a wet spot(s) indicates a
positive test (i.e., liquid release). Note results on data sheet.
7.20 Release toggle clamps and remove sample holder from baseplate.
Invert sample holder onto suitable surface and remove the knob screws holding the
bottom plate.
7.21 Remove the bottom plate and immediately examine the filter paper
for wet spots as described in Step 7.19. Note results on data sheet. Wet
spot(s) on either filter indicates a positive test.
8.0 QUALITY CONTROL
8.1 Duplicate samples should be analyzed every twenty samples or every
analytical batch, whichever is more frequent. Refer to Chapter One for
additional QC protocols.
9.0 METHOD PERFORMANCE
9.1 Precision and accuracy data are not available at this time.
10.0 REFERENCES
1. Hoffman, P., G. Kingsbury, B. Lesnik, M. Meyers, "Background Document for
the Liquid Release Test (LRT) Procedure"; document submitted to the Environmental
Protection Agency by Research Triangle Institute: Research Triangle Park, NC
under Contract No. 68-01-7075, Work Assignment 76 and Contract No. 68-WO-0032,
Work Assignment 12.
9096 - 5 Revision 0
September 1994
-------
FIGURE 1.
LRT DEVICE
Pressure
Application
Device
50 psi
Piston
:••••-•'••
•Sample-Holding Cylinder
Filter
Separator Plate
9096 - 6
Separator Plate
Filter
Bottom Plate
Revision 0
September 1994
-------
FIGURE 2.
76 MM DIAMETER FILTER PAPER
120*
9096 - 7
Revision 0
September 1994
-------
FIGURE 3.
GLASS GRID SPECIFICATIONS.
0.2b Inch (
I
(
\
1.8cm*
1.7cm
•^•^HB
I
4.
1
_l
t
0 cm
1
1
9096 - 8
Revision 0
September 1994
-------
FIGURE 4.
POSITIONING OF DYE ON GLASS PLATE
Methylene Blue
Anthraquinone
7.5 cm
7.5 cm
9096 - 9
Revision 0
September 1994
-------
METHOD 9096
LIQUID RELEASE TEST (LRT) PROCEDURE
C START J
I
7.& Add more
sample
7 1 Disassemble
LRTD to ensure
cleanliness end
dryness
7 2 Piece
screen, grid
and fi1ter
peper on
cylinder base
7 3 Secure
sample holder
7.4 - 7.5
Fill cylinder
with sample;
tap to remove
ai r
77 Smooth
sample
surface
7 8 Place
stainless-steel
and grid on top
of sample
7.9 Place
filter paper
on grid and
center in ths
device
7.10 Lower
piston into
sample holder
7 11 Place
sample holder
on base plate
and secure
7 12 Lock
pressure
device on top
of sample
holder
7 13 Connect
air line*
7 H Pressurise
LRTD and
maintain
pressure for 10
minutes
7.15 - 7.16
Depra»*urize
and remove
LRTD I torn
sample holder
7 .18 Remove
piston
7.19 - 7 21
Disassemble end
check filter
peper for wet
spot(s)
f STOP J
9096 - 10
Revision 0
September 1994
-------
APPENDIX A
LIQUID RELEASE TEST PRE-TEST
1.0 SCOPE AND APPLICATION
1.1 The LRT Pre-Test is an optional, 5 minute laboratory test designed
to determine whether or not liquids will be definitely released from sorbents
before applying the LRT. This test is performed to prevent unnecessary cleanup
and possible damage to the LRT device.
1.2 This test is purely optional and completely up to the discretion
of the operator as to when it should be used.
2.0 SUMMARY OF METHOD
A representative sample will be loaded into a glass grid that is placed on
a glass plate already stained with 2 dyes (one water soluble and one oil
soluble). A second glass plate will be placed on top and a 2 Ib. weight placed
on top for 5 minutes. At the end of 5 minutes the base of the glass grid is
examined for any dye running along the edges, this would indicate a liquid
release.
3.0 INTERFERENCES
A liquid release can be detected at lower Liquid Loading Levels with
extremely clean glassware. The glass plates and glass grid should be cleaned
with a laboratory detergent, rinsed with Deionized water, rinsed with acetone,
and thoroughly dried.
4.0 APPARATUS AND MATERIALS
4.1 Glass Plate: 2 glass plates measuring 7.5 cm x 7.5 cm.
4.2 Glass Grid: See Figure 3.
4.3 Paint Brush: Two small paint brushes for applying dyes.
4.4 Spatula: To assist in loading the sample.
4.5 Weight: 2.7 kg weight to apply pressure to the sample.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Methylene Blue dye in methanol.
9096 - 11 Revision 0
September 1994
-------
5.3 Anthraquinone dye in toluene.
6.0 SAMPLE COLLECTION, PRESERVATION AND HANDLING
See LRT Procedure.
7.0 PROCEDURE
7.1 Paint one strip, approximately 1 cm wide, of methylene blue dye
across the center of a clean and dry glass plate (see Figure 4). The dye is
allowed to dry.
7.2 Paint one strip, approximately 1 cm wide, of anthraquinone dye
across the center of the same glass plate (see Figure 4). This strip should be
adjacent to and parallel with the methylene blue strip. The dye is allowed to
dry.
7.3 Place the glass grid in the center of the dye-painted glass plate.
7.4 Place a small amount of sample into the glass-grid holes, pressing
down gently until the holes are filled to slightly above the grid top.
7.5 Place a second, clean and dry, glass plate on top of the sample and
grid.
7.6 Place a 2.7 kg weight on top of the glass for 5 minutes.
7.7 After 5 minutes remove the weight and examine the base of the grid
extending beyond the sample holes for any indication of dyed liquid. The entire
assembly may be turned upside down for observation. Any indication of liquid
constitutes a release and the LRT does not need to be performed.
8.0 QUALITY CONTROL
8.1 Refer to Chapter One for specific quality control procedures.
9.0 METHOD PERFORMANCE
9.1 Precision and accuracy data are not available at this time.
10.0 REFERENCES
1. Research Triangle Institute. "Background Document for the Liquid Release
Test: Single Laboratory Evaluation and 1988 Collaborative Study".
Submitted to the Environmental Protection Agency under Contract No. 68-01-
7075, Work Assignment 76 and Contract No. 68-WO-0032, Work Assignment 12.
September 18, 1991.
9096 - 12 Revision 0
September 1994
-------
METHOD 9096
APPENDIX A
START
7.1 Paint methy 1ene
b lue strip on
glass. dry
7 2 Paint
anthraquinone strip
on glass parallel
to first strip, dry
7 3 Place grad in
center of glass
plate
7 4 Fill
holes of
grid with sample
7 5 Place second
glass plale on lop
of sample
7.6 Apply weight on
glass for S minutes
7 7 Remove weight
and check for x«t
spot|sI
STOP
9096 - 13
Revision 0
September 1994
-------
SO
-------
METHOD 9131
TOTAL COLIFORM: MULTIPLE TUBE FERMENTATION TECHNIQUE
1.0 SCOPE AND APPLICATION
1.1 This method is used to determine the presence of a member of the
coliform group in ground water and surface water.
1.2 The coliform group, as analyzed for in this procedure, i.s defined as
all aerobic and facultative anaerobic, gram-negative, non-spore-forming, rod-
shaped bacteria that ferment lactose with gas formation within 48 hr at 35*C.
2.0 SUMMARY OF METHOD
2.1 The multiple-tube fermentation technique is a three-stage procedure
in which the results are statistically expressed in terms of the Most Probable
Number (MPN). These stages -- the presumptive stage, confirmed stage, and
completed test — are briefly summarized below. (For the analysis to be
accurate, a five-tube test is required.)
2.1.1 Presumptive Stage: A series of lauryl tryptose broth primary
fermentation tubes are inoculated with graduated quantities of the sample
to be tested. The inoculated tubes are incubated at 35 + 0.5*C for
24+2 hr, at which time the tubes are examined for gas formation. For
the tubes in which no gas is formed, continue incubation and examine for
gas formation at the end of 48 + 3 hr. Formation of gas in any amount
within 48 + 3 hr is a positive presumptive test.
2.1.2 Confirmed Stage: The confirmed stage is used on all primary
fermentation tubes showing gas formation during the 24-hr and 48-hr
periods. Fermentation tubes containing brilliant green lactose bile
broth are inoculated with medium from the tubes showing a positive result
in the presumptive test. Inoculation should be performed as soon as
possible after gas formation occurs. The inoculated tubes are incubated
for 48 + 3 hr at 35 + 0.5*C. Formation of gas at any time in the tube
indicates a positive confirmed test.
2.1.3 Completed Test: The completed test is performed on all
samples showing a positive result in the confirmed test. It can also be
used as a quality control measure on 20% of all samples analyzed. One or
more plates of eosin methylene blue are streaked with sample to be
analyzed. The streaked plates are incubated for 24 + 2 hr at 35 + 0.5*C.
After incubation, transfer one or more typical colonies (nucleated, with
or without metallic sheen) to a lauryl tryptose broth fermentation tube
and a nutrient agar slant. The fermentation tubes and agar slants are
incubated at 35 + 0.5'C for 24+2 hr, or for 48 + 3 hr if gas is not
produced. From the agar slants corresponding to the fermentation tubes
in which gas formation occurs, gram-stained samples are examined
9131 - 1
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microscopically. The formation of gas 1n the fermentation tube and the
presence of gram-negative, non-spore-forming, rod-shaped bacteria 1n the
agar culture may be considered a satisfactorily completed test,
demonstrating the positive presence of coHform bacteria 1n the analyzed
sample.
2.2 More detailed treatment of this method 1s presented In Standard
Methods for the Examination of Water and Wastewater and 1n Microbiological
Methods for Monitoring the Environment (see References, Section 10.0).
3.0 INTERFERENCES
3.1 The distribution of bacteria 1n water is irregular. Thus, a
five-tube test 1s required 1n this method for adequate statistical accuracy.
3.2 The presence of residual chlorine or other halogens can prevent the
continuation of bacterial action. To prevent this occurrence, sodium
thlosulfate should be added to the sterile sample container.
3.3 Water samples high 1n copper, zinc, or other heavy metals can be
toxic to bacteria. Chelatlng agents such as ethylened1am1netetraacet1c acid
(EDTA) should be added only when heavy metals are suspected of being present.
3.4 It 1s Important to keep 1n mind that MPN tables are probability
calculations and Inherently have poor precision. They Include a 23% positive
bias that generally results 1n high value. The precision of the MPN can be
Improved by Increasing the number of sample portions examined and the number
of samples analyzed from the same sampling point.
