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METHOD 504. 1,2-DIBROMOETHANE (EDB) AND l,2-DIBROKO-3-CHLOROPROPANE
(DBCP) IN WATER BY MICROEXTRACTION AND GAS CHRONATOGRAPHY
Revision 2.0
T. W. Winfield - Method 504, Revision 1.0 (1986)
T. W. Uinfield - Method 504, Revision 2.0 (1989)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 504
1,2-DIBROMOETHANE (EDB) AND l,2-DIBROMO-3-CHLOROPROPANE (DBCP)
IN WATER BY NICROEXTRACTION AND GAS CHROMATOGRAPHY
1. SCOPE AND APPLICATION
1.1 This method (1-4) is applicable to the determination of the
following compounds in finished drinking water and groundwater:
Chemical Abstract Services
Analvte Registry Number
1,2-Dibromoethane 106-93-4
l,2-Dibromo-3-Chloropropane 96-12-8
1.2 For compounds other than the above mentioned analytes, or for other
sample sources, the analyst must demonstrate the usefulness of the
method by collecting precision and accuracy data on actual samples
(5) and provide qualitative confirmation of results by gas
chromatography/mass spectrometry (GC/MS) (6).
1.3 The experimentally determined method detection limits (HDL) (7) for
EDB and DBCP were calculated to be 0.01 jjg/L. The method has been
shown to be useful for these analytes over a concentration range
from approximately 0.03 to 200 /*g/L. Actual detection limits are
highly dependent upon the characteristics of the gas chromatographic
system used.
2. SUMMARY OF METHOD
2.1 Thirty-five ml of sample are extracted with 2 ml of hexane. Two pL
of the extract are then injected into a gas chromatograph equipped
with a linearized electron capture detector for separation and
analysis. Aqueous calibration standards are extracted and analyzed
in an identical manner as the samples in order to compensate for
possible extraction losses.
2.2 The extraction and analysis time is 30 to 50 min per sample depending
upon the analytical conditions chosen.
2.3 Confirmatory evidence can be obtained using a dissimilar column. When
component concentrations are sufficiently high, Method 524.1 or
524.2 may be employed for improved specificity.
3. DEFINITIONS
3.1 Laboratory duplicates (LD1 and LD2) -- Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precisioi
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associated with laboratory procedures, but not with sample
collection, preservation, or storage procedures.
3.2 Field duplicates (FD1 and FD2) -- Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.3 Laboratory reagent blank (LRB) -- An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
that are used with other samples. The LRB is used to determine if
method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.4 Field reagent blank (FRB) -- Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage, preservation
and all analytical procedures. The purpose of the FRB is to determine
if method analytes or other interferences are present in the field
environment.
3.5 Laboratory performance check solution (LPC) -- A solution of method
analytes, surrogate compounds, and internal standards used to
evaluate the performance of the instrument system with respect to a
defined set of method criteria.
3.6 Laboratory fortified blank (LFB) -- An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.7 Laboratory fortified sample matrix (LFM) -- An aliquot of an
environmental sample to which known quantities of the method analytes
are added in the laboratory. The LFM is analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.8 Stock standard solution -- A concentrated solution containing a
single certified standard that is a method analyte, or a concentrated
solution of a single analyte prepared in the laboratory with an
assayed reference compound. Stock standard solutions are used to
prepare primary dilution standards.
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3.9 Primary dilution standard solution -- A solution of several analytf
prepared in the laboratory from stock standard solutions and dilute^
as needed to prepare calibration solutions and other needed analyte
solutions.
3.10 Calibration standard (CAL) -- a solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.11 Quality control sample (QCS) -- a sample matrix containing method
analytes or a solution of method analytes in a water miscible solvent
which is used to fortify reagent water or environmental samples. The
QCS is obtained from a source external to the laboratory, and is used
to check laboratory performance with externally prepared test
materials.
4. INTERFERENCES
4.1 Impurities contained in the extracting solvent usually account for
the majority of the analytical problems. Solvent blanks should be
analyzed on each new bottle of solvent before use. Indirect dally
checks on the extracting solvent are obtained by monitoring the
reagent water blanks (Sect. 7.3.4). Whenever an interference is
noted in the reagent water blank, the analyst should reanalyze the
extracting solvent. Low level interferences generally can be removed
by distillation or column chromatography (4). WARNING: When a
solvent is purified, stabilizers put into the solvent by the
manufacturer are removed thus potentially making the solvent
hazardous. Also, when a solvent is purified, preservatives put into
the solvent by the manufacturer are removed thus potentially making
the shelf-life short. However, it is generally more economical to
obtain a new source of solvent. Interference-free solvent is defined
as a solvent containing less than 0.1 /zg/L individual analyte
interference. Protect interference-free solvents by storing in an
area known to be free of organochlorine solvents.
4.2 This liquid/liquid extraction technique efficiently extracts a wide
boiling range of non-polar organic compounds and, in addition,
extracts polar organic components of the sample with varying
efficiencies.
4.3 Current column technology suffers from the fact that EDB at low
concentrations may be masked by very high levels of
dibromochloromethane (DBCM), a common disinfection by-product of
chlorinated drinking waters.
5. SAFETY
5.1 The toxicity and carcinogenicity of chemicals used in this method has
not been precisely defined; each chemical should be treated as a
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potential health hazard, and exposure to these chemicals should be
minimized. Each laboratory Is responsible for maintaining awareness
of OSHA regulations regarding safe handling of chemicals used in this
method. Additional references to laboratory safety are available
(7-9) for the information of the analyst.
5.2 EDB and DBCP have been tentatively classified as known or suspected
human or mammalian carcinogens. Pure standard materials and stock
standard solutions of these compounds should be handled in a hood or
glovebox. A NIOSH/MESA approved toxic gas respirator should be worn
when the analyst handles high concentrations of these toxic
compounds.
5.3 WARNING: When a solvent is purified, stabilizers put into the
solvent by the manufacturer are removed thus potentially making the
solvent hazardous.
6. APPARATUS AND EQUIPMENT
6.1 SAMPLE CONTAINERS - 40-mL screw cap vials (Pierce #13075 or
equivalent) each equipped with a size 24 cap with a flat, disc-like
PTFE-faced polyethelene film/foam extrusion (Fisher 002-883-3F or
equivalent). Individual vials shown to contain at least 40.0 ml can
be calibrated at the 35.0 ml mark so that volumetric, rather than
gravimetric, measurements of sample volumes can be performed. Prior
to use, wash vials and septa with detergent and rinse with tap and
distilled water. Allow the vials and septa to air dry at room
temperature, place in a 105°C oven for one hr, then remove and allow
to cool in an area known to be free of organic solvent vapors.
6.2 VIALS, auto sampler, screw cap with PTFE-faced septa, 1.8 mL, Varian
#96-000099-00 or equivalent.
6.3 MICRO SYRINGES - 10 and 100 /*L.
6.4 MICRO SYRINGE - 25 til with a 2-inch by 0.006-inch needle - Hamilton
#702N or equivalent.
6.5 PIPETTES - 2.0 and 5.0 mL transfer.
6.6 STANDARD SOLUTION STORAGE CONTAINERS - 15-mL bottles with PTFE-lined
screw caps.
6.7 GAS CHROMATOGRAPHY SYSTEM
6.7.1 The GC must be capable of temperature programming and should
be equipped with a linearized electron capture detector and a
capillary column splitless injector at 200"C.
6.7.2 Two gas chromatography columns are recommended. Column A
(Sect. 6.7.3) is a highly efficient column that provides
separations for EDB and DBCP without interferences from
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tribalomethanes (Sect. 4.4). Column A should be used as th»
primary analytical column unless routinely occurring analytes
are not adequately resolved. Column B (Sect. 6.7.4) is
recommended for use as a confirmatory column when GC/MS
confirmation is not viable. Retention times for EDB and DBCP
on these columns are presented in Table 1.
6.7.3 Column A - 0.32 mm ID x 30M long fused silica capillary with
dimethyl silicone mixed phase (Durawax-DX3, 0.25 tun film, or
equivalent). The linear velocity of the helium carrier gas
should be about 25 cm/sec at 100'C and 7 psi column head
pressure. The column temperature is programmed to hold at
40"C for 4 min, to increase to 190'C at 8°C/min, and hold at
190'C for 25 min or until all expected compounds have eluted.
(See Figure 1 for a sample chromatogram.)
6.7.4 Column B (alternative column) - 0.32mm ID x 30M long fused
silica capillary with methyl polysiloxane phase (DB-1, 1.0 *im
film, or equivalent). The linear velocity of the helium
carrier gas should be about 25 cm/sec at 100'C. The column
temperature is programmed to hold at 40*C for 4 min, to
increase to 270'C at 10°C/min, and hold at 270°C for 10 min
or until all expected compounds have eluted.
6.7.5 Column C (alternative column, wide bore) -- 0.53 mm ID x 30 M
long, 2.0 jum film thickness, Rtx-Volatiles (part #10902),
dimethyl diphenyl polysiloxane, bonded phase. The hydrogen
carrier gas flow is about 80 cm/sec linear velocity, measured
at 50°C with about 11.5 psi column head pressure. The oven
temperature is programmed to hold at 200°C until all expected
compounds have eluted. Injector temperature: 250°C.
Detector temperature: 250°C. NOTE: The above parameters
were obtained by Restek Corporation during preliminary
attempts to improve the separation of EDB and DBCM.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 REAGENTS
7.1.1 Hexane extraction solvent - UV Grade, Burdick and Jackson #216
or equivalent.
7.1.2 Methyl alcohol - ACS Reagent Grade, demonstrated to be free of
analytes.
7.1.3 Sodium chloride, Nad - ACS Reagent Grade - For pretreatment
before use, pulverize a batch of Nad and place in a muffle
furnace at room temperature. Increase the temperature to 400°C
for 30 min. Place in a bottle and cap.
7.1.4 Sodium thiosulfate, ^28203, ACS Reagent Grade -- For
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preparation of solution (0.04 g/mL), mix 1 g of ^$203 with
reagent water and bring to 25-mL volume in a volumetric flask.
7.2 STANDARD MATERIALS
7.2.1 1,2-Dibromoethane - 99%, available from Aldrich Chemical
Company.
7.2.2 l,2-Dibromo-3-chloropropane - 99%, available from USEPA, EMSL-
QARD, Cincinnati, Ohio 45268.
7.3 REAGENT WATER - Reagent water is defined as water free of
interference when employed in the procedure described herein.
7.3.1 Reagent water can be generated by passing tap water through a
filter bed containing activated carbon. Change the activated
carbon when there is evidence that volatile organic compounds
are breaking through the carbon.
7.3.2 A Millipore Super-Q Water System or its equivalent may be used
to generate deionized reagent water.
7.3.3 Reagent water may also be prepared by boiling water for
15 min. Subsequently, while maintaining the temperature at
90°C, bubble a contaminant-free inert gas through the water at
100 mL/min for 1 hr. While still hot, transfer the water to
a narrow mouth screw cap bottle with a Teflon seal.
7.3.4 Test reagent water each day it is used by analyzing it
according to Sect. 11.
7.4 STOCK STANDARD SOLUTIONS - These solutions may be purchased as
certified solutions or prepared from pure standard materials using
the following procedures:
7.4.1 Place about 9.8 mL of methanol into a 10-mL ground-glass
stoppered volumetric flask. Allow the flask to stand,
unstoppered, for about 10 min and weigh to the nearest 0.1
mg.
7.4.2 Use a 100-jiL syringe and immediately add two or more drops of
standard material to the flask. Be sure that the standard
material falls directly into the alcohol without contacting
the neck of the flask.
7.4.3 Reweigh, dilute to volume, stopper, then mix by inverting the
flask several times. Calculate the concentration in
micrograms per microliter from the net gain in weight.
7.4.4 Store stock standard solutions in 15-mL bottles equipped with
PTFE-lined screw caps. Methanol solutions prepared from
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liquid analytes are stable for at least four weeks when sto
at 4'C.
7.5 PRIMARY DILUTION STANDARD SOLUTIONS -- Use stock standard solutions
to prepare primary dilution standard solutions that contain both
analytes in methanol. The primary dilution standards should be
prepared at concentrations that can be easily diluted to prepare
aqueous calibration standards (Sect. 9.1.1) that will bracket the
working concentration range. Store the primary dilution standard
solutions with minimal headspace and check frequently for signs of
deterioration or evaporation, especially just before preparing
calibration standards. The storage time described for stock standard
solutions in Sect. 7.4.4 also applies to primary dilution standard
solutions.
7.6 LABORATORY FORTIFIED BLANK (LFB) SAMPLE CONCENTRATE (0.25 fig/ml) -
Prepare a LFB sample concentrate of 0.25 /xg/mL of each analyte from
the stock standard solutions prepared in Sect. 7.4.
7.7 MDL CHECK SAMPLE CONCENTRATE (0.02 /ig/mL) -- Dilute 2 mL of LFB
sample concentrate (Sect. 7.6) to 25 mL with methanol.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE COLLECTION
8.1.1 Replicate field reagent blanks (FRB) must be handled along
with each sample set, which is composed of the samples
collected from the same general sampling site at approximately
the same time. At the laboratory, fill a minimum of two
sample bottles with reagent water, seal, and ship to the
sampling site along with sample bottles. Wherever a set of
samples is shipped and stored, it must be accompanied by the
FRB.
8.1.2 Collect all samples in 40-mL bottles into which 3 mg of sodium
thiosulfate crystals have been added to the empty bottles just
prior to shipping to the sampling site. Alternately, 75 /iL of
freshly prepared sodium thiosulfate solution (0.04 g/mL may be
added to empty 40-mL bottles just prior to sample collection.
8.1.3 When sampling from a water tap, open the tap and allow the
system to flush until the water temperature has stabilized
(usually about 10 min). Adjust the flow to about 500 mL/min
and collect samples from the flowing stream.
8.1.4 When sampling from a well, fill a wide-mouth bottle or beaker
with sample, and carefully fill 40-mL sample bottles.
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8.2 SAMPLE PRESERVATION
8.2.1 The samples must be chilled to 4°C on the day of collection
and maintained at that temperature until analysis. Field
samples that will not be received at the laboratory on the day
of collection must be packaged for shipment with sufficient
ice to insure that they will be <4*C on arrival at the
laboratory.
8.2.2 The addition of sodium thiosulfate as a dechlorinating agent
and/or acidification to pH 2 with 1:1 HC1, common preservative
procedures for purgeable compounds, have been shown to have no
effect on EDB or DBCP (See Table 3). Nonetheless, sodium
thiosulfate must be added to avoid the possibility of
reactions which may occur between residual chlorine and
indeterminant contaminants present in some solvents, yielding
compounds which may subsequently interfere with the analysis.
The presence of sodium thiosulfate will arrest the formation
of DBCM (See Sect. 4.3). Also, samples should be acidified to
avoid the possibility of microbial degradation which may
periodically affect these analytes contained in other
groundwater matrices.
8.3 SAMPLE STORAGE
8.3.1 Store samples and field reagent blanks together at 4°C until
analysis. The sample storage area must be free of organic
solvent vapors.
8.3.2 Analyze all samples within 28 days of collection. Samples not
analyzed within this period must be discarded and replaced.
9. CALIBRATION AND STANDARDIZATION
9.1 CALIBRATION
9.1.1 At least three calibration standards are needed; five are
recommended. One should contain EDB and DBCP at a
concentration near to but greater than the method detection
limit (Table 1) for each compound; the other two should be at
concentrations that bracket the range expected in samples.
For example, if the MDL is 0.01 /zg/L, and a sample expected to
contain approximately 0.10 /jg/L is to be analyzed, aqueous
standards should be prepared at concentrations of 0.02 Mg/L,
0.10 jig/L, and 0.20 /zg/L.
9.1.2 To prepare a calibration standard (CAL), add an appropriate
volume of a primary dilution standard solution to an aliquot
of reagent water in a volumetric flask. If less than 20 /iL of
an alcoholic standard is added to the reagent water, poor
precision may result. Use a 25-jiL micro syringe and rapidly
inject the alcoholic standard into the expanded area of the
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filled volumetric flask. Remove the needle as quickly as
possible after injection. Mix by inverting the flask several
times. Discard the contents contained in the neck of the
flask. Aqueous standards should be prepared fresh and
extracted immediately after preparation unless sealed and
stored without headspace as described in Sect. 8.
9.1.3 Each day, analyze each calibration standard according to Sect.
11 and tabulate peak height or area response versus the
concentration in the standard. The results can be used to
prepare a calibration curve for each compound. Alternatively,
if the ratio of concentration to response (calibration factor)
is a constant over the working range (<20% relative standard
deviation), linearity through the origin can be assumed and
the average ratio or calibration factor can be used in place
of a calibration curve.
9.1.4 Single point calibration is a viable alternative to a
calibration curve. Prepare single point standards from the
secondary dilution standard solutions. The single point
calibration standard should be prepared at a concentration
that produces a response close to that of the unknowns, i.e.,
no more than 20% deviation between response of standard and
response of sample.
9.2 INSTRUMENT PERFORMANCE - Check the performance of the entire
analytical system daily using data gathered from analyses of reagent
water blanks, standards, and the QC check standard (Sect. 10.3).
9.2.1 Significant peak tailing in excess of that shown for the
target compounds in the method chromatogram (Figure 1) must be
corrected. Tailing problems are generally traceable to
active sites on the GC column, improper column installation,
or the operation of the detector.
9.2.2 Check the precision between replicate analyses. A properly
operating system should perform with an average relative
standard deviation of less than 10%. Poor precision is
generally traceable to pneumatic leaks, especially at the
injection port.
10. QUALITY CONTROL
10.1 Each laboratory that uses this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory detection limits
capability and an ongoing analysis of laboratory performance check
solutions (LPC), laboratory reagent blanks (LRB), laboratory
fortified blanks (LFB), laboratory fortified sample matrix (LFM), and
quality cor.rol samples (QCS) to evaluate and document data quality.
Ongoing d:-ta quality checks are compared with established performance
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criteria to determine if the results of analyses meet the performance
characteristics of the method.
10.1.1 The analyst must make an initial determination of the method
detection limits and demonstrate the ability to generate
acceptable precision with this method. This is established as
described in Sect. 10.2.
10.1.2 In recognition of advances that are occurring in chromato-
graphy, the analyst is permitted certain options to improve
the separations or lower the cost of measurements. Each time
such a modification is made to the method, the analyst is
required to repeat the procedure in Sect. 10.2.
10.1.3 Each day, the analyst must analyze a laboratory reagent blank
(LRB) and a field reagent blank, if applicable (Sect. 8.1.1),
to demonstrate that interferences from the analytical system
are under control before any samples are analyzed.
10.1.4 The laboratory must, on an ongoing basis, demonstrate through
the analyses of laboratory fortified blanks (LFB) that the
operation of the measurement system is in control. This
procedure is described in Sect. 10.3. The frequency of the
LFB analyses is equivalent to 10% of all samples analyzed.
10.1.5 On a weekly basis, the laboratory should demonstrate the
ability to analyze low level samples. The procedure for low
level LFB samples is described in Sect. 10.4.
10.2 To establish the ability to achieve low detection limits and generate
acceptable accuracy and precision, the analyst should perform the
following operations:
10.2.1 Prepare four to seven samples at 0.02 /ig/L by fortifying 35 /zL
of the MDL check sample concentrate (Sect. 7.7) into 35-mL
aliquots of reagent water in 40-mL bottles. Cap and mix
well.
10.2.2 Analyze the well-mixed MDL check samples according to the
method beginning in Sect. 11.
10.2.3 Calculate the average concentration found (X) in jig/L, and the
standard deviation of the concentrations(s) in /ig/L, for each
analyte. Then, calculate the MDL for each analyte.
10.2.4 For each analyte, X should be between 80% and 120% of the true
value. Additionally, the calculated MDL should meet data
quality objectives. If both analytes meet these criteria, the
system performance is acceptable and analysis of actual
samples can begin. If either analyte fails to meet the data
quality objectives on the basis of high variability, correct
the source of the problem and repeat the test. It is
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recommended that the laboratory repeat the MDL determinate
on a regular basis. CAUTION: No attempts to establish low
detection limits should be made before instrument optimization
and adequate conditioning of both the column and the GC
system. Conditioning includes the processing of LFB and LFM
samples containing moderate concentration levels of EDB and
DBCP.
10.3 The laboratory must demonstrate on a frequency equivalent to 10% of
the sample load that the measurement system is in control by
analyzing an LFB of both analytes at 0.25 pg/L concentration level.
10.3.1 Prepare an LFB sample (0.25 pg/L) by adding 35 pL of LFB
concentrate (Sect. 7.6) to 35 mL of reagent water in a 40-mL
bottle.
10.3.2 Immediately analyze the LFB sample according to Sect. 11 and
calculate the recovery for each analyte. The recovery should
be between 60% and 140% of the expected value.
10.3.3 If the recovery for either analyte falls outside the
designated range, the analyte fails the acceptance criteria.
A second LFB containing each analyte that failed must be
analyzed. Repeated failure, however, will confirm a general
problem with the measurement system. If this occurs, locate
and correct the source of the problem and repeat the test.
10.4 On a weekly basis, the laboratory should demonstrate the ability to
analyze low level samples.
10.4.1 Prepare an MDL check sample (0.02 jig/L) as outlined in Sect.
10.2.1 and immediately analyze according to the method in
Sect. 11.
10.4.2 The instrument response must indicate that the laboratory's
MDL is distinguishable from instrument background signal. If
not, correct the problem and repeat the MDL test in Sect.
10.2.
10.4.3 For each analyte, the recovery must be between 60% and 140% of
the expected value.
10.4.4 When either analyte fails the test, the analyst should repeat
the test for that analyte. Repeated failure, however, will
confirm a general problem with the measurement system or
faulty samples and/or standards. If this occurs, locate and
correct the source of the problem and repeat the test.
10.5 At least quarterly, a quality control sample from an external source
should be analyzed. If measured analyte concentrations are not of
acceptable accuracy, check the entire analytical procedure to locate
and correct the problem source.
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10.6 At least once in every 20 samples, fortify an aliquot of a randomly
selected routine sample with a known amount (see Sect. 4.3). The
added concentration should not be less than the background concentra-
tion of the sample selected for fortification. To simplify these
checks, it would be convenient to use LFM concentrations «10X MDL.
Over time, recovery should be evaluated on fortified samples from all
routine sources.
10.7 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific practices
that are most productive depend upon the needs of the laboratory and
the nature of the samples. Field duplicates may be analyzed to
assess the precision of the environmental measurements. Whenever
possible, the laboratory should analyze standard reference materials
and participate in relevant performance evaluation studies.
11. PROCEDURE
11.1 SAMPLE PREPARATION
11.1.1 Remove samples and standards from storage and allow them to
reach room temperature.
11.1.2 For samples and field reagent blanks, contained in 40-mL
bottles, remove the container cap. Discard a 5-mL volume
using a 5-mL transfer pipette or 10-mL graduated cylinder.
Replace the container cap and weigh the container with
contents to the nearest O.lg and record this weight for
subsequent sample volume determination (Sect. 11.3).
11.1.3 For calibration standards, laboratory fortified blanks and
laboratory reagent blanks, measure a 35-mL volume using a
50-mL graduated cylinder and transfer it to a 40-mL sample
container.
11.2 MICROEXTRACTION AND ANALYSIS
11.2.1 Remove the container cap and add 6 g NaCl (Sect. 7.1.3) to the
sample.
11.2.2 Recap the sample container and dissolve the NaCl by shaking by
hand for about 20 sec.
11.2.3 Remove the cap and, using a transfer pipette, add 2.0 mL of
hexane. Recap and shake vigorously by hand for 1 min. Allow
the water and hexane phases to separate. (If stored at this
stage, keep the container upside down.)
11.2.4 Remove the cap and carefully transfer 0.5 mL of the hexane
layer into an autoinjector using a disposable glass pipette.
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11.2.5 Transfer the remaining hexane phase, being careful not to
include any of the water phase, into a second autoinjector
vial. Reserve this second vial at 4°C for a reanalysis if
necessary.
11.2.6 Transfer the first sample vial to an autoinjector set up to
inject 2.0 /iL portions into the gas chromatograph for
analysis. Alternatively, 2 pL portions of samples, blanks and
standards may be manually injected, although an autoinjector
is recommended.
11.3 DETERMINATION OF SAMPLE VOLUME
11.3.1 For samples and field blanks, remove the cap from the sample
container.
11.3.2 Discard the remaining sample/hexane mixture. Shake off the
remaining few drops using short, brisk wrist movements.
11.3.3 Reweigh the empty container with original cap and calculate
the net weight of sample by difference to the nearest 0.1 g.
This net weight (in g) is equivalent to the volume of water
(in ml) extracted. (Sect. 12.3)
12. CALCULATIONS
12.1 Identify EDB and DBCP in the sample chromatogram by comparing the
retention time of the suspect peak to retention times generated by
the calibration standards and the laboratory control standard.
12.2 Use single point calibrations (Sect. 9.1.4) or use the calibration
curve or calibration factor (Sect. 9.1.3) to directly calculate the
unconnected concentration (Cj) of each analyte in the sample (e.g.,
calibration factor x response).
12.3 Calculate the sample volume (V$) as equal to the net sample weight:
Vs = gross weight (Sect. 11.1.2) - bottle tare (Sect. 11.3.3).
12.4 Calculate the corrected sample concentration as:
Concentration, /jg/L = C< x 35
Vs
12.5 Results should be reported with an appropriate number of significant
figures. Experience indicates that three significant figures may be
used for concentrations above 99 pg/L, two significant figures for
concentrations between 1-99 pg/L, and 1 significant figure for lower
concentrations.
13. ACCURACY AND PRECISION
13.1 Single laboratory and interlaboratory accuracy and precision at
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several concentrations in three waters are presented in Tables 2 and
4 (1). The method detection limits are presented in Table 1.
13.2 In a preservation study extending over a 4-week period, the average
percent recoveries and relative standard deviations presented in
Table 3 were observed for reagent water (acidified), tap water and
groundwater (1). The results for acidified and non-acidified samples
were not significantly different.
14. REFERENCES
1. Uinfield, T.W., J.E. Longbottom, R.L. Graves and A.L. Cohen,
"Analysis of Organohalide Pesticides and Commerical PCB Products in
Drinking Water by Microextraction and Gas Chromatography," U.S.
Environmental Protection Agency. Environmental Monitoring Systems
Laboratory, Cincinnati, Ohio.
2. Glaze, VI.VI., Lin, C.C., "Optimization of Liquid-Liquid Extraction
Methods for Analysis of Organics in Water," EPA-600/S4-83-052,
January 1984.
3. Henderson, J.E., Peyton, G.R. and Glaze, W.H.(1976). In Identifica-
tion and Analysis of Organic Pollutants in Water (L.H. Keith ed.),
pp. 105-111. Ann Arbor Sci. Publ., Ann Arbor, Michigan.
4. Richard, J.J., G.A. Junk, "Liquid Extraction for Rapid Determination
of Halomethanes in Water," Journal AWWA, 69, 62, January 1977.
5. Handbook for Analytical Quality Control in Water and Wastewater
Laboratories, EPA-600/4-79-019, U. S. Environmental Protection
Agency, Environmental Monitoring Systems Laboratory - Cincinnati,
Ohio 45268, March 1979.
6. Budde, W.L., J.W. Eichelberger, "Organic Analyses Using Gas Chromato-
graphy-Mass Spectrometry," Ann Arbor Science, Ann Arbor, Michigan
1979.
7. Glaser, J.A. D.L. Forest, G.D. McKee, S.A. Quave, and W.L. Budde,
"Trace Analyses for Wastewaters," Environmental Science and Technol-
ogy, 15, 1426 (1981).
8. "Carcinogens-Working with Carcinogens", Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute of Occupational Safety and Health,
Publication No. 77-206, August, 1977.
9. OSHA Safety and Health Standards,(29CFR1910), Occupational Safety and
Health Administration, OSHA 2206.
10. Safety in Academic Chemistry Laboratories,American Chemical Society
Publication, Committee on Chemical Safety, 4th Edition, 1985.
103
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TABLE 1. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
FOR 1,2-DIBROMOETHANE (EDB) AND l,2-DIBROMO-3-CHLOROPROPANE (DBCP)
Retention Time. Min MDL. uq/L
Analvte Column A Column B Column C*
EDB 9.5 8.9 4.1 0.01
DBCP 17.3 15.0 12.8 0.01
* The MDL experimentally observed by Resteck Corporation during
preliminary optimization was 0.3
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TABLE 2. SINGLE LABORATORY ACCURACY AND PRECISION
FOR EDB AND DBCP IN TAP WATER
Analvte
Number
of
Samples
Concen-
tration
(UQ/L)
Average
Accuracy
Relative
Standard
Deviation
m
EDB 7
7
7
DBCP 7
7
7
0.03
0.24
50.0
0.03
0.24
50.0
114
98
95
90
102
94
9.5
11.8
4.7
11.4
8.3
4.8
TABLE 3. ACCURACY AND PRECISION AT 2.0 /ig/L OVER A 4-WEEK STUDY PERIOD
Analvte
Matrix1
Average
Number
of Samples
Relative
Accuracy
(% Recovery)
Std. Dev.
EDB
DBCP
RW-A
GW
GW-A
TW
TW-A
RW-A
GW
GW-A
TW
TW-A
16
15
16
16
16
16
16
16
16
16
104
101
96
93
93
105
105
101
95
94
4.7
2.5
4.7
6.3
6.1
8.2
6.2
8.4
10.1
6.9
Matrix Identities
RW-A = Reagent water at pH 2
GW = Groundwater, ambient pH
GW-A = Groundwater at pH 2
TW = Tap water, ambient pH
A = Tap water at pH 2.
105
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TABLE 4. INTERLABORATORY STUDY OF METHOD 504
REGRESSION EQUATIONS FOR RECOVERY AND PRECISION
1,2-Dibromo-
Vlater Type 1.2-Dibromoethane 3-chloropropane
Applicable Cone. Range (0.05 - 6.68) pg/L (0.05 - 6.40) /zg/L
Reagent Water
Single-Analyst Precision SR = 0.041X + 0.004 SR = 0.065X + 0.000
Overall Precision S = 0.075X + 0.008 S = 0.143X - 0.000
Recovery X = 1.072C - 0.006 X = 0.987C - 0.000
Ground Water
Single-Analyst Precision SR = 0.046X + 0.002 SR = 0.076X - 0.000
Overall Precision S = 0.102X + 0.006 S = 0.160X + 0.006
Recovery X = 1.077C - 0.001 X = 0.972C + 0.007
X = Mean recovery
C = True value for the concentration
106
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Column: Fused silica capillary
Liquid Phast: Durawai-DX3
Film Thieknasa: 0.2S jim
Column Dimensions: 30 M10.317 mm 10
2 4 6 8 10 12 14 16 18. 20 22 24 26 28 30
Time (Min)
igure 1. Extract of reagent water spiked at 0.114 pg/L with EDB and DBCP.
107
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METHOD 505. ANALYSIS OF ORGANOHALIDE PESTICIDES AND
COMMERCIAL POLYCHLORINATED BIPHENYL (PCB) PRODUCTS
IN HATER BY MICROEXTRACTION AND GAS CHROMATOGRAPHY
Revision 2.0
T. U. Uinfield - Method 505, Revision 1.0 (1986)
T. U. Uinfield - Method 505, Revision 2.0 (1989)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
109
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METHOD 505
ANALYSIS OF ORGANOHALIDE PESTICIDES AND COMMERCIAL POLYCHLORINATED BIPHENYL
(PCB) PRODUCTS IN WATER BY MICROEXTRACTION AND GAS CHROMATOGRAPHY
1. SCOPE AND APPLICATION
1.1
This method (1,2,3) is applicable to the determination of the
following analytes in finished drinking water, drinking water during
intermediate stages of treatment, and the raw source water:
Analvte
Chemical Abstract Service
Registry Number
1.2
1.3
Alachlor 5972-60-8
Aldrin 309-00-2
Atrazine 1912-24-9
Chlordane 57-74-9
alpha-Chlorodane 5103-71-9
gamma-Chlorodane 5103-74-2
Dieldrin 60-57-1
Endrin 72-20-8
Heptachlor 76-44-8
Heptachlor Epoxide 1024-57-3
Hexachlorobenzene 118-74-1
Hexachlorocyclopentadiene 77-74-4
Lindane 58-89-9
Methoxychlor 72-43-5
cis-Nonachlor
trans-Nonachlor 39765-80-5
Simazine 122-34-9
Toxaphene 8001-35-2
Aroclor 1016 12674-11-2
Aroclor 1221 11104-28-2
Aroclor 1232 11141-16-5
Aroclor 1242 53469-21-9
Aroclor 1248 12672-29-6
Aroclor 1254 11097-69-1
Aroclor 1260 11096-82-5
For compounds other than the above mentioned analytes or for other
sample sources, the analyst must demonstrate the applicability of the
method by collecting precision and accuracy data on fortified samples
(i.e., groundwater, tap water) (4) and provide qualitative
confirmation of results by Gas Chromatography/Mass Spectrometry
(GC/MS) (5), or by GC analysis using dissimilar columns.
Method detection limits (MDL) (6) for the above organohalides and
Aroclors have been experimentally determined (Sect. 13.1). Actual
detection limits are highly dependent upon the characteristics of the
110
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gas chromatographic system used (e.g. column type, age, and proper
conditioning; detector condition; and injector mode and condition).
1.4 This method is restricted to use by or under the supervision of
analysts experienced in the use of GC and in the interpretation of
gas chromatograms. Each analyst must demonstrate the ability to
generate acceptable results with this method using the procedure
described in Sect. 11.
1.5 Analytes that are not separated chromatographically, i.e., analytes
which have very similar retention times, cannot be individually
identified and measured in the same calibration mixture or water
sample unless an alternative technique for identification and
quantitation is used (Sect. 11.4).
1.6 When this method is used to analyze unfamiliar samples for any or all
of the analytes above, analyte identifications should be confirmed by
at least one additional qualitative technique.
1.7 Degradation of Endrin, caused by active sites in the injection port
and GC columns, may occur. This is not as much a problem with new
capillary columns as with packed columns. However, high boiling
sample residue in capillary columns will create the same problem
after injection of sample extracts.
2. SUMMARY OF METHOD
2.1 Thirty-five ml of sample are extracted with 2 ml of hexane. Two jiL
of the extract are then injected into a gas chromatograph equipped
with a linearized electron capture detector for separation and
analysis. Aqueous calibration standards are extracted and analyzed
in an identical manner in order to compensate for possible extraction
losses.
2.2 The extraction and analysis time is 30 to 50 min per sample depending
upon the analytes and the analytical conditions chosen. (See Sect.
6.9.)
3. DEFINITIONS
3.1 Laboratory duplicates (LD1 and LD2) -- Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample
collection, preservation, or storage procedures.
3.2 Field duplicates (FD1 and FD2) -- Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
Ill
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3.3 Laboratory reagent blank (LRB) --An aliquot of reagent water thai
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
that are used with other samples. The LRB is used to determine if
method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.4 Field reagent blank (FRB) -- Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage, preservation
and all analytical procedures. The purpose of the FRB is to determine
if method analytes or other interferences are present in the field
environment.
3.5 Laboratory performance check solution (LPC) -- A solution of method
analytes, surrogate compounds, and internal standards used to
evaluate the performance of the instrument system with respect to a
defined set of method criteria.
3.6 Laboratory fortified blank (LFB) -- An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.7 Laboratory fortified sample matrix (LFM) -- An aliquot of an
environmental sample to which known quantities of the method analytes
are added in the laboratory. The LFM is analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.8 Stock standard solution -- A concentrated solution containing a
single certified standard that is a method analyte, or a concentrated
solution of a single analyte prepared in the laboratory with an
assayed reference compound. Stock standard solutions are used to
prepare primary dilution standards.
3.9 Primary dilution standard solution -- A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
3.10 Calibration standard (CAL) -- a solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
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3.11 Quality control sample (QCS) -- a sample matrix containing method
analytes or a solution of method analytes in a water miscible solvent
which is used to fortify reagent water or environmental samples. The
QCS is obtained from a source external to the laboratory, and is used
to check laboratory performance with externally prepared test
materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware and other sample processing apparatus that lead
to discrete artifacts or elevated baselines in gas chromatograms.
All reagents and apparatus must be routinely demonstrated to be free
from interferences under the conditions of the analysis by running
laboratory reagent blanks as described in Sect. 10.2.
4.1.1 Glassware must be scrupulously cleaned (2). Clean all
glassware as soon as possible after use by thoroughly rinsing
with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing wih tap and reagent
water. Drain dry, and heat in an oven or muffle furnace at
400eC for 1 hr. Do not heat volumetric ware. Thermally
stable materials might not be eliminated by this treatment.
Thorough rinsing with acetone may be substituted for the
heating. After drying and cooling, seal and store glassware
in a clean environment to prevent any accumulation of dust or
other contaminants. Store inverted or capped with aluminum
foil.
4.1.2 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. WARNING:
When a solvent is purified, stabilizers put into the solvent
by the manufacturer are removed thus potentially making the
solvent hazardous. Also, when a solvent is purified,
preservatives put into the solvent by the manufacturer are
removed thus potentially reducing the shelf-life.
4.2 Interfering contamination may occur when a sample containing low
concentrations of analytes is analyzed immediately following a sample
containing relatively high concentrations of analytes. Between-
sample rinsing of the sample syringe and associated equipment with
hexane can minimize sample cross contamination. After analysis of a
sample containing high concentrations of analytes, one or more
injections of hexane should be made to ensure that accurate values
are obtained for the next sample.
4.3 Matrix interferences may be caused by contaminants that are
coextracted from the sample. Also, note that all the analytes listed
in the scope and application section are not resolved from each other
on any one column, i.e., one anlayte of interest may be an
113
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interferent for another analyte of interest. The extent of matri
interferences will vary considerably from source to source, depending
upon the water sampled. Cleanup of sample extracts may be necessary.
Positive identifications should be confirmed (Sect. 11.4).
4.4 It is important that samples and working standards be contained in
the same solvent. The solvent for working standards must be the same
as the final solvent used in sample preparation. If this is not the
case, chromatographic comparability of standards to sample may be
affected.
4.5 Caution must be taken in the determination of endrin since it has
been reported that the splitless injector may cause endrin
degradation (7). The analyst should be alerted to this possible
interference resulting in an erratic response for endrin.
4.6 Variable amounts of pesticides and commercial PCB products from
aqueous solutions adhere to glass surfaces. It is recommended that
sample transfers and glass surface contacts be minimized.
4.7 Aldrin, hexachlorocyclopentadiene and methoxychlor are rapidly
oxidized by chlorine. Dechlorination with sodium thiosulfate at time
of collection will retard further oxidation of these compounds.
4.8 WARNING: An interfering, erratic peak has been observed within the
retention window of heptachlor during many analyses of reagent, tap,
and groundwater. It appears to be related to dibutyl phthalate;
however, the specific source has not yet been definitively
determined. The observed magnitude and character of this peak
randomly varies in numerical value from successive injections made
from the same vial.
5. SAFETY
5.1 The toxicity and carcinogenicity of chemicals used in this method
have not been precisely defined; each chemical should be treated as a
potential health hazard, and exposure to these chemicals should be
minimized. Each laboratory is responsible for maintaining awareness
of OSHA regulations regarding safe handling of chemicals used in this
method. Additional references to laboratory safety are available
(8-10) for the information of the analyst.
5.2 The following organohalides have been tentatively classified as known
or suspected human or mammalian carcinogens: aldrin, commercial PCB
products, chlordane, dieldrin, heptachlor, hexachlorobenzene, and
toxaphene. Pure standard materials and stock standard solutions of
these compounds should be handled in a hood or glovebox.
5.3 WARNING: When a solvent is purified, stabilizers put into the
solvent by the manufacturer are removed thus potentially making the
solvent hazardous.
114
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APPARATUS AND EQUIPMENT
6.1 SAMPLE CONTAINERS - 40-mL screw cap vials (Pierce #13075 or
equivalent) each equipped with a size 24 cap with a flat, disc-like
TFE facing backed with a polyethylene film/foam extrusion (Fisher
#02-883-3F or equivalent). Prior to use, wash vials and septa with
detergent and rinse with tap and distilled water. Allow the vials
and septa to air dry at room temperature, place the vials in a 400*C
oven for one hour, then remove and allow to cool in an area known to
be free of organics.
6.2 VIALS - auto sampler, screw cap with septa, 1.8 mL, Varian
#96-000099-00 or equivalent or any other autosampler vials not
requiring more than 1.8 mL sample volumes.
6.3 AUTO SAMPLER - Hewlett-Packard 7671A, or equivalent.
6.4 MICRO SYRINGES - 10 and 100 0L.
6.5 MICRO SYRINGE - 25 0L with a 2-inch by 0.006-inch needle - Hamilton
702N or equivalent.