4.0 APPARATUS AND MATERIALS
4.1 Incubatorsj
4.1.1 Incubators must maintain a uniform and constant temperature
at all times 1n all areas, that 1s, they must not vary more than +0.5*C
1n the areas used. Obtain such accuracy by using a water-jacketed, or
anhydrlc-type Incubator with thermostatically controlled low-temperature
electric heating units properly Insulated and located in or adjacent to
the walls or floor of the chamber and preferably equipped with mechanical
means of circulating air. If a hot-air type Incubator 1s used, humidity
must be maintained at 75-80%.
4.1.2 Alternatively, use special Incubating rooms well Insulated
and equipped with properly distributed heating units and with forced air
circulation, provided that they conform to desired temperature limits and
relative humidity. When such rooms are used, record the dally
temperature range 1n areas where plates or tubes are Incubated. Provide
Incubators with open metal wire or sheet shelves so spaced as to assure
temperature uniformity throughout the chamber. Leave a 2.5-cm space
between walls and stacks of dishes or baskets of tubes.
9131 - 2
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4.1.3 Maintain an accurate thermometer with the bulb Immersed 1n
liquid (glycerine, water, or mineral oil) on each shelf 1n use within the
Incubator and record dally temperature readings (preferably morning and
afternoon). It 1s desirable, 1n addition, to maintain a maximum and
minimum registering thermometer within the Incubator on the middle shelf
to record the gross temperature range over a 24-hr period. At Intervals,
determine temperature variations within the Incubator when filled to
maximum capacity. Install a recording thermometer, whenever possible, to
maintain a continuous and permanent record of temperature. Mercury
thermometers should be graduated 1n 0.5*C Increments and calibrated
annually against an NBS certified thermometer. Dial thermometers should
be calibrated quarterly.
4.1.4 Keep water depth 1n the water bath sufficient to Immerse
tubes to upper level of media.
4.2 Hot-a1r sterilizing ovens; Use hot-air sterilizing ovens of
sufficient size to prevent Internalcrowding, constructed to give uniform and
adequate sterilizing temperatures of 170 + 10*C and equipped with suitable
thermometers. As an alternative, use a temperature-recording Instrument.
4.3 Autoclaves;
4.3.1 Use autoclaves of sufficient size to prevent Internal
crowding, constructed to provide uniform temperatures within the chambers
(up to and Including the sterilization temperature of 121*C); equipped
with an accurate thermometer, the bulb of which 1s located properly on
the exhaust line so as to register minimum temperature within the
sterilizing chambers (temperature-recording Instrument 1s optional);
equipped with pressure gauge and properly adjusted safety valves
connected directly with saturated-steam power lines or directly to a
suitable special steam generator (do not use steam from a boiler treated
with amines for corrosion control); and capable of reaching the desired
temperature within 30 m1n.
4.3.2 Use of a vertical autoclave or pressure cooker 1s not
recommended because of difficulty 1n adjusting and maintaining
sterilization temperature and the potential hazard. If a pressure cooker
1s used .1n emergency or special circumstances, equip 1t with an efficient
pressure gauge and a thermometer, the bulb of which 1s 2.5 cm above the
water level.
4.4 Colony counters; Use Quebec-type colony counter, dark-field model
preferred, or one providing equivalent magnification (1.5 diameters) and
satisfactory visibility.
4.5 pH Equipment; Use electrometrlc pH meters, accurate to at least 0.1
pH units, for determining pH values of media. See Method 9040 for standardi-
zation of a pH meter.
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4.6 Balances; Use balances providing a sensitivity of at least 0.1 g at
a load of 150 gr with appropriate weights. Use an analytical balance having a
sensitivity of 1 mg under a load of 10 g for weighing small quantities (less
than 2 g) of materials. Single-pan rapid-weigh balances are most convenient.
4.7 Media preparation utensils; Use boroslHcate glass or other
suitable noncorroslve equipment such as stainless steel. Use glassware that
1s clean and free of residues, dried agar, or other foreign materials that may
contaminate media.
4.8 Plpets and graduated cylinders;
4.8.1 Use plpets of any convenient size, provided that they deliver
the required volume accurately and quickly. The error of calibration for
a given manufacturer's lot must not exceed 2.5%. Use plpets having
graduations distinctly marked and with unbroken tips. Bacteriological-
transfer plpets or plpets conforming to the APHA standards given 1n the
latest edition of Standard Methods for the Examination of Dairy Products
may be used. Optimally, protect themouth end of all plpets by a cotton
plug to eliminate hazards to the worker or possible sample contamination
by saliva,
4.8.2 Use graduated cylinders meeting ASTM Standards (D-86 and D-
216) and with accuracy limits established by the National Bureau of
Standards, where appropriate.
4.9 PIpet containers; Use boxes of aluminum or stainless steel, end
measurement 5 to 7.5 cm, cylindrical or rectangular, and length about 40 cm.
When these are not available, paper wrappings may be substituted. To avoid
excessive charring during sterilization, use best-quality sulfate pulp (Kraft)
paper. Do not use copper or copper alloy cans or boxes as plpet containers.
4.10 Dilution bottles or tubes;
4.10.1 Use bottles or tubes of resistant glass, preferably
boroslHcate glass, closed with glass stoppers or screw caps equipped
with liners that do not produce toxic or bacterlostatlc compounds on
sterilization.
4.10.2 Do not use cotton plugs as closures. Mark gradation levels
Indelibly on side of dilution bottle or tube. Plastic bottles of
nontoxlc material and acceptable size may be substituted for glass,
provided that they can be sterilized properly.
4.11 Petrl dishes; Use glass or plastic Petrl dishes about 100 x 15 mm.
Use dishes the bottoms of which are free from bubbles and scratches and flat
so that the medium will be of uniform thickness throughout the plate. For the
membrane-filter technique, use loose-Hd glass or plastic dishes, 60 x 15 mm,
or t1ght-Hd dishes, 50 x 12 mm. Sterilize Petrl dishes and store 1n metal
cans (aluminum or stainless steel, but not copper), or wrap 1n paper --
preferably best-quality sulfate pulp (Kraft) -- before sterilizing.
9131 - 4
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4.12 Fermentation tubes and vials; Use only 10-mm x 75-mm fermentation
tubes. When tubes areusedforatest of gas production, enclose a shell
vial, Inverted. Use a vial of such size that U will be filled completely
with medium and at least partly submerged 1n the tube.
4.13 Inoculating equipment; Use wire loops made of 22- or 24-gauge
nickel alloy (chromel, nlchrome, or equivalent) or plat1num-1r1d1um for flame
sterilization. Single-service transfer loops of aluminum or stainless steel
are satisfactory. Use loops at least 3 mm 1n diameter. Sterilize by dry heat
or steam. Single-service hardwood applicators also may be used. Make these
0.2 to 0.3 cm 1n diameter and at least 2.5 cm longer than the fermentation
tube; sterilize by dry heat and store 1n glass or other nontoxlc containers.
5.0 REAGENTS
5.1 ASTM Type II water (ASTM D1193); Water should be monitored for
Impurities.
5.2 Buffered water;
5.2.1 To prepare stock phosphate buffer solution, dissolve 34.0 g
potassium d1hydrogen phosphate (KHgPO^ 1n 500 ml Type II water, adjust
to pH 7.2 + 0.5 with 1 N sodium hydroxide (NaOH), and dilute to 1 liter
with Type II water.
5.2.2 Add 1.25 ml stock phosphate buffer solution and 5.0 ml
magnesium chloride solution (38 g MgClj/Uter Type II water or
81.1 g MgCl2'6H20/l1ter Type II water) to 1 liter Type II water.
Dispense 1n amounts that will provide 99 + 2.0 ml or 9 + 0.2 ml after
autoclavlng for 15 m1n.
5.2.3 Peptone water: Prepare a 10% solution of peptone 1n Type II
water. Dilute a measured volume to provide a final 0.1% solution. Final
pH should be 6.8.
5.2.4 Dispense In amounts to provide 99 + 2.0 ml or 9 + 0.2 ml
after autoclavlng for 15 m1n.
5.2.5 Do not suspend bacteria 1n any dilution water for more than
30 m1n at room temperature because death or multiplication may occur,
depending on the species.
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5.3 Lauryl tryptose broth;
5.3.1 Components of the broth are:
Tryptose 20.0 g
Lactose 5.0 g
D1phosphate hydrogen
phosphate, K2HP04 2.75 g
Potassium dlhydrogen
phosphate, KH2P04 2.75 g
Sodium chloride, NaCI 5.0 g
Sodium lauryl sulfate 0.1 g
Type II water 1 liter
Lauryl tryptose broth 1s also available 1n a prepackaged dry powder form.
5.3.2 Make lauryl tryptose broth of such strength that adding
100-mL or 10-mL portions of sample to medium will not reduce Ingredient
concentrations below those of the standard medium. Prepare 1n accordance
with Table 1.
TABLE 1. PREPARATION OF LAURYL TRYPTOSE BROTH
Inoculum
(mL)
1
10
10
100
100
100
Amount of
Medium In Tube
(mL)
10 or more
10
20
50
35
20
Volume of
Medium +
Inoculum
(mL)
11 or more
20
30
150
135
120
Dehydrated Lauryl
Tryptose Broth
Required
(g/Hter)
35.6
71.2
53.4
106.8
137.1
213.6
5.3.3 Dispense the broth Into fermentation tubes which contain
Inverted vials. Add an amount sufficient to cover the Inverted vial, at
least partially, after sterilization has taken place. Sterilize at 121*C
for 12 to 15 m1n. The pH should be 6.8 + 0.2 after sterilization.
5.4 Brilliant green lactose bile broth;
5.4.1 Components of the broth are:
Peptone 10.0 g
Lactose 10.0 g
Oxgall 20.0 g
Brilliant green 0.0133 g
Type II water 1 liter
9131 - 6
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Date September 1986
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This broth 1s also available 1n a prepackaged dry powder form.
5.4.2 Dispense the broth Into fermentation tubes which contain
Inverted vials. Add an amount sufficient to cover the Inverted vial, at
least partially, after sterilization has taken place. Sterilize at 121*C
for 12 to 15 min. The pH should be 7.2 + 0.2 after sterilization.
5.5 Ammonium oxalate-crystal violet (Hucker's); Dissolve 2 g crystal
violet (90%3yecontent)In20mL95%ethyl alcohol, dissolve 0.8 g
(NH4)£C204'H20 1n 80 mL Type II water, mix the two solutions, and age for
24 hr before use; filter through paper Into a staining bottle.
5,6 Lugo!'s solution, Gram's modification; Grind 1 g Iodine crystals
and 2 g KI 1ni a mortar. Add"Type II water, a few mill litters at a time, and
grind thoroughly after each addition until solution 1s complete. Rinse
solution Into an amber glass bottle with the remaining water (using a total of
300 ml).