6.6 PIPETTES - 2.0 and 5.0 mL transfer.
6.7 VOLUMETRIC FLASKS - 10 and 100 mL, glass stoppered.
6.8 STANDARD SOLUTION STORAGE CONTAINERS - 15-mL bottles with PTFE-lined
screw caps.
6.9 GAS CHROMATOGRAPH -- Analytical system complete with temperature
programmable GC suitable and split/splitless injector for use with
capillary columns and all required accessories including syringes,
analytical columns, gases, a linearized electron capture detector and
stripchart recorder. A data system is recommended for measuring peak
areas. Table 1 lists retention times observed for method analytes
using the columns and analytical conditions described below.
6.9.1 Three gas chromatographic columns are recommended. Column 1
(Sect. 6.9.2) should be used as the primary analytical column
unless routinely occurring analytes are not adequately
resolved. Validation data presented in this method were
obtained using this column. Columns 2 and 3 are recommended
for use as confirmatory columns when GC/MS confirmation is not
available. Alternative columns may be used in accordance with
the provisions described in Sect. 10.3.
6.9.2 Column 1 (Primary Column) - 0.32 mm ID x 30 M long fused
silica capillary with chemically bonded methyl polysiloxane
phase (DB-1, 1.0 pm film, or equivalent). Helium carrier gas
flow is about 25 cm/sec linear velocity, measured at 180°
with 9 psi column head pressure. The oven temperature is
programmed from 180eC to 260°C at 4'C/min and held at 260eC
115
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until all expected compounds have eluted. Injector
temperature: 200"C. Splitless Mode: 0.5 min. Detector
temperature: 290'C. Sample chromatograms for selected
pesticides are presented in Figures 1 and 2. Chromatograms of
the Aroclors, toxaphene, and technical chlordane are presented
in Figures 3 through 11.
6.9.3 Column 2 (alternative column 1) - 0.32mm ID x 30 M long fused
silica capillary with a 1:1 mixed phase of dimethyl silicone
and polyethylene glycol (Durawax-DX3, 0.25/im film, or
equivalent). Helium carrier gas flow is about 25 cm/sec
linear velocity and oven temperature 1s programmed from 100'C
to 210°C at 8'C/min, and held at 210°C until all expected
compounds have eluted. Then the post temperature is
programmed to 240flC at 8eC/min for 5 min.
6.9.4 Column 3 (alternative column 2) - 0.32mm ID x 25 M long fused
silica capillary with chemically bonded 50:50 Methyl-Phenyl
silicone (OV-17, 1.5/wi film thickness, or equivalent). Helium
carrier gas flow is about 40 cm/sec linear velocity and oven
temperature is programmed from 100'C to 260°C at 4'C/min and
held at 260°C until all expected compounds have eluted.
7. REAGENTS AND CONSUMABLE MATERIALS - - WARNING: When a solvent is purified'
stabilizers put into the solvent by the manufacturer are removed thus
potentially making the solvent hazardous. Also, when a solvent is
purified, preservatives put into the solvent by the manufacturer are
removed thus potentially making the shelf-life short.
7.1 REAGENTS
7.1.1 Hexane extraction solvent - UV Grade, Burdick and Jackson #216
or equivalent.
7.1.2 Methyl alcohol - ACS Reagent Grade, demonstrated to be free of
analytes.
7.1.3 Sodium chloride, NaCl - ACS Reagent Grade - For pretreatment
before use, pulverize a batch of NaCl and place in a muffle
furnace at room temperature. Increase the temperature to
400°C and hold for 30 min. Place in a bottle and cap.
7.1.4 Sodium thiosulfate, Na2S?03, ACS Reagent Grade- -For
preparation of solution (0.04 g/mL), mix 1 g of NagSgC^ with
reagent water and bring to 25-mL volume in a volumetric flask.
7.2 REAGENT WATER - Reagent water is defined as water free of
interference when employed in the procedure described herein.
7.2.1 A Millipore Super-Q Water System or its equivalent may be used
to generate deionized reagent water.
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7.2.2 Test reagent water each day it is used by analyzing it
according to Sect. 11.
7.3 STOCK STANDARD SOLUTIONS - These solutions may be obtained as
certified solutions or prepared from pure standard materials using
the following procedures:
7.3.1 Prepare stock standard solutions (5000 /ig/ml_) by accurately
weighing about 0.0500 g of pure material. Dissolve the
material in methanol and dilute to volume in a 10-mL
volumetric flask. Larger volumes can be used at the
convenience of the analyst. 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. Commercially prepared stock standards can be used
at any concentration if they are certified by the manufacturer
or by an independent source.
7.3.2 Transfer the stock standard solutions into Teflon-sealed
screw-cap bottles. 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.
7.3.3 Stock standard solutions must be replaced after six months, or
sooner if comparison with check standards indicates a problem.
7.4 PRIMARY DILUTION STANDARD SOLUTIONS -- Use stock standard solutions
to prepare primary dilution standard solutions that contain the
analytes in methanol. The primary dilution standards should be
prepared at concentrations that can be easily diluted to prepare
aqueous calibration standards (Sect. 9.1.1) that will bracket the
working concentration range. Store the primary dilution standard
solutions with minimal headspace and check frequently for signs of
deterioration or evaporation, especially just before preparing
calibration standards. The storage time described for stock standard
solutions in Sect. 7.3.3 also applies to primary dilution standard
solutions.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE COLLECTION
8.1.1 Collect all samples in 40-mL bottles into which 3 mg of sodium
thiosulfate crystals have been added to the empty bottles just
prior to shipping to the sampling site. Alternately, 75 /iL of
freshly prepared sodium thiosulfate solution (0.04 g/mL) may
be added to empty 40-mL bottles just prior to sample
collection.
8.1.2 When sampling from a water tap, open the tap and allow the
system to flush until the water temperature has stabilized
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(usually about 10 min). Adjust the flow to about 500 mL/min
and collect samples from the flowing stream.
8.1.3 When sampling from a well, fill a wide-mouth bottle or beaker
with sample, and carefully fill 40-mL sample bottles.
8.2 SAMPLE PRESERVATION
8.2.1 The samples must be chilled to 4°C at the time of collection
and maintained at that temperature until the analyst is
prepared for the extraction process. Field samples that will
not be received at the laboratory on the day of collection
must be packaged for shipment with sufficient ice to insure
that they will be maintained at 4'C until arrival at the
laboratory.
8.3 SAMPLE STORAGE
8.3.1 Store samples and extracts at 4eC until extraction and
analysis.
8.3.2 Extract all samples as soon as possible after collection.
Results of holding time studies suggest that all analytes with
the possible exception of heptachlor were adequately stable
for 14 days when stored under these conditions. In general,
heptachlor showed inconsistent results. If heptachlor is to
be determined, samples should be extracted within 7 days of
collection. Analyte stability may be affected by the matrix;
therefore, the analyst should verify that the preservation
technique is applicable to the samples under study.
9. CALIBRATION AND STANDARDIZATION
9.1 Establish GC operating parameters equivalent to those indicated in
Sect. 6.9. WARNING: Endrin is easily degraded in the injection port
if the injection port or front of the column is dirty. This is the
result of buildup of high boiling residue from sample injection.
Check for degradation problems by injecting a mid-level standard
containing only endrin. Look for the degradation products of endrin
(endrin ketone and endrin aldehyde). If degradation of endrin
exceeds 20%, take corrective action before proceeding with
calibration. Calculate percent breakdown as follows:
Total endrin degradation peak area (endrin aldehyde + endrin ketone)
Total endrin peak area (endrin + endrin aldehyde + endrin ketone) x
9.2 At least three calibration standards are needed; five are
recommended. One should contain analytes at a concentration near but
greater than the method detection limit for each compound; the other
two should be at concentrations that bracket the range expected in
samples. For example, if the MDL is 0.01 jtg/L, and a sample expected
to contain approximately 0.10 /*g/L is to be analyzed, aqueous
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standards should be prepared at concentrations of 0.02 /ig/L, 0.10
/ig/L, and 0.20 pg/L.
9.2.1 To prepare a calibration standard (CAL), add an appropriate
volume of a secondary dilution standard to a 35-mL aliquot of
reagent water in a 40-mL bottle. Do not add less than 20 /*L
of an alcoholic standard to the reagent water. Use a 25-jtL
micro syringe and rapidly inject the alcoholic standard into
the middle point of the water volume. Remove the needle as
quickly as possible after injection. Mix by inverting and
shaking the capped bottle several times. Aqueous standards
must be prepared fresh daily.
9.2.2 Starting with the standard of lowest concentration, prepare,
extract, and analyze each calibration standard beginning with
Sect. 11.2 and tabulate peak height or area response versus
the concentration in the standard. The results are to be used
to prepare a calibration curve for each compound by plotting
the peak height or area response versus the concentration.
Alternatively, if the ratio of concentration to response
(calibration factor) is a constant over the working range (20%
RSD or less), linearity to the origin can be assumed and the
average ratio or calibration factor can be used in place of a
calibration curve.
9.2.3 The working calibration curve or calibration factor must be
verified on each working day by the measurement of one or more
calibration standards. If the response for an analyte varies
from the predicted response by more than ±20%, the test must
be repeated using a fresh calibration standard. If the
results still do not agree, generate a new calibration curve
or use a single point calibration standard as described in
Sect. 9.2.4.
9.2.4 Single point calibration is an acceptable alternative to a
calibration curve. Prepare single point standards from the
secondary dilution standard solutions. The single point
calibration standard should be prepared at a concentration
that produces a response close (±20% or less) to that of the
unknowns. Do not use less than 20 /zL of the secondary
dilution standard solution to produce a single point
calibration standard in reagent water.
9.3 INSTRUMENT PERFORMANCE - Check the performance of the entire
analytical system daily using data gathered from analyses of
laboratory reagent blanks (LRB), (CAL), laboratory duplicate samples
(LD1 and LD2), and the laboratory performance check solution (LPC)
(Sect. 10.6).
9.3.1 Significant peak tailing in excess of that shown for the
target compounds in the method chromatograms (Figures 1-11)
must be corrected. Tailing problems are generally traceable
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to active sites on the GC column, improper column
installation, or operation of the detector.
9.3.2 Check the precision between replicate analyses. Poor
precision is generally traceable to pneumatic leaks,
especially at the injection port. If the GC system is
apparently performing acceptably but with decreased
sensitivity, it may be necessary to generate a new curve or
set of calibration factors to verify the decreased responses
before searching for the source of the problem.
9.3.3 Observed relative area responses of endrin (See 4.5) must meet
the following general criteria:
9.3.3.1 The breakdown of endrin into its aldo and keto forms
must be adequately consistent during a period in
which a series of analyses is made. Equivalent
relative amounts of breakdown should be demonstrated
in the LRB, LPC, LFB, CAL and QCS. Consistent
breakdown resulting in these analyses would suggest
that the breakdown occurred in the instrument system
and that the methodology is in control.
9.3.3.2 Analyses of laboratory fortified matrix (LFM) samples
must also be adequately consistent after corrections
for potential background concentrations are made.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration
of laboratory capability, analysis of laboratory reagent blanks
(LRB), laboratory fortified blanks (LFB), laboratory fortified sample
matrix (LFM), and quality control samples (QCS).
10.2 Laboratory Reagent Blanks. Before processing any samples, the
analyst must demonstrate that all glassware and reagent interferences
are under control. Each time a set of samples is extracted or
reagents are changed, an LRB must be analyzed. If within the
retention time window of any analyte the LRB produces a peak that
would prevent the determination of that analyte, determine the source
of contamination and eliminate the interference before processing
samples.
10.3 Initial Demonstration of Capability
10.3.1 Select a representative concentration (about 10 times MOL or
at the regulatory Maximum Contaminant Level, whichever is
lower) for each analyte. Prepare a primary dilution standard
solution (in methanol) containing each analyte at 1000 times
selected concentration. With a syringe, add 35 ill of the
concentrate to each of at least four 35-mL aliquots of
120
-------
reagent water, and analyze each aliquot according to
procedures beginning in Sect. 11.
10.3.2 For each analyte the recovery value should for at least three
out of four consecutively analyzed samples fall in the range
of R±30% (or within R±3SR if broader) using the values for R
and Sp for reagent water in Table 2. For those compounds that
meet the acceptance criteria, performance is considered
acceptable and sample analysis may begin. For those compounds
that fail these criteria, initial demonstration procedures
should be repeated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples via a
new, unfamiliar method prior to obtaining some experience with
it. It is expected that as laboratory personnel gain
experience with this method the quality of data will improve
beyond those required here.
10.4 The analyst is permitted to modify GC columns, GC conditions, or
detectors to improve separations or lower analytical costs. Each
time such method modifications are made, the analyst must repeat the
procedures in Sect. 10.3.
10.5 Assessing Laboratory Performance - Laboratory Fortified Blank (LFB)
10.5.1 The laboratory must analyze at least one laboratory fortified
blank (LFB) per sample set (all samples extracted within a
24-h period). If the sample set contains more than 20
samples, analyze one LFB for every 20 samples. The fortifying
concentration of each analyte in the LFB sample should be 10
times MDL or the MCL, whichever is less. Calculate accuracy
as percent recovery (Xj). If the recovery of any analyte
falls outside the control limits (see Sect. 10.5.2), that
analyte is judged out of control, and the source of the
problem should be identified and resolved before continuing
analyses.
10.5.2 Until sufficient data become available from within their own
laboratory, usually a minimum of results from 20 to 30
analyses, the laboratory may assess laboratory performance
against the control limits in Sect. 10.3.2 that are derived
from the data in Table 2. When sufficient internal
performance data becomes available, develop control limits
from the mean percent recovery (X) and standard deviation (S)
of the percent recovery. These data are used to establish
upper and lower control limits as follows:
UPPER CONTROL LIMIT = X + 3S
LOWER CONTROL LIMIT = X - 3S
121
-------
After each five to ten new recovery measurements, new control
limits should be calculated using only the most recent 20-30
data points.
10.5.3 It is recommended that the laboratory periodically determine
and document its detection limit capabilities for analytes of
interest. CAUTION: No attempts to establish low detection
limits should be made before instrument optimization and
adequate conditioning of both the column and the GC system.
Conditioning includes the processing of LFB and LFM samples
containing moderate concentration levels of these analytes.
10.5.4 At least each quarter the laboratory should analyze quality
control samples (QCS) (if available). If criteria provided
with the QCS are not met, corrective action should be taken
and documented.
10.6 Assessing Analyte Recovery - Laboratory Fortified Sample Matrix (LFM)
10.6.1 The laboratory must add a known concentration to a minimum of
10% of the routine samples or one LFM per set, whichever is
greater. The fortified concentration should not be less than
the background concentration of the sample selected for
fortification. Ideally the LFM concentration should be the
same as that used for the LFB (Sect. 10.5). Periodically,
samples from all routine sample sources should be fortified.
10.6.2 Calculate the percent recovery (R,) for each analyte,
corrected for background concentrations measured in the
unfortified sample, and compare these values to the control
limits established in Sect. 10.5.2 from the analyses of LFBs.
10.6.3 If the recovery of any such analyte falls outside the
designated range, and the laboratory performance for that
analyte is shown to be in control (Sect. 10.5), the recovery
problem encountered with the dosed sample is judged to be
matrix related, not system related. The result for that
analyte in the unfortified sample is labeled suspect/matrix to
inform the data user that the results are suspect due to
matrix effects.
10.7 The laboratory may adopt additional quality control practices for use
with this method. The specific practices that are most productive
depend upon the needs of the laboratory and the nature of the
samples. For example, field or laboratory duplicates may be analyzed
to assess the precision of the environmental measurements or field
reagent blanks may be used to assess contamination of samples under
site conditions, transportation and storage.
122
-------
i*. PROCEDURE
11.1 SAMPLE PREPARATION
11.1.1 Remove samples from storage and allow them to equilibrate to
room temperature.
11.1.2 Remove the container caps. Withdraw and discard a 5-mL volume
using a 10-mL graduated cylinder. Replace the containe;- caps
and weigh the containers with contents to the nearest 0.1 g
and record these weights for subsequent sample volume
determinations (Sect. 11.3).
11.2 EXTRACTION AND ANALYSIS
11.2.1 Remove the container cap of each sample, and add 6 g NaCl
(Sect. 7.1.3) to the sample bottle. Using a transfer or
automatic dispensing pipet, add 2.0 mL of hexane. Recap and
shake vigorously by hand for 1 min. Invert the bottle and
allow the water and hexane phases to separate.
11.2.2 Remove the cap and carefully transfer approximately 0.5 mL of
hexane layer into an autosampler vial using a disposable glass
pipet.
11.2.3 Transfer the remaining hexane phase, being careful not to
include any of the water phase, into a second autosampler
vial. Reserve this second vial at 4°C for an immediate
reanalysis if necessary.
11.2.4 Transfer the first sample vial to an autosampler set up to
inject 1-2 /iL portions into the gas chromatograph for analysis
(See Sect. 6.9 for GC conditions). Alternately, 1-2 mL
portions of samples, blanks, and standards may be manually
injected, although an autosampler is strongly recommended.
11.3 DETERMINATION OF SAMPLE VOLUME IN BOTTLES NOT CALIBRATED
11.3.1 Discard the remaining sample/hexane mixture from the sample
bottle. Shake off the remaining few drops using short, brisk
wrist movements.
11.3.2 Reweigh the empty container with original cap and calculate
the net weight of sample by difference to the nearest 0.1 g
(Sect. 11.1.2 minus Sect. 11.3.2). This net weight (in
grams) is equivalent to the volume (in mL) of water extracted
(Sect. 12.3). By alternately using 40-mL bottles
precalibrated at 35-mL levels, the gravimetric steps can be
omitted, thus increasing the speed and ease of this
extraction process.
123
-------
11.4 IDENTIFICATION OF ANALYTES
11.4.1 Identify a sample component by comparison of Its retention
time to the retention time of a reference chromatogram. If
the retention time of an unknown compound corresponds, within
limits, to the retention time of a standard compound, then
identifiction is considered positive.
11.4.2 The width of the retention time window used to make
identifications should be based upon 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.
11.4.3 Identification requires expert judgement when sample
components are not resolved chromatographically. When peaks
obviously represent more than one sample componenet (i.e.,
broadened peak with shoulder(s) or valley between two or more
maxima), or any time doubt exists over the identification of a
peak on a chromatogram, appropriate alternative techniques to
help confirm peak identification need be employed. For
example, more positive identification may be made by the use
of an alternative detector which operates on a
chemical/physical principle different from that originally
used, e.g., mass spectrometry, or the use of a second
chromatography column. Suggested alternative columns are
described in Sect. 6.9.
12. CALCULATIONS
12.1 Identify the organohalides in the sample chromatogram by comparing
the retention time of the suspect peak to retention times generated
by the calibration standards and the laboratory fortified blanks.
Identify the multicomponent compounds using all peaks that are
characteristic of the specific compound from chromatograms generated
with individual standards. Select the most sensitive and
reproducible peaks to obtain a sum for calculation purposes (See
Table 1).
12.2 Use the single point calibration (Sect. 9.2.4) or use the calibration
curve or calibration factor (Sect. 9.2.3) to directly calculate the
uncorrected concentration (Ci) of each analyte in the sample (e.g.,
calibration factor x response).
12.3 Calculate the sample volume (Vs) as equal to the net sample weight:
Vs = gross weight (Sect. 11.1.2) - bottle tare (Sect. 11.3.2).
124
-------
12.4 Calculate the corrected sample concentration as:
Concentration, jig/L = 35(Cj)
(V$l
12.5 Results should be reported with an appropriate number of significant
figures. Experience indicates that three significant figures may be
used for concentrations above 99 /zg/L, two significant figures for
concentrations between 1-99 /zg/L, and 1 significant figure for lower
concentrations.
13. ACCURACY AND PRECISION
13.1 Single laboratory (EMSL-Cincinnati) accuracy and precision at several
concentrations in reagent, ground, and tap water matrices are
presented in Table 2.(11). These results were obtained from data
generated with a DB-1 column.
13.2 This method has been tested by 10 laboratories using reagent water
and groundwater fortified at three concentration levels. Single
operator precision, overall precision, and method accuracy were found
to be directly related to the concentration of the analyte and
virtually independent of the sample matrix. Linear equations to
describe the relationships are presented in Table 3.(12)
14. REFERENCES
1. Glaze, W.W., Lin, C.C., Optimization of Liquid-Liquid Extraction
Methods for Analysis of Organics in Water, EPA-600/S4-83-052, January
1984.
2. Henderson, J.E., Peyton, G.R. and Glaze, VI.H. (1976). In
"Identification and Analysis of Organic Pollutants in Water" (L.H.
Keith ed.), pp. 105-111. Ann Arbor Sci. Publ., Ann Arbor, Michigan.
3. Richard, J.J., Junk, G.A., "Liquid Extraction for Rapid Determination
of Halomethanes in Water," Journal AWWA, 69, 62, January 1977.
4. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio, 45268, March 1979.
5. Budde, W.L., Eichelberger, J.W., "Organic Analyses Using Gas
Chromatography-Mass Spectrometry," Ann Arbor Science, Ann Arbor,
Michigan 1979.
6. Glaser, J.A. et al., "Trace Analyses for Wastewaters," Environmental
Science and Technology, 15, 1426 (1981).
125
-------
7. Bellar, T.A., Stemmer, P., Lichtenberg, J.J., "Evaluation of
Capillary Systems for the Analysis of Environmental Extracts,"
EPA-600/S4-84-004, March 1984.
8. "Carcinogens-Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute of Occupational Safety and Health,
Publication No. 77-206, August, 1977.
9. "OSHA Safety and Health Standards, General Industry," (29CFR1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
10. "Safety in Academic Chemistry Laboratories," American Chemical
Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.
11. Winfield, T., et al. "Analysis of Organohalide Pesticides and
Commercial PCB Products in Drinking Water by Microextraction and Gas
Chromatography." In preparation.
12. Multilaboratory Method Validation Study #40, conducted by the Quality
Assurance Branch, EMSL-Ci. Report in progress.
126
-------
TABLE 1. RETENTION TINES FOR METHOD ANALYTES
Analyte
Retention Time(a), Min
Primary Confirm. 1 Confirm. 2
Hexachlorocyclopentadiene 5.
Simazine 10.
Atrazine 11.
Hexachlorobenzene 11.
Lindane 12.
Alachlor 15.
Heptachlor 15.
Aldrin 17.
Heptachlor Epoxide 19.
gamma-Chlordane 19.
alpha-Chlordane 20.
trans-Nonachlor 21.
Dieldrin 22.
Endrin 23.
cis-Nonachlor 24.
Methoxychlor 30.
5
9
2
9
3
1
9
6
0
9
9
3
1
2
3
0
6.
25.
22.
13.
18.
19.
17.
18.
24.
25.
26.
24.
45.
33.
39.
58.
8
7
6
4
4
7
5
4
6
9
6
8
1
3
0
5
5.2
19.9
19.6
15.6
18.7
21.1
20.0
21.4
24.6
26.0
26.6
26.3
27.8
29.2
30.4
36.4
Primary(b)
Aroclor 1016
Aroclor 1221
Aroclor 1232
Aroclor 1242
Aroclor 1248
Aroclor 1254
Aroclor 1260
Chlordane
Toxaphene
13
7.
11
11
14
19
23
15
21
.6,
7,
.2,
.2,
.8,
.1,
.4,
.1,
.7,
14
9.0
14
13
16
21
24
15
22
.8
»
.7
.6
.2
.9
.9
.9
.5
, 15
15.9
, 13
, 14
, 17
, 23
, 26
, 20
, 26
.2
»
.6
.7
.1
.4
.7
.1
.7
, 16.
19.1,
, 15.
, 15.
, 17.
, 24.
, 28.
, 20.
, 27.
2,
24
2,
2,
7,
9,
2,
9,
2
17
.7
17
17
19
26
29
21
.7
.7
.7,
.8,
.7
.9,
.3
19.8
22.0
32.6
Columns and analytical conditions are described in Sect. 6.9.2, 6.9.3, and
6.9.4.
Column and conditions described in Sect. 6.9.2. More than one peak listed
does not implicate the total number of peaks characteristic of the
multi-component analyte. Listed peaks indicate only the ones chosen for
summation in the quantification.
127
-------
TABLE 2. SINGLE LABORATORY ACCURACY, PRECISION AND METHOD DETECTION LIMITS
(MDLS) FOR ANALYTES FROM REAGENT HATER, GROUNDWATER, AND TAP HATER*
Analvte
Accuracy and Standard Deviation Data
Concen-
MDLb tration* Reagent Water Groundwater Tap Water
UQ/L UQ/L R£ SRS R So R
Aldrin
Alachlor
Aldrin
Atrazine
alpha-Chlordane
gamma-Chlordane
Chlordane
Dieldrin
Endrin
Heptachlor
Heptachlor Epoxide
Hexachlorobenzene
Hexachlorocyclopentadiene
Lindane
Methoxychlor
cis-Nonachlor
trans-Nonachlor
Simazine
Toxaphene
Aroclor 1016
Aroclor 1221
Aroclor 1232
Aroclor 1242
Aroclor 1248
Aroclor 1254
Aroclor 1260
0.075
0.225
0.007
2.4
0.006
0.012
0.14
0.012
0.063
0.003
0.004
0.002
0.13
0.003
0.96
0.027
0.011
6.8
1.0
0.08
15.0
0.48
0.31
0.102
0.102
0.189
0.15
0.50
0.05
5.0
20.0
0.06
0.35
0.06
0.35
0.17
3.4
0.10
3.6
0.10
3.6
0.032
1.2
0.04
1.4
0.003
0.09
0.15
0.35
0.03
1.2
2.10
7.03
0.06
0.45
0.06
0.35
25
60
10
80
1.0
180
3.9
4.7
3.6
3.4
1.8
1.7
2.0
1.8
86
102
106
85
95
95
86
95
86
NA
NA
87
114
119
99
77
80
100
115
104
103
73
73
91
111
100
98
110
82
95
86
99
65
NA
NA
NA
NA
NA
NA
NA
.
NA
-
NA
NA
9.5
13.4
20.0
16.2
5.2
3.5
17.0
0.4
18.5
8.0
3.6
17.1
9.1
29.8
6.5
10.2
7.4
15.6
6.6
13.5
6.6
5.1
11.7
6.5
5.0
21.0
10.9
15.2
21.3
9.6
21.8
8.3
3.6
12.6
15.3
6.6
8.3
13.5
6.0
11.5
_
10.4
-
20.7
-
100
_
86
95
86
83
94
86
95
-
-
67
94
94
100
37
71
90
103
91
101
87
69
88
109
-
_
101
93
83
94
97
59
-
-
.
_
_
_
.
_
_
-
-
-
-K
11.0
16.3
7.3
9.1
4.4
10.2
5.3
14.5
.
-
10.1
8.6
20.2
11.3
6.8
9.8
14.2
6.9
10.9
4.4
5.1
4.8
7.7
3.4
-
_
7.2
18.3
7.1
17.2
9.2
18.0
-
-
_
_
_
_
.
.
-
-
-
.
69
_
108
91
85
91
83
91
105
95
92
81
106
85
200
106
112
81
100
88
191
109
103
93
-
.
93
87
73
86
102
67
110
114
97
92
86
96
_
84
-
85
.
88
— -K-
9.0
_
10.9
3.1
7.1
2.4
14.7
6.0
12.4
9.6
15.7
14.0
14.0
12.4
22.6
16.8
7.5
5.9
15.6
13.4
18.5
14.3
8.1
18.4
.
.
14.3
5.4
4.1
5.1
13.4
6.2
9.5
13.5
7.5
9.6
7.3
7.4
_
9.9
-
11.8
-
10 <»
128
-------
Table 2 (Continued)
NA = Not applicable. A separate set of aqueous standards was not analyzed, and
the response factor for reagent water was used to calculate a recovery for the tap
water matrix.
aData corrected for amount detected in blank and represent the mean of 5-8
samples.
bMDL = method detection limit in sample in pg/L; calculated by multiplying
standard deviation (S) times the students' t value appropriate for a 99%
confidence level and a standard deviation estimate with n-1 degrees of freedom.
CR = average percent recovery.
^SR = Standard deviation about percent recovery.
* Refers to concentration levels used to generate R and SR data for the three types
of water Matrices, not for MDL determinations.
- No analyses conducted.
129
-------
TABLE 3. METHOD ACCURACY AND PRECISION AS FUNCTIONS OF CONCENTRATION-METHOD 505
REAGENT HATER
CO
o
Parameter
Atrazlne
Siniazine
Hexachl orobenzene
Lindane
Alachlor
Heptachlor
Aldrin
Heptachlor epoxide
Dieldrin
Endrin
Methoxychlor
Chlordane
Toxaphene
PCB-1016
PCB-1254
Applicable
Cone. Range
(WJ/L)
(3.06-45.90)
(12.55-50.20)
(0.01-0.37)
(0.04-1.39)
(0.50-37.50)
(0.04-1.41)
(0.04-1.42)
(0.04-1.42)
(0.10-7.53)
(0.10-7.50)
(0.20-15.00)
(0.51-50.90)
(5.63-70.40)
(0.50-49.80)
(0.50-50.40)
Accuracy as
Recovery X
(M9/L)
1.122C+0.97
0.892C+1.446
1.028C-0.00
1.009C-0.00
1.004C-0.08
1.002C+0.02
1.066C+0.00
0.952C+0.00
1.027C+0.00
0.958C+0.01
0.950C+0.15
1.037C+0.06
1.087C+0.24
0.856C+0.31
0.872C-0.01
Single Analyst
Precision Sr
(Mg/L)
O.OOOx+1.21
-0.049X+3.52
O.lOSx+0.00
0.057X+0.01
0.077X+0.10
0.107X+0.01
0.031X+0.02
0.032X+0.02
0.091X+0.01
0.116X+0.01
O.llSx+0.12
0.084X+0.06
0.131X-0.31
0.106x+0.31
0.122X+0.11
Overall Precision
(W/L)
0.045X+2.23
0.209X+1.23
0.227X+0.00
0.142X+0.00
O.lOSx+0.16
0.211X+0.02
0.264X-0.00
0.129X+0.02
0.198X+0.02
0.136X+0.02
0.125X+0.20
0.125x+0.19
0.269X+0.69
0.147X+0.45
0.281X+0.05
-------
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Figure 1
18 20 22
TIME (MIN)
Hexane spiked at 7.71 ug/L with heptachlor and Undane; 9.14 ug/L with heptachlor epoxlde;
11.4 ug/L with aldrln and hexachlorobenzene; 23 ug/L with butachlor, chlorpyrlfos. chlorpyrlfos.
methyl, dlclobenll. dleldrln, endHn, metolochlor, and propachlor; and 44.9 ug/L with
metnoxychlor.
-------
COLUMN: Fused silica capillary
LIQUID PHASE: 06-1
FILM THICKNESS:. l.Oum
COLUMN DIMENSIONS: 0.32mm ID,
30 M long
10 15 20
TIME (MIN)
25
30
35
Figure 2.
Extract of reagent water spiked at 20 ug/L with atrazlne,
60 ug/L with s1maz1ne, 0.45 uq/L with ds-nonachlor, and
0.35 ug/L with hexachlorocyclopentadlene, heptachlor,
alpha chlordane, ganro chlordane, and trans-nonachlor.
132
-------
GO
GO
COLUMN: Fused silica capillary
LIQUID PHASE: DB-1
FILM THICKNESS: l.Oiim
COLUMN DIMENSIONS: 0.32nm ID, 30 M long
JL
JL
JL
2 4 6 8 10 12 14 16 18 20 22 24
TIME (MIN)
26 28 30 32 54 36 38 40 42
Figure 3. Hexane spiked at 11.4 ug/L with Arodor 1016,
-------
COLUMN: Fused silica capillary
LIQUID PHASE: OB-1
FILM THICKNESS: 1.0pm
COLUMN DIMENSIONS: 0.32mn ID. 30 M long
xjA-j*—Av
10 12 14 16 IB 20 22 24
TIME (MIN)
2S 28 30 32 34 36 38 40 42 44
,gure 4. Hexane spiked at 171.4 ug/L with Aroclor 1221.
-------
CO
Fused silica capillary
LIQUID PHASE: DB-1
FILM THICKNESS: l.Qum
COLUMN DIMENSIONS: 0.32wm ID, 30 N long
2 4 6 8 10 12 14 1« 18 20 22 24 26 28
TIME (MIN)
Figure 5. Hexane spiked at 57.1 ug/L with Aroclor 1232.
30 32 34 36 38 40 42 44
-------
00
COLUMN: Fused silica capillary
LIQUID PHASE: DB-1
FILM THICKNESS: I.Otim
COLUMN DIMENSIONS: 0.32nm ID, 30 M long
10 12 14 16
10 20 22
TIME (MIN)
24 26 28 30 32 34 36 38 4O 42 44
Figure 6. Hexane spiked at 57.1 ug/L with Aroclor 1242.
-------
GO
COLUMN: Fused silica
LIQUID PHASE: DB-1
FILM THICKNESS: I.OpHi
COLUMN DIMENSIONS: 0.32nm ID, 30 N long
JL
_L
10 12 14 16
18 20 22
TIME (MIN)
24 25 28 30 32 34 36 38 40 42 44
Figure?. Hexane spiked at 57.1 ug/L with Aroclor 1248.
-------
00
co
COLUMN: Fused silica capillary
LIQUID PHASE: DB-1
FILM THICKNESS: 1.0pm
COLUMN DIMENSIONS: 0.32nm ID, 30 N long
10 12 14 16
18 20 22
TIME (MIN)
24 28 28 30 32 34 3« 38 40 42 44
rlgure 8. Hexane spiked at 42.9 ug/L with Aroclor 1254.
-------
COLUMN: Fused silica capillary
LIQUID PHASE: DB-1
FILM THICKNESS: I.Oi*
COLUMN DIMENSIONS: 0,3Znw ID, 30 * long
Jl
2 4 • 9 10 12 14 16 IS 20 22 24 26 28
TIME (MIN)
Figure 9. Hexane spiked at 34.3 ug/L with Aroclor 1260.
30 32 54 36 38 4O 42 44
-------
COLUMN: Fused silica capillary
LIQUID PHASE: DB-1
FILM THICKNESS: I.Oum
COLUMN DIMENSIONS: 0.32mn ID, 30 M long
2 4 6 8 10 12 14 16 18 20 22 24 25 28
TIME (NIN)
Igure 10. Hexane spiked at 28.6 ug/L with chlordane.
30 32 34 98 38 4O 42 44
-------
COLUMN: Fused silica capillary
LIQUID PHASE: DB-1
FILM THICKNESS: 1.0w«
COLUMN DIMENSIONS: 0.32m ID, 30 M long
JL
a 4 o a 10 12 14 i« le 20 22
TIME (MIN)
Figure 11. Hexane spiked at 57.1 ug/L with toxaphene.
24 26 29 30 32 34 36 38 4O 42
-------
METHOD 507. DETERMINATION OF NITROGEN- AND PHOSPHORUS-CONTAINING PESTICIDES
IN WATER BY GAS CHROMATOGRAPHY WITH A NITROGEN-PHOSPHORUS DETECTOR
Revision 2.0
T. Engels (Battelle Columbus Laboratories) - National Pesticide Survey
Method 1, Revision 1.0 (1987)
R. L. Graves - Method 507, Revision 2.0 (1989)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
143
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HETHOO 507
DETERMINATION OF NITROGEN-AND PHOSPHORUS-CONTAINING PESTICIDES IN WATER
BY GAS CHRONATOGRAPKY WITH A NITROGEN-PHOSPHORUS DETECTOR
1. SCOPE AND APPLICATION
1.1 This is a gas chromatographic (GC) method applicable to the determi-
nation of certain nitrogen- and phosphorus-containing pesticides in
ground water and finished drinking water.(1) The following compounds
can be determined using this method:
Analvte
Chemical Abstract Services
Registry Number
Alachlor 15972-60-8
Ametryn 834-12-8
Atraton 1610-17-9
Atrazine 1912-24-9
Bromacil 314-40-9
Butachlor 23184-66-9
Butyl ate 2008-41-5
Carboxin 5234-68-5
Chlorpropham 101-21-3
Cycloate 1134-23-2
Diazinon(a)* 333-41-5
Dichlorvos 62-73-7
Diphenamid 957-51-7
Disulfoton* 298-04-4
Disulfoton sulfone* . 2497-06-5
Disulfoton sulfoxide(a) 2497-07-6
EPTC 759-94-4
Ethoprop 13194-48-4
Fenamiphos 22224-92-6
Fenarimol 60168-88-9
Fluridone 59756-60-4
Hexazinone 51235-04-2
Merphos* 150-50-5
Methyl paraoxon 950-35-6
Metolachlor 51218-45-2
Metribuzin 21087-64-9
Mevinphos 7786-34-7
MGK 264 113-48-4
Molinate 2212-67-1
Napropamide 15299-99-7
Norflurazon 27314-13-2
Pebulate 1114-71-2
Prometon 1610-18-0
Prometryn 7287-19-6
Pronamide(a)* 23950-58-5
Propazine 139-40-2
144
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Simazine 122-34-9
Simetryn 1014-70-6
Stirofos 22248-79-9
Tebuthiuron 34014-18-1
Terbacil 5902-51-2
Terbufos(a)* 13071-79-9
Terbutryn 886-50-0
Triademefon 43121-43-3
Tricyclazole 41814-78-2
Vernolate 1929-77-7
(a) Compound exhibits aqueous instability. Samples for which this
compound is an analyte of interest must be extracted
immediately (Sections 11.1 through 11.3).
* These compounds are only qualitatively identified in the
National Pesticides Survey (NFS) Program. These compounds are
not quantitated because control over precision has not been
accomplished.
1.2 This method has been validated in a single laboratory and estimated
detection limits (EDLs) have been determined for the analytes above
(Sect. 13). Observed detection limits may vary among waters,
depending upon the nature of interferences in the sample matrix and
the specific instrumentation used.
1.3 This method is restricted to use by or under the supervision of
analysts experienced in the use of GC and in the interpretation of
gas chromatograms. Each analyst must demonstrate the ability to
generate acceptable results with this method using the procedure
described in Sect. 10.3.
1.4 Analytes that are not separated chromatographically, i.e., analytes
which have very similar retention times, cannot be individually
identified and measured in the same calibration mixture or water
sample unless an alternative technique for identification and
quantitation exist (Section 11.5).
1.5 When this method is used to analyze unfamiliar samples for any or all
of the analytes above, analyte identifications should be confirmed by
at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 A measured volume of sample of approximately 1 L is extracted with
methylene chloride by shaking in a separatory funnel or mechanical
tumbling in a bottle. The methylene chloride extract is isolated,
dried and concentrated to a volume of 5 ml during a solvent exchange
to methyl tert-butyl ether (MTBE). Chromatographic conditions are
described which permit the separation and measurement of the analytes
in the extract by Capillary Column GC with a nitrogen-phosphorus
detector (NPD).
145
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3. DEFINITIONS
3.1 Internal standard -- A pure analyte(s) added to a solution in known
amount(s) and used to measure the relative responses of other method
analytes and surrogates that are components of the same solution.
The internal standard must be an analyte that is not a sample
component.
3.2 Surrogate analyte -- A pure analyte(s), which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in
known amount(s) before extraction and is measured with the same
procedures used to measure other sample components. The purpose of a
surrogate analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates (LD1 and LD2) -- Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of L01 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample
collection, preservation, or storage procedures.
3.4 Field duplicates (FD1 and FD2) -- Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.5 Laboratory reagent blank (LRB) -- An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
that are used with other samples. The LRB is used to determine if
method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.6 Field reagent blank (FRB) -- Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage, preservation
and all analytical procedures. The purpose of the FRB is to determine
if method analytes or other interferences are present in the field
environment.
3.7 Laboratory performance check solution (LPC) -- A solution of method
analytes, surrogate compounds, and internal standards used to
evaluate the performance of the instrument system with respect to a
defined set of method criteria.
3.8 Laboratory fortified blank (LFB) -- An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
146
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whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.9 Laboratory fortified sample matrix (LFM) -- An aliquot of an
environmental sample to which known quantities of the method analytes
are added in the laboratory. The LFM 1s analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.10 Stock standard solution -- A concentrated solution containing a
single certified standard that is a method analyte, or a concentrated
solution of a single analyte prepared in the laboratory with an
assayed reference compound. Stock standard solutions are used to
prepare primary dilution standards.
3.11 Primary dilution standard solution -- A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
3.12 Calibration standard (CAL) -- a solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.13 Quality control sample (QCS) -- a sample matrix containing method
analytes or a solution of method analytes in a water miscible solvent
which is used to fortify reagent water or environmental samples. The
QCS is obtained from a source external to the laboratory, and is used
to check laboratory performance with externally prepared test
materials.
INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware and other sample processing apparatus that lead
to discrete artifacts or elevated baselines in gas chromatograms.
All reagents and apparatus must be routinely demonstrated to be free
from interferences under the conditions of the analysis by running
laboratory reagent blanks as described in Sect. 10.2.
4.1.1 Glassware must be scrupulously cleaned. (2) Clean all glass-
ware as soon as possible after use by thoroughly rinsing with
the last solvent used in it. Follow by washing with hot water
and detergent and thorough rinsing with tap and reagent water.
Drain dry, and heat in an oven or muffle furnace at 400°C for
1 hour. Do not heat volumetric ware. Thermally stable
materials might not be eliminated by this treatment. Thorough
147
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rinsing with acetone may be substituted for the heating.
After drying and cooling, seal and store glassware in a cic«in
environment to prevent any accumulation of dust or other
contaminants. Store inverted or capped with aluminum foil.
4.1.2 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. WARNING:
When a solvent is purified, stabilizers added by the
manufacturer may be removed thus potentially making the
solvent hazardous. Also, when a solvent is purified,
preservatives added by the manufacturer are removed thus
potentially reducing the shelf-life.
4.2 Interfering contamination may occur when a sample containing low
concentrations of analytes is analyzed immediately following a sample
containing relatively high concentrations of analytes.
Between-sample rinsing of the sample syringe and associated equipment
with MTBE can minimize sample cross contamination. After analysis of
a sample containing high concentrations of analytes, one or more
injections of MTBE should be made to ensure that accurate values are
obtained for the next sample.
4.3 Matrix interferences may be caused by contaminants that are
coextracted from the sample. Also, note that all the analytes listed
in the scope and application section are not resolved from each other
on any one column, i.e., one analyte of interest may be an
interferant for another analyte of interest. The extent of matrix
interferences will vary considerably from source to source, depending
upon the water sampled. Further processing of sample extracts may be
necessary. Positive identifications should be confirmed (Sect.
11.5).
4.4 It is important that samples and working standards be contained in
the same solvent. The solvent for working standards must be the same
as the final solvent used in sample preparation. If this is not the
case, chromatographic comparability of standards to sample may be
affected.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound must
be treated as a potential health hazard. Accordingly, exposure to
these chemicals must be reduced to the lowest possible level. 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 and have been identified (3-5) for the information of the
analyst.
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5.2 WARNING: When a solvent is purified, stabilizers added by the
manufacturer may be removed thus potentially making the solvent
hazardous.
6. APPARATUS AND EQUIPMENT (All specifications are suggested. Catalog
numbers are included for illustration only.)
6.1 Sample bottle -- Borosilicate, 1-L volume with graduations (Wheaton
Media/Lab bottle 219820 or equivalent), fitted with screw caps lined
with TFE-fluorocarbon. Protect samples from light.The container must
be washed and dried as described in Sect. 4.1.1 before use to
minimize contamination. Cap liners are cut to fit from sheets
(Pierce Catalog No. 012736 or equivalent) and extracted with methanol
overnight prior to use.
6.2 GLASSWARE
6.2.1 Separatory funnel -- 2000-mL, with TFE-fluorocarbon stopcock,
ground glass or TFE-fluorocarbon stopper.
6.2.2 Tumbler bottle -- 1.7-L (Wheaton Roller Culture Vessel or
equivalent), with TFE-fluorocarbon lined screw cap. Cap
liners are cut to fit from sheets (Pierce Catalog No. 012736)
and extracted with methanol overnight prior to use.
6.2.3 Flask, Erlenmeyer -- 500-mL.
6.2.4 Concentrator tube, Kuderna-Danish (K-D) -- 10- or 25-mL,
graduated (Kontes K-570050-2525 or K-570050-1025 or
equivalent). Calibration must be checked at the volumes
employed in the test. Ground glass stoppers are used to
prevent evaporation of extracts.
6.2.5 Evaporative flask, K-D -- 500-mL (Kontes K-570001-0500 or
equivalent). Attach to concentrator tube with springs.
6.2.6 Snyder column, K-D -- three-ball macro (Kontes K-503000-0121
or equivalent).
6.2.7 Snyder column, K-D -- Two-ball micro (Kontes K-569001-0219 or
equivalent).
6.2.8 Vials -- glass, 5- to 10-mL capacity with TFE-fluorocarbon
lined screw cap.
6.3 Separatory funnel shaker (Optional) -- Capable of holding 2-L separa-
tory funnels and shaking them with rocking motion to achieve thorough
mixing of separatory funnel contents (available from Eberbach Co. in
Ann Arbor, MI or other suppliers).
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6.4 Tumbler -- Capable of holding tumbler bottles and tumbling them
end-over-end at 30 turns/min (Associated Design and Mfg. Co.,
Alexandria, VA. or other suppliers).
6.5 Boiling stones -- Carborundum, #12 granules (Arthur H. Thomas Co.
#1590-033 or equivalent). Heat at 400SC for 30 min prior to use.
Cool and store in desiccator.
6.6 Water bath -- Heated, capable of temperature control (± 2°C). The
bath should be used in a hood.
6.7 Balance -- Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.8 GAS CHROMATOGRAPH -- Analytical system complete with temperature
programmable GC suitable for use with capillary columns and all
required accessories including syringes, analytical columns, gases,
detector and stripchart recorder. A data system is recommended for
measuring peak areas. Table 1 lists retention times observed for
method analytes using the columns and analytical conditions described
below.
6.8.1 Column 1 (Primary column) -- 30 m long x 0.25 mm I.D. DB-5
bonded fused silica column, 0.25 urn film thickness (J&W
Scientific) or equivalent. Helium carrier gas flow is
established at 30 cm/sec linear velocity and oven temperature
is programmed from 60°C to 300eC at 4°C/min. Data presented
in this method were obtained using this column. The injection
volume was 2 til in splitless mode with a 45 s delay. The
injector temperature was 250°C and the detector temperature
was 300°C. Alternative columns may be used in accordance with
the provisions described in Sect. 10.4.
6.8.2 Column 2 (Confirmation column) -- 30 m long x 0.25 mm
I.D.DB-1701 bonded fused silica column, 0.25 jtm film thickness
(J&W Scientific) or equivalent. Helium carrier gas flow is
established at 30 cm/sec linear velocity and oven temperature
is programmed from 60C to 300°C at 4°C/min.
6.8.3 Detector -- Nitrogen-phosphorus (NPD). A NPD was used to
generate the validation data presented in this method.
Alternative detectors, including a mass spectrometer, may be
used in accordance with the provisions described in Sect.
10.4.
REAGENTS AND CONSUMABLE MATERIALS - - WARNING: When a solvent is
purified, stabilizers added by the manufacturer are removed thus
potentially making the solvent hazardous. Also, when a solvent is
purified, preservatives added by the manufacturer are removed thus
potentially reducing the shelf-life.
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7.1 Acetone, methylene chloride, methyl tert. -butyl ether (MTBE) --
Distilled-in-glass quality or equivalent.
7.2 Phosphate buffer, pH 7 -- Prepare by mixing 29.6 ml 0.1 N HC1 and 50
ml 0.1 M dipotassium phosphate.
7.3 Sodium chloride (NaCl), crystal, ACS grade -- Heat treat in a shallow
tray at 450*C for a minimum of 4 hours to remove interfering organic
substances.
7.4 Sodium sulfate, granular, anhydrous, ACS grade -- Heat treat in a
shallow tray at 450"C for a minimum of 4 hours to remove interfering
organic substances.
7.5 Sodium thiosulfate, granular, anhydrous, ACS grade.
7.6 Triphenyl phosphate (TPP) -- 98% purity, for use as internal standard
(available from Aldrich Chemical Co.).
7.7 l,3-Dimethyl-2-nitrobenzene -- 98% purity, for use as surrogate
standard (available from Aldrich Chemical Co.).
7.8 Mercuric Chloride -- ACS grade (Aldrich Chemical Co.) - for use as a
bactericide. If any other bactericide can be shown to work as well
as mercuric chloride, it may be used instead.
7.9 Reagent water -- Reagent water is defined as a water that is
reasonably free of contamination that would prevent the determination
of any analyte of interest. Reagent water used to generate the
validation data in this method was distilled water obtained from the
Magnetic Springs Water Co., Columbus, Ohio.
7.10 STOCK STANDARD SOLUTIONS (1.00 fig/fii) -- Stock standard solutions may
be purchased as certified solutions or prepared from pure standard
materials using the following procedure:
7.10.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material in MTBE and dilute to volume in a 10-mL volumetric
flask. The stock solution for simazine should be prepared in
methanol . Larger volumes may be used at the convenience of the
analyst. If compound purity is certified at 96% 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.
7.10.2 Transfer the stock standard solutions into
TFE-fluorocarbon-sealed screw cap amber vials. Store at room
temperature and protect from light.
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7.10.3 Stock standard solutions should be replaced after two months
or sooner if comparison with laboratory fortified blanks, or
QC samples indicate a problem.
7.11 INTERNAL STANDARD SOLUTION -- Prepare the internal standard solution
by accurately weighing approximately 0.0500 g of pure TPP. Dissolve
the TPP in MTBE and dilute to volume in a 100-ml volumetric flask.
Transfer the internal standard solution to a TFE-fluorocarbon-sealed
screw cap bottle and store at room temperature. Addition of 50 /iL of
the internal standard solution to 5 ml of sample extract results in a
final TPP concentration of 5.0 /ig/mL. Solution should be replaced
when ongoing QC (Sect. 10) indicates a problem. Note that TPP has
been shown to be an effective internal standard for the method
analytes (1), but other compounds may be used if the quality control
requirements in Sect. 10 are met.
7.12 SURROGATE STANDARD SOLUTION -- Prepare the surrogate standard
solution by accurately weighing approximately 0.0250 g of pure
l,3-dimethyl-2-nitrobenzene. Dissolve the 1,3-dimethyl-
2-nitrobenzene in MTBE and dilute to volume in a 100-mL volumetric
flask. Transfer the surrogate standard solution to a TFE-fluoro-
carbon-sealed screw cap bottle and store at room temperature.
Addition of 50 /iL of the surrogate standard solution to a 1-L sample
prior to extraction results in a l,3-dimethyl-2-nitrobenzene
concentration in the sample of 12.5 /jg/L. Solution should be
replaced when ongoing QC (Sect. 10) indicates a problem. Note that
l,3-dimethyl-2-nitrobenzene has been shown to be an effective
surrogate standard for the method analytes (1), but other compounds
may be used if the quality control requirements in Sect. 10 are met.
7.13 LABORATORY PERFORMANCE CHECK SOLUTION -- Prepare the laboratory
performance check solution by adding 5 fil of the vernolate stock
solution, 0.5 mL of the bromacil stock solution, 30 pL of the
prometon stock solution, 15 0L of the atrazine stock solution, 1.0 mL
of the surrogate solution, and 500 pL of the internal standard
solution to a 100-mL volumetric flask. Dilute to volume with MTBE
and thoroughly mix the solution. Transfer to a TFE-fluorocarbon-'
sealed screw cap bottle and store at room temperature. Solution
should be replaced when ongoing QC (Section 10) indicates a problem.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices (6) should be followed; however, the bottle must
not be prerinsed with sample before collection.
8.2 SAMPLE PRESERVATION AND STORAGE
8.2.1 Add mercuric chloride (See 7.8) to the sample bottle in
amounts to produce a concentration of 10 mg/L. Add 1 mL of a
solution containing 10 mg/mL of mercuric chloride in reagent
water to the sample bottle at the sampling site or in the
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laboratory before shipping to the sampling site. A major dis-
advantage of mercuric chloride is that it is a highly toxic
chemical; mercuric chloride must be handled with caution, and
samples containing mercuric chloride must be disposed of
properly.
8.2.2 If residual chlorine is present, add 80 mg of sodium
thiosulfate per liter of sample to the sample bottle prior to
collecting the sample.
8.2.3 After the sample is collected in a bottle containing preserv-
ative^), seal the bottle and shake vigorously for 1 min.
8.2.4 The samples must be iced or refrigerated at 4eC away from
light from the time of collection until extraction. Preserv-
ation study results indicated that most method analytes
present in samples were stable for 14 days when stored under
these conditions. (1). The analytes disulfoton sulfoxide,
diazinon, pronamide, and terbufos exhibited significant
aqueous instability, and samples to be analyzed for these
compounds must be extracted immediately. The analytes
carboxin, EPTC, fluridone, metolachlor, napropamide,
tebuthiuron, and terbacil exhibited recoveries of less than
60% after 14 days. Analyte stability may be affected by the
matrix; therefore, the analyst should verify that the
preservation technique is applicable to the samples under
study.
8.3 Extract Storage -- Extracts should be stored at 4"C away from light.
Preservation study results indicate that most analytes are stable for
28 days; however, a 14-day maximum extract storage time is
recommended. The analyst should verify appropriate extract holding
times applicable to the samples under study.
9. CALIBRATION
9.1 Establish GC operating parameters equivalent to those indicated in
Sect. 6.8. The GC system may be calibrated using either the internal
standard technique (Sect. 9.2) or the external standard technique
(Sect. 9.3). Be aware that NPDs may exhibit instability (i.e., fail
to hold calibration curves over time). The analyst may, when
analyzing samples for target analytes which are very rarely found,
prefer to analyze on a daily basis a low level (e.g. 5 to 10 times
detection limit or 1/2 times the regulatory limit, whichever is
less), sample (containing all analytes of interest) and require some
minimum sensitivity (e.g. 1/2 full scale deflection) to show that if
the analyte were present it would be detected. The analyst may then
quantitate using single point calibration (Sect. 9.2.5 or 9.3.4).
NOTE: Calibration standard solutions must be prepared such that no
unresolved analytes are mixed together.
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9.2 INTERNAL STANDARD CALIBRATION PROCEDURE -- To use this approach, U
analyst must select one or more internal standards compatible in
analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is
not affected by method or matrix interferences. TPP has been
identified as a suitable internal standard.
9.2.1 Prepare calibration standards at a minimum of three (recommend
five) concentration levels for each analyte of interest by
adding volumes of one or more stock standards to a volumetric
flask.If Merphos is to be determined, calibrate with DEF
(S,S,S-tributylphosphoro-trithioate). To each calibration
standard, add a known constant amount of one or more of the
internal standards, and dilute to volume with MTBE. The
lowest standard should represent analyte concentrations near,
but above, their respective EDLs. The remaining standards
should bracket the analyte concentrations expected in the
sample extracts, or should define the working range of the
detector.
9.2.2 Analyze each calibration standard according to the procedure
described in Sect. 11.4. Tabulate response (peak height or
area) against concentration for each compound and internal
standard. Calculate the response factor (RF) for each analyte
and surrogate using Equation 1.
RF = (AsHcis) Equation 1
(Ais)(Cs)
where :
As = Response for the analyte.
Ais = Response for the internal standard.
Cis = Concentration of the internal standard /ig/L.
Cs = Concentration of the analyte to be measured /ig/L.
9.2.3 If the RF value over the working range is constant (20% RSD or
less) the average RF can be used for calculations.
Alternatively, the results can be used to plot a calibration
curve of response ratios (As/Ais) vs. Cs.
9.2.4 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any analyte varies from the
predicted response by more than ±20%, the test must be
repeated using a fresh calibration standard. If the
repetition also fails, a new calibration curve must be
generated for that analyte using freshly prepared standards.
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9.2.5 Single point calibration is a viable alternative to a
calibration curve. Prepare single point standards from the
secondary dilution standards in MTBE. The single point
standard should be prepared at a concentration that produces a
response that deviates from the sample extract response by no
more than 20%.
9.2.6 Verify calibration standards periodically, recommend at least
quarterly, by analyzing a standard prepared from reference
material obtained from an independent source. Results from
these analyses must be within the limits used to routinely
check calibration.
9.3 EXTERNAL STANDARD CALIBRATION PROCEDURE
9.3.1 Prepare calibration standards at a minimum of three (recommend
five) concentration levels for each analyte of interest and
surrogate compound by adding volumes of one or more stock
standards to a volumetric flask. If Merphos is to be
determined, calibrate with DEF (S,S,S-tributylphosphoro-
trithioate). Dilute to volume with MTBE. The lowest standard
should represent analyte concentrations near, but above, their
respective EDLs. The remaining standards should bracket the
analyte concentrations expected in the sample extracts, or
should define the working range of the detector.
9.3.2 Starting with the standard of lowest concentration, analyze
each calibration standard according to Sect. 11.4 and tabulate
response (peak height or area) versus the concentration in the
standard. The results can be used to prepare a calibration
curve for each compound. Alternatively, if the ratio of
response to concentration (calibration factor) is a constant
over the working range (20% RSD or less), linearity through
the origin can be assumed and the average ratio or calibration
factor can be used in place of a calibration curve.
9.3.3 The working calibration curve or calibration factor must be
verified on each working day by the measurement of a minimum
of two calibration check standards, one at the beginning and
one at the end of the analysis day. These check standards
should be at two different concentration levels to verify the
calibration curve. For extended periods of analysis (greater
than 8 hrs.), it is strongly recommended that check standards
be interspersed with samples at regular intervals during the
course of the analyses. If the response for any analyte
varies from the predicted response by more than ±20%, the test
must be repeated using a fresh calibration standard. If the
results still do not agree, generate a new calibration curve.
9.3.4 Single point calibration is a viable alternative to a
calibration curve. Prepare single point standards from the
secondary dilution standards in MTBE. The single point
155
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standard should be prepared at a concentration that produce
response that deviates from the sample extract response by no
more than 20%.
9.3.5 Verify calibration standards periodically, recommend at least
quarterly, by analyzing a standard prepared from reference
material obtained from an independent source. Results from
these analyses must be within the limits used to routinely
check calibration.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration
of laboratory capability, determination of surrogate compound
recoveries in each sample and blank, monitoring internal standard
peak area or height In each sample and blank (when internal standard
calibration procedures are being employed), analysis of laboratory
reagent blanks, laboratory fortified samples, laboratory fortified
blanks, and QC samples.
10.2 Laboratory Reagent Blanks. Before processing any samples, the
analyst must demonstrate that all glassware and reagent Interferences
are under control. Each time a set of samples is extracted or
reagents are changed, a LRB must be analyzed. If within the
retention time window of any analyte of interest the LRB produces a
peak that would prevent the determination of that analyte, determine
the source of contamination and eliminate the interference before
processing samples.
10.3 Initial Demonstration of Capability.
10.3.1 Select a representative fortified concentration (about 10
times EDL or at the regulatory Maximum Contaminant Level,
whichever is lower) for each analyte. Prepare a sample
concentrate (in methanol) containing each analyte at 1000
times selected concentration. With a syringe, add 1 mL of the
concentrate to each of at least four 1-L aliquots of reagent
water, and analyze each aliquot according to procedures
beginning in Sect. 11.
10.3.2 For each analyte the recovery value for all four of these
samples must fall in the range of R ± 30% (or within R ± 3Sp
if broader) using the values for R and SR for reagent water in
Table 2. For those compounds that meet the acceptance
criteria, performance is considered acceptable and sample
analysis may begin. For those compounds that fail these
criteria, this procedure must be repeated using four fresh
samples until satisfactory performance has been demonstrated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples via a
new, unfamiliar method prior to obtaining some experience wi
156
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It. It Is expected that as laboratory personnel gain
experience with this method the quality of data will improve
beyond those required here.
10.4 The analyst is permitted to modify GC columns, GC detectors, GC
conditions, continuous extraction techniques, concentration
techniques (i.e. evaporation techniques), internal standards or
surrogate compounds. Each time such method modifications are made,
the analyst must repeat the procedures in Sect. 10.3.
10.5 Assessing Surrogate Recovery
10.5.1 When surrogate recovery from a sample or method blank is <70%
or >130%, check (1) calculations to locate possible errors,
(2) fortifying solutions for degradation, (3) contamination,
and (4) instrument performance. If those steps do not reveal
the cause of the problem, reanalyze the extract.
10.5.2 If a blank extract reanalysis fails the 70-130% recovery
criterion, the problem must be identified and corrected before
continuing.
10.5.3 If sample extract reanalysis meets the surrogate recovery
criterion, report only data for the reanalyzed extract. If
sample extract reanalysis continues to fail the recovery
criterion, report all data for that sample as suspect.
10.6 Assessing the Internal Standard
10.6.1 When using the internal standard calibration procedure, the
analyst is expected to monitor the IS response (peak area or
peak height) of all samples during each analysis day. The IS
response for any sample chromatogram should not deviate from
the daily calibration check standard's IS response by more
than 30%.
10.6.2 If >30% deviation occurs with an individual extract, optimize
instrument performance and inject a second aliquot of that
extract.
10.6.2.1 If the reinjected aliquot produces an acceptable
internal standard response report results for that
aliquot.
10.6.2.2 If a deviation of greater than 30% is obtained for
the reinjected extract, analysis of the sample
should be repeated beginning with Sect. 11, provided
the sample is still available. Otherwise, report
results obtained from the re-injected extract, but
annotate as suspect.
157
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10.6.3 If consecutive samples fall the IS response acceptance
criterion, immediately analyze a calibration check standard.
10.6.3.1 If the check standard provides a response factor (RF)
within 20% of the predicted value, then follow
procedures itemized in Sect. 10.6.2 for each sample
failing the IS response criterion.
10.6.3.2 If the check standard provides a response factor which
deviates more than 20% of the predicted value, then
the analyst must recalibrate, as specified in Sect. 9.
10.7 Assessing Laboratory Performance - Laboratory Fortified Blank
10.7.1 The laboratory must analyze at least one laboratory fortified
blank (LFB) sample with every twenty samples or one per sample
set (all samples extracted within a 24-h period) whichever is
greater. The fortified concentration of each analyte in the
LFB should be 10 times EDL or the MCL, whichever is less.
Calculate accuracy as percent recovery (Xj). If the recovery
of any analyte falls outside the control limits (see Sect.
10.7.2), that analyte is judged out of control, and the source
of the problem should be identified and resolved before
continuing analyses.
10.7.2 Until sufficient data become available from within their own
laboratory, usually a minimum of results from 20 to 30
analyses, the laboratory should assess laboratory performance
against the control limits in Sect. 10.3.2 that are derived
from the data in Table 2. When sufficient internal
performance data becomes available, develop control limits
from the mean percent recovery (X) and standard deviation (S)
of the percent recovery. These data are used to establish
upper and lower control limits as follows:
UPPER CONTROL LIMIT = X + 3S
LOWER CONTROL LIMIT = X - 3S
After each five to ten new recovery measurements, new control
limits should be calculated using only the most recent 20-30
data points. These calculated control limits should never
exceed those established in Sect. 10.3.2.
10.7.3 It is recommended that the laboratory periodically determine
and document its detection limit capabilities for analytes of
interest.
10.7.4 At least quarterly, analyze a QC sample from an outside
source.
158
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10.7.5 Laboratories are encouraged to participate in external
performance evaluation studies such as the laboratory
certification programs offered by many states or the studies
conducted by USEPA. Performance evaluation studies serve as
independent checks on the analyst's performance.
10.8 Assessing Analyte Recovery - Laboratory Fortified Sample Matrix
10.8.1 The laboratory must add a known concentration to a minimum of
5% of the routine samples or one sample concentration per set,
whichever is greater. The fortified concentration should not
be less then the background concentration of the sample
selected for fortification. Ideally, the concentration should
be the same as that used for the laboratory fortified blank
(Sect. 10.7). Over time, samples from all routine sample
sources should be fortified.
10.8.2 Calculate the percent recovery, P of the concentration for each
analyte, after correcting the analytical result, X, from the
fortified sample for the background concentration, b, measured
in the unfortified sample, i.e.,:
P = 100 (X - b) / fortifying concentration,
and compare these values to control limits appropriate for
reagent water data collected in the same fashion. If the
analyzed unfortified sample is found to contain NO background
concentrations, and the added concentrations are those
specified in Sect. 10.7, then the appropriate control limits
would be the acceptance limits in Sect. 10.7. If, on the other
hand, the analyzed unfortified sample is found to contain
background concentration, b, estimate the standard deviation at
the background concentration, 55, using regressions or
comparable background data and, similarly, estimate the mean,
Xa and standard deviation, sa, of analytical results at the
total concentration after fortifying. Then the appropriate
percentage control limits would be P ± 3sp , where:
P = 100 X / (b + fortifying concentration)
2 2 1/2
and Sp = 100 (s + Su ) /fortifying concentration
For example, if the background concentration for Analyte A was
found to be 1 jig/L and the added amount was also 1 0g/L, and
upon analysis the laboratory fortified sample measured 1.6 /i/L,
then the calculated P for this sample would be (1.6 ttg/L minus
1.0 tig/L)/l /zg/L or 60%. This calculated P is compared to
control limits derived from prior reagent water data. Assume
it is known that analysis of an interference free sample at 1
ttg/L yields an s of 0.12 jtg/L and similar analysis at 2.0 itg/L
yields X and s of 2.01 /zg/L and 0.20 jug/L, respectively. The
appropriate limits to judge the reasonableness of the percent
159
-------
recovery, 60%, obtained on the fortified matrix sample is
computed as follows:
[100 (2.01 jtg/L) / 2-0 «/L]
o
±3(100) [(0.12 Mg/L)2 + (0.20 /ig/L)2] /1.0/ig/L
100.5% ± 300 (0.233) =
100.5% ± 70% or 30% to 170% recovery of the added analyte.
10.9 ASSESSING INSTRUMENT SYSTEM - LABORATORY PERFORMANCE CHECK (LPC) -
Instrument performance should be monitored on a daily basis by
analysis of the LPC sample. The LPC sample contains compounds
designed to indicate appropriate instrument sensitivity, column
performance (primary column) and chromatographic performance. LPC
sample components and performance criteria are listed in Table 3.
Inability to demonstrate acceptable instrument performance indicates
the need for reevaluation of the instrument system. The sensitivity
requirements are set based on the EOLs published in this method. If
laboratory EDLs differ from those listed in this method,
concentrations of the instrument QC standard compounds must be
adjusted to be compatible with the laboratory EDLs.
10.10 The laboratory may adopt additional quality control practices for
use with this method. The specific practices that are most
productive depend upon the needs of the laboratory and the nature of
the samples. For example, field or laboratory duplicates may be
analyzed to assess the precision of the environmental measurements
or field reagent blanks may be used to assess contamination of
samples under site conditions, transportation and storage.
11. PROCEDURE
11.1 EXTRACTION (MANUAL METHOD)
11.1.1 Mark the water meniscus on the side of the sample bottle for
later determination of sample volume (Sect. 11.1.6). Add
preservative to blanks and QC check standards. Fortify the
sample with 50 jiL of the surrogate standard solution. Pour
the entire sample into a 2-L separatory funnel.
11.1.2 Adjust the sample to pH 7 by adding 50 mL of phosphate buffer.
11.1.3 Add 100 g NaCl to the sample, seal, and shake to dissolve
salt.
11.1.4 Add 60 mL methylene chloride to the sample bottle, seal, and
shake 30 s to rinse the inner walls. Transfer the solvent to
the separatory funnel and extract the sample by vigorously
shaking the funnel for 2 min with periodic venting to release
excess pressure. Allow the organic layer to separate from t'
160
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water phase for a minimum of 10 min. If the emulsion
interface between layers is more than one third the volume of
the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring,
filtration of the emulsion through glass wool,
centrifugation, or other physical methods. Collect the
methylene chloride extract in a 500-mL Erlenmeyer flask.
11.1.5 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time,
combining the extracts in the Erlenmeyer flask. Perform a
third extraction in the same manner.
11.1.6 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the water to a 1000-mL
graduated cylinder. Record the sample volume to the nearest
5 ml.
11.2 EXTRACTION (AUTOMATED METHOD) -- Data presented in this method were
generated using the automated extraction procedure with the
mechanical tumbler.
11.2.1 Mark the water meniscus on the side of the sample bottle for
later determination of sample volume (Sect. 11.2.6). Add
preservative to blanks and QC check standards. Fortify the
sample with 50 /tL of the surrogate standard solution. If the
mechanical separatory funnel shaker is used, pour the entire
sample into a 2-L separatory funnel. If the mechanical
tumbler is used, pour the entire sample into a tumbler bottle.
11.2.2 Adjust the sample to pH 7 by adding 50 ml of phosphate buffer.
11.2.3 Add 100 g NaCl to the sample, seal, and shake to dissolve
salt.
11.2.4 Add 300 ml methylene chloride to the sample bottle, seal, and
shake 30 s to rinse the inner walls. Transfer the solvent to
the sample contained in the separatory funnel or tumbler
bottle, seal, and shake for 10 s, venting periodically.
Repeat shaking and venting until pressure release is not
observed. Reseal and place sample container in appropriate
mechanical mixing device (separatory funnel shaker or
tumbler). Shake or tumble the sample for 1 hour. Complete
mixing of the organic and aqueous phases should be observed
within about 2 min after starting the mixing device.
11.2.5 Remove the sample container from the mixing device. If the
tumbler is used, pour contents of tumbler bottle into a 2-L
separatory funnel. Allow the organic layer to separate from
the water phase for a minimum of 10 min. If the emulsion
interface between layers is more than one third the volume of
161
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the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring,
filtration through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a
500-mL Erlenmeyer flask.
11.2.6 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the water to a 1000-mL
graduated cylinder. Record the sample volume to the nearest
5 ml.
11.3 EXTRACT CONCENTRATION
11.3.1 Assemble a K-D concentrator by attaching a 25-mL concentrator
tube to a 500-mL evaporative flask. Other concentration
devices or techniques may be used in place of the K-D if the
requirements of Sect. 10.3 are met.
11.3.2 Dry the extract by pouring it through a solvent-rinsed drying
column containing about 10 cm of anhydrous sodium sulfate.
Collect the extract in the K-D concentrator, and rinse the
column with 20-30 ml methylene chloride. Alternatively, add
about 5 g anhydrous sodium sulfate to the extract in the
Erlenmeyer flask; swirl flask to dry extract and allow to sit
for 15 min. Decant the methylene chloride extract into the K-D
concentrator. Rinse the remaining sodium sulfate with two
25-mL portions of methylene chloride and decant the rinses
into the K-D concentrator.
11.3.3 Add 1 to 2 clean boiling stones to the evaporative flask and
attach a macro 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, 65 to 70°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 15 to 20 min. 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 2 mL, remove the K-D apparatus and allow it to
drain and cool for at least 10 min.
11.3.4 Remove the Snyder column and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 mL of MTBE. Add
5-10 mL of MTBE and a fresh boiling stone. Attach a
micro-Snyder column to the concentrator tube and prewet the
column by adding about 0.5 mL of MTBE to the top. Place the
micrj K-D apparatus on the water bath so that the concentrator
ti'l-a is partially immersed in the hot water. Adjust the
• irtical position of the apparatus and the water temperature
162
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as required to complete concentration in 5 to 10 min. When
the apparent volume of liquid reaches 2 ml, remove the micro
K-D from the bath and allow it to drain and cool. Add 5-10 ml
MTBE to the micro K-D and reconcentrate to 2 ml. Remove the
micro K-D from the bath and allow it to drain and cool.
Remove the micro Snyder column, and rinse the walls of the
concentrator tube while adjusting the volume to 5.0 ml with
MTBE. NOTE: If methylene chloride is not completely removed
from the final extract, it may cause detector problems.
11.3.5 Transfer extract to an appropriate- sized TFE-fluorocarbon-
sealed screw-cap vial and store, refrigerated at 4"C, until
analysis by GC-NPD.
11.4 GAS CHROMATOGRAPHY
11.4.1 Sect. 6.8 summarizes the recommended operating conditions for
the gas chromatograph. Included in Table 1 are retention times
observed using this method. Other GC columns, chromatographic
conditions, or detectors may be used if the requirements of
Sect. 10.3 are met.
11.4.2 Calibrate the system daily as described in Sect. 9. The
standards and extracts must be in MTBE.
11.4.3 If the internal standard calibration procedure is used, add
50 /iL of the internal standard solution to the sample
extract, seal, and shake to distribute the internal standard.
11.4.4 Inject 2 /iL of the sample extract. Record the resulting peak
size in area units.
11.4.5 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
11.5 IDENTIFICATION OF ANALYTES
11.5.1 Identify a sample component by comparison of its retention
time to the retention time of a reference chromatogram. If
the retention time of an unknown compound corresponds, within
limits, to the retention time of a standard compound, then
identification is considered positive.
11.5.2 The width of the retention time window used to make
identifications should be based upon 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.
163
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11.5.3 Identification requires expert judgement when sample
components are not resolved chromatographically. When peaks
obviously represent more than one sample component (i.e.,
broadened peak with shoulder(s) or valley between two or more
maxima), or any time doubt exists over the identification of a
peak on a chromatogram, appropriate alternative techniques to
help confirm peak identification, need be employed. For
example, more positive identification may be made by the use
of an alternative detector which operates on a
chemical/physical principle different from that originally
used, e.g., mass spectrometry, or the use of a second
chromatography column. A suggested alternative column is
described in Sect. 6.8.
12. CALCULATIONS
12.1 Calculate analyte concentrations in the sample from the response for
the analyte using the calibration procedure described in Sect. 9.
12.2 If the internal standard calibration procedure is used, calculate the
concentration (C) in the sample using the response factor (RF)
determined in Sect. 9.2 and Equation 2, or determine sample
concentration from the calibration curve.
C (/zg/L) = (AS)(!s) Equation 2
(A1s)(RF)(Vo)
where:
As = Response for the parameter to be measured.
Ais = Response for the internal standard.
Is = Amount of internal standard added to each extract
Vo = Volume of water extracted (L).
12.3 If the external standard calibration procedure is used, calculate the
amount of material injected from the peak response using the
calibration curve or calibration factor determined in Sect. 9.3.2.
The concentration (C) in the sample can be calculated from
Equation 3.
= (AHvt) Equation 3
(Vi)(Vs)
164
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where:
A = Amount of material injected (ng).
V^ = Volume of extract injected (/iL).
Vj. = Volume of total extract (nl).
Vs = Volume of water extracted (ml).
13. PRECISION AND ACCURACY
13.1 In a single laboratory, analyte recoveries from reagent water were
determined at five concentration levels. Results were used to
determine analyte EDLs and demonstrate method range.(1) Analytes
were divided into five groups for recovery studies. Analyte EDLs and
analyte recoveries and standard deviation about the percent
recoveries at one concentration are given in Table 2.
13.2 In a single laboratory, analyte recoveries from two standard
synthetic ground waters were determined at one concentration level.
Results were used to demonstrate applicability of the method to
different ground water matrices.(1) Analyte recoveries from the two
synthetic matrices are given in Table 2.
14. REFERENCES
1. National Pesticide Survey Method No. 1: Determination of Nitrogen- and
Phosphorus-Containing Pesticides in Groundwater by Gas Chromatography
with a Nitrogen-Phosphorus Detector.
2. ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82,
"Standard Practice for Preparation of Sample Containers and for
Preservation", American Society for Testing and Materials, Philadel-
phia, PA, 1986.
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, Aug. 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. ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82, "Stan-
dard Practice for Sampling Water," American Society for Testing and
Materials, Philadelphia, PA, 1986.
165
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TABLE 1. RETENTION TINES FOR METHOD ANALYTES
Retention Time3
Analyte Col. 1 Col. 2
l,3-Dimethyl-2-nitrobenzene(surrogate)
Dichlorvos
Disulfoton sulfoxide
EPIC
Butyl ate
Mevinphos
Vernolate
Pebulate
Tebuthiuron
Molinate
Ethoprop
Cycloate
Chlorpropham
Atraton
Simazine
Prometon
Atrazine
Propazine
Terbufos
Pronamide
Diazinon
Disulfoton
Terbacil
Metribuzin
Methyl paraoxon
Simetryn
Alachlor
Ametryn
Prometryn
Terbutryn
Bromacil
Metolachlor
Triademefon
MGK 264 (c)
Diphenamid
Stirofos
Disulfoton sulfone
Butachlor
Fenamiphos
Napropamide
Tricyclazole
Merphos (d)
Carboxin
Norflurazon
Triphenyl phosphate (int. std.)
166
14.48
16.54
19.08
20.07
22.47
22.51
22.94
23.41
25.15
25.66
28.58
28.58
29.09
31.26
31.49
31.58
31.77
32.01
32.57
32.76
33.23
33.42
33.79
35.20
35.58
35.72
35.96
36.00
36.14
36.80
37.22
37.74
38.12
38.73
38.87
41.27
41.31
41.45
41.78
41.83
42.25
42.35
42.77
45.92
47
(b)
15.35
(b)
16.57
18.47
21.92
19.25
19.73
42.77
22.47
26.42
29.67
(b)
29.97
31.32
30
31.23
31.13
(b)
32.63
(b)
30.9
(b)
34.73
34.1
34.55
34.1
34.52
34.23
34.8
40
35.7
37
36.73
37.97
39.65
42.42
39
41
(b)
44.33
39.28
42.05
47.58
45.4
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TABLE 1 (CONTINUED)
Retention Time3
Analyte Col.l Col.2
Hexazinone 46.58 47.8
Fenarimol 51.32 50.02
Fluridone 56.68 59.07
a Columns and analytical conditions are described in Sect. 6.8.1 and 6.8.2,
b Data not available
c MGK 264 gives two peaks; peak identified in this table used for
quantification.
d Merphos is converted to S,S,S-tributylphosphoro-trithioate (DEF) in the
hot GC injection port; DEF is actually detected using these analyses
conditions.
167
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TABLE 2. SINGLE LABORATORY ACCURACY, PRECISION AND ESTIMATED DETECTION C'IMITS
(EDLS) FOR ANALYTES FROM REAGENT HATER AND SYNTHETIC GROUNDWATERS(A)
Analyte
Alachlor
Ametryn
Ametraton
Atrazine
Bromacil
Butachlor
Butyl ate
Carboxin
Chlorpropham
Cycloate
Diazinon
Dichlorvos
Diphenamid
Disulfoton
Disulfoton sulfone
Disulfoton sulfoxide
EPTC
Ethoprop
Fenamiphos
Fenarimol
Fluridone
Hexazinone
Merphos
Methyl paraoxon
Metolachlor
Metribuzin
Mevinphos
MGK 264
Molinate
Napropamide
Norflurazon
Pebulate
Prometon
Prometryn
Pronamide
Propazine
Simazine
Simetryn
Stirofos
Tebuthiuron
Terbacil
Terbufos
Terbutryn
EDLD
W/L
0.38
2
0.6
0.13
2.5
0.38
0.15
0.6
0.5
0.25
0.25
2.5
0.6
0.3
3.8
0.38
0.25
0.19
1
0.38
3.8
0.76
0.25
2.5
0.75
0.15
5
0.5
0.15
0.25
0.5
0.13
0.3
0.19
0.76
0.13
0.075
0.25
0.76
1.3
4.5
0.5
0.25
Cone.
M9/L
3.8
20
6
1.3
25
3.8
1.5
6
5
2.5
2.5
25
6
3
7.5
3.8
2.5
1.9
10
3.8
38
7.6
2.5
25
7.5
1.5
50
5
1.5
2.5
5
1.3
3
1.9
7.6
1.3
0.75
2.5
7.6
13
45
5
2.5
Reagent
Rc
95
91
91
92
91
96
97
102
93
89
115
97
93
89
98
87
85
103
90
99
87
90
96
98
93
101
95
100
98
101
94
94
78
93
91
92
100
99
98
84
97
97
94
Water
SRd
11
10
11
8
9
4
21
4
11
9
7
6
8
10
10
11
9
5
8
5
9
7
8
10
4
5
11
4
18
6
5
9
9
8
10
8
7
5
6
9
6
4
9
Synthetic
Water 1
R SR
82
102
84
89
81
93
36
98
82
97
83
86
88
107
92
88
83
91
87
89
91
86
90
97
92
99
93
91
83
89
101
80
89
91
84
89
86
88
84
85
86
80
91
6
11
7
6
5
15
8
13
7
14
8
6
4
12
5
22
5
7
5
6
11
6
4
8
10
10
6
11
8
5
15
6
5
8
7
6
5
4
6
10
5
6
8
Synthetic
Water 2
R SR
90
96
91
92
88
84
83
87
93
93
84
106
93
95
96
54
86
79
89
89
86
95
92
94
84
86
92
83
89
104
87
98
63
93
92
92
103
103
95
98
102
77
92
8
4
8
5
8
5
8
5
8
3
3
16
5
5
3
19
4
3
2
6
10
9
4
4
4
4
4
6
9
18
4
15
2
4
8
5
14
14
10
13
12
7
4
168
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TABLE 2. (CONTINUED)
EDLD Reagent Water
Analyte
W/L
Cone. Rc SR°
n/l
Synthetic
Water le
R SR
Synthetic
Water 2f
R SR
Triademefon
Tricyclazole
Vernolate
0.65
1
0.13
6.5
10
1.3
93 8
86 7
93 6
94
90
79
5
6
9
95 5
90 11
81 2
a Data corrected for blank and represent the analysis of 7-8 samples using
mechanical tumbling and internal standard calibration.
b EDL = estimated detection limit; defined as either MDL (Appendix B to 40 CFR
Part 136 - Definition and Procedure for the Determination of the Method
Detection Limit - Revision 1.11) or a level of compound in a sample yielding a
peak in the final extract with signal-to-noise ratio of approximately 5,
whichever value is higher. The concentration used in determining the EDL is
not the same as the concentration presented in this table.
c R = average percent recovery.
d S = standard deviation of the percent recovery.
e Corrected for amount found in blank; Absopure Nature Artesian Spring Water
Obtained from the Absopure Water Company in Plymouth, Michigan.
f Corrected for amount found in blank; reagent water fortified with fulvic acid
at the 1 mg/L concentration level. A well-character!zed fulvic acid, available
from the International Humic Substances Society (associated with the United
States Geological Survey in Denver, Colorado), was used.