5.7 Counterstaln; Dissolve 2.5 g safranln dye 1n 100 mL 95% ethyl
alcohol. Add 10 ml to 100 ml Type II water.
5.8 Acetone alcohol; Mix equal volumes of ethyl alcohol, 95%, with
acetone.
5.9 Gram staining kits; Commercially available kits may be substituted
for 5.5, 5.6, 5.7, and 5.8.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed 1n U.S. EPA, 1978.
6.2 Clean all glassware thoroughly with a suitable detergent and hot
water, rinse with hot water to remove all traces of residual washing compound,
and finally rinse with Type II water. If mechanical glassware washers are
used, equip them with Influent plumbing of stainless steel or other nontoxlc
material. Do not use copper piping to distribute Type II water. Use
stainless steel or other nontoxlc material for the rinse-water system.
6.2.1 Sterilize glassware, except when 1n metal containers, for not
less than 60 m1n at a temperature of 170*C, unless 1t 1s known from
recording thermometers that oven temperatures are uniform, under which
exceptional condition use 160*C. Heat glassware 1n metal containers to
170*C for not less than 2 hr.
6.2.2 Sterilize sample bottles not made of plastic as above, or 1n
an autoclave at 121*C for 15 min. Perform a sterility check on one
bottle per batch.
9131 - 7
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Date September 1986
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6.2.3 If water containing residual chlorine and other halogens 1s
to be collected, add sufficient ^$203 to clean sample bottle before
sterilization to give a concentration of about 100 mg/L in the sample.
To a 120-mL bottle add 0.1 mL 10% solution of Na2$203 (this will
neutralize a sample containing about 15 mg/L residual chlorine). Stopper
bottle, cap, and sterilize by either dry or moist heat, as directed
previously.
6.2.4 Collect water samples high 1n copper or zinc and wastewater
samples high 1n heavy metals 1n sample bottles containing a chelatlng
agent that will reduce metal toxldty. This 1s particularly significant
when such samples are 1n transit for 4 hr or more. Use 372 mg/L of the
tetrasodlum salt of ethylenedlaminetetraacetlc add (EDTA). Adjust EDTA
solution to pH 6.5 before use. Add EDTA separately to sample bottle
before bottle sterilization (0.3 mL 15% solution 1n a 120-mL bottle) or
combine It with the ^$203 solution before addition.
6.3 When the sample 1s collected, leave ample air space 1n the bottle
(at least 2.5 cm) to facilitate mixing by shaking, preparatory to examination.
Be careful to take samples that will be representative of the water being
tested and avoid sample contamination at time of collection or 1n period
before examination.
6.4 Keep sampling bottle closed until the moment 1t 1s to be filled.
Remove stopper and hood or cap as a unit, taking care to avoid soiling.
During sampling, do not handle stopper or cap and neck of bottle and protect
them from contamination. Hold bottle near base, fill 1t without rinsing,
replace stopper or cap Immediately, and secure hood around neck of bottle.
7.0 PROCEDURE
7.1 Presumptive stage:
7.1.1 Inoculate a series of fermentation tubes ("primary"
fermentation tubes) with appropriate graduated quantities (multiples and
submultlples of 1 mL) of sample. Be sure that the concentration of
nutritive Ingredients 1n the mixture of medium and added sample conforms
to the requirements given In Paragraph 5.3. Use a sterile plpet for
Initial and subsequent transfers from each sample container. If the
plpet becomes contaminated before transfers are completed, replace with a
sterile plpet. Use a separate sterile plpet for transfers from each
different dilution. Do not prepare dilutions 1n direct sunlight. Use
caution when removing sterile plpets from the container; to avoid
contamination, do not drag plpet tip across exposed ends of plpets or
across Ups and necks of dilution bottles. When removing sample, do not
Insert plpets more than 2.5 cm below the surface of sample or dilution.
When discharging sample portions, hold pipet at an angle of about 45*,
with tip touching the Inside neck of the tube. The portions of sample
used for. Inoculating lauryl-tryptose-broth fermentation tubes will vary
1n size and number with the character of the water under examination, but
9131 - 8
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Date September 1986
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1n general use decimal multiples and submultiples of 1 ml_. Use Figure 1
as a guide to preparing dilutions. After adding sample, mix thoroughly
by shaking the test tube rack. Do not invert the tubes.
7.1.2 Incubate inoculated fermentation tubes at 35 + 0.5'C. After
24 + 2 hr shake each tube gently and examine it and, if no gas has formed
and~been trapped in the inverted vial, reincubate and reexamine at the
end of 48 +3 hr. Record presence or absence of gas formation,
regardless of amount, at each examination of the tubes.
7.1.3 Formation of gas in any amount in the inner fermentation
tubes or vials within 48 + 3 hr constitutes a positive presumptive test.
Do not confuse the appearance of an air bubble 1n a clear tube with
actual gas production. If gas is formed as a result of fermentation, the
broth medium will become cloudy. Active fermentation may be shown by the
continued appearance of small bubbles of gas throughout the medium
outside the Inner vial when the fermentation tube is shaken gently.
7.1.4 The absence of gas formation at the end of 48+3 hr of
incubation constitutes a negative test. An arbitrary limit of 48 hr for
observation doubtless excludes from consideration occasional members of
the col 1 form group that form gas very slowly and generally are of limited
sanitary significance.
7.2 Confirmed stage;
7.2.1 Submit all primary fermentation tubes showing any amount of
gas within 24 hr of incubation to the Confirmed Test. If active
fermentation appears in the primary fermentation tube earlier than 24 hr,
transfer to the confirmatory medium without waiting for the full 24-hr
period to elapse. If additional primary fermentation tubes show gas
production at the end of 48-hr incubation, submit these to the Confirmed
Test.
7.2.2 Gently shake or rotate primary fermentation tube showing gas
and do one of two things: (a) with a sterile metal loop, 3 mm in
diameter, transfer one loopful of culture to a fermentation tube
containing brilliant green lactose bile broth, or (b) insert a sterile
wooden applicator at least 2.5 cm long Into the culture, remove it
promptly, and plunge it to the bottom of fermentation tube containing
brilliant green lactose bile broth. Remove and discard applicator.
7.2.3 Incubate the inoculated brilliant green lactose bile broth
tube for 48 + 3 hr at 35 + 0.5*C. Formation of gas 1n any amount in the
Inverted viaT of the brilliant green lactose bile broth fermentation tube
at any time within 48 + 3 hr constitutes a positive Confirmed Test.
7.3 Completed test;
7.3.1 Use the Completed Test on positive confirmed tubes to
establish definitely the presence of col 1 form bacteria and provide
quality control data for 20% of all samples analyzed.
9131 - 9
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Date September 1986
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Delivery
volume
Culture dishes
Actual volume
of cample in
diih
1 ml
0.1 ml
10"2 ml
10'3 ml
Figure 1. Preparation of dilutions.
9131 - 10
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Date September 1986
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7.3.2 Streak one or more eosln methylene blue plates from each tube
of brilliant green lactose bile broth showing gas as soon as possible
after the appearance of gas. Streak plates to ensure presence of some
discrete colonies separated by at least 0.5 cm. Observe the following
precautions when streaking plates to obtain a high proportion of
successful Isolations If coliform organisms are present: (a) use an
Inoculating needle slightly curved at the tip; (b) tap and Incline the
fermentation tube to avoid picking up any membrane or scum on the needle;
(c) Insert end of needle into the liquid in the tube to a depth of
approximately 5.0 mm; and (d) streak plate with curved section of the
needle 1n contact with the agar to avoid a scratched or torn surface.
7.3.3 Incubate plates (inverted) at 35 + 0.5'C for 24+2 hr.
7.3.4 The colonies developing on eosln methylene blue agar are
called: typical (nucleated, with or without metallic sheen); atypical
(opaque, unnucleated, mucoid, pink after 24-hr Incubation); or negative
(all others). From each of these plates, pick one or more typical well-
isolated coliform colonies or, if no typical colonies are present, pick
two or more colonies considered most likely to consist of organisms of
the coliform group and transfer growth from each isolate to a lauryl-
tryptose-broth fermentation tube and to a nutrient agar slant.
NOTE: If possible, when transferring colonies, choose well-isolated
colonies and barely touch the surface of the colony with a
flame-sterilized, air-cooled transfer needle to minimize the
danger of transferring a mixed culture.
7.3.5 Incubate secondary broth tubes at 35 + 0.5*C for 24+2 hr;
if gas is not produced within 24+2 hr, reincubate and examine again at
48+3 hr. Microscopically examine gram-stained preparations (see
Paragraph 7.4) from those 24-hr agar slant cultures corresponding to the
secondary tubes that show gas.
7.3.6 Formation of gas 1n the secondary tube of lauryl tryptose
broth within 48 + 3 hr and demonstration of gram-negative, non-spore-
forming, rod-shaped bacteria in the agar culture constitute a
satisfactory Completed Test, demonstrating the presence of a member of
the coliform group.
7.4 Gram-stain procedure:
7.4.1 Prepare a light emulsion of the bacterial growth from an agar
slant in a drop of Type II water on a glass slide. A1r-dry or fix by
passing the slide through a flame and stain for 1 m1n with the ammonium
oxalate-crystal violet solution. Rinse the slide in tap water; apply
Lugol's solution for 1 m1n. (See Paragraphs 5.5-5.8 for reagent.)
7.4.2 Rinse the stained slide in tap water. Decolorize for
approximately 15 to 30 sec with acetone alcohol by holding slide between
the fingers and letting acetone alcohol flow across the stained smear
until no more stain Is removed. Do not over-decolorize. Counterstain
with safranln (Paragraph 5.7) for 15 sec, then rinse with tap water, blot
dry with bibulous paper, and examine microscopically.
9131 - 11
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Date September 1986
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7.4.3 Cells that decolorize and accept the safranin stain are pink
and defined as gram-negative In reaction. Cells that do not decolorize
but retain the crystal violet stain are deep blue and are defined as
gram-positive.
7.5 Computing and recording of MPN;
7.5.1 The calculated density of conform bacteria in a sample can
be obtained from the MPN table, based on the number of positive tubes in
each dilution of the confirmed or completed test. Table 2 shows MPN
Indices and 95% confidence limits for potable water testing, and Table 3
describes the MPN indices and 95% confidence limits for general use.