169
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TABLE 3. LABORATORY PERFORMANCE CHECK SOLUTION
Test
Sensitivity
Chromatographic performance
Column performance
Analyte
Vernolate
Bromacil
Prometon
Atrazine
Cone,
/ig/mL
0.05
5.0
0.30
0.15
Requirements
Detection of
0.80 < PGF <
Resolution >
analyte; S/N > 3
1.20 (a)
0.7 (b)
a PGF - peak Gaussian factor. Calculated using the equation:
1.83 x W(l/2)
PGF
where W(l/2) is the peak width at half height and W(l/10) is the peak width at tenth height.
b Resolution between the two peaks as defined by the equation:
t
R = -.-
W
where t is the difference in elution times between the two peaks and W is the average peak
width, at the baseline, of the two peaks.
-------
METHOD 508. DETERMINATION OF CHLORINATED PESTICIDES IN WATER BY
GAS CHROMATOGRAPHY WITH AN ELECTRON CAPTURE DETECTOR
Revision 3.0
J. J. Llchtenberg, J. E. Longbottom, T. A. Bellar, J. H. Elchelberger,
and R. C. Dressman - EPA 600/4-81-053, Revision 1.0 (1981)
T. Engels (Battelle Columbus Laboratories) - National Pesticide
Survey Method 2, Revision 2.0 (1987)
R. L. Graves - Method 508, Revision 3.0 (1989)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
171
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METHOD 508
DETERMINATION OF CHLORINATED PESTICIDES IN WATER BY
GAS CHROMATOGRAPHY WITH AN ELECTRON CAPTURE DETECTOR
1. SCOPE AND APPLICATION
1.1 This is a gas chromatographlc (GC) method applicable to the
determination of certain chlorinated pesticides in groundwater and
finished drinking water.(1) The following compounds can be determined
using this method:
Compound
Aldrin
Chlordane-alpha
Chiordane-gamma
Chlorneb
Chlorobenzilate(a)
Chlorothalonil
DCPA
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Etridiazole
HCH-alpha
HCH-beta
HCH-delta(a)
HCH-gamma (Lindane)
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Methoxychlor
cis-Permethrin
trans-Permethrin
Propachlor
Trifluralin
Aroclor 1016*
Aroclor 1221*
Aroclor 1232*
Aroclor 1242*
Aroclor 1248*
Aroclor 1254*
Chemical Abstract Service
Registry Number
309-00-2
5103-71-9
5103-74-2
2675-77-6
501-15-6
2921-88-2
1897-45-6
72-54-8
72-55-9
50-29-3
60-57-1
959-98-8
33213-65-9
1031-07-8
72-20-8
7421-93-4
2593-15-9
319-84-6
319-85-7
319-86-8
58-89-9
76-44-8
1024-57-3
118-74-1
72-43-5
52645-53-1
52645-53-1
1918-16-7
1582-09-8
12674-11-2
11104-28-2
11141-16-5
53469-21-9
12672-29-6
11097-69-1
172
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Aroclor 1260* 11096-82-5
Toxaphene* 8001-35-2
Chlordane* 57-74-9
* The extraction conditions of this method are comparable to
USEPA Method 608, which does measure the multicomponent
constituents: commercial polychlorinated biphenyl (PCB)
mixtures (Aroclors), toxaphene, and chlordane. The extract
derived from this procedure may be analyzed for these
constituents by using the GC conditions prescribed in either
Method 608 (packed column) or Method 505 (capillary column).
The columns used in this method may well be adequate, however,
no data were collected for these constituents during methods
development.
(a) These compounds are only qualitatively identified 1n the
National Pesticides Survey (NPS) Program. These compounds are
not quantitated because control over precision has not been
accomplished.
1.2 This method has been validated 1n a single laboratory and estimated
detection limits (EDLs) have been determined for the analytes above
(Sect. 13). Observed detection limits may vary between waters,
depending upon the nature of interferences in the sample matrix and
the specific instrumentation used.
1.3 This method is restricted to use by or under the supervision of
analysts experienced in the use of GC and in the interpretation of gas
chromatograms. Each analyst must demonstrate the ability to generate
acceptable results with this method using the procedure described in
Sect. 10.3.
1.4 Degradation of DDT and Endrin caused by active sites in the injection
port and GC columns may occur. This is not as much a problem with new
capillary columns as with packed columns. However, high boiling
sample residue in capillary columns will create the same problem after
injection of sample extracts.
1.5 Analytes that are not separated chromatographically, i.e., analytes
which have very similar retention times cannot be individually
identified and measured in the same calibration mixture or water
sample unless an alternative technique for identification and
quantitation exist (Sect. 11.5).
1.6 When this method is used to analyze unfamiliar samples for any or all
of the analytes above, analyte identifications must be confirmed by at
least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 A measured volume of sample of approximately 1 L is solvent extracted
with methylene chloride by shaking in a separatory funnel or
173
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mechanical tumbling in a bottle. The methylene chloride extract i
isolated, dried and concentrated to a volume of 5 ml after solvent
substitution with methyl tert-butyl ether (MTBE). Chromatographic
conditions are described which permit the separation and measurement
of the analytes in the extract by capillary column/GC with an electron
capture detector (ECD).
3. DEFINITIONS
3.1 Internal standard -- A pure analyte(s) added to a solution in known
amount(s) and used to measure the relative responses of other method
analytes and surrogates that are components of the same solution. The
internal standard must be an analyte that is not a sample component.
3.2 Surrogate analyte -- A pure analyte(s), which is extremely unlikely to
be found in any sample, and which is added to a sample aliquot in
known amount(s) before extraction and is measured with the same
procedures used to measure other sample components. The purpose of a
surrogate analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates (LD1 and LD2) -- Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample collection,
preservation, or storage procedures.
3.4 Field duplicates (FD1 and FD2) -- Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures. Analyses
of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory
procedures.
3.5 Laboratory reagent blank (LRB) -- An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates that
are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.6 Field reagent blank (FRB) -- Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage, preservation
and all analytical procedures. The purpose of the FRB is to determine
if method analytes or other interferences are present in the field
environment.
3.7 Laboratory performance check solution (LPC) -- A solution of method
analytes, surrogate compounds, and internal standards used to evaluate
the performance of the instrument system with respect to a defined set
of method criteria.
174
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3.8 Laboratory fortified blank (LFB) -- An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.9 Laboratory fortified sample matrix (LFM) -- An aliquot of an
environmental sample to which known quantities of the method analytes
are added in the laboratory. The LFM is analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be determined
in a separate aliquot and the measured values in the LFM corrected for
background concentrations.
3.10 Stock standard solution -- A concentrated solution containing a single
certified standard that is a method analyte, or a concentrated
solution of a single analyte prepared in the laboratory with an
assayed reference compound. Stock standard solutions are used to
prepare primary dilution standards.
3.11 Primary dilution standard solution -- A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
3.12 Calibration standard (CAL) -- a solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are used
to calibrate the instrument response with respect to analyte
concentration.
3.13 Quality control sample (QCS) -- a sample matrix containing method
analytes or a solution of method analytes in a water miscible solvent
which is used to fortify reagent water or environmental samples. The
QCS is obtained from a source external to the laboratory, and is used
to check laboratory performance with externally prepared test
materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware and other sample processing apparatus that lead to
discrete artifacts or elevated baselines in gas chromatograms. All
reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running labora-
tory reagent blanks as described in Sect. 10.2.
4.1.1 Glassware must be scrupulously cleaned (2). Clean all glass-
ware as soon as possible after use by thoroughly rinsing with
the last solvent used in it. Follow by washing with hot water
175
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and detergent and thorough rinsing with tap and reagent wate
Drain dry, and heat in an oven or muffle furnace at 400°C for i
hour. Do not heat volumetric ware. Thermally stable materials
such as PCBs might not be eliminated by this treatment.
Thorough rinsing with acetone may be substituted for the
heating. After drying and cooling, seal and store glassware in
a clean environment to prevent any accumulation of dust or
other contaminants. Store inverted or capped with aluminum
foil.
4.1.2 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. WARNING:
When a solvent is purified, stabilizers added by the
manufacturer are removed thus potentially making the solvent
hazardous. Also, when a solvent is purified, preservatives
added by the manufacturer are removed thus potentially reducing
the shelf-life.
4.2 Interferences by phthalate esters can pose a major problem in pesti-
cide analysis when using the electron capture detector. These
compounds generally appear in the chromatogram as large peaks. Common
flexible plastics contain varying amounts of phthalates that are
easily extracted or leached during laboratory operations. Cross
contamination of clean glassware routinely occurs when plastics are
handled during extraction steps, especially when solvent-wetted
surfaces are handled. Interferences from phthalates can best be
minimized by avoiding the use of plastics in the laboratory. Exhaus-
tive cleanup of reagents and glassware may be required to eliminate
background phthalate contamination.(3,4)
4.3 Interfering contamination may occur when a sample containing low
concentrations of analytes is analyzed immediately following a sample
containing relatively high concentrations of analytes. Between-sample
rinsing of the sample syringe and associated equipment with MTBE can
minimize sample cross contamination. After analysis of a sample
containing high concentrations of analytes, one or more injections of
MTBE should be made to ensure that accurate values are obtained for
the next sample.
4.4 Matrix interferences may be caused by contaminants that are
coextracted from the sample. Also, note that all the analytes listed
in the Scope and Application Section are not resolved from each other
on any one column, i.e., one analyte of interest may be an interferant
for another analyte of interest. The extent of matrix interferences
will vary considerably from source to source, depending upon the water
sampled. Cleanup of sample extracts may be necessary. Positive
identifications should be confirmed (Sect. 11.5).
4.5 It is important that samples and standards be contained in the same
solvent, i.e., the solvent for final working standards must be the
same as the final solvent used in sample preparation. If this is not
176
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the case chromatographic comparability of standards to sample may be
affected.
4.6 WARNING: A dirty injector insert will cause the late eluting
compounds to drop off.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound must
be treated as a potential health hazard. Accordingly, exposure to
these chemicals must be reduced to the lowest possible level. 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 and have been identified (5-7) for the information of the
analyst.
5.2 WARNING: When a solvent is purified stabilizers added by the
manufacturer are removed thus potentially making the solvent
hazardous.
6. APPARATUS AND EQUIPMENT (All specifications are suggested. Catalog numbers
are included for illustration only.)
6.1 SAMPLE BOTTLE -- Borosilicate, 1-L volume with graduations (Wheaton
Media/Lab bottle 219820 or equivalent), fitted with screw caps lined
with TFE-fluorocarbon. Protect samples from light. The container
must be washed and dried as described in Sect. 4.1.1 before use to
minimize contamination. Cap liners are cut to fit from sheets (Pierce
Catalog No. 012736) and extracted with methanol overnight prior to
use.
6.2 GLASSWARE
6.2.1 Separatory funnel -- 2000-mL, with TFE-fluorocarbon stopcock,
ground glass or TFE-fluorocarbon stopper.
6.2.2 Tumbler bottle 1.7-L (Wheaton Roller Culture Vessel or
equivalent), with TFE-fluorocarbon lined screw cap. Cap liners
are cut to fit from sheets (Pierce Catalog No. 012736) and
extracted with methanol overnight prior to use.
6.2.3 Flask, Erlenmeyer -- 500-mL.
6.2.4 Concentrator tube, Kuderna-Danish (K-D) 10- or 25-mL, graduated
(Kontes K-570050-1025 or K-570050-2525 or equivalent).
Calibration must be checked at the volumes employed in the
test. Ground glass stoppers are used to prevent evaporation of
extracts.
177
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6.2.5 Evaporative flask, K-D 500-ml (Kontes K-570001-0500 or
equivalent). Attach to concentrator tube with springs.
6.2.6 Snyder column, K-D three-ball macro (Kontes K-503000-0121 or
equivalent).
6.2.7 Snyder column, K-D two-ball micro (Kontes K-569001-0219 or
equivalent).
6.2.8 Vials -- Glass, 5- to 10-mL capacity with TFE-fluorocarbon
lined screw cap.
6.3 SEPARATOR FUNNEL SHAKER -- Capable of holding 2-L separatory funnels
and shaking them with rocking motion to achieve thorough mixing of
separatory funnel contents (available from Eberbach Co. in Ann Arbor,
MI or other suppliers).
6.4 TUMBLER -- Capable of holding tumbler bottles and tumbling them
end-over-end at 30 turns/min (Associated Design and Mfg. Co.,
Alexandria, VA or other suppliers.).
6.5 BOILING STONES CARBORUNDUM, #12 granules (Arthur H. Thomas Co.
#1590-033 or equivalent). Heat at 400°C for 30 min prior to use.
Cool and store in a desiccator.
6.6 WATER BATH -- Heated, capable of temperature control (± 2°C). The
bath should be used in a hood.
6.7 BALANCE -- Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.8 GAS CHROMATOGRAPH -- Analytical system complete with temperature
programmable GC suitable for use with capillary columns and all
required accessories including syringes, analytical columns, gases,
detector and stripchart recorder. A data system is recommended for
measuring peak areas. Table 1 lists retention times observed for
method analytes using the columns and analytical conditions described
below.
6.8.1 Column 1 (Primary column) -- 30 m long x 0.25 mm I.D. DB-5
bonded fused silica column, 0.25 jun film thickness (J&VI
Scientific). Helium carrier gas flow is established at 30
cm/sec linear velocity and oven temperature is programmed from
60"C to 300'C at 4°C/min. Data presented in this method were
obtained using this column. The injection volume was 2 /*L
splitles's mode with a 45 sec. delay. The injector temperature
was 250*C and the detector temperature was 320°C. Column
performance criteria are presented in Table 3 (See Section
10.9). Alternative columns may be used in accordance with the
provisions described in Sect. 10.4.
178
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6.8.2 Column 2 (Alternative column) -- 30 m long x 0.25 mm
I.D.DB-1701 bonded fused silica column, 0.25 pm film thickness
(J&W Scientific). Helium carrier gas flow is established at
30 cm/sec linear velocity and oven temperature is programmed
from 60'C to 300'C at 4°C/min.
6.8.3 Detector -- Electron capture. This detector has proven
effective in the analysis of spiked reagent and artificial
ground waters. An ECD was used to generate the validation data
presented in this method. Alternative detectors, including a
mass spectrometer, may be used in accordance with the
provisions described in Sect. 10.4.
REAGENTS AND CONSUMABLE MATERIALS - - WARNING: When a solvent is purified,
stabilizers added by the manufacturer are removed thus potentially making
the solvent hazardous. Also, when a solvent is purified, preservatives
added by the manufacturer are removed thus potentially reducing the shelf -
life.
7.1 ACETONE, methylene chloride, MTBE -- Distilled-in-glass quality or
equivalent.
7.2 PHOSPHATE BUFFER, pH7 Prepare by mixing 29.6 mL 0.1 N HC1 and 50 mL
0.1 M dipotassium phosphate.
7.3 SODIUM CHLORIDE, crystal, ACS grade. Heat treat in a shallow tray at
450'C for a minimum of 4 hours to remove interfering organic sub-
stances.
7.4 SODIUM SULFATE, granular, anhydrous, ACS grade. Heat treat in a
shallow tray at 450°C for a minimum of 4 hours to remove interfering
organic substances.
7.5 SODIUM THIOSULFATE, granular, anhydrous, ACS grade.
7.6 PENTACHLORONITROBENZENE (PCNB) 98% purity, for use as internal
standard.
7.7 4,4'-DICHLOROBIPHENYL (DCB) 96% purity, for use as surrogate standard
(available from Chemicals Procurement Inc.).
7.8 MERCURIC CHLORIDE -- ACS grade -- for use as a bactericide. If any
other bactericide can be shown to work as well as mercuric chloride,
it may be used instead.
7.9 REAGENT WATER -- Reagent water is defined as water that is reasonably
free of contamination that would prevent the determination of any
analyte of interest. Reagent water used to generate the validation
data in this method was distilled water obtained from the Magnetic
Springs Water Co., Columbus, Ohio.
179
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7.10 STOCK STANDARD SOLUTIONS (1.00 /igM) -- Stock standard solutions ma.
be purchased as certified solutions or prepared from pure standard
materials using the following procedure:
7.10.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the material
in MTBE and dilute to volume in a 10-mL volumetric flask.
Larger volumes may be used at the convenience of the analyst.
If compound purity is certified at 96% 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.
7.10.2 Transfer the stock standard solutions into TFE-fluoro-
carbon-sealed screw cap amber vials. Store at room temperature
and protect from light.
7.10.3 Stock standard solutions should be replaced after two months or
sooner if comparison with laboratory fortified blanks, or QC
samples Indicate a problem.
7.11 INTERNAL STANDARD SOLUTION -- Prepare an internal standard fortifying
solution by accurately weighing approximately 0.0010 g of pure PCNB.
Dissolve the PCNB in MTBE and dilute to volume in a 10-mL volumetric
flask. Transfer the internal standard solution to a TFE-fluoro-
carbon-sealed screw cap bottle and store at room temperature.
Addition of 5 0L of the internal standard fortifying solution to 5 mL
of sample extract results in a final internal standard concentration
of 0.1 /jg/mL. Solution should be replaced when ongoing QC (Sect. 10)
indicates a problem. Note that PCNB has been shown to be an effective
internal standard for the method analytes (1), but other compounds may
be used if the quality control requirements in Section 10 are met.
7.12 SURROGATE STANDARD SOLUTION -- Prepare a surrogate standard
fortifying solution by accurately weighing approximately 0.0050 g of
pure DCB. Dissolve the DCB in MTBE and dilute to volume in a 10-mL
volumetric flask. Transfer the surrogate standard fortifying solution
to a TFE-fluorocarbon-sealed screw cap bottle and store at room
temperature. Addition of 50 fit of the surrogate standard fortifying
solution to a 1-L sample prior to extraction results in a surrogate
standard concentration in the sample of 25 pg/L and, assuming
quantitative recovery of OCB, a surrogate standard concentration in
the final extract of 5.0 /zg/mL. Solution should be replaced when
ongoing QC (Sect. 10) indicates a problem. Note DCB has been shown to
be an effective surrogate standard for the method analytes (1), but
other compounds may be used if the quality control requirements in
Section 10 are met.
7.13 LABORATORY PERFORMANCE CHECK SOLUTION -- Prepare by accurately
weighing 0.0010 g each of chlorothalonil, chlorpyrifos, DCPA, and HCH-
delta. Dissolve each analyte in MTBE and dilute to volume in
180
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individual 10-mL volumetric flasks. Combine 2 /zL of the chloropyrifos
stock solution, 50 /zL of the DCPA stock solution, 50 pL of the
chlorothalonil stock solution, and 40 pL of the HCH-delta stock
solution to a 100-mL volumetric flask and dilute to volume with MTBE.
Transfer to a TFE-fluorcarbon-sealed screw cap bottle and store at
room temperature. Solution should be replaced when ongoing QC
(Section 10) indicates a problem.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices (8) should be followed; however, the bottle must
not be prerinsed with sample before collection.
8.2 SAMPLE PRESERVATION
8.2.1 Add mercuric chloride (See 7.8) to the sample bottle in amounts
to produce a concentration of 10 mg/L. Add 1 ml of a 10 mg/mL
solution of mercuric chloride in reagent water to the sample
bottle at the sampling site or in the laboratory before
shipping to the sampling site. A major disadvantage of
mercuric chloride is that it is a highly toxic chemical;
mercuric chloride must be handled with caution, and samples
containing mercuric chloride must be disposed of properly.
8.2.2 If residual chlorine is present, add 80 mg of sodium
thiosulfate per liter of sample to the sample bottle prior to
collecting the sample.
8.2.3 After adding the sample to the bottle containing
preservative(s), seal the sample bottle and shake vigorously
for 1 min.
8.2.4 Samples must be iced or refrigerated at 4°C from the time of
collection until extraction. Preservation study results
indicate that most of the target analytes present in the
samples are stable for 7 days when stored under these
conditions (1). Preservation data for the analytes
chlorthalonil, alpha-HCH, delta-HCH, gamma-HCH, cis-permethrin,
trans-permethrin, and trifluralin are nondefinitive, and
therefore if these are analytes of interest, it is recommended
that the samples be analyzed immediately. Analyte stability
may be affected by the matrix; therefore, the analyst should
verify that the preservation technique is applicable to the
samples under study.
8.3 EXTRACT STORAGE
8.3.1 Sample extracts should be stored at 4*C away from light. A
14-day maximum extract storage time is recommended. However,
analyte stability may be affected by the matrix; therefore, the
181
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analyst should verify appropriate extract holding times
applicable to the samples under study.
9. CALIBRATION
9.1 Establish GC operating parameters equivalent to those indicated in
Sect. 6.8. The GC system must be calibrated using the internal
standard technique (Sect. 9.2) or the external standard technique
(Sect. 9.3). WARNING: DDT and endrin are easily degraded in the
injection port if the injection port or front of the column is dirty.
This is the result of buildup of high boiling residue from sample
injection. Check for degradation problems by injecting a mid-level
standard containing only 4,4'-DDT and endrin. Look for the
degradation products of 4,4'-DDT (4,4'-DDE and 4,4'-DDD) and endrin
(endrin ketone and endrin aldehyde). If degradation of either DDT or
endrin exceeds 20%, take corrective action before proceeding with
calibration. Calculate percent breakdown as follows:
% breakdown Total DDT degradation peak area (DDE + DDD) inn
for 4,4'-DDT ~ Total DDT peak area (DDT + DDE + DDD) x JUU
% breakdown
for Endrin
Total endrin degradation peak area (endrin aldehyde + endrin ketone) ,««
Total endrin peak area (endrin + endrin aldehyde + endrin ketone)
NOTE: Calibration standard solutions must be prepared such that no
unresolved analytes are mixed together.
9.2 INTERNAL STANDARD CALIBRATION PROCEDURE -- To use this approach, the
analyst must select one or more internal standards compatible in
analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is
not affected by method or matrix interferences. PCNB has been
identified as a suitable internal standard. Data presented in this
method were generated using the internal standard calibration
procedure.
9.2.1 Prepare calibration standards at a minimum of three (recommend
five) concentration levels for each analyte of interest and
surrogate compound by adding volumes of one or more stock
standards to a volumetric flask. To each calibration standard,
add a known constant amount of one or more of the internal
standards, and dilute to volume with MTBE. The lowest standard
should represent analyte concentrations near, but above, their
respective EDLs. The remaining standards should correspond to
the range of concentrations expected in the sample
concentrates, or should define the working range of the
detector. The calibration standards must bracket the analyte
concentrations found in the sample extracts.
9.2.2 Analyze each calibration standard according to the procedure
(Sect. 11.4). Tabulate response ( peak height or area) against
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concentration for each compound and Internal standard.
Calculate the response factor (RF) for each analyte and
surrogate using Equation 1.
(As)(C1s)
RF = Equation 1
(Ais)(Cs)
where
As = Response for the analyte to be measured.
AiS = Response for the internal standard.
Cis = Concentration of the internal standard (/*g/L).
Cs = Concentration of the analyte to be measured (/*g/L).
9.2.3 If the RF value over the working range is constant (20% RSD or
less) the average RF can be used for calculations.
Alternatively, the results can be used to plot a calibration
curve of response ratios (As/Ais) vs. Cs.
9.2.4 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any analyte varies from the
predicted response by more than ± 20%, the test must be
repeated using a fresh calibration standard. Alternatively, a
new calibration curve must be prepared for that analyte.
9.2.5 Single point calibration is a viable alternative to a
calibration curve. Prepare single point standards from the
secondary dilution standards in MTBE. The single point
standards should be prepared at a concentration that produces a
response that deviates from the sample extract response by no
more than 20%.
9.2.6 Verify calibration standards periodically, recommend at least
quarterly, by analyzing a standard prepared from reference
material obtained from an independent source. Results from
these analyses must be within the limits used to routinely
check calibration.
9.3 EXTERNAL STANDARD CALIBRATION PROCEDURE
9.3.1 Prepare calibration standards at a minimum of three (recommend
five) concentration levels for each analyte of interest and
surrogate compound by adding volumes of one or more stock
standards to a volumetric flask. Dilute to volume with MTBE.
The lowest standard should represent analyte concentrations
near, but above, their respective EDLs. The other
concentrations should correspond to the range of concentrations
expected in the sample concentrates, or should define the
working range of the detector. The calibration standards must
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bracket the analyte concentrations found In the sample
extracts.
9.3.2 Starting with the standard of lowest concentration, analyze
each calibration standard according to Sect. 11.4 and tabulate
response (peak height or area) versus the concentration in the
standard. The results can be used to prepare a calibration
curve for each compound. Alternatively, if the ratio of
response to concentration (calibration factor) is a constant
over the working range (20% RSD or less), linearity through the
origin can be assumed and the average ratio or calibration
factor can be used in place of a calibration curve.
9.3.3 The working calibration curve or calibration factor must be
verified on each working day by the measurement of a minimum of
two calibration check standards, one at the beginning and one
at the end of the analysis day. These check standards should be
at two different concentration levels to verify the calibration
curve. For extended periods of analysis (greater than 8 hrs.},
it is strongly recommended that check standards be interspersed
with samples at regular intervals during the course of the
analyses. If the response for any analyte varies from the
predicted response by more than ±20%, the test must be repeated
using a fresh calibration standard. If the results still do
not agree, generate a new calibration curve.
9.3.4 Single point calibration is a viable alternative to a
calibration curve. Prepare single point standards from the
secondary dilution standards in MTBE. The single point
standards should be prepared at a concentration that produces a
response that deviates from the sample extract response by no
more than 20%.
9.3.5 Verify calibration standards periodically, recommend at least
quarterly, by analyzing a standard prepared from reference
material obtained from an independent source. Results from
these analyses must be within the limits used to routinely
check calibration.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration
of laboratory capability, determination of surrogate compound
recoveries in each sample and blank, monitoring internal standard
peak area or height in each sample and blank (when internal standard
calibration procedures are being employed), analysis of laboratory
reagent blanks, laboratory fortified samples, laboratory fortified
blanks, and QC samples.
10.2 Laboratory Reagent Blanks -- Before processing any samples, the
analyst must demonstrate that all glassware and reagent interferences
are under control. Each time a set of samples is extracted or
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reagents are changed, a laboratory reagent blank (LRB) must be
analyzed. If within the retention time window of any analyte of
interest the LRB produces a peak that would prevent the determina-
tion of that analyte, determine the source of contamination and
eliminate the interference before processing samples.
10.3 INITIAL DEMONSTRATION OF CAPABILITY
10.3.1 Select a representative fortified concentration (about 10
times EDL or at the regulatory Maximum Contaminant Level,
whichever is lower) for each analyte. Prepare a sample
concentrate (in methanol) containing each analyte at 1000
times selected concentration. With a syringe, add 1 mL of the
concentrate to each of at least four 1-L aliquots of reagent
water, and analyze each aliquot according to procedures
beginning in Section 11.
10.3.2 For each analyte the recovery value for all four of these
samples must fall in the range of R ± 30% (or within R ± 3SR
if broader) using the values for R and SR for reagent water in
Table 2. For those compounds that meet the acceptance
criteria, performance is considered acceptable and sample
analysis may begin. For those compounds that fail these
criteria, this procedure must be repeated using four fresh
samples until satisfactory performance has been demonstrated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples via a
new, unfamiliar method prior to obtaining some experience with
it. It is expected that as laboratory personnel gain
experience with this method the quality of data will improve
beyond those required here.
10.4 The analyst is permitted to modify GC columns, GC conditions, GC
detectors, continuous extraction techniques, concentration
techniques (i.e. evaporation techniques), internal standards or
surrogate compounds. Each time such method modifications are made,
the analyst must repeat the procedures in Section 10.3.
10.5 ASSESSING SURROGATE RECOVERY
10.5.1 When surrogate recovery from a sample or method blank is <70%
or >130%, check (1) calculations to locate possible errors,
(2) fortifying solutions for degradation, (3) contamination or
other obvious abnormalities, and (4) instrument performance.
If those steps do not reveal the cause of the problem,
reanalyze the extract.
10.5.2 If a blank extract reanalysis fails the 70-130% recovery
criterion, the problem must be identified and corrected before
continuing.
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10.5.3 If sample extract reanalysis meets the surrogate recovery
criterion, report only data for the reanalyzed extract. If
sample extract reanalysis continues to fail the surrogate
recovery criterion, report all data for that sample as
suspect.
10.6 ASSESSING THE INTERNAL STANDARD
10.6.1 When using the internal standard calibration procedure, the
analyst is expected to monitor the IS response (peak area or
peak height) of all samples during each analysis day. The IS
response for any sample chromatogram should not deviate from
the daily calibration check standards IS response by more than
30%.
10.6.2 If >30% deviation occurs with an individual extract, optimize
instrument performance and inject a second aliquot of that
extract.
10.6.2.1 If the reinjected aliquot produces an acceptable
internal standard response report results for that
aliquot.
10.6.2.2 If a deviation of greater than 30% is obtained for the
re-injected extract, analysis of the sample should be
repeated beginning with Section 11, provided the
sample is still available. Otherwise, report results
obtained from the re-injected extract, but annotate
as suspect.
10.6.3 If consecutive samples fail the IS response acceptance
criterion, immediately analyze a calibration check standard.
10.6.3.1 If the check standard provides a response factor (RF)
within 20% of the predicted value, then follow
procedures itemized in Section 10.6.2 for each sample
failing the IS response criterion.
10.6.3.2 If the check standard provides a response factor which
deviates more than 20% of the predicted value, then
the analyst must recalibrate, as specified in
Section 9.
10.7 ASSESSING LABORATORY PERFORMANCE - LABORATORY FORTIFIED BLANK
10.7.1 The laboratory must analyze at least one laboratory fortified
blank (LFB) sample with every twenty samples or one per sample
set (all samples extracted within a 24-h period) whichever is
greater. The fortified concentration of each analyte in the
LFB should be 10 times EDL or the MCL, whichever is less.
Calculate accuracy as percent recovery (Xi). If the recovery
of any analyte falls outside the control limits (see Sect.
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10.7.2), that analyte Is judged out of control, and the source
of the problem should be identified and resolved before
continuing analyses.
10.7.2 Until sufficient data become available from within their own
laboratory, usually a minimum of results from 20 to 30
analyses, the laboratory should assess laboratory performance
against the control limits in Sect. 10.3.2 that are derived
from the data in Table 2. When sufficient internal performance
data becomes available, develop control limits from the mean
percent recovery (X) and standard deviation (S) of the percent
recovery. These data are used to establish upper and lower
control limits as follows:
UPPER CONTROL LIMIT = X + 3S
LOWER CONTROL LIMIT = X - 3S
After each five to ten new recovery measurements, new control
limits should be calculated using only the most recent 20-30
data points. These calculated control limits should never
exceed those established in Sect. 10.3.2.
10.7.3 It is recommended that the laboratory periodically document and
determine its detection limit capabilities for the analytes of
interest.
10.7.4 At least quarterly, analyze a QC sample from an outside source.
10.7.5 Laboratories are encouraged to participate in external
performance evaluation studies such as the labroatory
certification programs offered by many states or the studies
conducted by USEPA. Performance evaluation studies serve as
independent checks on the analyst's performance.
10.8 ASSESSING METHOD PERFORMANCE - LABORATORY FORTIFIED SAMPLE MATRIX
10.8.1 The laboratory must add a known concentration to a minimum of
10% of the routine samples or one sample concentration per set,
whichever is greater. The added concentration should not be
less then the background concentration of the sample selected
for fortification. Ideally, the fortified analyte
concentrations should be the same as that used for the LFB
(Section 10.7). Over time, samples from all routine sample
sources should be fortified.
10.8.2 Calculate the percent recovery, P of the concentration for each
analyte, after correcting the analytical result, X, from the
fortified sample for the background concentration, b, measured
in the unfortified sample, i.e.,:
P « 100 (X - b) / fortifying concentration,
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and compare these values to control limits appropriate for
reagent water data collected in the same fashion. If the
analyzed unfortified sample is found to contain NO background
concentrations, and the added concentrations are those
specified in Sect. 10.7, then the appropriate control limits
would be the acceptance limits in Sect. 10.7. If, on the other
hand, the analyzed unfortified sample is found to contain
background concentration, b, estimate the standard deviation at
the background concentration, SD, using regressions or
comparable background data and, similarly, estimate the mean,
Xa and standard deviation, sa, of analytical results at the
total concentration after fortifying. Then the appropriate
percentage control limits would be P t 3sp , where:
P = 100 X / (b + fortifying concentration)
2 2 !/2
and Sp = 100 (s + sb ) /fortifying concentration
For example, if the background concentration for Analyte A was
found to be 1 fig/I and the added amount was also 1 /ig/L, and
upon analysis the laboratory fortified sample measured 1.6 p/L,
then the calculated P for this sample would be (1.6 /ig/L minus
1.0 jtg/L)/l pg/L or 60%. This calculated P is compared to
control limits derived from prior reagent water data. Assume
it is known that analysis of an interference free sample at 1
fig/I yields an s of 0.12 /ig/L and similar analysis at 2.0 /ig/L
yields X and s of 2,01 pg/L and 0.20 /ig/L, respectively. The
appropriate limits to judge the reasonableness of the percent
recovery, 60%, obtained on the fortified matrix sample is
computed as follows:
[100 (2.01 /zg/L) / 2.0 /ig/L]
, V2
± 3 (100) [(0.12 /ig/L)2 + (0.20 /ig/L)2] / 1.0 /ig/L =
100.5% ± 300 (0.233) =
100.5% ± 70% or 30% to 170% recovery of the added analyte.
10.8.3 If the recovery of any such analyte falls outside the
designated range, and the laboratory performance for that
analyte is shown to be in control (Sect. 10.7), the recovery
problem encountered with the dosed sample is judged to be
matrix related, not system related. The result for that
analyte in the unfortified sample is labeled suspect/matrix to
inform the data user that the results are suspect due to matrix
effects.
10.9 ASSESSING INSTRUMENT SYSTEM - LABORATORY PERFORMANCE CHECK SAMPLE -
Instrument performance should be monitored on a daily basis by
analysis of the LPC sample. The LPC sample contains compounds
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designed to Indicate appropriate instrument sensitivity, column
performance (primary column) and chromatographic performance. LPC
sample components and performance criteria are listed in Table 3.
Inability to demonstrate acceptable instrument performance indicates
the need for reevaluation of the instrument system. The sensitivity
requirements are set based on the EDLs published in this method. If
laboratory EDLs differ from those listed in this method,
concentrations of the instrument QC standard compounds must be
adjusted to be compatible with the laboratory EDLs.
10.10 The laboratory may adopt additional quality control practices for use
with this method. The specific practices that are most productive
depend upon the needs of the laboratory and the nature of the
samples. For example, field or laboratory duplicates may be analyzed
to asses the precision of the environmental measurements or filed
reagent blanks may be used to asses contamination of samples under
site conditions, transportation and storage.
11. PROCEDURE
11.1 EXTRACTION (MANUAL METHOD)
11.1.1 Mark the water meniscus on the side of the sample bottle for
later determination of sample volume (Sect. 11.1.6). Add
preservative to blanks and QC check standards. Fortify the
sample with 50 pi of the surrogate standard fortifying
solution. Pour the entire sample into a 2-L separatory funnel.
11.1.2 Adjust the sample to pH 7 by adding 50 mL of phosphate buffer.
Check pH: add ^804 or NaOH if necessary.
11.1.3 Add 100 g Nad to the sample, seal, and shake to dissolve
salt.
11.1.4 Add 60 mL methylene chloride to the sample bottle, seal, and
shake 30 s to rinse the inner walls. Transfer the solvent to
the separatory funnel and extract the sample by vigorously
shaking the funnel for 2 min with periodic venting to release
excess pressure. Allow the organic layer to separate from the
water phase for a minimum of 10 min. If the emulsion
interface between layers is more than one third the volume of
the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring,
filtration of the emulsion through glass wool, centrifugation,
or other physical methods. Collect the methylene chloride
extract in a 500-mL Erlenmeyer flask.
11.1.5 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time,
combining the extracts in the Erlenmeyer flask. Perform a
third extraction in the same manner.
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11.1.6 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the water to a 1000-mL
graduated cylinder. Record the sample volume to the nearest
5 ml.
11.2 AUTOMATED EXTRACTION METHOD -- Data presented in this method were
generated using the automated extraction procedure with the
mechanical tumbler.
11.2.1 Mark the water meniscus on the side of the sample bottle for
later determination of sample volume (Sect. 11.2.6). Add
preservative to blanks and QC check standards. Fortify the
sample with 50 0L of the surrogate standard fortifying
solution. If the mechanical separatory funnel shaker is used,
pour the entire sample into a 2-L separatory funnel. If the
mechanical tumbler is used, pour the entire sample into a
tumbler bottle.
11.2.2 Adjust the sample to pH 7 by adding 50 ml of phosphate buffer.
Check pH: add ^$04 or NaOH if necessary.
11.2.3 Add 100 g NaCl to the sample, seal, and shake to dissolve
salt.
11.2.4 Add 300 ml methylene chloride to the sample bottle, seal, and
shake 30 s to rinse the inner walls. Transfer the solvent to
the sample contained in the separatory funnel or tumbler
bottle, seal, and shake for 10 s, venting periodically.
Repeat shaking and venting until pressure release is not
observed during venting. Reseal and place sample container in
appropriate mechanical mixing device (separatory funnel shaker
or tumbler). Shake or tumble the sample for 1 hour. Complete
mixing of the organic and aqueous phases should be observed
within about 2 min after starting the mixing device.
11.2.5 Remove the sample container from the mixing device. If the
tumbler is used, pour contents of tumbler bottle into a 2-L
separatory funnel. Allow the organic layer to separate from
the water phase for a minimum of 10 min. If the emulsion
interface between layers is more than one third the volume of
the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring,
filtration through glass wool, centrifugation, or other
physical methods. Collect the methylene chloride extract in a
500-mL Erlenmeyer flask.
11.2.6 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the water to a 1000-mL
graduated cylinder. Record the sample volume to the nearest
5 ml.
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11.3 EXTRACT CONCENTRATION
11.3.1 Assemble a K-D concentrator by attaching a 25-mL concentrator
tube to a 500-ml evaporative flask. Other concentration
devices or techniques may be used in place of the K-D if the
requirements of Sect. 10.3 are met.
11.3.2 Dry the extract by pouring it through a solvent-rinsed drying
column containing about 10 cm of anhydrous sodium sulfate.
Collect the extract in the K-D concentrator, and rinse the
column with 20-30 ml methylene chloride. Alternatively, add
about 5 g anhydrous sodium sulfate to the extract in the
Erlenmeyer flask; swirl flask to dry extract and allow to sit
for 15 min. Decant the methylene chloride extract into the
K-D concentrator. Rinse the remaining sodium sulfate with two
25-mL portions of methylene chloride and decant the rinses
into the K-D concentrator.
11.3.3 Add 1 to 2 clean boiling stones to the evaporative flask and
attach a macro 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, 65 to 70°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 15 to 20 min. 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 2 ml, remove the K-D apparatus and allow it to
drain and cool for at least 10 min.
11.3.4 Remove the Snyder column and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 ml of MTBE. Add
5-10 ml of MTBE and a fresh boiling stone. Attach a
micro-Snyder column to the concentrator tube and prewet the
column by adding about 0.5 ml of MTBE to the top. Place the
micro 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 in 5 to 10 min. When
the apparent volume of liquid reaches 2 ml, remove the micro
K-D from the bath and allow it to drain and cool. Add 5-10 ml
MTBE to the micro K-D and reconcentrate to 2 ml. Remove the
micro K-D from the bath and allow it to drain and cool.