TABLE 2. MPN INDEX AND 95% CONFIDENCE LIMITS FOR VARIOUS COMBINATIONS OF
POSITIVE AND NEGATIVE RESULTS WHEN FIVE 10-mL PORTIONS ARE USED
Number of Tubes
Giving Positive
Reaction out of
5 of 10 mL each
0
1
2
3
4
5
MPN
Index per
100 mL
<2.2
2.2
5.1
9.2
16
>16
95% Confidence
Lower
0
0.1
0.5
1.6
3.3
8.0
Limits
Upper
6.0
12.6
19.2
29.4
52.9
Infinite
7.5.2 Three dilutions are necessary for the determination of the
MPN Index. For example (see Table 3), 1f five 10-mL, five 1.0-mL, and
five 0.1-mL portions of the samples are used as Inocula and four of the
10-mL, two of the 1-mL, and none of the 0.1-mL portions of inocula give
positive results, the coded result is 4-2-0 and the MPN index is 22 per
100 mL.
7.5.3 In cases when the serial decimal dilution is other than 10,
1, and 0.1 mL, or when more than three sample volumes are used in the
series, refer to the sources cited 1n Section 10.0, References, for the
necessary density determination procedures.
7.5.4 All MPN values for water samples should be reported on the
basis of a 100-mL sample.
8.0 QUALITY CONTROL
8.1 Extensive quality control procedures are provided in Part IV of U.S.
EPA, 1978 (see Section 10.0, References). These procedures should be adhered
to at all times.
9131 - 12
Revision 0
Date September 1986
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TABLE 3. MPN INDEX FOR SERIAL DILUTIONS OF SAMPLE
Number of Tubes
Giving Positive
Reaction out of
5 of
10 mL
each
0
0
0
0
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
3
4
4
4
4
4
5 of
1 mL
each
0
0
1
2
0
0
1
1
2
0
0
1
1
2
3
0
0
1
1
2
2
3
0
0
1
1
1
5 of
0.1 mL
each
0
1
0
0
0
1
0
1
0
0
1
0
1
0
0
0
1
0
1
0
1
0
0
1
0
1
2
MPN
Index
per
100 mL
<2
2
2
4
2
4
4
6
6
5
7
7
9
9
12
8
11
11
14
14
17
17
13
17
17
21
26
95%
Confidence
Limits
Lower
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
1
1
2
2
3
1
2
2
4
4
5
5
3
5
5
7
9
Upper
7
7
11
7
11
11
15
15
13
17
17
21
21
28
19
25
25
34
34
46
46
31
46
46
63
78
Source: U.S. EPA, 1978.
(Continued on next page)
9131 - 13
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Date September 1986
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TABLE 3. MPN INDEX FOR SERIAL DILUTIONS OF SAMPLE
(Continued)
Number of Tubes
Giving Positive
Reaction out of
5 of
10 mL
each
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5 of
1 mL
each
2
2
3
3
4
0
0
0
1
1
1
2
2
2
3
3
3
3
4
4
4
4
4
5
5
5
5
5
5
5 of
0.1 mL
each
0
1
0
1
0
0
1
2
0
1
2
0
1
2
0
1
2
3
0
1
2
3
4
0
1
2
3
4
5
MPN
Index
per
100 mL
22
26
27
33
34
23
31
43
33
46
63
49
70
94
79
110
140
180
130
170
220
280
350
240
350
540
920
1600
^2400
95%
Confidence
Limits
Lower
7
9
9
11
12
7
11
15
11
16
21
17
23
28
25
31
37
44
35
43
57
90
120
68
120
180
300
640
Upper
67
78
80
93
93
70
89
110
93
120
150
130
170
220
190
250
340
500
300
490
700
850
1000
750
1000
1400
3200
5800
Source: U.S. EPA, 1978.
9131 - 14
Revision 0
Date September 1986
-------
8.2 Samples must be maintained as closely as possible to original
condition by careful handling and storage. Sample sites and sampling
frequency should provide data representative of characteristics and
variability of the water quality at that site. Samples should be analyzed
Immediately. They should be refrigerated at a temperature of 1-4*C and
analyzed within 6 hr.
8.3 Quality control of culture media is critical to the validity of
microbiological analysis. Some important factors to consider are summarized
below:
8.3.1 Order media to last for only 1 yr; always use oldest stock
first. Maintain an inventory of all media ordered, Including a visual
Inspection record.
8.3.2 Hold unopened media for no longer than 2 yr. Opened media
containers should be discarded after 6 mo.
8.3.3 When preparing media keep containers open as briefly as
possible. Prepare media 1n delonlzed or distilled (Type II) water of
proven quality. Check the pH of the media after solution and
sterilization; it should be within 0.2 units of the stated value.
Discard and remake if it is not.
8.3.4 Autoclave media for the minimal time specified by the
manufacturer because the potential for damage increases with increased
exposure to heat. Remove sterile media from the autoclave as soon as
pressure 1s zero. Effectiveness of the sterilization should be checked
weekly, using strips or ampuls of Bacillus stearothemophelus.
8.3.5 Agar plates should be kept slightly open for 15 min after
pouring or removal from refrigeration to evaporate free moisture. Plates
must be free of lumps, uneven surfaces, pock marks, or bubbles, which can
prevent good contact between the agar and medium.
8.3.6 Avoid shaking fermentation tubes, which can entrap air 1n the
Inner vial and produce a false positive result.
8.3.7 Store fermentation tube media in the dark at room temperature
or 4*C. If refrigerated, Incubate overnight at room temperature to
detect false positive gas bubbles.
8.3.8 Quality control checks of prepared media should include the
incubation of 5% of each batch of medium for 2 days at 35*C to inspect
for growth and positive/negative checks with pure culture.
8.4 Analytical quality control procedures should include:
8.4.1 Duplicate analytical runs on at least 10% of all known
positive samples analyzed.
9131 - 15
Revision 0
Date September 1986
-------
8.4.2 At least one positive control sample should be run each month
for each parameter tested.
8.4.3 At least one negative (sterile) control should be run with
each series of samples using buffered water and the medium batch used at
the beginning of the test series and following every tenth sample. When
sterile controls Indicate contamination, new samples should be obtained
and analyzed.
8.4.4 The Type II water used should be periodically checked for
contamination.
8.4.5 For routine MPN tests, at least 5% of the positive confirmed
samples should be tested by the complete test.
9.0 METHOD PERFORMANCE
9.1 No data provided.
10.0 REFERENCES
1. Standard Methods for the Examination of Water and Wastewater, 15th ed.
(1980).
2. U.S. Environmental protection Agency, Microbiological Methods for
Monitoring the Environment, EPA 600/8-78-017, December 1978.
9131 - 16
Revision
Date September 1986
-------
•"£ Tec; 9:2-.
MULTIPLE TUBE FE«MENT»TIOS TECWNIOJ£
Stage
oiif Irmcc
St«ge
7.1.1
Inoculate • series of
ferment•11 on tuDes
-Ifi cr*Ouat.eO
ooj^tltus o' sample
7.1.5
7.2.1
SuOmlt tuDes
for «hic" oas
has formao. Co
Confirmee Tesi
XncuDate
inoculatea
fermtntat Son
tuett
RcincuCate
reaxamine at
no of -4B hours
7.2.2
Shake
tube:
place culture
In tuDe »1t^
green lactose
Bill brotn
7.2.3
Incubate t» le
Orotn tuOe for
4B nours
9131 - 17
Revision 0
Date September 1986
-------
TOTAL
Mttrioo 913:
" MULTIPLE TyeE FERMENTATION TECHNIQUE
(Cent inuca!
Comoleted
Te*t
7.3.1
Prepare
emultion
o' bacterial
growtn fro^
agar slant 'or
Submit
tublt
g*< n«*
fo>-(n«o to
Completed Test
7.3.8
7. 4 il
A>r-dry or fix
Stre»K «o«ln
»i«tnyl»n« blue Ol»te«
from ««cn tuO« of
Cllt orotn
cnocino g»*
7.-I.2
ncuDate
examine »9«ln
•t 8 hour*
ttclneo preo-
• r»tlons froir
»)»nt cultures
(see 7.4)
Decolorize.
countercta in
wltn »»fr»nln:
7.3.3
Incubate
inverted plate*
7.3
Gram
negative cells
are olnt: gram
positive cells
are oeeo blue
typical
colonies: transfer
orowtn to
fermentation tube
•no aosr slant
7.3.5
7.5
density of
col 1 form
bacteria from
MPN table
Incubate
secondary
Broth tubes
for 24 hours
Stop
)
( Stop J
9131 - 18
Revision 0
Date September 1986
-------
in
o
-------
METHOD 9250
CHLORIDE (COLORIMETRIC. AUTOMATED FERRICYANIDE AAI)
1.0 SCOPE AND APPLICATION
1.1 This automated method is applicable to ground water, drinking,
surface, and saline waters, and domestic and Industrial wastes. The
applicable range 1s 1 to 250 mg Cl per liter of sample.
2.0 SUMMARY OF METHOD
2.1 Thlocyanate 1on (SCN) 1s liberated from mercuric thlocyanate through
sequestration of mercury by chloride 1on to form un-1on1zed mercuric chloride.
In the presence of ferric 1on, the liberated SCN forms highly colored ferric
thiocyanate 1n a concentration proportional to the original chloride
concentration.
3.0 INTERFERENCES
3.1 No significant interferences.
4.0 APPARATUS AND MATERIALS
4.1 Automated continuous-flow analytical instrument;
4.1.1 Sampler I.
4.1.2 Continuous filter.
4.1.3 Manifold.
4.1.4 Proportioning purap.
4.1.5 Colorimeter: equipped with 15-mm tubular flowcell and 480-nm
filters.
4.1.6 Recorder.
5.0 REAGENTS
5.1 ASTM Type II water (ASTM D1193): Water should be monitored for
impurities.
5.2 Ferric ammonium sulfate; Dissolve 60 g of FeNH4(S04)2'12H20 in
approximately 500 mL TypeII water. Add 355 mL of concentrated HNOs and
dilute to 1 liter with Type II water. Filter.
9250 - 1
Revision 0
Date September 1986
-------
5.3 Saturated mercuric thigcyanate; Dissolve 5 g of Hg(SCN)2 1n 1 liter
of Type II water.Decantand"f1Hera portion of the saturated supernatant
liquid to use as the reagent and refill the bottle with distilled water.
5.4 Sodium chloride stock solution (0.0141 N NaCl): Dissolve 0.8241 g
of pre-drled (140*C) NaCl 1n Type II water. Dilute to 1 liter in a volumetric
flask (1 ml = 0.5 mg Cl).
5.4.1 Prepare a series of standards by diluting suitable volumes of
stock solution to 100.0 ml with Type II water. The following dilutions
are suggested:
Stock
Solution (ml) Concentration (mg/L)
1.0 5.0
2.0 10.0
4.0 20.0
8.0 40.0
15.0 75.0
20.0 100.0
30.0 150.0
40.0 200.0
50.0 250.0
Choose three of the nine standard concentrations 1n such a way that the
chosen standards will bracket the expected concentration range of the
sample.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed in Chapter Nine of this manual.
6.2 No special requirements for preservation.
7.0 PROCEDURE
7.1 No advance sample preparation is required. Set up manifold, as
shown 1n Figure 1. For water samples known to be consistently low in chloride
content, it is advisable to use only one Type II water Intake line.
7.2 Allow both colorimeter and recorder to warm up for 30 mln. Run a
baseline with all reagents, feeding Type II water through the sample line.
Adjust dark current and operative opening on colorimeter to obtain stable
baseline.
9250 - 2
Revision 0
Date September 1986
-------
ro
01
o
I
CA!
o ;o
o/ n
r* <
to o
3
a
cr
n
CONTINUOUS FILTFR
COLORIMETER
15 mm TUBULAR f/c
I mm FILTERS
SAMPUNG TIME: 2 ° MINUTES
oo
FIGURE 1. CHLORIDE MANIFOLD AA I
-------
7.3 Place Type II water wash tubes 1n alternate openings in sampler and
set sample timing at 2.0 m1n.
7.4 Place working standards 1n sampler 1n order of decreasing
concentrations. Complete filling of sampler tray with unknown samples.
7.5 Switch sample line from Type II water to sampler and begin analysis.
7.6 Calculation;
7.6.1 Prepare standard curve by plotting peak heights of processed
standards against known concentrations. Compute concentration of samples
by comparing sample peak heights with standard curve.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or inspection.
8.2 Calibration curves must be composed of a minimum of a blank and
three standards. Employ a minimum of one blank per sample batch to Determine
if contamination has occurred.
8.3 Dilute samples if they are mor.e concentrated than the highest
standard or if they fall on the plateau of a calibration curve.
8.4 Verify calibration with an independently prepared check standard
every 15 samples.
8.5 Run one spike duplicate sample for every 10 samples. A duplicate
sample is a sample brought through the whole sample preparation and analytical
process.
9.0 METHOD PERFORMANCE
9.1 Precision and accuracy data are available in Method 325.1 of Methods
for Chemical Analysis of Water and Wastes.
10.0 REFERENCES
1. O'Brien, J.E., "Automatic Analysis of Chlorides in Sewage," Waste Engr.,
33, 670-672 (Dec. 1962).
2, Standard Methods for the Examination of Water and Wastewater, 14th ed.,
p. 613, Method 602 (1975).
9250 - 4
Revision
Date September 1986
-------
CMLOBIOE ICOLOBIMCTRIC. AUTOMATED re«WICVANIDE AAI)
C •— )
7.1
Set uo *enirala
•ft enown in
Figure 1
7.8
W»r» up
colorl»«t«r
•no r«coro«r;
obtain • «t»ol«
bavtlln*
Switch ••»ol«
line to
• •mpl«r; •nelyzi
7.6.1
Preoere
•tenaero
curve: ceneute
coneentretIon
of e«»D\«m
f Stop J
9250 - 5
Revision 0
Date September 1986
-------
C/l
-------
METHOD 9251
CHLORIDE (COLORIMETRIC, AUTOMATED FERRICYANIDE AAII)
1.0 SCOPE AND APPLICATION
1.1 This automated method 1s applicable to ground water, drinking,
surface, and saline waters, and domestic and Industrial wastes. The
applicable range 1s 1-200 mg Cl~ per liter of sample.
2.0 SUMMARY OF METHOD
2.1 Thlocyanate 1on (SCN) Is liberated from mercuric thlocyanate through
sequestration of mercury by chloride 1on to form un-1on1zed mercuric chloride.
In the presence of ferric 1on, the liberated SCN forms highly colored ferric
thlocyanate 1n a concentration proportional to the original chloride
concentration.
3.0 INTERFERENCES
3.1 No significant Interferences.
4.0 APPARATUS AND MATERIALS
4.1 Automated continuous-flow analytical Instrument;
4.1.1 Sampler I.
4.1.2 Analytical cartridge.
4.1.3 Proportioning pump.
4.1.4 Colorimeter: Equipped with 15-mm tubular flowcell and 480-nm
filters.
4.1.5 Recorder.
4.1.6 Digital printer (optional).
5.0 REAGENTS
5.1 ASTM Type II water (ASTM D1193): Water should be monitored for
Impurities.
5.2 Mercuric thlocyanate solution; Dissolve 4.17 g of HgfSCN)? 1n 500
mL methanoTDilute to 1 literwith methanol, mix, and filter through filter
paper.
9251 - 1
Revision 0
Date September 1986
-------
5.3 Ferric nitrate solution. 20.2%: Dissolve 202 g of Fe(N03)3-9H20 1n
500 ml of Type II water.Add 31.5 ml concentrated nitric acid, mix, and
dilute to 1 liter with Type II water.
5.4 Color reagent; Add 150 ml of mercuric thlocyanate solution
(Paragraph 5.2) to 150 ml of ferric nitrate solution (Paragraph 5.3), mix, and
dilute to 1 liter with Type II water. A combined color reagent is commer-
cially available.
5.5 Sodium chloride stock solution (0.0141 N NaCl): Dissolve 0.8241 g
of pre-dr1ed (140*C) NaCl 1n Type II water. Dilute to 1 liter 1n a volumetric
flask (1 mL = 0.5 mg CT).
5.5.1 Prepare a series of standards by diluting suitable volumes of
stock solution to 100.0 ml with Type II water. The following dilutions
are suggested:
Stock
Solution (ml) Concentration (mq/L)
1.0 5.0
2.0 10.0
4.0 20.0
8.0 40.0
15.0 75.0
20.0 100.0
30.0 150.0
40.0 200.0
Choose three of the nine standard concentrations In such a way that the
chosen standards will bracket the expected concentration range of the
sample.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed 1n Chapter Nine of this manual.
6.2 No special requirements for preservation.
7.0 PROCEDURE
7.1 When particulate matter 1s present, the sample must be filtered
prior to the determination. The sample may be centrlfuged 1n place of
filtration. Set up the manifold, as shown 1n Figure 1.
9251 - 2
Revision
Date September 1986
-------
WASTE
<£>
ro
en
I
to
O 30
o> eo
r+ <.
n> -*•
CO
O)
•a
DILUTION
LOOP
WASTE
i
i
d_
W
If
&
TO SAMPLER
170
• 0
5
146 0152 02
14 TURNS t
• UlOJ i
1 1 1 1 • t
TURNS t
*• TO F/C
COLORIMETER PUMP TUBE
480 NM
15mmF/C
TO WHT/WHT
. ^ WASTE TUBE
*^
1/0 0103 1
•tllllt t
5 TURNS |
WASTE »
PUR ORG.
BLK BLK.
PUR PUR.
ORG. CRN.
BLK. BLK.
BLK BLK.
GRY. GRY.
ORG. CRN.
GRY. GRY.
WHT. WHT
GRY. GRY
PROPOR
mi
M1/MIN
340 OIL WATER
032 AIR
250 OIL. WATER
010 SAMPLE
032 AIR
032 AIR
1.00 RE SAMPLE
010 OIL. WATER
1 00 COLOR REAGENT
0 60 SAMPLE WASTE
100 FROMF/C
TIONING
MP
A4
GO
CTi
Figure 1. Chloride Manifold A A IT 0-200 mg C1/L.
-------
7.2 Allow both colorimeter and recorder to warm up for 30 min. Run a
baseline with all reagents, feeding Type II water through the sample line.
7.3 Place working standards 1n sampler 1n order of decreasing
concentrations. Complete filling of sampler tray with unknown samples.
7.4 When a stable baseline has been obtained, start the sampler.
7.5 Calculation; Prepare standard curve by plotting peak heights of
processed standards against known concentrations. Compute concentration of
samples by comparing sample peak heights with standard curve. Note that this
1s not a linear curve, but a second order curve. (See Paragraph 8.2.)
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Calibration curves must be composed of a minimum of a blank and
three standards. Employ a minimum of one blank per sample batch to determine
if contamination has occurred.
8.3 Dilute samples if they are more concentrated than the highest
standard or 1f they fall on the plateau of a calibration curve.
8.4 Verify calibration with an Independently prepared check standard
every 15 samples.
8.5 Run one spike duplicate sample for every 10 samples. A duplicate
sample 1s a sample brought through the whole sample preparation and analytical
process.
9.0 METHOD PERFORMANCE
9.1 Precision and accuracy data are not available at this time.
10.0 REFERENCES
1. O'Brien, O.E., "Automatic Analysis of Chlorides in Sewage," Waste Engr.,
33, 670-672 (Dec. 1962).
2. Technlcon AutoAnalyzer II, Industrial Method No. 99-70W, Technicon
Industrial Systems, Tarrytown, New York, 10591 (Sept. 1973).
9251 - 4
Revision
Date September 1986
-------
METHOD 9251
CHLORIDE (COUOBIMETRIC. AUTOMATED FERRICYANIOE AA II)
Ye«
7.2
w«rn UD
color-
imcter »nd
recorder; run •
baseline with
•11 reae«nts
Fllt«r
7.3
star
unknov
In sen
Place
wortclng
in ••mole*
ipler tray
7.4
Obtain stable
baccllnc start
•ampler
7.5.1
I Prepare
standard
curve; compute
concentration
of sample*
f Stop J
9251 - 5
Revision 0
Date September 1986
-------
M?
-------
METHOD 9252A
CHLORIDE (TITRIMETRIC. MERCURIC NITRATE)
1.0 SCOPE AND APPLICATION
1.1 This method is applicable to ground water, drinking, surface, and
saline waters, and domestic and industrial wastes.
1.2 The method is suitable for all concentration ranges of chloride
content; however, in order to avoid large titration volume, a sample aliquot
containing not more than 10 to 20 mg Cl" per 50 mL is used.
1.3 Automated titration may be used.
2.0 SUMMARY OF METHOD
2.1 An acidified sample is titrated with mercuric nitrate in the
presence of mixed diphenylcarbazone-bromophenol blue indicator. The end point
of the titration is the formation of the blue-violet mercury diphenylcarbazone
complex.
3.0 INTERFERENCES
3.1 Anions and cations at concentrations normally found in surface
waters do not interfere. However, at the higher concentration often found in
certain wastes, problems may occur.
3.2 Sulfite interference can be eliminated by oxidizing the 50 ml of
sample solution with 0.5-1 mL of H202.
3.3 Bromide and iodide are also titrated with mercuric nitrate in the same
manner as chloride.