Remove the micro Snyder column, and rinse the walls of the
concentrator tube while adjusting the volume to 5.0 ml with
MTBE.
11.3.5 Transfer extract to an appropriate-si zed TFE-fluorocarbon-
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sealed screw-cap vial and store, refrigerated at 4°C, until
analysis by GC-NPD.
11.4 GAS CHROMATOGRAPHY
11.4.1 Sect. 6.8 summarizes the recommended operating conditions for
the gas chromatograph. Included in Table 1 are retention
times observed using this method. Other GC columns,
chromatographic conditions, or detectors may be used if the
requirements of Sect. 10.3 are met.
11.4.2 Calibrate the system daily as described in Sect. 9. The
standards and extracts must be in MTBE.
11.4.3 If the internal standard calibration procedure is used, add
5 nl of the internal standard fortifying solution to the
sample extract, seal, and shake to distribute the internal
standard.
11.4.4 Inject 2 til of the sample extract. Record the resulting peak
size in area units.
11.4.5 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
11.5 IDENTIFICATION OF ANALYTES
11.5.1 Identify a sample component by comparison of its retention
time to the retention time of a reference chromatogram. If
the retention time of an unknown compound corresponds, within
limits, to the retention time of a standard compound, then
identification is considered positive.
11.5.2 The width of the retention time window used to make
identifications should be based upon 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.
11.5.3 Identification requires expert judgment when sample
components are not resolved chromatographically. When GC peaks
obviously represent more than one sample component (i.e.,
broadened peak with shoulder(s) or valley between two or more
maxima), or any time doubt exists over the identification of a
peak on a chromatogram, appropriate alternate techniques, to
help confirm peak identification, need to be employed. For
example, more positive identification may be made by the use
of an alternative detector which operates on a
chemical/physical principle different from that originally
used; e.g., mass spectrometry, or the use of a second
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chromatography column. A suggested alternative column is
described in Sect. 6.8.
12. CALCULATIONS
12.1 Calculate analyte concentrations in the sample from the response for
the analyte using the calibration procedure described in Sect. 9.
12.2 If the internal standard calibration procedure is used, calculate the
concentration (C) in the sample using the calibration curve or
response factor (RF) determined in Sect. 9.2 and Equation 2.
(As)ds)
C (jtg/L) = Equation 2
(A1s)(RF)(V0)
where:
As = Response for the parameter to be measured.
Ais = Response for the internal standard.
Is = Amount of internal standard added to each extract (/ig).
V0 = Volume of water extracted (L).
12.3 If the external standard calibration procedure is used, calculate the
amount of material injected from the peak response using the
calibration curve or calibration factor determined in Section 9.3.
The concentration (C) in the sample can be calculated from Equation 3.
(A)(Vt)
C (pg/L) = Equation 3
(Vi)(Vs)
where:
A = Amount of material injected (ng).
Vi = Volume of extract injected (pL).
Vt = Volume of total extract (/zL).
Vs = Volume of water extracted (mL).
13. PRECISION AND ACCURACY
13.1 In a single laboratory, analyte recoveries from reagent water were
determined at five concentration levels. Results were used to
determine analyte EDLs and demonstrate method range (1). Analytes
were divided into two fortified groups for recovery studies. Analyte
EDLs and analyte recoveries and standard deviation about the percent
recoveries at one concentration are given in Table 2.
13.2 In a single laboratory, analyte recoveries from two standard
synthetic ground waters were determined at one concentration level.
Results were used to demonstrate applicability of the method to
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different ground water matrices (1). Analyte recoveries from the two
synthetic matrices are given in Table 2.
14. REFERENCES
1. National Pesticide Survey Method No. 2: Determination of Chlorinated
Pesticides in Groundwater by Gas Chromatography with a Electron
Capture Detector.
2. ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82,
"Standard Practice for Preparation of Sample Containers and for
Preservation", American Society for Testing and Materials, Philadel-
phia, PA, 1986.
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, Aug. 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. ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82,
"Standard Practice for Sampling Water," American Society for Testing
and Materials, Philadelphia, PA, 1986.
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TABLE 1. RETENTION TINES FOR METHOD ANALYTES
Primarv
Retention Time3
(minutes)
Alternative
Etridiazole
Chlorneb
Propachlor
Trifluralin
HCH-alpha
Hexachlorobenzene
HCH-beta
HCH-gamma
PCNB (internal std.)
HCH-delta
Chlorthalonil
Heptachlor
Aldrin
Chlorpyrifos
DC PA
Heptachlor epoxide
Chlordane-gamma
Endosulfan I
Chlordane-alpha
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
Chi orobenzi late
4, 4' -ODD
Endrin aldehyde
Endosulfan sulfate
4,4'-DDT
Methoxychlor
cis-Permethrin
trans-Permethrin
DCB
23.46
25.50
28.90
31.62
31.62
31.96
33.32
33.66
34
35.02
35.36
37.74
40.12
40.6
41.14
42.16
43.52
44.20
44.54
45.90
45.90
46.92
47.60
47.94
48.28
48.62
49.98
50.32
53.38
58.48
58.82
64.1
22.78
26.18
30.94
(b)
32.98
(b)
40.12
35.36
34
41.48
39.78
36.72
38.08
(b)
41.14
42.16
43.86
43.52
44.54
44.88
45.90
(b)
51.68
48.28
46.92
46.92
49.30
50.32
53.72
(b)
(b)
(b)
* Columns and analytical conditions are described in Sect. 6.8.1 and 6.8.2.
D Data not available.
195
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TABLE 2. SINGLE LABORATORY ACCURACY, PRECISION AND ESTIMATED DETECTION LIMITS
(EDLS) FOR ANALYTES FROM REAGENT WATER AND SYNTHETIC GROUNDWATERSA
vo
Analvte
Aldrin
Chlordane-alpha
Chlordane-gamma
Chlorneb
Chi orobenzi late
Chlorthalonil
DC PA
4, 4' -ODD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan sulfate
Endrin
Endrin aldehyde
Endosulfan II
Etridlazole
HCH-alpha
HCH-beta
HCH-delta
HCH-gamma
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Methoxychlor
cis-Permethrin
trans-Permethrin
Propachlor
Trifluralln
EDLB
ua/L
0.075
0.0015
0.0015
0.5
5
0.025
0.025
0.0025
0.01
0.06
0.02
0.015
0.015
0.015
0.025
0.024
0.025
0.025
0.01
0.01
0.015
0.01
0.015
0.0077
0.05
0.5
0.5
0.5
0.025
Cone.
ua/L
0.15
0.15
0.15
5
10
0.25
0.25
0.25
0.1
0.6
0.2
0.15
0.15
0.15
0.25
0.15
0.25
0.05
0.1
0.1
0.15
0.1
0.15
0.05
0.5
5
5
5
0.25
Reagent
Re
86
99
99
97
108
91
103
107
99
112
87
87
102
88
88
92
103
92
95
102
89
98
87
99
105
91
111
103
103
Water
SRd
9.5
11.9
11.9
11.6
5.4
8.2
12.4
6.4
11.9
16.8
8.7
8.7
15.3
8.8
7.9
10.1
6.2
10.1
6.7
11.2
9.8
11.8
8.7
21.8
13.7
9.1
6.7
9.3
5.2
Synthetic
R
100
96
96
95
98
103
100
96
96
98
103
102
94
98
103
98
91
106
92
99
115
85
103
82
101
96
97
116
86
Water le
SR
11.0
12.5
12.5
6.7
10.8
10.3
13.0
8.6
12.5
11.8
9.3
8.2
1.3
9.8
11.3
10.8
6.4
7.4
5.5
11.9
6.9
11.1
7.2
9.8
10.1
11.5
9.7
4.6
10.3
Synthetic
R
69
99
99
75
102
71
101
101
99
84
82
84
72
104
84
76
98
86
100
103
85
85
82
68
104
86
102
95
87
Water 2f
SP
9.0
7.9
6.9
8.3
9.2
9.2
6.1
7.1
6.9
8.4
7.4
8.4
12.2
9.4
9.2
6.8
3.9
7.7
6.0
6.2
7.7
7.7
9.8
4.8
6.2
9.5
7.1
7.6
9.6
-------
TABLE 2. (Continued)
a Data corrected for amount detected in blank and represent the mean of 7-8 samples.
b EDL = estimated detection limit; defined as either MDL (Appendix B to 40 CFR Part 136
- Definition and Procedure for the Determination of the Method Detection Limit -
Revision 1.11) or a level of compound in a sample yielding a peak in the final extract
with signal-to-noise ratio of approximately 5, whichever value is higher. The
concentration level used in determining the EDL is not the same as the concentration
level presented in this table.
c R = average percent recovery.
d SR = standard deviation of the percent recovery.
e Corrected for amount found in blank; Absopure Nature Artesian Spring Water Obtained
from the Absopure Water Company in Plymouth, Michigan.
f Corrected for amount found in blank; reagent water fortified with fulvic acid at the 1
mg/L concentration level. A well-characterized fulvic acid, available from the
International Humic Substances Society (associated with the United States Geological
Survey in Denver, Colorado), was used.
-------
TABLE 3. LABORATORY PERFORMANCE CHECK SOLUTION
Test
Sensitivity
Chromatographic performance
Column performance
Analyte
Chlorpyrifos
DCPA
Chlorothalonil
HCH-delta
Cone,
M9/L
0.0020
0.0500
0.0500
0.0400
Requirements
Detection of
PSF between 0
Resolution >
analyte; S/N > 3
.80 and 1.15*
0.50b
00
a PGF - peak Gaussian factor. Calculated using the equation:
PGF , 1.83 x
where W(l/2) is the peak width at half height and W(l/10) is the peak width at tenth height.
b Resolution between the two peaks as defined by the equation:
t
R =
W
where t is the difference in elution times between the two peaks and W is the average peak
width, at the baseline, of the two peaks.
-------
METHOD 508A. SCREENING FOR POLYCHLORINATED BIPHENYLS
BY PERCHLORINATION AND GAS CHROMATOGRAPHY
Revision 1.0
T. A. Bellar - Method 508A, Revision 1.0 (1989)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
199
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METHOD 508A
SCREENING FOR POLYCHLORINATED BIPHENYLS
BY PERCHLORINATION/GAS CHROMATOGRAPHY
1. SCOPE AND APPLICATION
1.1. This procedure may be used for screening finished drinking water,
raw source water, or drinking water in any treatment stage for
polychlorinated biphenyls (PCBs). This procedure is applicable to
samples containing PCBs as single congeners or as complex mixtures
such as weathered, intact, or mixtures of commercial Aroclors. The
procedure is incapable of identifying the parent PCBs because the
original PCBs are chemically converted to a common product,
decachlorobiphenyl (DCB). The procedure has only been evaluated
using Aroclors and 2-chlorobiphenyl as a source of PCBs.
1.2. This procedure is primarily designed to function as a pass/fail test
for DCB at 0.5 /jg/L. However, it will accurately measure DCB from
the method detection limit (MDL) to 5.0 pg/L. It is prone to false
positive interferences and can result in a calculated weight of PCBs
significantly greater than that of PCB originally present in the
sample. If DCB is detected at 0.5 pg/L or above, then an approved
method for the analysis of PCBs should be used to accurately identify
the source and measure the concentration of the PCBs.
1.3. This procedure can be used to help confirm the presence of PCBs for
other methods using electron capture or halogen specific detectors
whenever chromatographic patterns are not representative of those
described in the method.
2. SUMMARY OF PROCEDURE
2.1. A 1-L water sample is placed into a separatory funnel and extracted
with methylene chloride or one of several optional solvents. The
extract is dried, concentrated, and the solvent is exchanged to
chloroform. The PCBs are then reacted with antimony pentachloride
(SbClc) (in the presence of an iron catalyst and heat) to form DCB.
The DCB is extracted with hexane from the reaction mixture; after
the extract is purified, an aliquot is injected into a gas
chromatograph (GC) equipped with an electron capture detector (ECD)
for separation and measurement. The GC is calibrated using DCB as
the standard.
3. DEFINITIONS
3.1. Calibration Standard (CAL) -- a solution of DCB used to calibrate
the ECD.
3.2. Congener Number -- Throughout this procedure, individual PCBs are
described with the number assigned by Ballschmiter and Zell (1).
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(This number is also used to describe PCB congeners in catalogs
produced by Ultra Scientific, Hope, RI.)
3.3. Laboratory Duplicates (LD1 and LD2) -- Two sample aliquots taken in
the analytical laboratory are analyzed with identical procedures.
Analysis of laboratory duplicates indicates precision associated
with laboratory procedures, but not with sample collection,
preservation or storage procedures.
3.4. Laboratory Performance Check Solution (LPC) -- A solution of method
analytes used to evaluate the analytical system performance with
respect to a defined set of criteria.
3.5. Laboratory Reagent Blank (LRB) -- An aliquot of reagent water that
is treated as a sample. It is exposed to all glassware and
apparatus, and all method solvents and reagents are used. The
extract is concentrated to the final volume used for samples and is
analyzed the same as a sample extract.
3.6. Laboratory Fortified Sample Matrix (LFM) -- An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.7. Quality Control (QC) Sample -- A sample containing known
concentrations of analytes that is analyzed by a laboratory to
demonstrate that it can obtain acceptable identifications and
measurements with procedures to be used to analyze environmental
samples containing the same or similar analytes. Analyte
concentrations are known by the analyst. Preparation of the QC
check sample by a laboratory other than the laboratory performing
the analysis is highly desirable.
4. INTERFERENCES
4.1. Interferences may be caused by contaminants in solvents reagents,
glassware, and other sample processing equipment. Laboratory
reagent blanks (LRBs) are analyzed routinely to demonstrate that
these materials are free of interferences under the analytical
conditions used for samples.
4.2. To minimize interferences, glassware (including sample bottles)
should be meticulously cleaned. As soon as possible after use,
rinse glassware with the last solvent used. Then wash with
detergent in hot water and rinse with tap water followed by
distilled water. Drain dry and heat in a muffle furnace at 450°C
for a few hours. After cooling, store glassware inverted or covered
with aluminum foil. Before using, rinse each piece with an
201
-------
appropriate solvent. Volumetric glassware should not be heated In
muffle furnace.
4.3. In addition to PCBs, several compounds and classes of compounds will
form OCB with varying yields when extracted and perchlorinated
according to this procedure. Based upon a literature search (2)
such compounds include biphenyl, polyhalogenated biphenyls,
hydrogenated biphenyls, and polyhalogenated terphenyls. If such
compounds are present in the extract, false positive or positively
biased data will be generated.
4.4. A splitless injection capillary column GC can be used but standards
and samples should be contained in the same solvent, or results may
be significantly biased.
4.5. PCBs are converted to DCB on a mole for mole basis. Converting DCB
concentrations back to the original PCB concentration is beyond the
scope of this method. For informational purposes and in order to
demonstrate the degree of increased weight of PCBs generated by the
procedure, Table 1 lists the conversion of 0.5 /ig/L of DCB back to
various sources of PCBs assuming 100% method recovery.
5. SAFETY
5.1. Chloroform and methyl ene chloride have been tentatively classified
as known or suspected human or mammalian carcinogens. The toxicity
or carcinogenicity of the remaining chemicals used in this method
has not been precisely defined. Therefore, each should be treated
as a potential health hazard, and exposure should be reduced to the
lowest feasible level. Each laboratory is responsible for safely
disposing materials and for maintaining awareness of OSHA
regulations regarding safe handling of the chemicals used in this
method. A reference file of material data handling sheets should be
made available to all personnel involved in analyses. Additional
information on laboratory safety is available (3-5).
5.2. Polychlorinated biphenyls have been classified as known or suspected
human or mammalian carcinogens. Primary standards of these compounds
should be prepared in an area specifically designed to handle
carcinogens. It is recommended that primary dilutions be obtained
from certified sources such as the EPA repository.
5.3. SbCls is a corrosive reagent that reacts violently with water. This
compound must be used with extreme caution. All operations
involving the pure reagent must be performed in a hood because
appreciable quantities of volatile, potentially harmful materials
will be lost to the atmosphere.
5.4. The perchlorination reaction described in this procedure requires
that the sample extract be heated to 205°C for about 30 min while
hermetically sealed in a glass test tube. The solvents and volumes
described in the procedure should be carefully reproduced; otherwise
202
-------
dangerous pressures may be generated during perchlorination. The
following safety precautions are strongly recommended.
5.4.1. Use only the prescribed perchlorination glassware and
visually check for flaws such as chips, strains, or
scratches. Discard if any abnormalities are noted.
5.4.2. After cooling the perchlorinated product is still under
slight pressure and should be carefully vented in a hood
(Sect. 11.2.8.).
5.4.3. The SbCls neutralization step involves an exothermic
reaction and should be performed in a hood (Sect. 11.2.9.).
5.4.4. An explosion shield should be used during the
perchlorination and neutralization procedures along with
additional eye protection such as an 8-in. face shield. An
oil bath heater should not be substituted for the block
digester.
5.5. Storage, labelling and disposal of PCBs must conform to all
applicable laws and regulations. See (6) for USEPA requirements.
Call the Toxics Substances Control Act hotline for further
assistance (1-800-424-9065).
5.6. Methylene chloride is described in the procedure (Sect. 11.1.2) as
the extraction solvent; however, hexane, hexane + 15% methylene
chloride or hexane + 15% ethylether may be substituted to minimize
laboratory personnel exposure to methylene chloride.
5.7. Chloroform is described in the procedure (Sect. 11.2.1) as the
solvent for the perchlorination reaction. Other less toxic solvents
including methylene chloride and hydrocarbons were evaluated but were
found to be unsuitable. Prior to implementing this procedure, all
laboratory personnel must be trained in safe handling practices for
chloroform.
6. APPARATUS AND EQUIPMENT
6.1. Sampling equipment
6.1.1. Water sample bottles -- meticulously cleaned 1-L glass
bottles fitted with Teflon-lined screw caps.
6.2. Glassware
6.2.1. Separatory Funnel -- 2-L with Teflon stopcock.
6.2.2. Drying Column -- glass column approximately 400 mm long x
19 mm i.d. with coarse frit filter disc.
203
-------
6.2.3. Concentrator Tube -- 10-mL graduated Kuderna-Danish desig"-
with ground-glass stopper.
6.2.4. Evaporative Flask -- 500-mL Kuderna-Danish design.
6.2.5. Snyder Column -- three-ball macro Kuderna-Danish design.
6.2.6. Snyder Column -- three-ball micro Kuderna-Danish design.
6.2.7. Vials -- 10- to 15-mL amber glass with Teflon-lined screw
caps.
6.2.8. Screw cap culture test tubes -- 100 mm x 13 mm i.d. Pyrex
with a Teflon-lined screw cap, Sargent-Welch 0S-79533A or
equivalent.
6.2.9. Disposable Pasteur pipettes -- 9-in. heavy wall.
6.2.10. Screw cap test tube -- 15 ml with Teflon-lined screw cap.
6.3. GC System -- Packed column or capillary column.
6.3.1. Isothermal packed column GC equipped with an on-column
injector and a linearized ECD capable of generating a linear
response for DCB from at least 0.005 to 1.0 ng injected.
6.3.2. Programmable capillary column GC equipped with an on-column
or splitless injector and a linearized ECD capable of
generating a linear response for DCB from at least 0.005 to
1.0 ng injected. The column oven temperature programmer
should have multi-ramp capabilities from at least 60°C to
300°C. For most precise data, an autoinjector should be
used.
6.4. GC Columns
6.4.1. Packed Column -- a 2 mm i.d. x 3 m, glass column packed with
3% OV-1 on 80-100 mesh Supelcoport or equivalent.
6.4.2. Capillary Column -- a 30 m x 0.32 mm i.d. fused silica
capillary coated with a bonded 0.25 ion film of cross linked
phenyl methyl silicone such as Durabond-5 (DB-5).
6.5. Miscellaneous Equipment
6.5.1. Volumetric flask -- 5-mL, 10-mL, and 100 ml with ground
glass stoppers.
6.5.2. Microsyringes -- various standard sizes.
6.5.3. Boiling Chips -- approximately 10/40 mesh. Heat at 400°C
204
-------
for 30 min or extract with methylene chloride in a Soxhlet
apparatus.
6.5.4. Water Bath -- heated, with concentric ring cover, capable of
temperature control with ± 2°C.
6.5.5. Analytical Balance -- capable of accurately weighing to
0.0001 g.
6.5.6. 1-L graduated cylinder.
6.5.7. Block digestor -- 1.4 cm i.d. x 5 cm deep holes. Operated
at 205°C ± 5°C. Note: A Technicon Model BD-40 block
digestor with specially fabricated aluminum insert bushings
was used to conduct the procedure development research.
Block digestors with holes of other dimensions may adversely
influence recoveries.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1. Solvents -- high purity, distilled in glass toluene, hexane,
methylene chloride, chloroform and methyl alcohol.
7.2. Sodium sulfate -- ACS granular, anhydrous. Purify by heating at
400°C for 4 h in a shallow dish. Store in a glass bottle with a
Teflon-lined screw cap.
7.3. SbCl5 > 98%.
7.4. Iron powder - 99.1%.
7.5. PCB Solutions.
7.5.1. Prepare a stock solution of Aroclor 1260 at 5.00 ng/nl in
methyl alcohol or obtain a similar mixture from a certified
source.
7.5.2. Prepare a stock solution of DCB at 1.00 /*g//iL in toluene or
obtain a similar mixture from a certified source.
7.5.3. PCB fortification solution. Dilute an aliquot of the
Aroclor 1260 stock solution in methyl alcohol to produce
about 10 ml of a solution containing 50.0 ng/pL. Store in a
50-90% filled glass bottle with a Teflon-lined screw cap.
7.5.4. Calibration standards. Five calibration solutions
containing DCB from 0.01 ng/pL to 1.0 ng//iL in hexane are
required to calibrate the detector response. Prepare
standards at 0.010, 0.080, 0.10, 0.25 and 1.0 ng//iL in
hexane (see 4.4) from the stock solution of DCB. Store in
50-90% filled glass bottles with Teflon-lined screw caps.
Monitor for solvent loss due to evaporation.
205
-------
7.5.5. Extract matrix evaluation solution. Dilute an aliquot of
the DCB stock solution to produce about 10 ml of a solution
containing 50.0 ng/pL in hexane. Store in a 50-90% filled
glass bottle with a Teflon-lined screw cap.
7.6. Hydrochloric Acid Solution 1+1 - Dilute one part concentrated
hydrochloric acid with one part distilled water.
7.7. 0.1N Sodium Bicarbonate (NaHC03) Solution - Dilute 0.84 g of ACS
grade NaHC03 to 100 ml with reagent water.
7.8. Reagent water - Water in which DCB is found to be less than 0.1 /ig/L
as analyzed by this procedure. Distilled water met this criterion.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1. Sample Collection
8.1.1. Collect duplicate samples in clean 1-L glass containers and
seal with a Teflon-lined screw cap. Fill the bottles to
about 90-95% full.
8.1.2. Because PCBs are hydrophobic they are likely to be adsorbed
on suspended solids. If suspended solids are present in the
source, a representative portion of solids must be included
in the water sample.
8.1.3. When sampling from a water tap, open the tap and allow the
system to flush until the water temperature has stabilized
(about 10 min). Adjust the flow to about 1 L/min and
collect the duplicate samples from the flowing stream.
8.1.4. When sampling from an open body of water, fill a 1-gal
wide-mouth bottle from a representative area. Carefully
fill the duplicate sample bottles from the 1-gal bottle.
8.2. Sample Preservation -- No chemical preservation reagents are
recommended. Store the samples at 4°C to retard microbial action
until analysis.
8.3. Sample Storage -- Extract samples within 14 days of collection (7).
Extracts and perchlorinated extracts may be stored for up to 30 days
if protected from solvent volatilization.
9. CALIBRATION -- Demonstration and documentation of acceptable initial
calibration is required before any samples are analyzed and is required
intermittently throughout sample analyses as dictated by results of
continuing calibration checks. After initial calibration is successfully
performed, a continuing calibration check is required at the beginning and
end of each set of samples or 8-hour period during which analyses are
performed.
206
-------
9.1. Initial Calibration
9.1.1. Inject duplicate aliquots (1-3 /iL ) of each calibration
solution into the GC. (Autoinjectors are preferred,
especially with splitless injectors.) Inject five
additional aliquots of the 0.10 ng//iL standard.
9.1.2. Accurately determine the DCB retention time (RT) and peak
area or peak height for each injection.
9.1.3. Determine the average RT and the standard deviation (SD) of
RTs for all 15 injections. To be acceptable, the RSD of the
RTs should be less than 0.2%.
9.1.4. Determine the response factor (RF) for each of the
injections by dividing the amount (ng) injected into the
resulting area or peak height or integrator units.
9.1.5. Determine the average RF and its SD and RSD for the seven
injections at the 0.10 ng//zL level.
9.1.6. The RSD of the RF should be less than 6% for the seven
injections at the 0.1 ng//zL level.
9.1.7. Compare the RF determined for the 0.01, 0.08, 0.25, and
1.0 ng standards to the average RF calculated in 9.1.5 ± 3
SD. If any value falls outside of this range, then the
instrument is not being operated within an acceptable linear
range and the sample volume injected must be adjusted
accordingly. Alternatively, the linear dynamic range can be
clearly defined by injecting standards at other
concentrations. To be marginally acceptable, the system
should function from 0.08 to 0.25 ng injected.
Table II shows typical values obtained during method
developmnt.
9.2. For an acceptable continuing calibration check, the 0.1 ng/jzL
calibration standard must be analyzed before and after a series of
samples or at least once after each 8 hours of operation. The RF
must be within ± 20% of the mean value determined in 9.1.5, or a
new calibration curve must be generated. Additionally, the RT must
fall within the mean value + 3 SD determined in 9.1.3, or a new
calibration curve must be generated or the reason for the RT
variance must be found and rectified.
9.3. Extract matrix effect evaluation -- It has been found that there may
be a matrix effect from the perchlorinated extract which can bias the
response on certain GC systems. Until this problem is understood, an
extract matrix effect evaluation should be performed on each gas
chromatographic system to determine if the system can be used for
207
-------
this procedure. This test should be repeated each time a
modification or change is made to the system.
9.3.1. Extract, perch!orinate, and cleanup duplicate drinking water
samples or laboratory reagent blanks according to the
procedure halting at step 11.2.13.
9.3.2. Combine the two extracts together in a 25-mL beaker or flask
and mix.
9.3.3. Immediately place 5.0 mL in a volumetric flask and seal.
Place the remaining solution in a second hermetically sealed
container and label MS-1 (mixed sample 1).
9.3.4. Analyze MS-1 in duplicate. If the value for the DCB is
< 0.05 ng//iL, proceed to 9.3.5. If > 0.05 ng//tL, proceed to
9.3.6.
9.3.5. Fortify the contents of the volumetric flask with 10.0 /iL
of the 50.0 ng/0L extract matrix evaluation solution (Sect.
7.5.5) and label SE-1 (fortified extract 1). Analyze SE-1
in duplicate, then proceed to 9.3.
9.3.6. Fortify the contents of the volumetric flask at three to ten
times the concentration found in 9.3.4. If the fortified
value plus the MS-1 value found in 9.3.4 exceeds the linear
dynamic range of the detector (Sect. 9.1.7), then terminate
the test and select another sample. Do not dilute extract
matrices to perform this test.
9.3.7. Determine the extract matrix bias according to the following
calculation:
(SE-1 no/al) - (MS-1 na/uLl x 100 _ „ recoverv
(Fortified value ng//iL) " * recovery
Recoveries between 80 and 120% are acceptable. If the
recovery is < 80%, the test should be repeated. If the
recovery remains < 80%, then another GC system should be
used.
10. QUALITY CONTROL
10.1. Laboratory Reagent Blank (LRB) -- Perform all steps in the
analytical procedure (Sect. 11) using all glassware, reagents,
standards, equipment, apparatus, and solvents that would be used
for a sample analysis using 1 L of reagent water.
10.1.1. Prepare and analyze a LRB before any samples are extracted
and analyzed.
208
-------
10.1.2. Prepare and analyze additional LRB whenever new batches or
sources of reagents are Introduced into the analysis
scheme.
10.1.3. Prepare a LRB each time samples are perchlorinated. If
large batches of samples are perchlorinated, then prepare
and analyze 1 LRB per 10 samples.
10.1.4. An acceptable LRB contains < 0.025 ng/0L of DCB.
10.1.5. Corrective action for unacceptable LRB -- Systematically
check solvents, reagents (particularly the SbCls and
methylene chloride), apparatus and glassware to locate and
eliminate the source of contamination before any samples
are extracted, perchlorinated, and analyzed. Purify or
discard contaminated reagents and solvents.
10.2. Calibration -- Included among initial and continuing calibration
procedures are numerous QC checks to ensure that valid data are
being acquired (See Sect. 9). Continuing calibration checks are
accomplished with results from analysis of one solution, the
0.10 ng//iL calibration solution.
10.2.1. If some criteria are not met for a continuing calibration
check after an 8-h period or after a series of samples are
analyzed, then those samples must be reanalyzed. Those
criteria include the RF criteria and the RT criteria
described in Sect. 9.2.
10.3. All sample concentrations must be bracketed by the calibration
curve and must be within the linear dynamic range of the detector.
(See Sect. 9.1.7.)
10.3.1. Samples that fall outside the linear dynamic range due to
excessive concentration must be reanalyzed after
appropriate dilution if accurate values for DCB are
required.
10.4. All GC systems must be evaluated for extract matrix effect bias
according to Sect. 9.3.
10.4.1. Systems that exhibit a bias in excess of + or - 20% should
not be used for this determination.
10.5. Initial demonstration of laboratory capability for water analysis.
10.5.1. Prepare one or more solutions containing representative PCB
mixtures at a concentration that falls within the linear
dynamic range of the instrument. Reagent water fortified
with Aroclor 1260 is recommended for this test.
209
-------
10.5.2. Fortify four to seven 1-L portions of reagent water with
10.0 nl of the 50 ng//iL PCB solution (Sect. 7.5.3).
Extract and analyze the fortified water samples according
to the procedure (Sect. 11).
10.5.3. Calculate the recovery according to the following formula:
y Recovery = f Total ng found in extract) x 100
where 691- 500 r,g
„
aSee Table 1 for the molecular weights of other Aroclors.
10.5.4. Determine the average concentration and the relative SD of
the five measurements. Average recovery should be 100% ±20
with a RSD of < 10%.
10.6. Fortify reagent water with varying quantities of the 50 ng/jiL PCB
solution (Sect. 7.5.3). Analyze at least one fortified sample for
each batch of 20 samples. Calculate recovery according to Sect.
10.5.3. Maintain QC charts of these data. Until interlaboratory
data are available, the recovery of the fortified sample should be
equivalent to that determined in 10.5.4.
10.7. Sample matrix effects have been observed with this procedure and
they are significant. Check for sample matrix effects by analyzing
one laboratory fortified sample matrix (LFM) for every 20 samples.
10.8. At least quarterly, analyze a quality control sample (QCS) from an
external source. If measured analyte concentrations are not of
acceptable accuracy (Sect. 10.5.4), check the entire analytical
procedure to locate and correct the problem source.
10.9. Qualitative identification of DCB in the samples is based on the
average RT for DCB determined in Sect. 9.1.3. For a positive
identification, the DCB peak must elute within the window bracketed
by the average retention ± 3 SD. If DCB appears to fall outside of
this window, then further analyses of samples should be halted and
Sect. 9.2 initiated.
10.10. It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to assess the precision of the environmental measurements.
Whenever possible, the laboratory should analyze standard reference
materials and participate in relevant performance evaluation
studies.
210
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I. PROCEDURE
11.1. Sample Extraction
11.1.1. Mark the sample meniscus on the side of the sample bottle for
later determination of sample volume. Pour the entire sample
into a 2-L separatory funnel.
11.1.2. Add 60 ml of methylene chloride (See Sect. 5.6} to the sample
bottle, seal, and shake 30 s to rinse the inner surface.
Transfer the solvent to the separatory funnel and extract the
sample by shaking the funnel for 2 min with periodic venting to
release excess pressure. Wait at least 10 min to allow the
organic layer to separate from the water phase. If the emulsion
interface between layers is more than one-third the volume of
the solvent layer, use mechanical techniques (such as stirring,
filtration of emulsion through glass wool, or centrifugation) to
complete phase separation. Collect the methylene chloride
extract in a 250-mL Erlenmeyer flask. Add a second 60-mL volume
of methylene chloride to the sample bottle and repeat the
extraction procedure a second time, combining the extracts in
the Erlenmeyer flask. Perform a third extraction in the same
manner.
11.1.3. Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-
mL concentrator tube to a 500-mL evaporative flask.
11.1.4. Pour the combined extract into a solvent-rinsed drying column
containing about 10 cm of anhydrous sodium sulfate. Rinse the
Erlenmeyer flask with a 20 to 30 ml portion of methylene
chloride adding the rinse to the drying column. Collect the
combined extract in the K-D concentrator.
11.1.5. Add one or two clean boiling chips to the evaporative flask and
attach a three-ball Snyder column. Prewet the Snyder column by
adding about 1 ml of methylene chloride to the top. Place the
K-D 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 with hot
vapor. Adjust the vertical position of the apparatus and the
water temperature as required to complete the concentration in
15-20 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 1 ml, remove the K-D apparatus from the water bath and
allow it to drain and cool for at least 10 min.
11.1.6. Remove the 10-mL concentrator tube from the 500-mL evaporative
flask and attach a 3-ball micro Snyder column. After wetting
the column with about 0.5 ml of methylene chloride, continue
concentrating the extract down to about 2 ml.
211
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11.1.7. Determine the original sample volume by refilling the sample
bottle with water to the mark and transferring the liquid to a
1000-mL graduated cylinder. Record the sample volume to the
nearest 5 ml.
11.2. Perchlorination (8,9)
11.2.1. Quantitatively transfer the extract to a 100 mm x 13 mm i.d.
screw cap test tube. Rinse the KD ampul three times with 250 ill
of chloroform adding the rinse to the test tube.
11.2.2. Concentrate the extract to about 0.1 mL (0.1 ml is about the
volume of one drop of water) by directing a stream of nitrogen
flowing at about 100 mL/m Into the test tube while warming the
base of the test tube in a 50°C water bath.
11.2.2.1. Do not allow to go to dryness.
11.2.2.2. Disposable pipettes are a convenient means of
directing the nitrogen into the test tube. In
an effort to minimize cross contamination, a
new pipette should be used for each sample.
11,2.3. Add an additional 2 ml of chloroform and again concentrate
to 0.1 mL using the nitrogen blow-down technique.
11.2.4. Add 100 mg of iron powder to the extract.
11.2.5. Using a disposable pipette, carefully add 25 drops of
to the extract. (See Sect. 5.3). Seal immediately.
11.2.6. Heat to 205°C ± 5°C for a minimum of 30 min but do not
exceed 45 min. Perform the reaction in the hood behind an
explosion shield.
11.2.7. Allow the mixture to cool to room temperature.
11.2.8. Carefully open in a hood. (The extract will be under a
slight pressure.)
11.2.9. Slowly add 0.5 mL of 1+1 diluted hydrochloric acid to the
perch! or inated extract in a hood. Caution: The remaining
SbClc will react exothermally with the HC1 . If a white
precipitate is present, add additional hydrochloric acid
solution until it dissolves.
11.2.10. Add 2.0 ml of hexane to the contents of the test tube.
Seal and shake for 2 min. Allow the two phases to
separate. Decant the top layer into a 5.0-mL volumetric
flask. Reextract the mixture two additional times: first
with 2.0 mL of hexane, then with 1.0 mL of hexane, adding
212
-------
the extracts to the 5.0-mL volumetric flask. Carefully
adjust the volume to 5.0 ml using hexane.
11.2.11. Add 4 ml of 0.1 N NaHC03 to a 15-mL test tube with a
Teflon-lined screw cap. Pour the contents of the 5-mL
volumetric flask into the test tube. (Note! Do not rinse
the volumetric flask with additional solvent.) Seal and
shake for 1 min. Allow the two phases to separate.
11.2.12. Decant the top layer into a second 15-mL test tube. Add
4 ml of reagent water. Seal and shake for 1 min.
11.2.13. Decant the top layer and store in a hermetically sealed
container for GC analysis.
11.3. GC -- Packed - on-column injection ECD, capillary - on-column
Injection electron capture and capillary splUless Injection ECD GC
systems have been evaluated and found to generate acceptable data
for DCB as long as Sect. 10.4 criteria are met. The following
conditions were used to generate the single-laboratory accuracy and
precision data listed 1n Sect. 13. The values given are for
guidance because slight modifications may be necessary to optimize
specific GC systems.
11.3.1. The packed column GC was operated with a glass column 3 m
long with an 1.d. of 2 mm. The column was packed with 3%
OV-1 coated on 80-100 mesh Supelcoport. 3.0 nl volumes
of each sample was Injected directly on column using an
autosampler. The injection port was held at 200°C while
the column was maintained isothermally at 235°C with an
Argon +5% methane carrier gas flowing at 50 mL/min. The
ECD was maintained at 300°C with no auxiliary make-up gas.
Under these conditions, the average RT for DCB was 9.49
min with a SD of 0.014. DCB was adequately resolved from
other perchlorination reaction byproducts to generate
accurate data for drinking water samples. Highly
contaminated raw source water generated complex chromato-
grams with late eluting components that interfered with
DCB measurements.
11.3.2. The capillary column on-column GC was operated with a DB-
5 fused silica column 30 m long with a 0.32 mm i.d. and a
0.25 /im film thickness. The helium carrier gas was
adjusted to flow at 29 cm/sec at 60°C. Three microliter
sample volumes were injected on-column into a 0.5 mm i.d.
x 10 cm fused silica retention gap using an autoinjector.
The retention gap was maintained at 60°C during injection.
The capillary column was maintained at 60°C until one
minute after injection, then programmed at 20°/min to
180°C. After a 2 minute hold, the column was again
213
-------
programmed at 20°C/min to 290°C and held there until all
compounds eluted. The ECD was operated at 300"C with an
Argon +5% methane makeup gas flowing at 20 mL/min.
Under these conditions the average RT for DCB was 21.85
min with a SO of 0.021. DCB was adequately resolved from
other perchlorination byproducts to generate accurate data
for both finished drinking water and raw source water
samples.
11.3.3. The capillary column splitless injection GC was operated
with a DB-5 fused silica column 30 m long with an i.d. of
0.32 mm and a 0.25 urn film thickness. The helium carrier
gas was adjusted to flow at 29 cm/sec at 180°C. Three pL
injection volumes were delivered by an autoinjector into
the splitless injector operated at 250°C. The splitless
time was set for 30 sec.
The capillary column was maintained at 180°C until one
minute after injection, then programmed at 20°C/min to
290°C and held for 20 min or until all late eluting
compounds eluted. The electron capture was operated at
300°C with an argon + 5% methane makeup gas flowing at
20 mL/min.
Under these conditions the average RT for DCB was 24.75
min with a SO of 0.009. DCB was adequately resolved from
other perchlorination byproducts to generate accurate data
for both finished drinking water and raw source water
samples.
12. CALCULATIONS
12.1. Calculate the concentration of the DCB found in each extract using
an automated data system or according to the formula.
12.1.1. Extract concentration ngM = ^Injected
RF
,_,,»-, * ..• it (Concentration no/uLl (5000)
12.1.2. Sample concentration ng/L = J volume of sample (L)
where: area sample = area, peak height or
integrator units
/il_ injected = volume of sample injected
into GC
5000 = final volume of extract in /*L
(Sect. 11.2.10)
214
-------
Volume of sample (L) = volume of sample
extracted in liters (Sect. 11.1.7)
RF = average RF {9.1.4} for
the 0.1 ng/jiL standard.
12.1.3. Calculations should utilize all available digits of
precision, but final reported concentrations should be
rounded to an appropriate number of significant figures
(one digit of uncertainty). Experience indicates that
three significant figures may be used for concentrations
above 99 Mg/U two significant figures for concentrations
between 0.1-99 pg/L, and one significant figure for lower
concentrations.
12.1.4. Do not subtract method blanks from the sample data unless
otherwise required in the procedure.
13. METHOD PERFORMANCE -- To obtain single-laboratory accuracy and precision
data for method analytes, seven 1-L aliquots of chlorinated tap water,
groundwater and river water were fortified with 500 ng of PCBs from
several sources. The samples were extracted, perchlorinated and analyzed
according to Sect. 11. Tables 3 and 4 list the resulting data.
14. REFERENCES
1. Ballschmiter, K. and M. Zell, Fresenius Z. Anal. Chem.. 302, 20,
1980.
2. DeKok, A., et al., Intern. J. Environ. Anal. Chem.. Vol. 11, pp.
17-41, 1982.