3.4 Ferric and chromate ions interfere when present in excess of 10 mg/L.
4.0 APPARATUS AND MATERIALS
4.1 Standard laboratory titrimetric equipment, including 1 mL or 5 mL
microburet with 0.01 mL gradations.
4.2 Class A volumetric flasks: 1 L'and 100 mL.
4.3 pH Indicator paper.
4.4 Analytical balance: capable of weighing to 0.0001 g.
5.0 REAGENTS
5.1 Reagent-grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
9252A - 1 Revision 1
September 1994
-------
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Standard sodium chloride solution, 0.025 N: Dissolve 1.4613 g
± 0.0002 g of sodium chloride (dried at 600°C for 1 hr) in chloride-free water
in a 1 liter Class A volumetric flask and dilute to the mark with reagent water.
5.4 Nitric acid (HN03) solution: Add 3.0 ml concentrated nitric acid
to 997 mL of reagent water ("3 + 997" solution).
5.5 Sodium hydroxide (NaOH) solution (lOg/L): Dissolve approximately
10 g of NaOH in reagent water and dilute to 1 L with reagent water.
5.6 Hydrogen peroxide (H202): 30%.
5.7 Hydroquinone solution (10 g/L): Dissolve 1 g of purified
hydroquinone in reagent water in a 100 mL Class A volumetric flask and dilute to
the mark.
5.8 Mercuric nitrate titrant (0.141 N): Dissolve 24.2 g Hg(N03)2 • H20
in 900 ml of reagent water acidified with 5.0 ml concentrated HN03 in a 1 liter
volumetric flask and dilute to the mark with reagent water. Filter, if
necessary. Standardize against standard sodium chloride solution (Step 5.3)
using the procedures outlined in Sec. 7.0. Adjust to exactly 0.141 N and check.
Store in a dark bottle. A 1.00 ml aliquot is equivalent to 5.00 mg of chloride.
5.9 Mercuric nitrate titrant (0.025 N): Dissolve 4.2830 g Hg(N03)2 •
H20 in 50 ml of reagent water acidified with 0.05 ml of concentrated
HN03 (sp. gr. 1.42) in a 1 liter volumetric flask and dilute to the mark with
reagent water. Filter, if necessary. Standardize against standard sodium
chloride solution (Sec. 5.3) using the procedures outlined in Sec. 7.0. Adjust
to exactly 0.025 N and check. Store in a dark bottle.
5.10 Mercuric nitrate titrant (0.0141 N): Dissolve 2.4200 g Hg(N03)2 •
H20 in 25 mL of reagent water acidified with 0.25 mL of concentrated HN03 (sp.
gr. 1.42) in a 1 liter Class A volumetric flask and dilute to the mark with
reagent water. Filter, if necessary. Standardize against standard sodium
chloride solution (Sec. 5.3) using the procedures outlined in Sec. 7.0. Adjust
to exactly 0.0141 N and check. Store in a dark bottle. A 1 mL aliquot is
equivalent to 500 jug of chloride.
5.11 Mixed indicator reagent: Dissolve 0.5 g crystalline diphenylcar-
bazone and 0.05 g bromophenol blue powder in 75 mL 95% ethanol in a 100 mL Class
A volumetric flask and dilute to the mark with 95% ethanol. Store in brown
bottle and discard after 6 months.
9252A - 2 Revision 1
September 1994
-------
5.12 Alphazurine indicator solution: Dissolve 0.005 g of alphazurine
blue-green dye in 95% ethanol or isopropanol in 100 ml Class A volumetric flask
and dilute to the mark with 95% ethanol or isopropanol.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed in Chapter Nine of this manual.
6.2 There are no special requirements for preservation.
7.0 PROCEDURE
7.1 Place 50 mL of sample in a vessel for titration. If the concentra-
tion is greater than 20 mg/L chloride, use 0.141 N mercuric nitrate titrant (Sec.
5.8) in Sec. 7.6, or dilute sample with reagent water. If the concentration is
less than 2.5 mg/L of chloride, use 0.0141 N mercuric nitrate titrant (Sec. 5.10)
in Sec. 7.6. Using a 1 mL or 5 mL microburet, determine an indicator blank on
50 mL chloride-free water using Sec. 7.6. If the concentration is less than
0.1 mg/L of chloride, concentrate an appropriate volume to 50 mL.
7.2 Add 5 to 10 drops of mixed indicator reagent (Sec. 5.11); shake or
swirl solution.
7.3 If a blue-violet or red color appears, add HN03 solution (Sec, 5.4)
dropwise until the color changes to yellow. Proceed to Sec. 7.5.
7.4 If a yellow or orange color forms immediately on addition of the
mixed indicator, add NaOH solution (Sec. 5.5) dropwise until the color changes
to blue-violet; then add HN03 solution (Sec. 5.4) dropwise until the color
changes to yellow.
7.5 Add 1 mL excess HN03 solution (Sec. 5.4).
7.6 Titrate with 0.025 N mercuric nitrate titrant (Sec. 5.9) until a
blue-violet color persists throughout the solution. If volume of titrant exceeds
10 mL or is less than 1 mL, use the 0.141 N or 0.0141 N mercuric nitrate
solutions, respectively. If necessary, take a small sample aliquot. Alphazurine
indicator solution (Sec. 5.12) may be added with the indicator to sharpen the end
point. This will change color shades. Practice runs should be made.
Note: The use of indicator modifications and the presence of heavy
metal ions can change solution colors without affecting the
accuracy of the determination. For example, solutions containing
alphazurine may be bright blue when neutral, grayish purple when
basic, blue-green when acidic, and blue-violet at the chloride end
point. Solutions containing about 100 mg/L nickel ion and normal
mixed indicator are purple when neutral, green when acidic, and
gray at the chloride end point. When applying this method to
samples that contain colored ions or that require modified
indicator, it is recommended that the operator become familiar with
the specific color changes involved by experimenting with solutions
prepared as standards for comparison of color effects.
9252A - 3 Revision 1
September 1994
-------
7.6.1 If chromate is present at <100 mg/L and iron is not
present, add 5-10 drops of alphazurine indicator solution (Sec. 5.12) and
acidify to a pH of 3 (indicating paper). End point will then be an olive-
purple color.
7.6.2 If chromate is present at >100 mg/L and iron is not
present, add 2 ml of fresh hydroquinone solution (Sec. 5.7).
7.6.3 If ferric ion is present use a volume containing no more
than 2.5 mg of ferric ion or ferric ion plus chromate ion. Add 2 ml fresh
hydroquinone solution (Sec. 5.7).
7.6.4 If sulfite ion is present, add 0.5 mL of H202 solution
(Sec. 5.6) to a 50 ml sample and mix for 1 min.
7.7 Calculation:
(A - B)N x 35,450
mg chloride/liter
ml of sample
where:
A = ml titrant for sample;
B - mi titrant for blank; and
N = normality of mercuric nitrate titrant.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for
easy reference or inspection. Refer to Chapter One for specific quality control
guidelines.
8.2 Analyze a standard reference material to ensure that correct
procedures are being followed and that all standard reagents have been prepared
properly.
8.3 Employ a minimum of one blank per analytical batch or twenty
samples, whichever is more frequent, to determine if contamination has occurred.
8.4 Run one matrix spike and matrix duplicate every analytical batch
or twenty samples, whichever is more frequent. Matrix spikes and duplicates are
brought through the whole sample preparation and analytical process.
9.0 METHOD PERFORMANCE
9.1 Water sarnples--A total of 42 analysts in 18 laboratories analyzed
synthetic water samples containing exact increments of chloride, with the results
shown in Table 1. In a single laboratory, using surface water samples at an
average concentration of 34 mg Cl/L, the standard deviation was +1.0. A
9252A - 4 Revision 1
September 1994
-------
synthetic unknown sample containing 241 mg/L chloride, 108 mg/L Ca, 82 mg/L Mg,
3.1 mg/L K, 19.9 mg/L Na, 1.1 mg/L nitrate N, 0.25 mg/L nitrate N, 259 mg/L
sulfate and 42.5 mg/L total alkalinity (contributed by NaHC03) in reagent water
was analyzed in 10 laboratories by the mercurimetric method, with a relative
standard deviation of 3.3% and a relative error of 2.9%.
9.2 Oil combustates--These data are based on 34 data points obtained by
five laboratories who each analyzed four used crankcase oils and three fuel oil
blends with crankcase oil in duplicate. The samples were combusted using Method
5050. A data point represents one duplicate analysis of a sample. One data
point was judged to be an outlier and was not included in these results.
9.2.1 Precision and bias.
9.2.1.1 Precision. The precision of the method as determined
by the statistical examination of interlaboratory test results is as
follows:
Repeatability - The difference between successive results
obtained by the same operator with the same apparatus under constant
operating conditions on identical test material would exceed, in the
long run, in the normal and correct operation of the test method, the
following values only in 1 case in 20 (see Table 2):
Repeatability = 7.61
*where x is the average of two results in M9/9-
Reproducibility - The difference between two single and
independent results obtained by different operators working in
different laboratories on identical test material would exceed, in
the long run, the following values only in 1 case in 20:
Reproducibility = 20.02 /**
*where x is the average value of two results in vg/g.
9.2.1.2 Bias. The bias of this method varies with
concentration, as shown in Table 3:
Bias = Amount found - Amount expected
9252A - 5 Revision 1
September 1994
-------
10.0 REFERENCES
1. Annual Book of ASTM Standards, Part 31, "Water," Standard D512-67, Method
A, p. 270 (1976).
2. Standard Methods for the Examination of Uater and Wastewater, 15th ed.,
(1980).
3. U.S. Environmental Protection Agency, Methods for Chemical Analysis of
Water and Wastes, EPA 600/4-79-020 (1983), Method 325.3.
9252A - 6 Revision 1
September 1994
-------
TABLE 1. ANALYSES OF SYNTHETIC WATER SAMPLES
FOR CHLORIDE BY MERCURIC NITRATE METHOD
Increment as Precision as Accuracy as
Chloride Standard Deviation Bias Bias
(mg/L) (ntg/L) (%} (mg/L)
17
18
91
97
382
398
1.54
1.32
2.92
3.16
11.70
11.80
+2.16
+3.50
+0.11
-0.51
-0.61
-1.19
+0.4
+0.6
+0.1
-0.5
-2.3
-4.7
TABLE 2. REPEATABILITY AND REPRODUCIBILITY
FOR CHLORINE IN USED OILS BY BOMB
OXIDATION AND MERCURIC NITRATE TITRATION
Average value, Repeatability, Reproducibility,
M9/9 MS/9 M9/9
500
1,000
1,500
2,000
2,500
3,000
170
241
295
340
381
417
448
633
775
895
1,001
1,097
9252A - 7 Revision 1
September 1994
-------
TABLE 3. RECOVERY AND BIAS DATA FOR CHLORINE IN
USED OILS BY BOMB OXIDATION AND
MERCURIC NITRATE TITRATION
Amount Amount
expected, found, Bias, Percent
M9/9 M9/9 M9/9 bias
320 460 140 +44
480 578 98 +20
920 968 48 +5
1,498 1,664 166 +11
1,527 1,515 - 12 - 1
3,029 2,809 -220 - 7
3,045 2,710 -325 -11
9252A - 8 Revision 1
September 1994
-------
METHOD 9252A
CHLORIDE (TITRIMETRIC, MERCURIC NITRATE)
START
7 I Place SO »,L
aample in litration
vei»el, determine
concentra tion of
mercuric nitrale
111 rant to use in
Step 7 6. determine
an indicator blank
7 2 Add
to *ampl
indicator
•; ahak*
7 4 Add *odiun
hydroxide unti1
3*tnpl« if
blue•viol el. add
nxtrie acid until
»ampie i« yel1o«
7
3 Add nitric acid
unti 1 sample is
yellow
7 S Add I mL nitric
acid
7 6 Titrate -ith
mercuric nitrate
until blue-violet
color p«Tai»te
7 7 Cjlculate
concent ration of
chloride in »ample
STOP
9252A - 9
Revision 1
September 1994
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on
-------
METHOD 9253
CHLORIDE (TITRIMETRIC. SILVER NITRATE)
1.0 SCOPE AND APPLICATION
1.1 This method is intended primarily for oxygen bomb combustates or
other waters where the chloride content is 5 mg/L or more and where interferences
such as color or high concentrations of heavy metal ions render Method 9252
impracticable.
2.0 SUMMARY OF METHOD
2.1 Water adjusted to pH 8.3 is titrated with silver nitrate solution
in the presence of potassium chromate indicator. The end point is indicated by
persistence of the orange-silver chromate color.
3.0 INTERFERENCES
3.1 Bromide, iodide, and sulfide are titrated along with the chloride.
Orthophosphate and polyphosphate interfere if present in concentrations greater
than 250 and 25 mg/L, respectively. Sulfite and objectionable color or turbidity
must be eliminated. Compounds that precipitate at pH 8.3 (certain hydroxides)
may cause error by occlusion.
3.2 Residual sodium carbonate from the bomb combustion may react with
silver nitrate to produce the precipitate, silver carbonate. This competitive
reaction may interfere with the visual detection of the end point. To remove
carbonate from the test solution, add small quantities of sulfuric acid followed
by agitation.
4.0 APPARATUS AND MATERIALS
4.1 Standard laboratory titrimetric equipment, including 1 mL or 5 mL
microburet with 0.01 mL gradations, and 25 mL buret.
4.2 Analytical balance: capable of weighing to 0.0001 g.
4.3 Class A volumetric flask: 1 L.
5.0 REAGENTS
5.1 Reagent grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination.
5.2 Reagent water. All references to water in this method refer to
reagent water, as defined in Chapter One.
5.3 Hydrogen peroxide (30%), H202.
9253 - 1 Revision 0
September 1994
-------
5.4 Phenolphthalein indicator solution (10 g/L).
5.5 Potassium chromate indicator solution. Dissolve 50 g of potassium
chromate (K2Cr04) in 100 mL of reagent water and add silver nitrate (AgN03) until
a slightly red precipitate is produced. Allow the solution to stand, protected
from light, for at least 24 hours after the addition of AgN03. Then filter the
solution to remove the precipitate and dilute to 1 L with reagent water.
5.6 Silver nitrate solution, standard (0.025N). Crush approximately
5 g of silver nitrate (AgNOJ crystals and dry to constant weight at 40°C.
Dissolve 4.2473 + 0.0002 g of the crushed, dried crystals in reagent water and
dilute to 1 L with reagent water. Standardize against the standard NaCl
solution, using the procedure given in Section 7.0.
5.7 Sodium chloride solution, standard (0.025N). Dissolve 1.4613 g
± 0.0002 g of sodium chloride (dried at 600°C for 1 hr) in chloride-free water
in a 1 liter Class A volumetric flask and dilute to the mark with reagent water.
5.8 Sodium hydroxide solution (0.25N). Dissolve approximately 10 g of
NaOH in reagent water and dilute to 1 L with reagent water.
5.9 Sulfuric acid (1:19), H2S04, Carefully add 1 volume of concentrated
sulfuric acid to 19 volumes of reagent water, while mixing.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected using a sampling plan that
addresses the considerations discussed in Chapter Nine of this manual.
6.2 There are no special requirements for preservation.
7.0 PROCEDURE
7.1 Pour 50 mL or less of the sample, containing between 0.25 mg and
20 mg of chloride ion, into a white porcelain container. Dilute to approximately
50 mL with reagent water, if necessary. Adjust the pH to the phenolphthalein end
point (pH 8.3) using H2S04 (Sec. 5.9) or NaOH solution (Sec. 5.8).
7.2 Add approximately 1.0 mL of K-CH^ indicator solution and mix. Add
standard AgN03 solution dropwise from a z5 mL buret until the orange color
persists throughout the sample when illuminated with a yellow light or viewed
with yellow goggles. Be consistent with endpoint recognition.
7.3 Repeat the procedure described in Sees. 7.1 and 7.2 using exactly
one-half as much original sample, diluted to 50 mL with halide-free water.
7.4 If sulfite ion is present, add 0.5 mL of H202 to the samples
described in Sees. 7.2 and 7.3 and mix for 1 minute. Adjust tne pH, then proceed
as described in Sees. 7.2 and 7.3.
9253 - 2 Revision 0
September 1994
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7.5 Calculation
7.5.1 Calculate the chloride ion concentration in the original
sample, in milligrams per liter, as follows:
Chloride (mg/L) - [(Vt - V2) x N x 71,000] / S
where:
Vx * Milliliters of standard AgNO, solution added in titrating
the sample prepared in Sec. 7.1.
V2 = Milliliters of standard AgNO, solution added in titrating
the sample prepared in Sec. 7.3.
N = Normality of standard AgN03 solution.
S - Milliliters of original sample in the 50 ml test sample
prepared in Sec. 7.1.
71,000 = 2 x 35,500 mg CT/equivalent, since Vj - 2V2.
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for
easy reference or inspection. Refer to Chapter One for specific quality control
guidelines.
8.2 Analyze a standard reference material to ensure that correct
procedures are being followed and that all standard reagents have been prepared
properly.
8.3 Employ a minimum of one blank per analytical batch or twenty
samples, whichever is more frequent, to determine if contamination has occurred.
8.4 Run one matrix spike and matrix duplicate every analytical batch
or twenty samples, whichever is more frequent. Matrix spikes and duplicates are
brought through the whole sample preparation and analytical process.
9.0 METHOD PERFORMANCE
9.1 These data are based on 32 data points obtained by five
laboratories who each analyzed four used crankcase oils and three fuel oil blends
with crankcase in duplicate. The samples were combusted using Method 5050. A
data point represents one duplicate analysis of a sample. Three data points were
judged to be outliers and were not included in these results.
9.1.1 Precision. The precision of the method as determined by
the statistical examination of inter-laboratory test results is as
follows:
9253 - 3 Revision 0
September 1994
-------
Repeatability - The difference between successive results obtained
by the same operator with the same apparatus under constant operating
conditions on identical test material would exceed, in the long run, in
the normal and correct operation of the test method, the following values
only in 1 case in 20 (see Table 1):
Repeatability = 0.36 x*
*where x is the average of two results in
Reproducibilitv - The difference between two single and independent
results obtained by different operators working in different laboratories
on identical test material would exceed, in the long run, the following
values only in 1 case in 20:
Reproducibility = 0.71 x*
where x is the average of two results in
9.1.2 Bias. The bias of this method varies with concentration,
as shown in Table 2:
Bias - Amount found - Amount expected
10.0 REFERENCES
1. Rohrbough, W.G.; et al. Reagent Chemicals. American Chemical Society
Specifications. 7th ed.; American Chemical Society: Washington, DC, 1986.
2. 1985 Annual Book of ASTM Standards. Vol. 11.01; "Standard Specification for
Reagent Water"; ASTM: Philadelphia, PA, 1985; D1193-77.
3. Gaskill, A.; Estes, E. D.; Hardison, D. L.; and Myers, L. E. "Validation
of Methods for Determining Chlorine in Used Oils and Oil Fuels," Prepared for
U.S. Environmental Protection Agency, Office of Solid Waste. EPA Contract No.
68-01-7075, WA 80. July 1988.
9253 - 4 Revision 0
September 1994
-------
TABLE 1.
REPEATABILITY AND REPRODUCIBILITY FOR CHLORINE IN USED
OILS BY BOMB OXIDATION AND SILVER NITRATE TITRATION
Average value Repeatability Reproducibility
(M9/9) (M9/9) (M9/9)
500
1,000
1,500
2,000
2,500
3,000
180
360
540
720
900
1,080
355
710
1,065
1,420
1,775
2,130
TABLE 2.
RECOVERY AND BIAS DATA FOR CHLORINE IN USED OILS BY
BOMB OXIDATION AND SILVER NITRATE TITRATION
Amount
expected
{M9/9)
320
480
920
1,498
1,527
3,029
3,045
Amount
found
(A9/9)
645
665
855
1,515
1,369
2,570
2,683
Bias,
(M9/9)
325
185
-65
17
-158
-460
-362
Percent
bias
+102
+39
-7
+1
-10
-15
-12
9253 - 5 Revision 0
September 1994
-------
METHOD 9253
CHLORIDE (TITRIMETRIC, SILVER NITRATE)
START
7 1 Place SO mL
•ample in porcelain
container
7 4 Add hydrogen
peroxide; mix for 1
minute
7 1 Adiu.t pH to
83
7 2 Add 1 0 mL
potajiiura chromate;
•tir; add 111v«r
nitrate until
orange color
penistt
7 3 Repeat »t«p«
7 1 and 7 2 with
1/2 ai much sample
diluted to SO mL
7 S Calculate
concentration of
chloride in lampli
STOP
9253 - 6
Revision 0
Septaiter 1994
-------
VO
-------
METHOD 9315
ALPHA-EMITTING RADIUM ISOTOPES
1.0 SCOPE AND APPLICATION
1.1 This method covers the measurement of the total soluble alpha-
emitting radlolsotopes of radium, namely rad1um-223, rad1um-224, and radlum-
226, 1n surface and ground waters.