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," (29 CFR 1910), Occupational
Safety and Health Administration, OSHA 2206.
5. "Safety in Academic Chemistry Laboratories," American Chemical
Society Publication, Committee on Chemical Safety, 4th Edition,
1985.
6. 40 CFR Part 761.60; .65; .40; .45 40 CFR Part 761, Polychlorinated
Biphenyls (PCBs) Manufacturing, Processing, Distribution in
Commerce and Use Prohibitions.
7. Bellar, T.A. and Lichtenberg, 0. J., "Some Factors Affecting
Recovery of Polychlorinated Biphenyls from Water and Bottom
Samples," ASTH, STP 573, Water Quality Parameters, 1975.
215
-------
8. H. Steinwandter, Brune, H. Fresenius Z. Anal. Chem. 3H, 160,
1983.
9. H. Steinwandter, Fresenius Z. Anal. Chem. 317, 869-871, 1984.
10. Armour, J., JOAC. 56, 4, 987-993, 1973.
11. Glaser, J.A., D.L. Foerst, 6.D. McKee, S.A. Quave, and W.L. Budde,
"Trace Analyses for Wastewaters," Environ. Sci. Techno!. 15 1426,
1981.
216
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TABLE 1. DECACHLOROBIPHENYL EQUIVALENT OF
COMMON PCB SOURCES
Decachloro-
Comoound
2-Chlorobiphenyl
Aroclor 1221
Aroclcr 1232
Aroclor 1242
Aroclor 1016
Aroclor 1248
Aroclor 1254
Aroclor 1260
Dechlorobiphenyl
Congener
Number
1
N/A
N/A
N/A
N/A
N/A
N/A
N/A
209
Molecular
Uelaht3
188.5
188.5
223
257.5
257.5
292
326.4
361
499
Concentration
(ua/L)
0.19
0.19
0.23
0.26
0.26
0.30
0.33
0.36
0.50
D biphenyl
Eauivalentmc
263
263
217
192
192
167
152
139
100
? Values from (10).
b fig/I of various PCBs required to generate a value of 0.50 /ig/L DCB (assuming
100% method recovery).
c The decachlorobiphenyl produced by perchlorination will be this percentage
greater than the original concentration of the PCB/Aroclor listed.
217
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TABLE 2. CALIBRATION CURVE LINEARITY TEST
AND RETENTION DATA
Standard
Concentration
(na//zL)
0.01
0.01
0.08
0.08
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.25
0.25
1.0
1.0
Average RT =
SD =
Relative Standard Deviation =
Retention
Time
(min)
24.74
24.74
24.74
24.73
24.75
24.75
24.75
24.75
24.75
24.74
24.74
24.76
24.76
24.76
24.76
24.75
0.009
0.038%
Response
Factor
(area/nq)
48790
50650
48240
47260
48300
49550
51170
49160
43220
47490
47320
49960
48240
47230
48410
Average 48030
Standard
Deviation 2500
Relative
Standard 5.2%
Deviation
218
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TABLE 3. SPLITLESS CAPILLARY COLUMN SINGLE LABORATORY
ACCURACY AND PRECISION FOR FORTIFIED TAP WATER
Source of
PCBs
2-Chlorobiphenyl
Aroclor 1221
Aroclor 1232'
Aroclor 1242
Aroclor 1248
Aroclor 1254
Aroclor 1260
Biphenyld
MDLdD
ua/L
0.08
0.14
0.23
0.21
0.15
0.14
0.14
Concentration
fua/L)
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
Accuracy36
85; (96)b
99
124
82
136
122; (137)c
113; (96)b
109; (75)c
Precision36
RSD. (%)
5.0; (9.9)b
8.4
11.3
13.1
8.6
6.4; (7.6JJ
6.5; (6.9)b
4.8; (5.8)c
aData corrected for source water background. Average value over
study =0.11
bData collected by on-column capillary column GC.
C0ata collected by packed column GC.
^Potential method interference compound.
6Fortified matrix effect bias (See Sect. 9.3)
Splitless capillary column 103, 113
Packed column 93, 95
Splitless on-column (not performed)
219
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TABLE 4. SPLITLESS CAPILLARY COLUMN SINGLE LABORATORY ACCURACY
AND PRECISION FOR RAW SOURCE HATERS
Raw
Source
Water
Ohio River
Spring
Ohio River
Little Miami
River
Source
of PCBs
Aroclor
1221
1260
1221
1260
Concen-
tration
lUQ/L]
0.50
0.50
0.50
5.0
Extraction
Solvent
CH2C12
CH2C12
Hexane
Hexane
Source Water
Background
fUQ/L)
0.54
0.19
0.16
0.14
Accuracy
1%)
114
101
123
91
Precision
RSD 1%)
8.4
7.9
7.5
5.8
Ohio River
1260
5.0
Hexane
0.29
100
5.4
220
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METHOD 515.1. DETERMINATION OF CHLORINATED ACIDS IN HATER
BY GAS CHROMATOGRAPHY WITH AN ELECTRON CAPTURE DETECTOR
Revision 4.0
R.C. Dressman and J.J. Lichtenberg - EPA 600/4-81-053, Revision 1.0 (1981)
J.W. Hodgeson - Method 515, Revision 2.0 (1986)
T. Engels (Battelle Columbus Laboratories) - National Pesticide Survey Method 3
Revision 3.0 (1987)
R.L. Graves - Method 515.1, Revision 4.0 (1989)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
221
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METHOD 515.1
DETERMINATION OF CHLORINATED ACIDS IN HATER BY GAS
CHROHATOGRAPHY KITH AN ELECTRON CAPTURE DETECTOR
1. SCOPE AND APPLICATION
1.1 This is a gas chromatographic (GC) method applicable to the
determination of certain chlorinated acids in ground water and
finished drinking water.(1) The following compounds can be
determined by this method:
Chemical Abstract Services
Analvte Registry Number
Acifluorfen* 50594-66-6
Bentazon 25057-89-0
Chloramben* 133-90-4
2,4-D 94-75-7
Dalapon* 75-99-0
2,4-DB 94-82-6
OCPA acid metabolites(a)
Dicamba 1918-00-9
3,5-Dichlorobenzoic acid 51-36-5
Dichlorprop 120-36-5
Oinoseb 88-85-7
5-Hydroxydicamba 7600-50-2
4-Nitrophenol* 100-02-7
Pentachlorophenol (PCP) 87-86-5
Picloram 1918-02-1
2,4,5-T 93-76-5
2,4,5-TP 93-72-1
(a)DCPA monoacid and diacid metabolites Included in method scope;
DCPA diacid metabolite used for validation studies.
"These compounds are only qualitatively identified in the National
Pesticides Survey (NPS) Program. These compounds are not quantitated
because control over precision has not been accomplished.
1.2 This method may be applicable to the determination of salts and
esters of analyte acids. The form of each acid is not distinguished
by this method. Results are calculated and reported for each listed
analyte as the total free acid.
1.3 This method has been validated in a single laboratory and estimated
detection limits (EDLs) have been determined for the analytes above
(Sect.13). Observed detection limits may vary between ground waters,
depending upon the nature of interferences in the sample matrix and
the specific instrumentation used.
222
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1.4 This method is restricted to use by or under the supervision of
analysts experienced in the use of GC and in the interpretation of
gas chromatograms. Each analyst must demonstrate the ability to
generate acceptable results with this method using the procedure
described in Sect. 10.3.
1.5 Analytes that are not separated chromatographically i.e., which have
very similar retention times, cannot be individually identified and
measured in the same calibration mixture or water sample unless an
alternate technique for identification and quantitation exist (Sect.
11.8).
1.6 When this method is used to analyze unfamiliar samples for any or all
of the analytes above, analyte identifications must be confirmed by
at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 A measured volume of sample of approximately 1 L is adjusted to pH 12
with 6 N sodium hydroxide and shaken for 1 hr to hydrolyze
derivatives. Extraneous organic material is removed by a solvent
wash. The sample is acidified, and the chlorinated acids are
extracted with ethyl ether by shaking in a separatory funnel or
mechanical tumbling in a bottle. The acids are converted to their
methyl esters using diazomethane as the derivatizing agent. Excess
derivatizing reagent is removed, and the esters are determined by
capillary column/GC using an electron capture detector (ECD).
2.2 The method provides a Florisil cleanup procedure to aid in the
elimination of interferences that may be encountered.
3. DEFINITIONS
3.1 Internal standard -- A pure analyte(s) added to a solution in known
amount(s) and used to measure the relative responses of other method
analytes and surrogates that are components of the same solution.
The internal standard must be an analyte that is not a sample
component.
3.2 Surrogate analyte -- A pure analyte(s), which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in
known amount(s) before extraction and is measured with the same
procedures used to measure other sample components. The purpose of a
surrogate analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates (LD1 and LD2) -- Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample
collection, preservation, or storage procedures.
223
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3.4 Field duplicates (FD1 and FD2) -- Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.5 Laboratory reagent blank (LRB) -- An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
that are used with other samples. The LRB is used to determine if
method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.6 Field reagent blank (FRB) -- Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage, preservation
and all analytical procedures. The purpose of the FRB is to determine
if method analytes or other interferences are present in the field
environment.
3.7 Laboratory performance check solution (LPC) -- A solution of method
analytes, surrogate compounds, and internal standards used to
evaluate the performance of the instrument system with respect to a
defined set of method criteria.
3.8 Laboratory fortified blank (LFB) -- An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.9 Laboratory fortified sample matrix (LFM) -- An aliquot of an
environmental sample to which known quantities of the method analytes
are added in the laboratory. The LFM is analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.10 Stock standard solution -- A concentrated solution containing a
single certified standard that is a method analyte, or a concentrated
solution of a single analyte prepared in the laboratory with an
assayed reference compound. Stock standard solutions are used to
prepare primary dilution standards.
3.11 Primary dilution standard solution -- A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
224
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3.12 Calibration standard (CAL) -- a solution prepared from the primary
dilution standard solution and stock standard solutions of the
Internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.13 Quality control sample (QCS) -- a sample matrix containing method
analytes or a solution of method analytes in a water miscible solvent
which is used to fortify reagent water or environmental samples. The
QCS is obtained from a source external to the laboratory, and is used
to check laboratory performance with externally prepared test
materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware and other sample processing apparatus that lead
to discrete artifacts or elevated baselines in gas chromatograms.
All reagents and apparatus must be routinely demonstrated to be free
from interferences under the conditions of the analysis by running
laboratory reagent blanks as described in Sect. 10.2.
4.1.1 Glassware must be scrupulously cleaned.(2) Clean all glass-
ware as soon as possible after use by thoroughly rinsing with
the last solvent used in it. Follow by washing with hot water
and detergent and thorough rinsing with dilute acid, tap and
reagent water. Drain dry, and heat in an oven or muffle
furnace at 400°C for 1 hr. Do not heat volumetric ware.
Thermally stable materials such as PCBs might not be
eliminated by this treatment. Thorough rinsing with acetone
may be substituted for the heating. After drying and
cooling, seal and store glassware in a clean environment to
prevent any accumulation of dust or other contaminants.
Store inverted or capped with aluminum foil.
4.1.2 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.
WARNING: When a solvent is purified, stabilizers added by the
manufacturer are removed, thus potentially making the solvent
hazardous. Also, when a solvent is purified, preservatives
added by the manufacturer are removed, thus potentially
reducing the shelf-life.
4.2 The acid forms of the analytes are strong organic acids which react
readily with alkaline substances and can be lost during sample
preparation. Glassware and glass wool must be acid-rinsed with IN
hydrochloric acid and the sodium sulfate must be acidified with
sulfuric acid prior to use to avoid analyte losses due to adsorption.
225
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4.3 Organic acids and phenols, especially chlorinated compounds, cause
the most direct interference with the determination. Alkaline
hydrolysis and subsequent extraction of the basic sample removes many
chlorinated hydrocarbons and phthalate esters that might otherwise
interfere with the electron capture analysis.
4.4 Interferences by phthalate esters can pose a major problem in pesti-
cide analysis when using the ECD. These compounds generally appear
in the chromatogram as large peaks. Common flexible plastics contain
varying amounts of phthalates, that are easily extracted or leached
during laboratory operations. Cross contamination of clean glassware
routinely occurs when plastics are handled during extraction steps,
especially when solvent-wetted surfaces are handled. Interferences
from phthalates can best be minimized by avoiding the use of plastics
in the laboratory. Exhaustive purification of reagents and glassware
may be required to eliminate background phthalate contamination.(3,4)
4.5 Interfering contamination may occur when a sample containing low
concentrations of analytes is analyzed immediately following a sample
containing relatively high concentrations of analytes.
Between-sample rinsing of the sample syringe and associated equipment
with methyl-t-butyl-ether (MTBE) can minimize sample cross
contamination. After analysis of a sample containing high
concentrations of analytes, one or more injections of MTBE should be
made to ensure that accurate values are obtained for the next sample.
4.6 Matrix interferences may be caused by contaminants that are
coextracted from the sample. Also, note that all analytes listed in
the Scope and Application Section are not resolved from each other on
any one column, i.e., one analyte of interest may be an interferant
for another analyte of interest. The extent of matrix interferences
will vary considerably from source to source, depending upon the
water sampled. The procedures in Sect. 11 can be used to overcome
many of these interferences. Positive identifications should be
confirmed (Sect. 11.8).
4.7 It is important that samples and working standards be contained in
the same solvent. The solvent for working standards must be the same
as the final solvent used in sample preparation. If this is not the
case, chromatographic comparability of standards to sample may be
affected.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound must
be treated as a potential health hazard. Accordingly, exposure to
these chemicals must be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of
OSHA regulations regarding the safe handling of the chemicals
specified -n this method. A reference file of material safety data
sheets ?' ould also be made available to all personnel involved in the
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chemical analysis. Additional references to laboratory safety are
available and have been identified (6-8) for the information of the
analyst.
5.2 DIAZOMETHANE -- A toxic carcinogen which can explode under certain
conditions. The following precautions must be followed:
5.2.1 Use only a well ventilated hood --do not breath vapors.
5.2.2 Use a safety screen.
5.2.3 Use mechanical pipetting aides.
5.2.4 Do not heat above 90°C -- EXPLOSION may result.
5.2.5 Avoid grinding surfaces, ground glass joints, sleeve
bearings, glass stirrers -- EXPLOSION may result.
5.2.6 Store away from alkali metals -- EXPLOSION may result.
5.2.7 Solutions of diazomethane decompose rapidly in the presence
of solid materials such as copper powder, calcium chloride,
and boiling chips.
5.2.8 The diazomethane generation apparatus used in the
esterification procedures (Sect. 11.4 and 11.5) produces
micromolar amounts of diazomethane to minimize safety
hazards.
5.3 ETHYL ETHER -- Nanograde, redistilled in glass, if necessary.
5.3.1 Ethyl ether is an extremely flammable solvent. If a
mechanical device is used for sample extraction, the device
should be equipped with an explosion-proof motor and placed
in a hood to avoid possible damage and injury due to an
explosion.
5.3.2 Must be free of peroxides as indicated by EM Quant test
strips (available from Scientific Products Co., Cat. No.
PI 126-8, and other suppliers).
5.4 WARNING: When a solvent is purified, stabilizers added by the
manufacturer are removed, thus potentially making the solvent
hazardous.
APPARATUS AND EQUIPMENT (All specifications are suggested. Catalog
numbers are included for illustration only.)
6.1 SAMPLE BOTTLE -- Borosilicate, 1-L volume with graduations (Wheaton
Media/Lab bottle 219820 or equivalent), fitted with screw caps lined
with TFE-fluorocarbon. Protect samples from light. The container
must be washed and dried as described in Sect. 4.1.1 before use to
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minimize contamination. Cap liners are cut to fit from sheets
(Pierce Catalog No. 012736) and extracted with methanol overnight
prior to use.
6.2 GLASSWARE
6.2.1 Separatory funnel -- 2000-mL, with TFE-fluorocarbon stop-
cocks, ground glass or TFE-fluorocarbon stoppers.
6.2.2 Tumbler bottle -- 1.7-L (Wheaton Roller Culture Vessel or
equivalent), with TFE-fluorocarbon lined screw cap. Cap
liners are cut to fit from sheets {Pierce Catalog No. 012736)
and extracted with methanol overnight prior to use.
6.2.3 Concentrator tube, Kuderna-Danish (K-D) -- 10- or 25-mL,
graduated (Kontes K-570050-2525 or Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes
employed in the test. Ground glass stoppers are used to
prevent evaporation of extracts.
6.2.4 Evaporative flask, K-D -- 500-mL (Kontes K-570001-0500 or
equivalent). Attach to concentrator tube with springs.
6.2.5 Snyder column, K-D -- three-ball macro (Kontes K-503000-0121
or equivalent).
6.2.6 Snyder column, K-D -- two-ball micro (Kontes K-569001-0219 or
equivalent).
6.2.7 Flask, round-bottom -- 500-mL with 24/40 ground glass joint.
6.2.8 Vials -- glass, 5- to 10-mL capacity with TFE-fluorocarbon
lined screw cap.
6.2.9 Disposable pipets -- sterile plugged borosilicate glass, 5-mL
capacity (Corning 7078-5N or equivalent).
6.3 SEPARATORY FUNNEL SHAKER -- Capable of holding 2-L separatory funnels
and shaking them with rocking motion to achieve thorough mixing of
separatory funnel contents (available from Eberbach Co. in Ann Arbor,
MI or other suppliers).
6.4 TUMBLER -- Capable of holding tumbler bottles and tumbling them
end-over-end at 30 turns/min (Associated Design and Mfg. Co.,
Alexandria, VA and other suppliers).
6.5 BOILING STONES -- Teflon, Chemware (Norton Performance Plastics No.
015021 and other suppliers).
6.6 WATER BATH -- Heated, capable of temperature control (± 2°C). The
bath should be used in a hood.
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6.7 BALANCE -- Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.8 DIAZOMETHANE GENERATOR -- Assemble from two 20 x 150 mm test tubes,
two Neoprene rubber stoppers, and a source of nitrogen as shown in
Figure 1 (available from Aldrich Chemical Co.). When esterification
is performed using diazomethane solution, the diazomethane collector
is cooled in an approximately 2-1 thermos for ice bath or a
cryogenically cooled vessel (Thermoelectrics Unlimited Model SK-12 or
equivalent).
6.9 GLASS WOOL -- Acid washed (Supelco 2-0383 or equivalent) and heated
at 450eC for 4 hr.
6.10 GAS CHROMATOGRAPH -- Analytical system complete with temperature
programmable GC suitable for use with capillary columns and all
required accessories including syringes, analytical columns, gases,
detector and stripchart recorder. A data system is recommended for
measuring peak areas. Table 1 lists retention times observed for
method analytes using the columns and analytical conditions described
below.
6.10.1 Column 1 (Primary column) -- 30 m long x 0.25 mm I.D. DB-5
bonded fused silica column, 0.25 tm film thickness (J&W
Scientific). Helium carrier gas flow is established at 30
cm/sec linear velocity and oven temperature is programmed
from 60°C to 300'C at 4°C/min. Data presented in this method
were obtained using this column. The injection volume was
2 0L splitless mode with 45 second delay. The injector
temperature was 250°C and the detector was 320°C. Alterna-
tive columns may be used in accordance with the provisions
described in Sect. 10.2.
6.10.2 Column 2 (Confirmation column) -- 30 m long x 0.25 mm I.D.
DB-1701 bonded fused silica column, 0.25 urn film thickness
(J&W Scientific). Helium carrier gas flow is established at
30 cm/sec linear velocity and oven temperature is programmed
from 60°C to 300'C at 4'C/min.
6.10.3 Detector -- Electron capture. This detector has proven
effective in the analysis of fortified reagent and artificial
ground waters. An ECO was used to generate the validation
data presented in this method. Alternative detectors,
including a mass spectrometer, may be used in accordance with
the provisions described in Sect. 10.3.
REAGENTS AND CONSUMABLE MATERIALS - WARNING: When a solvent is purified,
stabilizers added by the manufacturer are removed, thus potentially making
the solvent hazardous. Also, when a solvent is purified, preservatives
added by the manufacturer are removed, thus potentially reducing the
shelf-life.
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7.1 ACETONE, METHANOL, METHYLENE CHLORIDE, MTBE -- Pesticide quality
equivalent.
7.2 ETHYL ETHER, UNPRESERVED -- Nanograde, redistilled in glass if
necessary. Must be free of peroxides as indicated by EM Quant test
strips (available from Scientific Products Co., Cat. No. PI126-8, and
other suppliers). Procedures recommended for removal of peroxides
are provided with the test strips.
7.3 SODIUM SULFATE, GRANULAR, ANHYDROUS, ACS GRADE -- Heat treat in a
shallow tray at 450°C for a minimum of 4 hr to remove interfering
organic substances. Acidify by slurrying 100 g sodium sulfate with
enough ethyl ether to just cover the solid. Add 0.1 mL concentrated
sulfuric acid and mix thoroughly. Remove the ether under vacuum.
Mix 1 g of the resulting solid with 5 mL of reagent water and measure
the pH of the mixture. The pH must be below pH 4. Store at 130°C.
7.4 SODIUM THIOSULFATE, GRANULAR, ANHYDROUS -- ACS grade.
7.5 SODIUM HYDROXIDE (NAOH), PELLETS -- ACS grade.
7.5.1 NaOH, 6 N -- Dissolve 216 g NaOH in 900 mL reagent water.
7.6 SULFURIC ACID, CONCENTRATED -- ACS grade,sp. gr. 1.84.
7.6.1 Sulfuric acid, 12 N -- Slowly add 335 mL concentrated
sulfuric acid to 665 mL of reagent water.
7.7 POTASSIUM HYDROXIDE (KOH), PELLETS -- ACS grade.
7.7.1 KOH, 37% (w/v) -- Dissolve 37 g KOH pellets in reagent water
and dilute to 100 mL.
7.8 CARBITOL (DIETHYLENE GLYCOL MONOETHYL ETHER) -- ACS grade. Available
from Aldrich Chemical Co.
7.9 DIAZALD, ACS grade -- available from Aldrich Chemical Co.
7.10 DIAZALD SOLUTION -- Prepare a solution containing 10 g Diazald in 100
mL of a 50:50 by volume mixture of ethyl ether and carbitol. This
solution is stable for one month or longer when stored at 4°C in an
amber bottle with a Teflon-lined screw cap.
7.11 SODIUM CHLORIDE (NACL), CRYSTAL, ACS GRADE -- Heat treat in a shallow
tray at 450°C for a minimum of 4 hr to remove interfering organic
substances.
7.12 4,4'-DIBROMOOCTAFLUOROBIPHENYL (DBOB) -- 99% purity, for use as
internal standard (available from Aldrich Chemical Co).
7.13 2,4-DICHLOROPHENYLACETIC ACID (DCAA) -- 99% purity, for use as
surrogate standard (available from Aldrich Chemical Co).
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7.14 MERCURIC CHLORIDE - ACS grade (Aldrich Chemical Co.) - for use as a
bacterloclde. If any other bactericide can be shown to work as well
as mercuric chloride, it may be used instead.
7.15 REAGENT WATER -- Reagent water is defined as water that is reasonably
free of contamination that would prevent the determination of any
analyte of interest. Reagent water used to generate the validation
data in this method was distilled water obtained from the Magnetic
Springs Water Co., Columbus, Ohio.
7.16 SILICIC ACID, ACS GRADE.
7.17 FLORISIL -- 60-100/PR mesh (Sigma No. F-9127). Activate by heating
in a shallow container at 150°C for at least 24 and not more than 48
hr.
7.18 STOCK STANDARD SOLUTIONS (1.00 /tgM) -- Stock standard solutions may
be purchased as certified solutions or prepared from pure standard
materials using the following procedure:
7.18.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material in MTBE and dilute to volume in a 10-mL volumetric
flask. Larger volumes may be used at the convenience of the
analyst. If compound purity is certified at 96% 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.
7.18.2 Transfer the stock standard solutions into TFE-fluoro-
carbon-sealed screw cap amber vials. Store at room tempera-
ture and protect from light.
7.18.3 Stock standard solutions should be replaced after two months
or sooner if comparison with laboratory fortified blanks, or
QC samples indicate a problem.
7.19 INTERNAL STANDARD SOLUTION -- Prepare an internal standard solution
by accurately weighing approximately 0.0010 g of pure DBOB.
Dissolve the DBOB in MTBE and dilute to volume in a 10-mL volumetric
flask. Transfer the internal standard solution to a TFE-fluoro-
carbon-sealed screw cap bottle and store at room temperature.
Addition of 25 /iL of the internal standard solution to 10 mL of
sample extract results in a final internal standard concentration of
0.25 itg/mL. Solution should be replaced when ongoing QC (Sect. 10)
indicates a problem. Note that DBOB has been shown to be an
effective internal standard for the method analytes(l), but other
compounds may be used if the quality control requirements in Sect. 10
are met.
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7.20 SURROGATE STANDARD SOLUTION -- Prepare a surrogate standard solut"1
by accurately weighing approximately 0.0010 g of pure DCAA.
Dissolve the DCAA in MTBE and dilute to volume in a 10-mL volumetric
flask. Transfer the surrogate standard solution to a TFE-fluoro-
carbon-sealed screw cap bottle and store at room temperature.
Addition of 50 /iL of the surrogate standard solution to a 1-L sample
prior to extraction results in a surrogate standard concentration in
the sample of 5 /ig/L and, assuming quantitative recovery of DCAA, a
surrogate standard concentration in the final extract of 0.5 /ig/mL.
Solution should be replaced when ongoing QC (Sect. 10) indicates a
problem. Note DCAA has been shown to be an effective surrogate
standard for the method analytes(l), but other compounds may be used
if the quality control requirements in Sect. 10.4 are met.
7.21 LABORATORY PERFORMANCE CHECK SOLUTIONS -- Prepare a diluted dinoseb
solution by adding 10 /iL of the 1.0 /ig//iL dinoseb stock solution to
the MTBE and diluting to volume in a 10-mL volumetric flask. To
prepare the check solution, add 40 /iL of the diluted dinoseb
solution, 16 /iL of the 4-nitrophenol stock solution, 6 /iL of the 3,5-
dichlorobenzoic acid stock solution, 50 /iL of the surrogate standard
solution, 25 /iL of the internal standard solution, and 250 /iL of
methanol to a 5-mL volumetric flask and dilute to volume with MTBE.
Methylate sample as described in Sects. 11.4 or 11.5. Dilute the
sample to 10 mL in MTBE. Transfer to a TFE-fluorocarbon-sealed screw
cap bottle and store at room temperature. Solution should be
replaced when ongoing QC (Sect. 10) indicates a problem.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices (8) should be followed; however, the bottle must
not be prerinsed with sample before collection.
8.2 SAMPLE PRESERVATION AND STORAGE
8.2.1 Add mercuric chloride (See 7.14) to the sample bottle in
amounts to produce a concentration of 10 mg/L. Add 1 mL of a
10 mg/mL solution of mercuric chloride in water to the sample
bottle at the sampling site or in the laboratory before
shipping to the sampling site. A major disadvantage of
mercuric chloride is that it is a highly toxic chemical;
mercuric chloride must be handled with caution, and samples
containing mercuric chloride must be disposed of properly.
8.2.2 If residual chlorine is present, add 80 mg of sodium
thiosulfate per liter of sample to the sample bottle prior to
collecting the sample.
8.2.3 After the sample is collected in the bottle containing
preservative(s), seal the bottle and shake vigorously for
1 min.
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8.2.4 The samples must be iced or refrigerated at 4°C away from
light from the time of collection until extraction.
Preservation study results indicate that the analytes
(measured as total acid) present in samples are stable for 14
days when stored under these conditions.(1) However, analyte
stability may be affected by the matrix; therefore, the
analyst should verify that the preservation technique is
applicable to the samples under study.
8.3 EXTRACT STORAGE
8.3.1 Extracts should be stored at 4°C away from light.
Preservation study results indicate that most analytes are
stable for 28 days(l); however, the analyst should verify
appropriate extract holding times applicable to the samples
under study.
9. CALIBRATION
9.1 Establish GC operating parameters equivalent to those indicated in
Sect. 6.10. The GC system may be calibrated using either the
internal standard technique (Sect. 9.2) or the external standard
technique (Sect. 9.3). NOTE: Calibration standard solutions must be
prepared such that no unresolved analytes are mixed together.
9.2 INTERNAL STANDARD CALIBRATION PROCEDURE -- To use this approach, the
analyst must select one or more internal standards compatible in
analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard is
not affected by method or matrix interferences. DBOB has been
identified as a suitable internal standard.
9.2.1 Prepare calibration standards at a minimum of three (recommend
five) concentration levels for each analyte of interest by
adding volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant
amount of one or more of the internal standards and 250 pL
methanol, and dilute to volume with MTBE. Esterify acids with
diazomethane as described in Sect. 11.4 or 11.5. The lowest
standard should represent analyte concentrations near, but
above, the respective EDLs. The remaining standards should
bracket the analyte concentrations expected in the sample
extracts, or should define the working range of the detector.
9.2.2 Analyze each calibration standard according to the procedure
(Sect. 11.7). Tabulate response (peak height or area)
against concentration for each compound and internal
standard. Calculate the response factor (RF) for each
analyte and surrogate using Equation 1.
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(As) (cis)
RF = Equation 1
(Au) (Cs)
where:
As = Response for the analyte to be measured.
ATs = Response for the internal standard.
Cis = Concentration of the internal standard (/ig/L).
Cs = Concentration of the analyte to be measured (/tg/L).
9.2.3. If the RF value over the working range is constant (20% RSD or
less) the average RF can be used for calculations. Alterna-
tively, the results can be used to plot a calibration curve
of response ratios (AS/A,S) vs. Cs.
9.2.4 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any analyte varies from the
predicted response by more than ±20%, the test must be
repeated using a fresh calibration standard. If the repeti-
tion also fails, a new calibration curve must be generated for
that analyte using freshly prepared standards.
9.3.5 Single point calibration is a viable alternative to a
calibration curve. Prepare single point standards from the
secondary dilution standards in MTBE. The single point
standards should be prepared at a concentration that produces
a response that deviates from the sample extract response by
no more than 20%.
9.2.6 Verify calibration standards periodically, recommend at least
quarterly, by analyzing a standard prepared from reference
material obtained from an independent source. Results from
these analyses must be within the limits used to routinely
check calibration.
9.3 EXTERNAL STANDARD CALIBRATION PROCEDURE
9.3.1 Prepare calibration standards at a minimum of three
(recommend five) concentration levels for each analyte of
interest and surrogate compound by adding volumes of one or
more stock standards and 250 /*L methanol to a volumetric
flask. Dilute to volume with MTBE. Esterify acids with
diazomethane as described in Sect. 11.4 or 11.5. The best
standard should represent analyte concentrations near, but
above, the respective EDL. The remaining standards should
bracket the analyte concentrations expected in the sample
extracts, or should define the working range of the detector.
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9.3.2 Starting with the standard of lowest concentration, analyze
each calibration standard according to Sect. 11.7 and tabulate
response (peak height or area) versus the concentration in the
standard. The results can be used to prepare a calibration
curve for each compound. Alternatively, if the ratio of
response to concentration (calibration factor) is a constant
over the working range (20% RSD or less), linearity through
the origin can be assumed and the average ratio or calibration
factor can be used in place of a calibration curve.
9.3.3 The working calibration curve or calibration factor must be
verified on each working day by the measurement of a minimum
of two calibration check standards, one at the beginning and
one at the end of the analysis day. These check standards
should be at two different concentration levels to verify the
calibration curve. For extended periods of analysis (greater
than 8 hr), it is strongly recommended that check standards
be interspersed with samples at regular intervals during the
course of the analyses. If the response for any analyte
varies from the predicted response by more than + 20%, the
test must be repeated using a fresh calibration standard. If
the results still do not agree, generate a new calibration
curve or use a single point calibration standard as described
in Sect. 9.3.3.
9.3.4 Single point calibration is a viable alternative to a
calibration curve. Prepare single point standards from the
secondary dilution standards in MTBE. The single point
standards should be prepared at a concentration that produces
a response that deviates from the sample extract response by
no more than 20%.
9.2.5 Verify calibration standards periodically, recommend at least
quarterly, by analyzing a standard prepared from reference
material obtained from an independent source. Results from
these analyses must be within the limits used to routinely
check calibration.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration
of laboratory capability, determination of surrogate compound
recoveries in each sample and blank, monitoring internal standard
peak area or height in each sample and blank (when internal standard
calibration procedures are being employed), analysis of laboratory
reagent blanks, laboratory fortified samples, laboratory fortified
blanks, and QC samples.
10.2 LABORATORY REAGENT BLANKS (LRB). Before processing any samples, the
analyst must demonstrate that all glassware and reagent
interferences are under control. Each time a set of samples is
extracted or reagents are changed, a LRB must be analyzed. If within
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the retention time window of any analyte the LRB produces a peak '"at
would prevent the determination of that analyte, determine the i :e
of contamination and eliminate the interference before processing
samples.
10.3 Initial Demonstration of Capability.
10.3.1 Select a representative fortified concentration (about 10
times EDL) for each analyte. Prepare a sample concentrate
(in methanol) containing each analyte at 1000 times selected
concentration. With a syringe, add 1 ml of the concentrate to
each of at least four 1-L aliquots of reagent water, and
analyze each aliquot according to procedures beginning in
Sect. 11.
10.3.2 For each analyte the recovery value for all four of these
samples must fall in the range of R ± 30% (or within R + 3SR
if broader) using the values for R and SR for reagent water in
Table 2. For those compounds that meet the acceptable
criteria, performance is considered acceptable and sample
analysis may begin. For those compounds that fail these
criteria, this procedure must be reported using five fresh
samples until satisfactory performance has been demonstrated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples via a
new, unfamiliar method prior to obtaining some experience
with it. It is expected that as laboratory personnel gain
experience with this method the quality of data will improve
beyond those required here.
10.4 The analyst is permitted to modify GC columns, GC conditions,
detectors, continuous extraction techniques, concentration
techniques (i.e., evaporation techniques), internal standard or
surrogate compounds. Each time such method modifications are made,
the analyst must repeat the procedures in Sect. 10.3
10.5 ASSESSING SURROGATE RECOVERY.
10.5.1 When surrogate recovery from a sample or method blank is <70%
or >130%, check (1) calculations to locate possible errors,
(2) spiking solutions for degradation, (3) contamination, and
(4) instrument performance. If those steps do not reveal the
cause of the problem, reanalyze the extract.
10.5.2 If a blank extract reanalysis fails the 70-130% recovery
criterion, the problem must be identified and corrected
before continuing.
10.5.3 If sample extract reanalysis meets the surrogate recovery
criterion, report only data for the analyzed extract. If
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sample extract continues to fail the recovery criterion,
report all data for that sample as suspect.
10.6 ASSESSING THE INTERNAL STANDARD
10.6.1 When using the internal standard calibration procedure, the
analyst is expected to monitor the IS response (peak area or
peak height) of all samples during each analysis day. The IS
response for any sample chromatogram should not deviate from
the daily calibration check standard's IS response by more
than 30%.
10.6.2 If >30% deviation occurs with an individual extract, optimize
instrument performance and inject a second aliquot of that
extract.
10.6.2.1 If the reinjected aliquot produces an acceptable
internal standard response, report results for that
aliquot.
10.6.2.2 If a deviation of greater than 30% 1s obtained for
the reinjected extract, analysis of the samples
should be repeated beginning with Sect. 11,
provided the sample 1s still available. Otherwise,
report results obtained from the reinjected
extract, but annotate as suspect.
10.6.3 If consecutive samples fall the IS response acceptance
criterion, Immediately analyze a calibration check standard.
10.6.3.1 If the check standard provides a response factor
(RF) within 20% of the predicted value, then follow
procedures itemized in Sect. 10.6.2 for each sample
failing the IS response criterion.
10.6.3.2 If the check standard provides a response factor
which deviates more than 20% of the predicted
value, then the analyst must recalibrate, as
specified in Sect. 9.
10.7 ASSESSING LABORATORY PERFORMANCE - LABORATORY FORTIFIED BLANK
10.7.1 The laboratory must analyze at least one laboratory fortified
blank (LFB) sample with every 20 samples or one per sample set
(all samples extracted within a 24-hr period) whichever is
greater. The concentration of each analyte in the LFB should
be 10 times EDL or the MCL, whichever is less. Calculate
accuracy as percent recovery (Xi). If the recovery of any
analyte falls outside the control limits (see Sect. 10.7.2),
that analyte is judged out of control, and the source of the
problem should be identified and resolved before continuing
analyses.
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10.7.2 Until sufficient data become available from within their own
laboratory, usually a minimum of results from 20 to 30
analyses, the laboratory should assess laboratory performance
against the control limits in Sect. 10.3.2 that are derived
from the data in Table 2. When sufficient internal
performance data becomes available, develop control limits
from the mean percent recovery (X) and standard deviation (S)
of the percent recovery. These data are used to establish
upper and lower control limits as follows:
UPPER CONTROL LIMIT = X + 3S
LOWER CONTROL LIMIT = X - 3S
After each five to ten new recovery measurements, new control
limits should be calculated using only the most recent 20-30
data points. These calculated control limits should never
exceed those established in Section 10.3.2.
10.7.3 It is recommended that the laboratory periodically determine
and document its detection limit capabilities for the
analytes of interest.
10.7.4 At least quarterly, analyze a QC sample from an outside
source.
10.7.5 Laboratories are encouraged to participate in external
performance evaluation studies such as the laboratory
certification programs offered by many states or the studies
conducted by USEPA. Performance evaluation studies serve as
independent checks on the analyst's performance.
10.8 ASSESSING ANALYTE RECOVERY - LABORATORY FORTIFIED SAMPLE MATRIX
10.8.1 The laboratory must add a known concentration to a minimum of
10% of the routine samples or one sample concentration per
set, whichever is greater. The concentration should not be
less then the background concentration of the sample selected
for fortification. Ideally, the concentration should be the
same as that used for the laboratory fortified blank (Sect.
10.7). Over time, samples from all routine sample sources
should be fortified.
10.8.2 Calculate the percent recovery, P of the concentration for each
analyte, after correcting the analytical result, X, from the
fortified sample for the background concentration, b, measured
in the unfortified sample, i.e.,:
P = 100 (X - b) / fortifying concentration,
and compare these values to control limits appropriate for
reagent water data collected in the same fashion. If the
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analyzed unfortified sample is found to contain NO background
concentrations, and the added concentrations are those
specified in Sect. 10.7, then the appropriate control limits
would be the acceptance limits in Sect. 10.7. If, on the other
hand, the analyzed unfortified sample is found to contain
background concentration, b, estimate the standard deviation at
the background concentration, s^, using regressions or
comparable background data and, similarly, estimate the mean,
Xa and standard deviation, sa, of analytical results at the
total concentration after fortifying. Then the appropriate
percentage control limits would be P ± 3sp , where:
P = 100 X / (b + fortifying concentration)
2 2 1/2
and Sp = 100 (s + s. ) /fortifying concentration
For example, if the background concentration for Analyte A was
found to be 1 /ig/L and the added amount was also 1 /*g/L, and
upon analysis the laboratory fortified sample measured 1.6 /i/L,
then the calculated P for this sample would be (1.6 /ig/L minus
1.0 /ig/L)/l /ig/L or 60%. This calculated P is compared to
control limits derived from prior reagent water data. Assume
it is known that analysis of an interference free sample at 1
/ig/L yields an s of 0.12 /ig/L and similar analysis at 2.0 /ig/L
yields X and s of 2.01 /ig/L and 0.20 /ig/L, respectively. The
appropriate limits to judge the reasonableness of the percent
recovery, 60%, obtained on the fortified matrix sample is
computed as follows:
[100 (2.01 /ig/L) / 2.0 /ig/L]
9 9 1/2
± 3 (100) [(0.12 /ig/L)2 + (0.20 /tg/L)2] / 1.0 /ig/L =
100.5% ± 300 (0.233) =
100.5% ± 70% or 30% to 170% recovery of the added analyte.
10.8.3 If the recovery of any such analyte falls outside the
designated range, and the laboratory performance for that
analyte is shown to be in control (Sect. 10.7), the recovery
problem encountered with the fortified sample is judged to be
matrix related, not system related. The result for that
analyte in the unfortified sample is labeled suspect/matrix to
inform the data user that the results are suspect due to
matrix effects.
10.9 ASSESSING INSTRUMENT SYSTEM - LABORATORY PERFORMANCE CHECK SAMPLE -
Instrument performance should be monitored on a daily basis by
analysis of the LPC sample. The LPC sample contains compounds
designed to indicate appropriate instrument sensitivity, column
performance (primary column) and chromatographic performance. LPC
sample components and performance criteria are listed in Table 3.