1.2 Although the method does not always give an accurate measurement of
the rad1um-226 content of the sample (when other radium alpha emitters are
present), 1t can be used to screen samples. When the total radium alpha
activity of a drinking water sample 1s greater than 5 pC1/L, then the radlum-
226 analysis 1s required. If the level of rad1um-226 exceeds 3 pC1/Lr the
sample must also be measured for rad1um-228 (Method 9320).
1.3 Because this method provides for the separation of radium from other
water-dissolved sol Ids 1n the sample, the sensitivity of the method 1s a
function of sample size, reagent and Instrument background, counting
efficiency, and counting time.
1.4 Absolute measurement can be made by calibrating the alpha detector
with standard rad1um-226 1n the geometry obtained with the final precipitate.
2.0 SUMMARY OF METHOD
2.1 The radium 1n the surface water or ground water sample 1s collected
by coprec1p1tat1on with barium and lead sulfate and purified by repredpl-
tatlon from EDTA solution. Citric add 1s added to the water sample to assure
that complete Interchange occurs before the first precipitation step. The
final BaS04 precipitate, which Includes rad1um-226, rad1um-224, and radlum-
223, 1s alpha counted to determine the total disintegration rate of the radium
Isotopes.
2.2 The radium activities are counted In an alpha counter where
efficiency for determining rad1um-226 has been calibrated with a standard of
known rad1um-226 activity. By making a correction for the Ingrowth of alpha
activity 1n rad1um-226 for the elapsed time after separation, one can
determine radium activity 1n the sample. Because some daughter Ingrowth can
occur before the separated radium 1s counted, It Is necessary to make activity
corrections for the count rate. A table of Ingrowth factors for various times
after radium separation 1s provided 1n Paragraph 7.14.
3.0 INTERFERENCES
3.1 Inasmuch as the radlochemlcal yield of the radium activity 1s based
on the chemical yield of the BaS04 precipitate, the presence of significant
natural barium In the sample will result In a falsely high chemical yield.
9315 - 1
Revision 0
Date September 1986
-------
3.2 Radium Isotopes are separated from other alpha-emitting
radlonucltdes by this method.
3.3 The alpha count of the separated radium must be corrected for Us
partially Ingrown alpha-emitting daughters.
4.0 APPARATUS AND MATERIALS
4.1 Alpha scintillation or a gas-flow proportional alpha particle
counting system with low background «1 com).
4.2 Stainless steel counting planchets.
4.3 Electric hot plate.
4.4 Drying oven and/or drying lamp.
4.5 Glass desiccator.
4.6 Analytical balance.
4.7 Centrifuge.
4.8 Glassware.
5.0 REAGENTS
5.1 Distilled or delonlzed water (Type II).
5.2 Acetic add. 17.4 N: glacial CHaCOOH (cone.), sp. gr. 1.05, 99. 8%.
5.3 Ammonium sulfate, 200 mg/mL: Dissolve 20 g (NH4)2$04 1n a minimum
of water and dilute to 100 ml.
5.4 Barium carrier. 16 mg/mL, standardized:
5.4.1 Dissolve 2.846 g BaCl2'2H20 1n water, add 0.5 mL 16 N HN03,
and dilute to 100 mL with water.
5.4.2 To perform standardization (1n triplicate): Pipette 2.0 mL
carrier solution Into a centrifuge tube containing 15 mL water. Add 1 mL
18 N HoSOd with stirring and digest precipitate 1n a water bath for 10
m1n. Cool, centrifuge, and decant the supernatant. Wash precipitate
with 15 mL water. Transfer the precipitate to a tared stainless steel
planchet with a minimum of water. Dry under Infrared lamp, store 1n
desiccator, and weigh as BaS04.
5.5 Citric acid. 1 M: Dissolve 19.2 g CgHgO;'^ 1n water and dilute to
100 mL.
9315 - 2
Revision
Date September 1986
-------
5.6 EDTA reagent, basic (0.25 M): Dissolve 20 g NaOH 1n 750 ml water,
heat and slowly add 93 g d1sodium ethylened1n1tr1loacetate dlhydrate
(Na2Ci0Hi408N2'2H20). Heat and stir until dissolved? filter through coarse
filter paper and dilute to 1 liter.
5.7 Lead carrier. 15 mg/mL: Dissolve 2.397 g Pb(N03)2 1n water, add 0.5
ml 16 N HNOa, and dilute to 100 ml with water.
5.8 Sodium hydroxide. 6 N: Dissolve 24 g NaOH 1n 80 ml water and dilute
to 100 ml.
5.9 Sulfurlc add, 18 N: Cautiously mix 1 volume 36 N H2S04 (concen-
trated) with 1 volume of water.
5.10 Sulfurlc add. 0.1 N: Mix 1 volume 18 N H2S04 with 179 volumes
of water.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 All samples must have been collected 1n a manner which addresses the
considerations discussed 1n Chapter Nine of this manual.
6.2 It 1s recommended that samples be preserved at the time of
collection by adding enough 1 N HN03 to the sample to bring It to pH 2 (15 ml
1 N HN03 per liter of sample Is usually sufficient). If samples are to be
collected without preservation, they should be brought to the laboratory
within 5 days and then preserved and held 1n the original container for a
minimum of 16 hr before analysis or transfer of the sample.
6.3 The container choice should be plastic rather than glass to prevent
loss due to breakage during transportation and handling.
7.0 PROCEDURE
7.1 Calibration;
7.1.1 The counting efficiency for radium alpha particles with
barium sulfate carrier present must be determined using a standard
(known) radium alpha activity and 32 mg of barium carrier as BaS04 (same
carrier amount used 1n samples). This 1s done with spiked distilled
water samples, and the procedure for regular samples 1s followed. Note
the time of the Ra-BaS04 precipitation.
7.1.2 The radium alpha counting efficiency, E, 1s calculated as
follows:
E (cpm/dpm) « --
9315 - 3
Revision
Date September 1986
-------
where:
C » sample net cpm (gross counts minus background divided
by the counting time 1n minutes).
A » dpm of rad1um-226 added to sample.
I • Ingrowth factor for the elapsed time from Ra-BaS04,
precipitation to midpoint of counting time.
7.2 To a 1,000-mL surface water or ground water sample, add 5 ml 1 M
, 1 ml lead carrier, and 2.0 ml barium carrier, and heat to boiling.
7.3 Cautiously, with vigorous stirring, add 20 ml 18 N ^Stty. Digest 5
to 10 m1n and let the mixed BaS04-PbS04 precipitate settle overnight. Decant
and discard supernate.
7.4 Transfer the precipitate to a centrifuge tube with a minimum amount
of 0.1 N H2$04. Centrifuge and discard supernate.
7.5 Wash the precipitate twice with 0.1 N HgSCty. Centrifuge and discard
washes.
7.6 Dissolve the precipitate by adding 15 ml basic EDTA reagent; heat 1n
a hot-water bath and add a few drops 6 N NaOH until solution 1s complete.
7.7 Add 1 ml (MU^SCH (200 mg/mL) and stir thoroughly. Add 17.4 N
CH3COOH dropwlse until precipitation begins and then add 2 ml extra. Digest 5
to 10 m1n.
7.8 Centrifuge, discard the supernate, and record time.
NOTE: At this point, the separation of the BaS04 1s complete, and the
Ingrowth of radon (and daughters) commences.
7.9 Wash the BaS04 precipitate with 15 ml water, centrifuge, and discard
wash.
7.10 Transfer the precipitate to a tared stainless steel planchet with a
minimum of water and dry under Infrared lamps.
NOTE: Drying should be rapid, but not too vigorous, to minimize any loss
of radon-222 that has already grown Into the precipitate.
7.11 Cool, weigh, and store 1n desiccator.
7.12 Count 1n a gas-flow Internal proportional counter or an alpha
scintillation counter to determine the alpha activity.
9315 - 4
Revision
Date September 1986
-------
7.13 Calculation;
7.13.1 Calculate the radlum-226 concentration, 0 (which would
Include any rad1um-224 and rad1um-223 that 1s present), 1n plcocurles per
liter as follows:
2.22 xExVxRxI
where:
C - net count rate, cpm.
E » counter efficiency, for rad1ura-226 1n BaS(>4 predetermined
for this procedure (see Paragraph 7.1.2).
V * liters of sample used.
R > fractional chemical yield.
I « Ingrowth correction factor (see Paragraph 7.14).
2.22 - conversion factor from dpw/pCI.
7.14 It Is not always possible to count the BaS04 precipitate
Immediately after separation; therefore, corrections must be made for the
Ingrowth of the rad1ura-226 daughters between the time of separation and
counting, according to the following table:
Hours from separation Ingrowth correction
to countingfactor
0 1.00
1 1.02
2 1.04
3 1.06
4 1.08
5 1.10
6 1.12
24 1.49
48 1.91
72 2.25
96 2.54
120 2.78
144 2.99
192 3.29
240 3.51
9315 - 5
Revision
Date September 1986
-------
8.0 QUALITY CONTROL
8.1 All quality control data should be maintained and available for easy
reference or Inspection.
8.2 Employ a minimum of one blank per sample batch to determine 1f
contamination or any memory effects are occurring.
8.3 Run one duplicate sample for every 10 samples. A duplicate sample
1s a sample brought through the whole sample-preparation process.
8.4 Spiked samples or standard reference materials.shall be periodically
employed to ensure that correct procedures are being followed and that all
equipment 1s operating properly.
9.0 METHOD PERFORMANCE
9.1 No data provided.
10.0 REFERENCES
10.1 None required.
9315 - 6
Revision
Date September 1986
-------
METHOD 9315
ALPHA-CM!TT2NC FUOIUM ISOTOPES
Celibret*
detector* far
rediuit elph*
•eeeurencnt
7.2
-LLJ
Olaaolve
precipitate
In EOT*: neat:
edd N«OH
AOd C«H.O,' MJ.O. leed
• no beriux c»rrlir-«
to Meter leapt*;
neet to eoillng
7.7
• tlr; edo
CHjCOOK digeet
7.3
»oa
stirring: dlgeet:
precipitate:
dlacard auparnete
7.
7.1
Centrifuge:
aiacord
auoarnote:
record tl*e
Centrifuge:
ditcard
euptrnete
7.9
Mean
oracloitete:
centrifuge:
dlechero Mean
Heah
precipitate:
centrifuge:
dlacard weanee
7.101
Tranafar
precipitate
to plencnet:
dry
7.tt|
Cool, we ten.
•no store In
Oeelccetor
7.ia
Uat counter to
determine alpha
ectivity
7.13.1
Calculate
radluej-2Z6
concentration
7.14
Correct
for tngrowtn
of radiuiR-226
daugntera
o
9315 - 7
Revision 0
Date September 1986
------- |