239
-------
Inability to demonstrate acceptable instrument performance indicates
the need for revaluation of the instrument system. The sensitivity
requirements are set based on the EDLs published in this method. If
laboratory EDLs differ from those listed in this method, concentrations
of the instrument QC standard compounds must be adjusted to be
compatible with the laboratory EDLs.
10.10 The laboratory may adopt additional quality control practices for use
with this method. The specific practices that are most productive
depend upon the needs of the laboratory and the nature of the samples.
For example, field or laboratory duplicates may be analyzed to assess
the precision of the environmental measurements or field reagent blanks
may be used to assess contamination of samples under site conditions,
transportation and storage.
11. PROCEDURE
11.1 MANUAL HYDROLYSIS, PREPARATION, AND EXTRACTION.
11.1.1 Add preservative to blanks and QC check standards. Mark the
water meniscus on the side of the sample bottle for later deter-
mination of sample volume (Sect. 11.1.9). Pour the entire
sample into a 2-L separatory funnel. Fortify sample with 50 pi*
of the surrogate standard solution.
11.1.2 Add 250 g NaCl to the sample, seal, and shake to dissolve salt.
11.1.3 Add 17 mL of 6 N NaOH to the sample, seal, and shake. Check
the pH of the sample with pH paper; if the sample does not have
a pH greater than or equal to 12, adjust the pH by adding more
6 N NaOH. Let the sample sit at room temperature for 1 hr,
shaking the separatory funnel and contents periodically.
11.1.4 Add 60 mL methylene chloride to the sample bottle to rinse the
bottle, transfer the methylene chloride to the separatory
funnel and extract the sample by vigorously shaking the funnel
for 2 min with periodic venting to release excess pressure.
Allow the organic layer to separate from the water phase for a
minimum of 10 min. If the emulsion interface between layers is
more than one-third the volume of the solvent layer, the
analyst must employ mechanical techniques to complete the phase
separation. The optimum technique depends upon the sample, but
may include stirring, filtration through glass wool,
centrifugation, or other physical methods. Discard the
methylene chloride phase.
11.1.5 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time,
discarding the methylene chloride layer. Perform a third
extraction in the same manner.
240
-------
11.1.6 Add 17 ml of 12 N ^04 to the sample, seal, and shake to mix.
Check the pH of the sample with pH paper; If the sample does
not have a pH less than or equal to 2, adjust the pH by adding
more 12 N
11.1.7 Add 120 ml ethyl ether to the sample, seal, and extract the
sample by vigorously shaking the funnel for 2 min with periodic
venting to release excess pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 min. If the
emulsion interface between layers is more than one third the
volume of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring,
filtration through glass wool, centrifugation, or other physical
methods. Remove the aqueous phase to a 2-L Erlenmeyer flask and
collect the ethyl ether phase in a 500-mL round-bottom flask
containing approximately 10 g of acidified anhydrous sodium
sulfate. Periodically, vigorously shake the sample and drying
agent. Allow the extract to remain in contact with the sodium
sulfate for approximately 2 hours.
11.1.8 Return the aqueous phase to the separatory funnel, add a 60-mL
volume of ethyl ether to the sample, and repeat the extraction
procedure a second time, combining the extracts in the 500-mL
erlenmeyer flask. Perform a third extraction with 60 ml of
ethyl ether in the same manner.
11.1.9 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the water to a 1000 -ml
graduated cylinder. Record the sample volume to the nearest
5 ml.
11.2 AUTOMATED HYDROLYSIS, PREPARATION, AND EXTRACTION. -- Data presented in
this method were generated using the automated extraction procedure
with the mechanical separatory funnel shaker.
11.2.1 Add preservative (Sect. 8.2) to any samples not previously
preserved, e.g., blanks and QC check standards. Mark the water
meniscus on the side of the sample bottle for later deter-
mination of sample volume (Sect. 11.2.9). Fortify sample with
50 nl of the surrogate standard solution. If the mechanical
separatory funnel shaker is used, pour the entire sample into a
2-L separatory funnel. If the mechanical tumbler is used, pour
the entire sample into a tumbler bottle.
11.2.2 Add 250 g NaCl to the sample, seal, and shake to dissolve salt.
11.2.3 Add 17 mL of 6 N NaOH to the sample, seal, and shake. Check
the pH of the sample with pH paper; if the sample does not have
a pH greater than or equal to 12, adjust the pH by adding more
6 N NaOH. Shake sample for 1 hr using the appropriate
mechanical mixing device.
241
-------
11.2.4 Add 300 ml methylene chloride to the sample bottle to rinse ,.,e
bottle, transfer the methylene chloride to the separatory
funnel or tumbler bottle, seal, and shake for 10 s, venting
periodically. Repeat shaking and venting until pressure
release is not observed during venting. Reseal and place
sample container in appropriate mechanical mixing device.
Shake or tumble the sample for 1 hr. Complete and thorough
mixing of the organic and aqueous phases should be observed at
least 2 min after starting the mixing device.
11.2.5 Remove the sample container from the mixing device. If the
tumbler is used, pour contents of tumbler bottle into a 2-L
separatory funnel. Allow the organic layer to separate from
the water phase for a minimum of 10 min. If the emulsion
interface between layers is more than one third the volume of
the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring,
filtration through glass wool, centrifugation, or other
physical methods. Drain and discard the organic phase. If the
tumbler is used, return the aqueous phase to the tumbler
bottle.
11.2.6 Add 17 mL of 12 N H2S04 to the sample, seal, and shake to mix.
Check the pH of the sample with pK paper; if the sample does
not have a pH less than or equal to 2, adjust the pH by adding
more 12 N H2S04. '
11.2.7 Add 300 mi ethyl ether to the sample, seal, and shake for 10 s,
venting periodically. Repeat shaking and venting until
pressure release is not observed during venting. Reseal and
place sample container in appropriate mechanical mixing device.
Shake or tumble sample for 1 hr. Complete and thorough mixing
of the organic and aqueous phases should be observed at least 2
min after starting the mixing device.
11.2.8 Remove the sample container from the mixing device. If the
tumbler is used, pour contents of tumbler bottle into a 2-L
separatory funnel. Allow the organic layer to separate from
the water phase for a minimum of 10 min. If the emulsion
interface between layers is more than one third the volume of
the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring,
filtration through glass wool, centrifugation, or other
physical methods. Drain and discard the aqueous phase.
Collect the extract in a 500-ml round-bottom flask containing
about 10 g of acidified anhydrous sodium sulfate. Periodically
vigorously shake the sample and drying agent. Allow the
extract to remain in contact with the sodium sulfate for
approximately 2 hr.
242
-------
11.2.9 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the water to a 1000-ml
graduated cylinder. Record the sample volume to the nearest
5 ml.
11.3 EXTRACT CONCENTRATION
11.3.1 Assemble a K-0 concentrator by attaching a concentrator tube to
a 500-mL evaporative flask.
11.3.2 Pour the dried extract through a funnel plugged with acid
washed glass wool, and collect the extract in the K-D
concentrator. Use a glass rod to crush any caked sodium
sulfate during the transfer. Rinse the round-bottom flask and
funnel with 20 to 30 ml of ethyl ether to complete the
quantitative transfer.
11.3.3 Add 1 to 2 clean boiling stones to the evaporative flask and
attach a macro Snyder column. Prewet the Snyder column by
adding about 1 ml ethyl ether to the top. Place the K-D
apparatus on a hot water bath, 60 to 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 with
hot vapor. 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 1 ml, remove the K-D
apparatus and allow it to drain and cool for at least 10 min.
11.3.4 Remove the Snyder column and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 ml of ethyl ether.
Add 2 ml of MTBE and a fresh boiling stone. Attach a
micro-Snyder column to the concentrator tube and prewet the
column by adding about 0.5 ml of ethyl ether to the top. Place
the micro 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 in 5 to 10
min. When the apparent volume of liquid reaches 0.5 ml, remove
the micro K-D from the bath and allow it to drain and cool.
Remove the micro Snyder column and add 250 /zL of methanol. If
the gaseous diazomethane procedure (Sect. 11.4) is used for
esterification of pesticides, rinse the walls of the concen-
trator tube while adjusting the volume to 5.0 ml with MTBE. If
the pesticides will be esterified using the diazomethane
solution (Sect. 11.5), rinse the walls of the concentrator tube
while adjusting the volume to 4.5 ml with MTBE.
11.4 ESTERIFICATION OF ACIDS USING GASEOUS DIAZOMETHANE -- Results presented
in this method were generated using the gaseous diazomethane
derivatization procedure. See Section 11.5 for an alternative
procedure.
243
-------
11.4.1 Assemble the diazomethane generator (Figure 1) in a hood.
11.4.2 Add 5 mL of ethyl ether to Tube 1. Add 1 ml of ethyl ether, 1
ml of carbitol, 1.5 mL of 37% aqueous KOH, and 0.2 grams
Diazald to Tube 2. Immediately place the exit tube into the
concentrator tube containing the sample extract. Apply
nitrogen flow (10 mL/min) to bubble diazomethane through the
extract for 1 min. Remove first sample. Rinse the tip of the
diazomethane generator with ethyl ether after methyl at ion of
each sample. Bubble diazomethane through the second sample
extract for 1 min. Diazomethane reaction mixture should be
used to esterify only two samples; prepare new reaction mixture
in Tube 2 to esterify each two additional samples. Samples
should turn yellow after addition of diazomethane and remain
yellow for at least 2 m1n. Repeat methyl ation procedure if
necessary.
11.4.3 Seal concentrator tubes with stoppers. Store at room tempera-
ture 1n a hood for 30 min.
11.4.4 Destroy any unreacted diazomethane by adding 0.1 to 0.2 grams
silicic add to the concentrator tubes. Allow to stand until
the evolution of nitrogen gas has stopped (approximately 20
m1n). Adjust the sample volume to 5.0 ml with MTBE.
11.5 ESTERIFICATION OF ACIDS USING DIAZOMETHANE SOLUTION -- Alternative
procedure.
11.5.1 Assemble the diazomethane generator (Figure 2) in a hood. The
collection vessel is a 10- or 15-mL vial, equipped with a
Teflon-lined screw cap and maintained at 0-5C.
11.5.2 Add a sufficient amount of ethyl ether to tube 1 to cover the
first impinger. Add 5 ml of MTBE to the collection vial. Set
the nitrogen flow at 5-10 mL/min. Add 2 mL Diazald solution
(Sect. 7.10} and 1.5 ml of 37% KOH solution to the second
impinger. Connect the tubing as shown and allow the nitrogen
flow to purge the diazomethane from the reaction vessel into
the collection vial for 30 min. Cap the vial when collection
is complete and maintain at 0-5'C. When stored at 0-5'C this
diazomethane solution may be used over a period of 48 hr.
11.5.3 To each concentrator tube containing sample or standard, add
0.5 mL diazomethane solution. Samples should turn yellow after
addition of the diazomethane solution and remain yellow for at
least 2 min. Repeat methylation procedure if necessary.
11.5.4 Seal concentrator tubes with stoppers. Store at room tempera-
ture in a hood for 30 min.
244
-------
11.5.5 Destroy any unreacted diazomethane by adding 0.1 to 0.2 grains
silicic acid to the concentrator tubes. Allow to stand until
the evolution of nitrogen gas has stopped (approximately 20
min). Adjust the sample volume to 5.0 ml with MTBE.
11.6 FLORISIL SEPARATION
11.6.1 Place a small plug of glass wool into a 5-mL disposable glass
pipet. Tare the pipet, and measure 1 g of activated Florisil
into the pipet.
11.6.2 Apply 5 ml of 5 percent methanol in MTBE to the Florisil.
Allow the liquid to just reach the top of the Florisil. In
this and subsequent steps, allow the liquid level to just reach
the top of the Florisil before applying the next rinse,
however, do not allow the Florisil to go dry. Discard eluate.
11.6.3 Apply 5 mL methylated sample to the Florisil leaving silicic
acid in the tube. Collect eluate in K-D tube.
11.6.4 Add 1 ml of 5 percent methanol in MTBE to the sample container,
rinsing walls. Transfer the rinse to the Florisil column
leaving silicic acid 1n the tube. Collect eluate in a K-D tube.
Repeat with 1-mL and 3-mL aliquots of 5 percent methanol in
MTBE, collecting eluates in K-D tube.
11.6.5 If necessary, dilute eluate to 10 ml with 5 percent methanol in
MTBE.
11.6.6 Seal the vial and store in a refrigerator if further processing
will not be performed immediately. Analyze by GC-ECD.
11.7 GAS CHROMATOGRAPHY
11.7.1 Sect. 6.10 summarizes the recommended operating conditions for
the GC. Included in Table 1 are retention times observed using
this method. Other GC columns, chromatographic conditions, or
detectors may be used if the requirements of Sect. 10.4 are
met.
11.7.2 Calibrate the system daily as described in Sect. 9. The
standards and extracts must be in MTBE.
11.7.3 If the internal standard calibration procedure is used, fortify
the extract with 25 pi of internal standard solution.
Thoroughly mix sample and place aliquot in a GC vial for
subsequent analysis.
11.7.4 Inject 2 jtL of the sample extract. Record the resulting peak
size in area units.
245
-------
11.7.5 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
11.8 IDENTIFICATION OF ANALYTES
11.8.1 Identify a sample component by comparison of its retention time
to the retention time of a reference chromatogram. If the
retention time of an unknown compound corresponds, within
limits, to the retention time of a standard compound, then
identification is considered positive.
11.8.2 The width of the retention time window used to make
identifications should be based upon 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.
11.8.3 Identification requires expert judgement when sample components
are not resolved chromatographically. When GC peaks obviously
represent more than one sample component (i.e., broadened peak
with shoulder(s) or valley between two or more maxima, or any
time doubt exists over the identification of a peak on a
chromatogram, appropriate alternative techniques, to help
confirm peak identification, need to be employed. For example,
more positive identification may be made by the use of an
alternative detector which operates on a chemical/physical
principle different from that originally used, e.g., mass
spectrometry, or the use of a second chromatography column. A
suggested alternative column in described in Sect. 6.10.
12. CALCULATIONS
12.1 Calculate analyte concentrations in the sample from the response for
the analyte using the calibration procedure described in Sect. 9.
12.2 If the internal standard calibration procedure is used, calculate the
concentration (C) in the sample using the response factor (RF)
determined in Sect. 9.2 and Equation 2, or determine sample
concentration from the calibration curve.
r i /,» (As)(Is)
C (M/L) = Equation 2.
(Ais)(RF)(V0)
where:
As = Response for the parameter to be measured.
Ais = Response for the internal standard.
Is - Amount of internal standard added to each extract
V0 = Volume of water extracted (L).
246
-------
12.3 If the external standard calibration procedure Is used, calculate the
amount of material Injected from the peak response using the
calibration curve or calibration factor determined in Sect. 9.3. The
concentration (C) in the sample can be calculated from Equation 3.
(A}(Vt)
C (/jg/L) = Equation 3.
(Vi)(Vs)
where:
A = Amount of material injected (ng).
Vj = Volume of extract injected (jiL).
Vt = Volume of total extract {/iL).
Vs = Volume of water extracted (ml).
13. PRECISION AND ACCURACY
13.1 In a single laboratory, analyte recoveries from reagent water were
determined at five concentration levels. Results were used to
determine analyte EDLs and demonstrate method range.(1) Analyte EDLs
and analyte recoveries and standard deviation about the percent
recoveries at one concentration are given in Table 2.
13.2 In a single laboratory, analyte recoveries from one standard synthetic
ground waters were determined at one concentration level. Results were
used to demonstrate applicability of the method to different ground
water matrices.(1) Analyte recoveries from the one synthetic matrix
are given in Table 2.
14. REFERENCES
1. National Pesticide Survey Method No. 3: "Determination of Chlorinated
Acids in Water by Gas Chromatography with an Electron Capture
Detector."
2. "Pesticide Methods Evaluation," Letter Report #33 for EPA Contract No.
68-03-2697. Available from U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio
45268.
3. ASTH Annual Book of Standards, Part 11, Volume 11.02, D3694-82,
"Standard Practice for Preparation of Sample Containers and for
Preservation", American Society for Testing and Materials,
Philadelphia, PA, p. 86, 1986.
4. Giam, C. S., Chan, H. S., and Nef, G. S. "Sensitive Method for Deter-
mination of Phthalate Ester Plasticizers in Open-Ocean Biota Samples,"
Analytical ChemistryT 47, 2225 (1975).
247
-------
5. Giam, C. S., and Chan, H. S. "Control of Blanks In the Analysis of
Phthalates in Air and Ocean Biota Samples," U.S. National Bureau of
Standards, Special Publication 442, pp. 701-708, 1976.
6. "Carcinogens - Working with Carcinogens," Department of Health, Educa-
tion, and Welfare, Public Health Service, Center for Disease Control,
National Institute for Occupational Safety and Health, Publication No.
77-206, Aug. 1977.
7. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
8. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
9. ASTM Annual Book of Standards, Part 11, Volume 11.01, 03370-82, "Stan-
dard Practice for Sampling Water," American Society for Testing and
Materials, Philadelphia, PA, p. 130, 1986.
248
-------
TABLE 1. RETENTION TINES FOR METHOD ANALYTES
Retention Time3
(minutes)
Analvte Primary Confirmation
Oalapon
3,5-Dichlorobenzoic acid
4-Nitrophenol
DCAA (surrogate)
Dicamba
Dichlorprop
2,4-D
DBOB (int. std.)
Pentachlorophenol (PCP)
Chloramben
2,4,5-TP
5-Hydroxydicamba
2,4,5-T
2,4-DB
Oinoseb
Bentazon
Picloram
DCPA acid metabolites
Acifluorfen
3.4
18.6
18.6
22.0
22.1
25.0
25.5
27.5
28.3
29.7
29.7
30.0
30.5
32.2
32.4
33.3
34.4
35.8
41.5
4.7
17.7
20.5
14.9
22.6
25.6
27.0
27.6
27.0
32.8
29.5
30.7
30.9
32.2
34.1
34.6
37.5
37.8
42.8
a Columns and analytical conditions are described in Sect. 6.10.1
and 6.10.2.
249
-------
TABLE 2. SINGLE LABORATORY ACCURACY, PRECISION AND ESTIMATED DETECTION LIMITS (EDLS)
FOR ANALYTES FROM REAGENT HATER AND SYNTHETIC GROUNDWATERS(A>
ro
wi
o
Analvte
Acifluorfen
Bentazon
Chloramben
2,4-D
Oalapon
2,4-DB
DCPA acid metabolites
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
4-Nitrophenol
Pentachlorophenol (PCP)
Picloram
2,4,5-T
2,4,5-TP
EDLh
uq/Lb
0.096
0.2
0.093
0.2
1.3
0.8
0.02
0.081
0.061
0.26
0.19
0.04
0.13
0.076
0.14
0.08
0.075
a Data corrected for amount detected
D FDI = ectimatoH Hotoi-1
(••inn 1 imi t • At
Concentration Reagent
ua/L Rc
0.2
1
0.4
1
10
4
0.2
0.4
0.6
2
0.4
0.2
1
0.04
0.6
0.4
0.2
in blank and
if i norl ac oi tl
121
120
111
131
100
87
74
135
102
107
42
103
131
130
91
117
134
represent the
iav> MRI ( Annan/4
Water
SRd
15.7
16.8
14.4
27.5
20.0
13.1
9.7
32.4
16.3
20.3
14.3
16.5
23.6
31.2
15.5
16.4
30.8
mean of
iv D *«
Synthetic
R
103
82
112
110
128
0
81
92
82
106
89
88
127
84
97
96
105
7-8 samples.
in rco D*V.+ TJC
Water le
SP
20.6
37.7
10.1
5.5
30.7
0
21.9
17.5
7.4
5.3
13.4
5.3
34.3
9.2
23.3
3.8
6.3
_ nA£ini + ^*\M
c
d
and Procedure for the Determination of the Method Detection Limit - Revision 1.11) or a level of
compound in a sample yielding a peak in the final extract with signal-to-noise ratio of
approximately 5, whichever value is higher. The concentration used in determining the EDL is not
the same as the concentration presented in this table.
R = average percent recovery.
Sr = standard deviation of the percent recovery
e Corrected for amount found in blank; Absopure Nature Artesian Spring Water Obtained from the
Absopure Water Company in Plymouth, Michigan.
-------
TABLE 3. LABORATORY PERFORMANCE CHECK SOLUTION
ro
en
Test
Sensitivity
Chromatographic performance
Column performance
Anal vte
Dinoseb
4-Nitrophenol
3,5-Dichlorobenzoic acid
4-Nitrophenol
Cone,
UQ/mL
0.004
1.6
0.6
1.6
Reauirements
Detection of analyte;
0.70 0.40D
S/N > 3
peak Gaussian factor. Calculated using the equation:
1.83 x Wfl/21
PGF
where W(l/2) is the peak width at half height and U(l/10) is the peak width at tenth height.
Resolution between the two peaks as defined by the equation:
R -1-
R = H
where t is the difference in elution times between the two peaks and U is the average peak width, at the
baseline, of the two peaks.
-------
Nitrogen
Tubel
Tub* 2
Sampl*
Tub*
FIGURE 1. GASEOUS DIAZOHETHANE GENERATOR
252
-------
nitrogen
THE
•« • t-
rubber stopper
ro
in
oo
••
O
s
tube 1
tube 2
qlass tublnq
Collection
\
Thermos or
r.ryoqenlc cooler
FIGURE 2. DIAZOMETHANE SOLUTION GENERATOR
-------
METHOD 524.1. MEASUREMENT OF PURGEABLE ORGANIC COMPOUNDS
IN HATER BY PACKED COLUMN GAS CHROMATOGRAPHY/MASS SPECTROMETRY
Revision 3.0
A. Alford-Stevens, J. H. Eichelberger, W. L. Budde - Method 524, Revision 1.0
(1983)
J. E. Longbottom, R. W. Slater, Jr. - Method 524.1, Revision 2.0 (1986)
J. W. Eichelberger, H. L. Budde - Method 524.1, Revision 3.0 (1989)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
255
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METHOD 524.1
MEASUREMENT OF PURGEABLE ORGANIC COMPOUNDS IN HATER BY
PACKED COLUMN GAS CHROMATOGRAPHY/MASS SPECTROHETRY
1. SCOPE AND APPLICATION
1.1 This Is a general purpose method for the Identification and simul-
taneous measurement of purgeable volatile organic compounds In
finished drinking water, raw source water, or drinking water in any
treatment stage (1). The method 1s applicable to a wide range of
organic compounds, including the four trihalomethane disinfection
by-products, that have sufficiently high volatility and low water
solubility to be efficiently removed from water samples with purge and
trap procedures. The following compounds are method analytes, and
single-laboratory accuracy, precision, and method detection limit data
have been determined with this method for 31 of them3.
Chemical Abstract Service
Compound Registry Number
Benzene 71-43-2
Bromobenzene 108-86-1
* Bromochloromethane 74-97-5
Bromodichloromethane 75-27-4
Bromoform 75-25-2
* Bromomethane 74-83-9
Carbon tetrachloride 56-23-5
Chlorobenzene 108-90-7
* Chloroethane 75-00-3
Chloroform 67-66-3
* Chloromethane 74-87-3
* 2-Chlorotoluene 95-49-8
* 4-Chlorotoluene 106-43-4
Dibromochloromethane 124-48-1
l,2-Dibromo-3-chloropropane 96-12-8
1,2-Dibromoethane 106-93-4
Dibromomethane 74-95-3
1,2-Dichlorobenzene 95-50-1
* 1,3-Dichlorobenzene 541-73-1
1,4-Dichlorobenzene 106-46-7
Dichlorodifluoromethane 75-71-8
1,1-Dichloroethane 75-34-3
1,2-Dichloroethane 107-06-2
1,1-Dichloroethene 75-35-4
* cis-l,2-Dichloroethene 156-59-4
trans-l,2-Dichloroethene 156-60-5
1,2-Dichloropropane 78-87-5
1,3-Dichloropropane 142-28-9
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* 2,2-Dichloropropane 590-20-7
* 1,1-Dichloropropene 563-58-6
* cis-l,3-Dichloropropene 10061-01-5
* trans-l,3-Dichloropropene 10061-02-6
* Ethylbenzene 100-41-4
Methylene chloride 75-09-2
Styrene 100-42-5
* 1,1,1,2-Tetrachloroethane 630-20-6
1,1,2,2-Tetrachloroethane 79-34-5
Tetrachloroethene 127-18-4
Toluene 108-88-3
1,1,1-Trichloroethane 71-55-6
* 1,1,2-Trichloroethane 79-00-5
Trichloroethene 79-01-6
Trichlorofluoromethane 75-69-4
* 1,2,3-Trichloropropane 96-18-4
Vinyl chloride 75-01-4
o-Xylene 95-47-6
* m-Xylene 108-38-3
p-Xylene 106-42-3
a Compounds preceded by an asterisk are known to be amenable to purge
and trap extraction (see Method 524.2), and chromatography on the
packed gas chromatography column used in this method, but precision,
accuracy, retention time, and method detection limit data is not
provided in this method.
1.2 Method detection limits (MDLs) (2) are compound and instrument
dependent and vary from approximately 0.1-2 /tg/L. The applicable
concentration range of this method is also compound and instrument
dependent and is approximately 0.1 to 200 ng/L. Analytes that are
inefficiently purged from water will not be detected when present at
low concentrations, but they can be measured with acceptable accuracy
and precision when present in sufficient amounts.
1.3 Analytes that are not separated chromatographically, but which have
different mass spectra and non-interfering quantisation ions, can be
identified and measured in the same calibration mixture or water
sample (Sect. 11.9.2). Table 1 lists primary and secondary quantita-
tion ions for each analyte. Analytes which have very similar mass
spectra cannot be individually identified and measured in the same
calibration mixture or water sample unless they have different
retention times (Sect.11.9.3). Coeluting compounds with very similar
mass spectra, typically many structural isomers, must be reported as
an isomeric group or pair. Cis- and trans-l,2-dichloroethene, two of
the three isomeric xylenes, and two of the three dichlorobenzenes are
three examples of structural isomers that cannot be explicitly
identified if more than one member of the isomeric group is present.
These groups of isomers must be reported as isomeric pairs (see Method
524.2 for an alternative approach).
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2. SUMMARY OF METHOD
2.1 Volatile organic compounds and surrogates with low water solubility
are extracted (purged) from the sample matrix by bubbling an inert
gas through the aqueous sample. Purged sample components are trapped
in a tube containing suitable sorbent materials. When purging is
complete, the sorbent tube is heated and backflushed with helium to
desorb the trapped sample components into a packed gas chromatography
(GC) column interfaced to a mass spectrometer (MS). The column is
temperature programmed to separate the method analytes which are then
detected with the MS. Compounds eluting from the GC column are
identified by comparing their measured mass spectra and retention
times to reference spectra and retention times in a data base.
Reference spectra and retention times for analytes are obtained by the
measurement of calibration standards under the same conditions used
for samples. The concentration of each identified component is
measured by relating the MS response of the quantitation ion produced
by that compound to the MS response of the quantitation ion produced
by a compound that is used as an internal standard. Surrogate
analytes, whose concentrations are known in every sample, are measured
with the same internal standard calibration procedure.
3. DEFINITIONS
3.1 Internal standard -- A pure analyte(s) added to a solution in known
amount(s) and used to measure the relative responses of other method
analytes and surrogates that are components of the same solution.
The internal standard must be an analyte that is not a sample
component.
3.2 Surrogate analyte -- A pure analyte(s), which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in
known amount(s) before extraction and is measured with the same
procedures used to measure other sample components. The purpose of a
surrogate analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates (LD1 and LD2) -- Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample
collection, preservation, or storage procedures.
3.4 Field duplicates (FD1 and FD2) -- Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.5 Laboratory recent blank (LRB) -- An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
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that are used with other samples. The LRB Is used to determine If
method analytes or other Interferences are present In the laboratory
environment, the reagents, or the apparatus.
3.6 Field reagent blank (FRB) -- Reagent water placed 1n a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage, preservation
and all analytical procedures. The purpose of the FRB 1s to determine
if method analytes or other interferences are present 1n the field
environment.
3.7 Laboratory performance check solution (LPC) -- A solution of one or
more compounds used to evaluate the performance of the Instrument
system with respect to a defined set of method criteria.
3.8 Laboratory fortified blank (LFB) -- An aliquot of reagent water to
which known quantities of the method analytes are added 1n the
laboratory. The LFB 1s analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.9 Laboratory fortified sample matrix (LFM) -- An aliquot of an environ-
mental sample to which known quantities of the method analytes are
added in the laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the
analytes in the sample matrix must be determined in a separate aliquot
and the measured values in the LFM corrected for background concentra-
tions.
3.10 Stock standard solution -- A concentrated solution containing a
single certified standard that is a method analyte, or a concentrated
solution of a single analyte prepared in the laboratory with an
assayed reference compound. Stock standard solutions are used to
prepare primary dilution standards.
3.11 Primary dilution standard solution -- A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
3.12 Calibration standard (CAL) -- a solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.13 Quality control sample (QCS) -- a sample matrix containing method
analytes or a solution of method analytes in a water miscible solvent
which is used to fortify reagent water or environmental samples. The
QCS is obtained from a source external to the laboratory, and is used
259
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to check laboratory performance with externally prepared test
materials.
4. INTERFERENCES
4.1 During analysis, major contaminant sources are volatile materials
in the laboratory and impurities in the inert purging gas and in
the sorbent trap. The use of non-polytetrafluoroethylene (PTFE)
plastic tubing, non-PTFE thread sealants, or flow controllers with
rubber components in the purging device should be avoided since
such materials out-gas organic compounds which will be concentrated
in the trap during the purge operation. Analyses of laboratory
reagent blanks provide information about the presence of
contaminants. When potential interfering peaks are noted in
laboratory reagent blanks, the analyst should change the purge gas
source and regenerate the molecular sieve purge gas filter.
Subtracting blank values from sample results is not permitted.
4.2 Interfering contamination may occur when a sample containing low
concentrations of volatile organic compounds is analyzed immediately
after a sample containing relatively high concentrations of volatile
organic compounds. A preventive technique is between-sample rinsing
of the purging apparatus and sample syringes with two portions of
reagent water. After analysis of a sample containing high concentra-
tions of volatile organic compounds, one or more laboratory reagent
blanks should be analyzed to check for cross contamination.
4.3 Special precautions must be taken to determine methylene chloride.
The analytical and sample storage area should be isolated from all
atmospheric sources of methylene chloride, otherwise random
background levels will result. Since methylene chloride will
permeate through PTFE tubing, all gas chromatography carrier gas
lines and purge gas plumbing should be constructed of stainless steel
or copper tubing. Laboratory worker's clothing should be cleaned
frequently since clothing previously exposed to methylene chloride
fumes during common liquid/liquid extraction procedures can
contribute to sample contamination.
5. SAFETY
5.1 The toxicity or carcinogenicity of chemicals used in this method
has not been precisely defined; each chemical should be treated as
a potential health hazard, and exposure to these chemicals should
be minimized. Each laboratory is responsible for maintaining
awareness of OSHA regulations regarding safe handling of chemicals
used in this method. Additional references to laboratory safety
are available (3-5) for the information of the analyst.
5.2 The following method analytes have been tentatively classified as
known or suspected human or mammalian carcinogens: benzene, carbon
tetrachloride, 1,4-dichlorobenzene, 1,2-dichlorethane, 1,1,2,2-tetra-
chloroethane, 1,1,2-trichloroethane, chloroform, 1,2-dibromoethane.
260
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tetrachloroethene, trichloroethene, and vinyl chloride. Pure standard
materials and stock standard solutions of these compounds should be
handled in a hood. A NIOSH/MESA approved toxic gas respirator should
be worn when the analyst handles high concentrations of these toxic
compounds.
6. APPARATUS AND EQUIPMENT
6.1 SAMPLE CONTAINERS -- 60-mL to 120-mL screw cap vials (Pierce #19832
or equivalent) each equipped with a PTFE-faced silicone septum
(Pierce #12718 or equivalent). Prior to use, wash vials and septa
with detergent and rinse with tap and distilled water. Allow the
vials and septa to air dry at room temperature, place in a 105°C
oven for 1 hr, then remove and allow to cool in an area known
to be free of organics.
6.2 PURGE AND TRAP SYSTEM -- The purge and trap system consists of three
separate pieces of equipment: purging device, trap, and desorber.
Systems are commercially available from several sources that meet
all of the following specifications.
6.2.1 The all glass purging device (Figure 1) should be designed to
accept 25-mL samples with a water column at least 5 cm deep.
A smaller (5-mL) purging device is recommended if the GC/MS
system has adequate sensitivity to obtain the method detection
limits required. Gaseous volumes above the sample must be kept
to a minimum (<15 mL) to eliminate dead volume effects. A
glass frit should be installed at the base of the sample
chamber so the purge gas passes through the water column as
finely divided bubbles with a diameter of <3 mm at the origin.
Needle spargers may be used, however, the purge gas must be
introduced at a point about 5 mm from the base of the water
column.
6.2.2 The trap (Figure 2) must be at least 25 cm long and have an
inside diameter of at least 0.105 in. Starting from the
inlet, the trap should contain 1.0 cm of methyl silicone
coated packing and the following amounts of adsorbents: 1/3
of 2,6-diphenylene oxide polymer, 1/3 of silica gel, and 1/3
of coconut charcoal. If it is not necessary to determine
dichlorodifluoromethane, the charcoal can be eliminated and
the polymer increased to fill 2/3 of the trap. Before
initial use, the trap should be conditioned overnight at
180°C by backflushing with an inert gas flow of at least
20 mL/min. Vent the trap effluent to the room, not to the
analytical column. Prior to daily use, the trap should be
conditioned for 10 min at 180°C with backflushing. The trap
may be vented to the analytical column during daily
conditioning; however, the column must be run through the
temperature program prior to analysis of samples.
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6.2.3 The use of the methyl silicone coated packing is recommende'1
but not mandatory. The packing serves a dual purpose of
protecting the Tenax adsorbant from aerosols, and also of
insuring that the Tenax is fully enclosed within the heated
zone of the trap thus eliminating potential cold spots.
Alternatively, silanized glass wool may be used as a spacer at
the trap inlet.
6.2.4 The desorber (Figure 2) must be capable of rapidly heating the
trap to 180°C. The polymer section of the trap should not
be heated higher than 200°C or the life expectancy of the trap
will decrease. Trap failure is characterized by a pressure
drop in excess of 3 pounds per square inch across the trap
during purging or by poor bromoform sensitivities. The
desorber design illustrated in Figure 2 meets these criteria.
6.3 GAS CHROMATOGRAPHY/MASS SPECTROMETER/DATA SYSTEM (GC/MS/DS)
6.3.1 The GC must be capable of temperature programming and should
be equipped with variable-constant differential flow
controllers so that the column flow rate will remain
constant throughout desorption and temperature program
operation. The column oven may require cooling to <30°C;
therefore, a subambient oven controller may be required. The
GC usually is interfaced to the MS with an all-glass
enrichment device and an all-glass transfer line, but any
enrichment device or transfer line can be used if the
performance specifications described in this method can be
achieved.
6.3.2 Gas Chromatography Column -- 1.5 to 2.5 m x 2 mm ID stainless
steel or glass, packed with 1% SP-1000 on Carbopack-B (60/80
mesh) or the equivalent.
6.3.3 The mass spectrometer must be capable of electron ionization
at a nominal electron energy of 70 eV. The spectrometer must
be capable of scanning from 35 to 260 amu with a complete scan
cycle time (including scan overhead) of 7 seconds or less.
(Scan cycle time = Total MS data acquisition time in seconds
divided by number of scans in the chromatogram). The spectro-
meter must produce a mass spectrum that meets all criteria in
Table 2 when 50 ng or less of 4-bromofluorobenzene (BFB) is
introduced into the GC. An average spectrum across the BFB GC
peak may be used to test instrument performance.
6.3.4 An interfaced data system is required to acquire, store,
reduce, and output mass spectral data. The computer software
should have the capability of processing stored GC/MS data by
recognizing a GC peak within any given retention time window,
comparing the mass spectra from the GC peak with spectral data
in a user-created data base, and generating a list of tenta-
tively identified compounds with their retention times and r"
262
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numbers. We software must allow integration of the ion
abundance of any specific ion between specified time or scan
number limits. The software should also allow calculation of
response factors as defined in Sect. 9.2.6 (or construction of
a second or third order regression calibration curve),
calculation of response factor statistics (mean and standard
deviation), and calculation of concentrations of analytes using
either the calibration curve or the equation in Sect. 12.
6.4 SYRINGE AND SYRINGE VALVES
6.4.1 Two 5-mL or 25-mL glass hypodermic syringes with Luer-Lok tip
(depending on sample volume used).
6.4.2 Three 2-way syringe valves with Luer ends.
6.4.3 One 25-0L micro syringe with a 2 in x 0.006 in ID, 22° bevel
needle (Hamilton 0702N or equivalent).
6.4.4 Micro syringes - 10, 100 nl.
6.4.5 Syringes - 0.5, 1.0, and 5-mL, gas tight with shut- off valve.
6.5 MISCELLANEOUS
6.5.1 Standard solution storage containers - 15-mL bottles with
PTFE-lined screw caps.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 TRAP PACKING MATERIALS
7.1.1 2,6-Diphenylene oxide polymer, 60/80 mesh, chromatographic
grade (Tenax GC or equivalent).
7.1.2 Methyl silicone packing (optional) - OV-1 (3%) on Chromo-
sorb W, 60/80 mesh, or equivalent.
7.1.3 Silica gel -- 35/60 mesh, Davison, grade 15 or equivalent.
7.1.4 Coconut charcoal -- Prepare from Barnebey Cheney, CA-580-26
lot 0M-2649 by crushing through 26 mesh screen.
7.2 COLUMN PACKING MATERIALS
7.2.1 1% SP-1000 on 60/80 mesh Carbopack-B or equivalent.
7.3 REAGENTS
7.3.1 Methanol -- Demonstrated to be free of analytes.
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7.3.2 Reagent water -- Prepare reagent water by passing tap water
through a filter bed containing about 0.5 kg of activated
carbon, by using a water purification system, or by boiling
distilled water for 15 min followed by a 1-h purge with
inert gas while the water temperature is held at 90°C.
Store in clean, narrow-mouth bottles with PTFE-lined septa
and screw caps.
7.3.3 Hydrochloric acid (1+1) -- Carefully add measured volume of
cone. HC1 to equal volume of reagent water.
7.3.4 Vinyl chloride -- Certified mixtures of vinyl chloride in
nitrogen and 99.9% pure vinyl chloride are available from
several sources (for example, Matheson, Ideal Gas Products,
and Scott Gases).
7.3.5 Ascorbic Acid -- ACS reagent grade, granular.
7.4 STOCK STANDARD SOLUTIONS -- These solutions may be purchased as
certified solutions or prepared from pure standard materials using
the following procedures. One of these solutions is required for
every analyte of concern, every surrogate, and the internal standard.
A useful working concentration is about 1-5 mg/mL.
7.4.1 Place about 9.8 ml of methanol into a 10-mL ground-glass
stoppered volumetric flask. Allow the flask to stand,
unstoppered, for about 10 min or until all alcohol-wetted
surfaces have dried and weigh to the nearest 0.1 mg.
7.4.2 If the analyte is a liquid at room temperature, use a 100-pL
syringe and immediately add two or more drops of reference
standard to the flask. Be sure that the reference standard
falls directly into the alcohol without contacting the neck
of the flask. If the analyte is a gas at room temperature,
fill a 5-mL valved gas-tight syringe with the standard to
the 5.0-mL mark, lower the needle to 5 mm above the methanol
meniscus, and slowly inject the standard into the neck area
of the flask. The gas will rapidly dissolve in the methanol.
7.4.3 Reweigh, dilute to volume, stopper, then mix by inverting the
flask several times. Calculate the concentration in /ig//iL
from the net gain in weight. When compound purity is
certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock
standard.
7.4.4 Store stock standard solutions in 15-mL bottles equipped
with PTFE-lined screw caps. Methanol solutions prepared
from liquid analytes are stable for at least four weeks when
stored at 4°C. Methanol solutions prepared from gaseous
analytes are not stable for more than one week when stored
264
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at <0°C; at room temperature, they must be discarded after one
day.
7.5 PRIMARY DILUTION STANDARDS -- Use stock standard solutions to prepare
primary dilution standard solutions that contain all the analytes of
concern and the surrogates (but not the Internal standard!) In
methanol. The primary dilution standards should be prepared at
concentrations that can be easily diluted to prepare aqueous calibra-
tion solutions that will bracket the working concentration range.
Store the primary dilution standard solutions with minimal headspace
and check frequently for signs of deterioration or evaporation,
especially just before preparing calibration solutions. Storage times
described for stock standard solutions in Sect. 7.4.4 also apply to
primary dilution standard solutions.
7.6 FORTIFICATION SOLUTIONS FOR INTERNAL STANDARD AND SURROGATES
7.6.1 A solution containing the internal standard and surrogates is
required to prepare laboratory reagent blanks (also used as a
laboratory performance check solution), and to fortify each
sample. Prepare a fortification solution containing fluoro-
benzene (internal standard), 1,2- dichlorobenzene-d4
(surrogate), and BFB (surrogate) in methanol at concentrations
of 5 /jg/mL of each. A 10-0L aliquot of this solution added to
a 25-mL water sample volume gives concentrations of 2 /tg/L of
each. A 10-/JL aliquot of this solution added to a 5-mL water
sample volume gives a concentration of 10 /ig/L of each.
Additional internal standards and surrogate analytes are
optional.
7.6.2 A solution of the internal standard alone is required to
prepare calibration standards, laboratory fortified blanks,
etc. The internal standard should be in methanol at a concen-
tration of 5 /Kj/mL.
7.7 PREPARATION OF LABORATORY REAGENT BLANK -- Fill a 25-mL (or 5-mL)
syringe with reagent water and adjust to the mark (no air bubbles).
Inject 10 nl of the fortification solution containing the internal
standard and surrogates through the Luer Lok valve into the reagent
water. Transfer the LRB to the purging device. See Sect. 11.1.2.
7.8 PREPARATION OF LABORATORY FORTIFIED BLANK -- Prepare this exactly
like a calibration standard. See Sect. 7.9.
7.9 PREPARATION OF CALIBRATION STANDARDS
7.9.1 The number of calibration solutions (CALs) needed depends on
the calibration range desired. A minimum of three CAL
solutions is required to calibrate a range of a factor of 20
in concentration. For a factor of 50 use at least four
standards, and for a factor of 100 at least five standards.
One calibration standard should contain each analyte of
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concern and each surrogate at a concentration of 2-10 times
the method detection limit (Table 3) for that compound. The
other CAL standards should contain each analyte of concern and
each surrogate at concentrations that define the range of the
method. Every CAL solution contains the internal standard at
the same concentration (10 /zg/L suggested).
7.9.2 To prepare a calibration standard, add an appropriate volume
of a primary dilution standard (containing analytes and surro-
gates) to an aliquot of reagent water in a volumetric flask.
Use a microsyringe and rapidly inject the methanol solutions
into the expanded area of the filled volumetric flask. Remove
the needle as quickly as possible after injection. Mix by
inverting the flask three times only. Discard the contents
contained in the neck of the flask. Aqueous standards are not
stable in a volumetric flask and should be discarded after
1 hr. unless transferred to a sample bottle and sealed
immediately.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE COLLECTION, DECHLORINATION, AND PRESERVATION
8.1.1 Collect all samples in duplicate. If samples contain residual
chlorine, and measurements of the concentrations of disinfec-
tion by-products (trihalomethanes, etc.) at the time of sample
collection are desired, add about 25 mg of ascorbic acid to the
sample bottle before filling. Fill sample bottles to overflow-
ing, but take care not to flush out the rapidly dissolving
ascorbic acid. No air bubbles should pass through the sample
as the bottle is filled, or be trapped in the sample when the
bottle is sealed. Adjust the pH of the duplicate samples to <2
by carefully adding one drop of 1:1 HC1 for each 20 mL of
sample volume. Seal the sample bottles, PFTE-face down, and
shake vigorously for 1 min.
8.1.2 When sampling from a water tap, open the tap and allow the
system to flush until the water temperature has stabilized
(usually about 10 min). Adjust the flow to about 500 mL/min
and collect duplicate samples from the flowing stream.
8.1.3 When sampling from an open body of water, fill a 1-quart
wide-mouth bottle or 1-liter beaker with sample from a
representative area, and carefully fill duplicate sample
bottles from the 1-quart container.
8.1.4 The samples must be chilled to 4°C on the day of collection
and maintained at that temperature until analysis. Field
samples that will not be received at the laboratory on the day
of collection must be packaged for shipment with sufficient ice
to ensure that they will be at 4°C on arrival at the
laboratory.
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8.2 SAMPLE STORAGE
8.2.1 Store samples at 4°C until analysis. The sample storage area
must be free of organic solvent vapors.
8.2.2 Analyze all samples within 14 days of collection. Samples
not analyzed within this period must be discarded and
replaced.
8.3 FIELD REAGENT BLANKS
8.3.1 Duplicate field reagent blanks must be handled along with each
sample set, which is composed of the samples collected from the
same general sample site at approximately the same time. At
the laboratory, fill field blank sample bottles with reagent
water, seal, and ship to the sampling site along with empty
sample bottles and back to the laboratory with filled sample
bottles. Wherever a set of samples is shipped and stored, it
is accompanied by appropriate blanks.
8.3.2 Use the same procedures used for samples to add ascorbic acid
and HC1 to blanks (Sect. 8.1.1).
9. CALIBRATION
9.1 Demonstration and documentation of acceptable initial calibration is
required before any samples are analyzed and is required intermit-
tently throughout sample analysis as dictated by results of
continuing calibration checks. After initial calibration is
successful, a continuing calibration check is required at the
beginning of each 8 hr period during which analyses are performed.
Additional periodic calibration checks are good laboratory practice.
9.2 INITIAL CALIBRATION
9.2.1 Calibrate the mass and abundance scales of the MS with
calibration compounds and procedures prescribed by the
manufacturer with any modifications necessary to meet the
requirements in Sect. 9.2.2.
9.2.2 Introduce into the GC (either by purging a laboratory reagent
blank or making a syringe injection) 50 ng of BFB and acquire
mass spectra for m/z 35-260 at 70 eV (nominal). Use the
purging procedure and/or GC conditions given in Sect. 11. If
the spectrum does not meet all criteria in Table 2, the MS
must be retuned and adjusted to meet all criteria before
proceeding with calibration. An average spectrum across the
GC peak may be used to evaluate the performance of the system.
9.2.3 Purge a medium CAL solution, for example 10-20 pg/L, using the
procedure given in Sect. 11.
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9.2.4 Performance criteria for the medium calibration. Examine
stored GC/MS data with the data system software. Figure .
shows an acceptable total ion chromatogram.
9.2.4.1 GC performance. Good column performance will produce
symmetrical peaks with minimum tailing for most
compounds. If peaks are broad, or sensitivity poor,
replace or repack the column. During handling,
packing, and programming, active sites can be exposed
on the Carbopack-B packing which can result in tailing
peak geometry and poor resolution of many constitu-
ents. Pneumatic shocks and rough treatment of packed
columns will cause excessive fracturing of the
packing. If pressure in excess of 60 psi is required
to obtain 40 mL/min carrier flow, the column should be
repacked. With the column connected to the MS
interface, a pressure below about 10~5 mm of Hg
indicates the jet separator is clogged.
9.2.4.2 MS sensitivity. The GC/MS peak identification
software should be able to recognize a GC peak in the
appropriate retention time window for each of the
compounds in calibration solution, and make correct
tentative identifications. If fewer than 99% of the
compounds are recognized, system maintenance is
required. See Sect. 9.3.6.
9.2.5 If all performance criteria are met, purge an aliquot of each
of the other CAL solutions using the same GC/MS conditions.
9.2.6 Calculate a response factor (RF) for each analyte, surrogate,
and isomer pair, for each CAL solution using the internal
standard fluorobenzene. Table 1 contains suggested quantita-
tion ions for all compounds. This calculation is supported in
acceptable GC/MS data system software (Sect. 6.3.4), and many
other software programs. RF is a unitless number, but units
used to express quantities of analyte and internal standard
must be equivalent.
RF . (AxHQis)
(AisMQx)
where: Ax = integrated abundance of the quantitation ion
of the analyte.
A,s = integrated abundance of the quantitation ion
of the internal standard.
Qx = quantity of analyte purged in ng or
concentration units.
Qis = quantity of internal standard purged in ng
or concentration units.
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9.2.6.1 For each analyte and surrogate, calculate the mean RF
from the analyses of the CAL solutions. Calculate the
standard deviation (SD) and the relative standard
deviation (RSD) from each mean: RSD = 100 (SD/M). If
the RSD of any analyte or surrogate mean RF exceeds
20%, either analyze additional aliquots of appropriate
CAL solutions to obtain an acceptable RSD of RFs over
the entire concentration range, or take action to
improve GC/MS performance. See Sect. 9.2.7.
9.2.7 As an alternative to calculating mean response factors and
applying the RSD test, use the GC/MS data system software or
other available software to generate a second or third order
regression calibration curve.
9.3 Continuing calibration check. Verify the MS tune and Initial
calibration at the beginning of each 8 hr work shift during which
analyses are performed using the following procedure.
9.3.1 Introduce Into the GC (either by purging a laboratory reagent
blank or making a syringe Injection) 50 ng of BFB and acquire
a mass spectrum that Includes data for m/z 35-260. If the
spectrum does not meet all criteria (Table 2), the MS must be
retuned and adjusted to meet all criteria before proceeding
with the continuing calibration check.
9.3.2 Purge a medium concentration CAL solution and analyze with the
same conditions used during the initial calibration.
9.3.3 Demonstrate acceptable performance for the criteria shown in
Sect. 9.2.4.
9.3.4 Determine that the absolute areas of the quantitation ions of
the internal standard and surrogates have not decreased by
more than 30% from the areas measured in the most recent
continuing calibration check, or by more than 50% from the
areas measured during initial calibration. If these areas
have decreased by more than these amounts, adjustments must be
made to restore system sensitivity. These adjustments may
require cleaning of the MS ion source, or other maintenance as
indicated in Sect. 9.3.6, and recalibration. Control charts
are useful aids in documenting system sensitivity changes.
9.3.5 Calculate the RF for each analyte and surrogate from the data
measured in the continuing calibration check. The RF for each
analyte and surrogate must be within 30% of the mean value
measured in the initial calibration. Alternatively, if a
second or third order regression is used, the point from the
continuing calibration check for each analyte and surrogate
must fall, within the analyst's judgement, on the curve from
the initial calibration. If these conditions do not exist,
269
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remedial action must be taken which may require reinitial
calibration.
9.3.6 Some possible remedial actions. Major maintenance such as
cleaning an ion source, cleaning quadrupole rods, etc. require
returning to the initial calibration step.
9.3.6.1 Check and adjust GC and/or MS operating conditions;
check the MS resolution, and calibrate the mass
scale.
9.3.6.2 Prepare fresh CAL solutions, and repeat the initial
calibration step.
9.3.6.3 Clean the MS ion source and rods (if a quadrupole).
9.3.6.4 Replace the MS electron multiplier, or any other
faulty components.
9.4 Optional calibration for vinyl chloride using a certified gaseous
mixture of vinyl chloride in nitrogen can be accomplished by the
following steps.
9.4.1 Fill the purging device with 25.0 ml of reagent
water or aqueous calibration standard.
9.4.2 Start to purge the aqueous mixture. Inject a known volume
(between 100 and 2000 /zL) of the calibration gas (at room
temperature) directly into the purging device with a gas tight
syringe. Slowly inject the gaseous sample through a septum
seal at the top of the purging device at 2000 pL/min. If the
injection of the standard is made through the aqueous sample
inlet part, flush the head volume with several ml of room air
or carrier gas. Inject the gaseous standard before 5 min of
the 11-min purge time have elapsed.
9.4.3 Determine the aqueous equivalent concentration of vinyl
chloride standard, in /zg/L, injected with the equation:
S = 0.102 (C)(V)
where S = Aqueous equivalent concentration
of vinyl chloride standard in /ig/L;
C = Concentration of gaseous standard in ppm
(v/v);
V = Volume of standard injected in milliliters.
270
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QUALITY CONTROL
10.1 Quality control (QC) requirements are the initial demonstration of
laboratory capability followed by regular analyses of laboratory
reagent blanks, field reagent blanks, and laboratory fortified
blanks. The laboratory must maintain records to document the
quality of the data generated. Additional quality control practices
are recommended.
10.2 Initial demonstration of low system background. Before any samples
are analyzed, it must be demonstrated that a laboratory reagent
blank (LRB) is reasonably free of contamination that would prevent
the determination of any analyte of concern. Sources of background
contamination are glassware, purge gas, sorbants, and equipment.
Background contamination must be reduced to an acceptable level
before proceeding with the next section. In general background from
method analytes should be below the method detection limit.
10.3 Initial demonstration of laboratory accuracy and precision. Analyze
four to seven replicates of a laboratory fortified blank containing
each analyte of concern at a concentration in the range of 0.2-5 /ig/L
(see regulations and maximum contaminant levels for guidance on
appropriate concentrations).
10.3.1 Prepare each replicate by adding an appropriate aliquot of a
quality control sample to reagent water. If a quality
control sample containing the method analytes is not
available, a primary dilution standard made from a source of
reagents different than those used to prepare the calibration
standards may be used. Also add the appropriate amounts of
internal standard and surrogates if they are being used.
Analyze each replicate according to the procedures described
in Section 11, and on a schedule that results in the analyses
of all replicates over a period of several days.
10.3.2 Calculate the measured concentration of each analyte in each
replicate, the mean concentration of each analyte in all
replicates, and mean accuracy (as mean percentage of true
value) for each analyte, and the precision (as relative
standard deviation, RSD) of the measurements for each
analyte. Calculate the MDL of each analyte using the
procedures described in Sect. 13.2 (2).
10.3.3 For each analyte and surrogate, the mean accuracy, expressed
as a percentage of the true value, should be 80-120% and the
RSD should be <20%. Some analytes, particularly the early
eluting gases and late eluting higher molecular weight
compounds, are measured with less accuracy and precision than
other analytes. The method detection limits must be suffici-
ent to detect analytes at the required levels. If these
criteria are not met for an analyte, take remedial action and
271
-------
repeat the measurements for that analyte to demonstrate
acceptable performance before samples are analyzed.
10.3.4 Develop and maintain a system of control charts to plot the
precision and accuracy of analyte and surrogate measurements
as a function of time. Charting of surrogate recoveries is
an especially valuable activity since these are present in
every sample and the analytical results will form a signi-
ficant record of data quality.
10.4 Monitor the integrated areas of the quantitation ions of the
internal standards and surrogates in continuing calibration checks.
These should remain reasonably constant over time. A drift of more
than 50% in any area is indicative of a loss in sensitivity, and the
problem must be found and corrected. These integrated areas should
also be reasonably constant in laboratory fortified blanks and
samples.
10.5 LABORATORY REAGENT BLANKS. With each batch of samples processed as
a group within a work shift, analyze a laboratory reagent blank to
determine the background system contamination. A FRB (Sect. 10.7)
may be used in place of an LRB.
10.5 With each batch of samples processed as a group within a work shift,
analyze a single laboratory fortified blank (LFB) containing each
analyte of concern at a concentration as determined in 10.3. If more
than 20 samples are included in a batch, analyze one LFB for every 20
samples. Use the procedures described in 10.3.3 to evaluate the
accuracy of the measurements, and to estimate whether the method
detection limits can be obtained. If acceptable accuracy and method
detection limits cannot be achieved, the problem must be located and
corrected before further samples are analyzed. Add these results to
the on-going control charts to document data quality.
10.7 With each set of field samples a field reagent blank (FRB) should be
analyzed. The results of these analyses will help define contamina-
tion resulting from field sampling and transportation activities. If
the FRB shows unacceptable contamination, a LRB must be measured to
define the source of the impurities.
10.8 At least quarterly, replicates of laboratory fortified blanks should
be analyzed to determine the precision of the laboratory measure-
ments. Add these results to the on-going control charts to document
data quality.
10.9 At least quarterly, analyze a quality control sample (QCS) from an
external source. If measured analyte concentrations are not of
acceptable accuracy, check the entire analytical procedure to locate
and correct the problem source.
10.10 Sample matrix effects have not been observed when this method is used
with distilled water, reagent water, drinking water, and ground
272
-------
water. Therefore, analysis of a laboratory fortified sample matrix
(LFM) is not required. It is recommended that sample matrix effects
be evaluated at least quarterly using the QCS described in 10.9.
10.11 Numerous other quality control measures are incorporated into other
parts of this procedure, and serve to alert the analyst to potential
problems.
11. PROCEDURE
11.1 SAMPLE INTRODUCTION AND PURGING
11.1.1 This method is designed for a 25-mL sample volume, but a
smaller (5 ml) sample volume is recommended if the GC/MS
system has adequate sensitivity to achieve the required method
detection limits. Adjust the purge gas (nitrogen or helium)
flow rate to 40 mL/min. Attach the trap inlet to the purging
device and open the syringe valve on the purging device.
11.1.2 Remove the plungers from two 25-mL (or 5-mL depending on
sample size) syringes and attach a closed syringe valve to
each. Warm the sample to room temperature, open the sample
bottle, and carefully pour the sample into one of the syringe
barrels to just short of overflowing. Replace the syringe
plunger, invert the syringe, and compress the sample. Open the
syringe valve and vent any residual air while adjusting the
sample volume to 25.0 ml (or 5-mL). For samples and blanks,
add 10 /zL of the fortification solution containing the internal
standard and the surrogates to the sample through the syringe
valve. For calibration standards and laboratory fortified
blanks, add 10 nl of the fortification solution containing the
internal standard only. Close the valve. Fill the second
syringe in an identical manner from the same sample bottle.
Reserve this second syringe for a reanalysis if necessary.
11.1.3 Attach the sample syringe valve to the syringe valve on the
purging device. Be sure that the trap is cooler than 25°C,
then open the sample syringe valve and inject the sample into
the purging chamber. Close both valves and initiate purging.
Purge the sample for 11.0 min at ambient temperature.
11.4 SAMPLE DESORPTION -- After the 11-min purge, place the purge and trap
system in the desorb mode. Introduce the trapped materials to the GC
column by rapidly heating the trap to 180°C while backflushing the
trap with an inert gas at 15 mL/min for about 4. min. Simultaneously
with the start of desorption, begin the temperature program of the gas
chromatograph, and start data acquisition. While the extracted sample
is being introduced into the gas chromatograph, empty the purging
device using the sample syringe and wash the chamber with two 25-mL
flushes of reagent water. After the purging device has been emptied,
leave syringe valve open to allow the purge gas to vent through the
sample introduction needle.
273
-------
11.5 GAS CHROMATOGRAPHY/MASS SPECTROMETRY -- Acquire and store data fro-
m/z 35-260 with a total cycle time (including scan overhead time) ,
7 sec or less. Cycle time should be adjusted to measure at least five
or more spectra during the elution of each GC peak. Adjust the helium
carrier gas flow rate to about 40 mL/min. The column temperature is
programmed to hold at 45°C for three min, increase to 220°C at
8°C/min, and hold at 220°C for 15 min or until all expected compounds
have eluted.
11.6 TRAP RECONDITIONING -- After desorbing the sample for 4 min,
recondition the trap by returning the purge and trap system to the
purge mode. Wait 15 sec, then close the syringe valve on the purging
device to begin gas flow through the trap. Maintain the trap
temperature at 180°C. After approximately 7 min, turn off the trap
heater and open the syringe valve to stop the gas flow through the
trap. When the trap is cool, the next sample can be analyzed.
11.7 TERMINATION OF DATA ACQUISITION -- When all the sample components
have eluted from the GC, terminate MS data acquisition. Use
appropriate data output software to display full range mass spectra
and appropriate plots of ion abundance as a function of time. If any
ion abundance exceeds the system working range, dilute the sample
aliquot in the second syringe with reagent water and analyze the
diluted aliquot.
11.8 IDENTIFICATION OF ANALYTES -- Identify a sample component by compar-
ison of its mass spectrum (after background subtraction) to a
reference spectrum in the user-created data base. The GC retention
time of the sample component should be within three standard
deviations of the mean retention time of the compound in the
calibration mixture.
11.8.1 In general, all ions that are present above 10% relative
abundance in the mass spectrum of the standard should be
present in the mass spectrum of the sample component and
should agree within absolute 20%. For example, if an ion has a
relative abundance of 30% in the standard spectrum, its
abundance in the sample spectrum should be in the range of 10
to 50%. Some ions, particularly the molecular ion, are of
special importance, and should be evaluated even if they are
below 10% relative abundance.
11.8.2 Identification requires expert judgement when sample
components are not resolved chromatographically and produce
mass spectra containing ions contributed by more than one
analyte. When GC peaks obviously represent more than one
sample component (i.e., broadened peak with shoulder(s) or
valley between two or more maxima), appropriate analyte
spectra and background spectra can be selected by examining
plots of characteristic ions for tentatively identified
components. When analytes coelute (i.e., only one GC peak is
apparent), the identification criteria can be met but each
274
-------
analyte spectrum will contain extraneous ions contributed by
the coeluting compound. Because purgeable organic compounds
are relatively small molecules and produce comparatively simple
mass spectra, this is not a significant problem for most method
analytes.
11.8.3 Structural isomers that produce very similar mass spectra can
be explicitly identified only if they have sufficiently
different GC retention times. Acceptable resolution is
achieved if the height of the valley between two peaks is less
than 25% of the average height of the two peaks. Otherwise,
structural isomers are identified as isomeric pairs. Cis- and
trans-l,2-dichloroethene, two of the three isomeric xylenes,
and two of the three dichlorobenzenes are three examples of
structural isomers that cannot be explicitly identified if both
members of the isomeric pair are present. These groups of
isomers must be reported as isomeric pairs (see Method 524.2
for an alternative approach).
11.8.4 Methylene chloride and other background components appear in
variable quantities in laboratory and field reagent blanks,
and generally cannot be accurately measured. Subtraction of
the concentration in the blank from the concentration in the
sample is not acceptable because the concentration of the
background in the blank is highly variable.
12. CALCULATIONS
12.1 Complete chromatographic resolution is not necessary for accurate and
precise measurements of analyte concentrations if unique ions with
adequate intensities are available for quantitation. For example,
although two listed analytes, carbon tetrachloride and bromodichloro-
methane, were not resolved with the GC conditions used, concentrations
were calculated by measuring the non-interfering quantitation ions.
12.1.1 Calculate analyte and surrogate concentrations.
c = (Ax)(Qjs) 1000
X (A1s) RF V
where: Cx = concentration of analyte or surrogate in
pg/L in the water sample.
Ax = integrated abundance of the quantitation ion
of the analyte in the sample.
Ais = integrated abundance of the quantitation ion
of the internal standard in the sample.
Qis = total quantity (in micrograms) of internal
standard added to the water sample.
V ° original water sample volume in ml.
RF = mean response factor of analyte from the
initial calibration.
275
-------
12.1.2 Alternatively, use the GC/MS system software or other
available proven software to compute the concentrations of
the analytes and surrogates from the second or third order
regression curves.
12.1.3 Calculations should utilize all available digits of precision,
but final reported concentrations should be rounded to an
appropriate number of significant figures (one digit of
uncertainty). Experience indicates that three significant
figures may be used for concentrations above 99 /ig/L, two
significant figures for concentrations between 1-99 pg/L. and
one significant figure for lower concentrations.
12.1.4 Calculate the total trihalomethane concentration by summing
the four individual trihalomethane concentrations in /ig/L.
13. ACCURACY AND PRECISION
13.1 Single laboratory accuracy and precision data were obtained for 31 of
the method analytes using laboratory fortified blanks with analytes at
concentrations between 1 and 5 0g/L, and these data are shown in Table
13.2 With these data, method detection limits were calculated using the
formula (2):
MDL = S t(n_lfl.aipha = o.99)
= Si
degrees of freedom
where: tin_i i-alpha = 0.99) • Student's t value for the 99%
confidence level with n-1
n = number of replicates
S = the standard deviation of the replicate analyses.
14. REFERENCES
Alford-Stevens, A., J.W. Eichelberger, W.L. Budde, "Purgeable Organic
Compounds in Water by Gas Chromatography/ Mass Spectrometry, Method
524." Environmental Monitoring and Support Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio, February 1983.
Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde,
"Trace Analyses for Wastewaters," Environ. Sci. Technol., 15, 1426,
1981.
"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.
276
-------
4. "OSHA Safety and Health Standards, General Industry," (29CFR1910),
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.
277
-------
TABLE 1. MOLECULAR HEIGHTS, RETENTION TINE DATA,
AND QUANTITATION IONS FOR METHOD ANALYTES
Compound
MU*
Retention'' Primary
Time Quantitation
(minrsec) Ions
Secondary
Quantitation
Ions
Internal standard
Fluorobenzene 96 16:34 96
Surrogates
4-Bromofluorobenzene 174 26:53 95
l,2-Dichlorobenzene-d4 150 35:55 152
Target Analvtes
Benzene 78 15:31 78
Bromobenzene 156 25:12 156
Bromochloromethane 128 9:20 128
Bromodichloromethane 162 12:24 83
Bromoform 250 17:17 173
Bromomethane 94 94
Carbon tetrachloride 152 12:19 117
Chlorobenzene 112 22:14 112
Chloroethane 64 64
Chloroform 118 9:41 83
Chloromethane 50 50
2-Chlorotoluene 126 91
4-Chlorotoluene 126 91
Dibromochloromethane 206 14:53 129
l,2-Dibromo-3-Chloropropane 234 23:55 75
1,2-Dibromoethane 186 16:10 107
Dibromomethane 172 10:38 93
1,2-Dichlorobenzene 146 35:07 146
1,3-DiChlorobenzene 146 35:55 146
1,4-DiChlorobenzene 146 35:55 146
Dichlorodifluoromethane 120 4:14 85
1,1-Dichloroethane 98 9:02 63
1,2-Dichloroethane 98 10:43 62
1,1-Dichloroethene 96 7:50 96
cis-l,2-Dichloroethene 96 96
trans-l,2-Dichloroethene 96 9:55 96
1,2-Dichloropropane 112 13:55 63
1,3-Dichloropropane 112 16:28 76
2,2-Dichloropropane 112 77
1,1-Dichloropropene 110 75
cis-l,3-dichloropropene 110 75
trans-l,3-dichloropropene 110 75
Ethyl benzene 106 91
p-Isopropyltoluene 134 119
77
174,176
115,150
77
77,158
49,130
85,127
175,252
96
119
77,114
66
85
52
126
126
127
155,157
109,188
95,174
111,148
111,148
111,148
87
65,83
98
61,63
61,98
61,98
112
78
97
110,77
106
134,91
278
-------
Compound
TABLE 1. (Continued)
MWa
Retention'3 Primary
Time Quantitation
(mi n: sec) Ions
Secondary
Quantitation
Ions
Methyl ene chloride
Styrene
1 , 1, 1 ,2-Tetrachloroethane
1,1,2, 2-Tetrachl oroethane
Tetrachloroethene
Toluene
1,1, 1 -Trichl oroethane
1,1, 2-Trichl oroethane
Trichl oroethene
Tr i chl orof 1 uoromethane
1,2,3-Trichloropropane
Vinyl Chloride
o-Xylene
m-Xylene
p-Xylene
84
104
166
166
164
92
132
132
130
136
146
62
106
106
106
5:21
29:02
19:31
20:00
21:22
11:41
14:43
7:22
4:00
30:34
30:48
30:48
84
104
131
83
166
92
97
S3
95
101
75
62
106
106
106
86,49
78
133,119
131,85
168,129
91
99,61
97,85
130,132
103
77
64
91
91
91
a Monoisotopic molecular weight calculated from the atomic masses of the
isotopes with the smallest masses.
D Retention time measured from the beginning of the thermal desorption step.
Compounds with no retention data are known to be amenable to purge and trap
extraction (see Method 524.2), and chromatography on the packed gas
chromatography column used in this method, but no retention time data is
available for this method.
279
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TABLE 2. ION ABUNDANCE CRITERIA FOR 4-BRONOFLUOROBENZENE (BFB)
Mass
ttt/z} Relative Abundance Criteria
50 15 to 40% of mass 95
75 30 to 80% of mass 95
95 Base Peak, 100% Relative Abundance
96 5 to 9% of mass 95
173 < 2% of mass 174
174 > 50% of mass 95
175 5 to 9% of mass 174
176 > 95% but < 101% of mass 174
177 5 to 9% of mass 176
280
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TABLE 3. ACCURACY AND PRECISION DATA FROM SEVEN TO NINE DETERMINATIONS
OF THE METHOD ANALYTES IN REAGENT WATER3
Mean
True Observed
Cone. Cone.
Comoound (pa/L) (ua/L)
Benzene
Bromobenzene
Bromod * chl oromethane
Bromoform ;
Carbon tetrachloride
Chlorobenzene
Chloroform
D1 bromochl oromethane
1.0 0.97
1.0 0.92
1.0 1.0
>.5 2.4
1.0 0.88
1.0 1.02
1.0 1.03
1.0 0.92
l,2-Dibromo-3-chloropropane 3.5 3.5
1,2-Oibromoethane
Dibromomethane
1.0 0.93
1.0 0.94
1,2-Dichlorobenzene 5.0 5.0
1,4-Dichlorobenzene 5.0 5.6
Di chl orodi f 1 uoromethane
1,1-Dichloroethane
1,2-Dichloroethane
1,1-Dlchloroethene
trans- 1,2-Di chl oroethene
1 , 2-Di chl oropropane
1,3-Dichloropropane
Methyl ene chloride
Styrene
1 , 1 ,2,2-Tetrachloroethane
Tetrachl oroethene
Toluene
1,1,1 -Tri chl oroethane
Trichloroethene
Tri chl orof 1 uoromethane
Vinyl chloride
o-Xylene
p-Xylene
1.0 0.96
1.0 1.05
L.O 0.97
1.0 1.09
1.0 0.98
L.O 1.01
.0 1.00
.0 0.99
.0 1.2
.0 1.11
.0 0.93
.0 1.05
.0 1.05
.0 0.90
.0 1.09
.0 0.98
.0 1.02
.0 1.11
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Rel . Mean Method
Std. Std. Accuracy Dect.
Dev. Dev. (% of True Limit
(ua/L) (%) Value) (ua/L)
.036
.042
.17
.23
.098
.047
.086
.14
.63
.13
.11
.35
.73
.11
.060
.077
.066
.066
.060
.033
.45
.072
.14
.10
.043
.093
.12
.072
.11
.068
.047
3.
4.
17.
9.
11.
4.
8.
15.
18.
14.
12.
7.
13.
12.
5.
7.
6.
6.
5.
3.
46.
6.
13.
11.
4.
8.
13.
6.
11.
6.
4.
7
6
6
6
3
0
7
9
1
7
9
3
0
1
9
6
7
2
97
92
100
100
88
102
103
92
100
93
94
100
112
96
105
97
109
98
101
100
99
120
111
93
105
105
90
109
98
102
111
0
0
0
0
0
0
0
0
2
0
0
1
2
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
.1
.1
.5
.7
.3
.1
.2
.4
.
.4
.3
•
.
.3
.2
.2
.2
.2
.2
.1
.
.2
.4
.3
.1
.3
.4
.2
.3
.2
.3
* Data obtained by Robert VI. Slater with a 25-mL sample size and the
compounds divided into two groups to minimize coelution.
281
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OPTIONAL
FOAM
TRAP
K IN. _
0. D. EXIT
•EXIT % IN.
0. D.
I—14MM 0. D.
INLET X IN.
0.0.
10MM GLASS FRIT
MEDIUM POROSITY
SAMPLE INLET
2-WAY SYRINGE VALVE
•17CM. 20 GAUGE SYRINGE NEEDLE
6MM. 0. D. RUBBER SEPTUM
~10MM. 0. D. 1/16 IN. O.D.
y STAINLESS ST
•INLET
% IN. 0. D.
13X MOLECULAR
SIEVE PURGE
GAS FILTER
PURGE GAS
CONTROL
FIGURE 1. PURGING DEVICE'
282
-------
PACKING PROCEDURE
CONSTRUCTION
WOOL
ACTIVATED,
CHARCOAL 7-7
GRADE 15 I
SIUCA GEL7'7 -
TENAX 7.7
**OV'1 10li
GLASS WOOL10"
aw
7A/FQOT
RESISTANCE
WIRE WRAPPED
SOLID
(DOUBLE LAYER)
7^/FOOT.
RESISTANCE
WIRE WRAPPED
SOLID
(SINGLE LAYER)
8CMH
TRAP INLET
. COMPRESSION
FITTING NUT
AND FERRULES
THERMOCOUPLE/
CONTROLLER
SENSOR
. / TUBING 25CM
r -LJ. 0.105 IN. I.D.
^ 7/0.125 IN. O.D,
C" */ STAINLESS STB
FIGURE 2. TRAP PACKINGS AND CONSTRUCTION TO INCLUDE
DESORB CAPABILITY
283
-------
COLUMN: 1% SP-1000 ON CARBOPACK:B
PROGRAM 45°C FOR 3 MIN. 8°C/MIN TO 220°C
DETECTOR: MASS SPECTROMETER
UJ
10 12 14 16 18 20
RETENTION TIME. MIN.
FIGURE 3. GAS CHROMATOGRAM
22
24
26 28
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METHOD 524.2. MEASUREMENT OF PURGEABLE ORGANIC COMPOUNDS IN
HATER BY CAPILLARY COLUMN GAS CHROMATOGRAPHY/MASS SPECTROMETRY
Revision 3.0
A. Alford-Stevens, J. U. Eichelberger, W. L. Budde - Method 524, Revision 1.0
(1983)
R. W. Slater, Jr. - Method 524.2, Revision 2.0 (1986)
J. W. Eichelberger, W. L. Budde - Method 524.2, Revision 3.0 (1989)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 524.2
MEASUREMENT OF PURGEABLE ORGANIC COMPOUNDS IN WATER BY
CAPILLARY COLUMN GAS CHROMATOGRAPHY/MASS SPECTROMETRY
1. SCOPE AND APPLICATION
1.1 This Is a genera] purpose method for the Identification and
simultaneous measurement of purgeable volatile organic compounds In
finished drinking water, raw source water, or drinking water in any
treatment stage (1-2). The method is applicable to a wide range of
organic compounds, including the four trihalomethane disinfection
by-products, that have sufficiently high volatility and low water
solubility to be efficiently removed from water samples with purge and
trap procedures. The following compounds can be determined by this
method.
Chemical Abstract Service
Compound Registry Number
Benzene 71-43-2
Bromobenzene 108-86-1
Bromochloromethane 74-97-5
Bromodichloromethane 75-27-4
Bromoform 75-25-2
Bromomethane 74-83-9
n-Butylbenzene 104-51-8
sec-Butyl benzene 135-98-8
tert-Butylbenzene 98-06-6
Carbon tetrachloride 56-23-5
Chlorobenzene 108-90-7
Chloroethane 75-00-3
Chloroform 67-66-3
Chloromethane 74-87-3
2-Chlorotoluene 95-49-8
4-Chlorotoluene 106-43-4
Dibromochloromethane 124-48-1
l,2-Dibromo-3-chloropropane 96-12-8
1,2-Dibromoethane 106-93-4
Dibromomethane 74-95-3
1,2-Dichlorobenzene 95-50-1
1,3-Dichlorobenzene 541-73-1
1,4-Dichlorobenzene 106-46-7
Dichlorodifluoromethane 75-71-8
1,1-Dichloroethane 75-34-3
1,2-Dichloroethane 107-06-2
1,1-Dichloroethene 75-35-4
cis-l,2-Dichloroethene 156-59-4
trans-1,2-Dichloroethene 156-60-5
1,2-Dichloropropane 78-87-5
1,3-Dichloropropane 142-28-9
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2,2-Dichloropropane 590-20-7
1,1-Dichloropropene 563-58-6
cis-l,3-Dichloropropene 10061-01-5
trans-1,3-Dichloropropene 10061-02-6
Ethyl benzene 100-41-4
Hexachlorobutadiene 87-68-3
Isopropylbenzene 98-82-8
4-Isopropyltoluene 99-87-6
Methylene chloride 75-09-2
Naphthalene 91-20-3
n-Propylbenzene 103-65-1
Styrene 100-42-5
1,1,1,2-Tetrachloroethane 630-20-6
1,1,2,2-Tetrachloroethane 79-34-5
Tetrachloroethene 127-18-4
Toluene 108-88-3
1,2,3-Trichlorobenzene 87-61-6
1,2,4-Trichlorobenzene 120-82-1
1,1,1-Trichloroethane 71-55-6
1,1,2-Trichloroethane 79-00-5
Trichloroethene 79-01-6
Trichlorofluoromethane 75-69-4
1,2,3-Trichloropropane 96-18-4
1,2,4-Trimethylbenzene 95-63-6
1,3,5-Trimethylbenzene 108-67-8
Vinyl chloride 75-01-4
o-Xylene 95-47-6
m-Xylene 108-38-3
p-Xylene 106-42-3
1.2 Method detection limits (MDLs) (3) are compound and instrument
dependent and vary from approximately 0.02-0.35 pg/L. The applicable
concentration range of this method is primarily column dependent and is
approximately 0.02 to 200 /zg/L for the wide-bore thick-film columns.
Narrow-bore thin-film columns may have a capacity which limits the
range to about 0.02 to 20 /jg/L. Analytes that are inefficiently purged
from water will not be detected when present at low concentrations, but
they can be measured with acceptable accuracy and precision when
present in sufficient amounts.
1.3 Analytes that are not separated chromatographically, but which have
different mass spectra and non-interfering quantitation ions, can be
identified and measured in the same calibration mixture or water sample
(Sect 11.6.2). Analytes which have very similar mass spectra cannot be
individually identified and measured in the same calibration mixture or
water sample unless they have different retention times (Sect.11.6.3).
Coeluting compounds with very similar mass spectra, typically many
structural isomers, must be reported as an isomeric group or pair. Two
of the three isomeric xylenes and two of the three dichlorobenzenes are
examples of structural isomers that may not be resolved on the
capillary column, and if not, must be reported as isomeric pairs.
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2. SUMMARY OF METHOD
2.1 Volatile organic compounds and surrogates with low water solubility are
extracted (purged) from the sample matrix by bubbling an inert gas
through the aqueous sample. Purged sample components are trapped in a
tube containing suitable sorbent materials. When purging is complete,
the sorbent tube is heated and backflushed with helium to desorb the
trapped sample components into a capillary gas chromatography (GC)
column interfaced to a mass spectrometer (MS). The column is tempera-
ture programmed to separate the method analytes which are then detected
with the MS. Compounds eluting from the GC column are identified by
comparing their measured mass spectra and retention times to reference
spectra and retention times in a data base. Reference spectra and
retention times for analytes are obtained by the measurement of
calibration standards under the same conditions used for samples. The
concentration of each identified component is measured by relating the
MS response of the quantitation ion produced by that compound to the MS
response of the quantitation ion produced by a compound that is used as
an internal standard. Surrogate analytes, whose concentrations are
known in every sample, are measured with the same internal standard
calibration procedure.
3. DEFINITIONS
3.1 Internal standard -- A pure analyte(s) added to a solution in known
amount(s) and used to measure the relative responses of other method
analytes and surrogates that are components of the same solution. The
internal standard must be an analyte that is not a sample component.
3.2 Surrogate analyte -- A pure analyte(s), which is extremely unlikely to
be found in any sample, and which is added to a sample aliquot in known
amount(s) before extraction and is measured with the same procedures
used to measure other sample components. The purpose of a surrogate
analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates (LD1 and LD2) -- Two sample aliquots taken in the
analytical laboratory and analyzed separately with identical proce-
dures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample collection,
preservation, or storage procedures.
3.4 Field duplicates (FD1 and FD2) -- Two separate samples collected at the
same time and place under identical circumstances and treated exactly
the same throughout field and laboratory procedures. Analyses of FD1
and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory
procedures.
3.5 Laboratory reagent blank (LRB) -- An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates that
are used with other samples. The LRB is used to determine if method
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analytes or other interferences are present in the laboratory environ-
ment, the reagents, or the apparatus.
3.6 Field reagent blank (FRB) -- Reagent water placed in a sample container
in the laboratory and treated as a sample in all respects, including
exposure to sampling site conditions, storage, preservation and all
analytical procedures. The purpose of the FRB is to determine if
method analytes or other interferences are present in the field
environment.
3.7 Laboratory performance check solution (LPC) -- A solution of one or
more compounds (analytes, surrogates, internal standard, or other test
compounds) used to evaluate the performance of the instrument system
with respect to a defined set of method criteria.
3.8 Laboratory fortified blank (LFB) -- An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its purpose
is to determine whether the methodology is in control, and whether the
laboratory is capable of making accurate and precise measurements at
the required method detection limit.
3.9 Laboratory fortified sample matrix (LFM) -- An aliquot of an environ-
mental sample to which known quantities of the method analytes are
added in the laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the
analytes in the sample matrix must be determined in a separate aliquot
and the measured values in the LFM corrected for background concentra-
tions.
3.10 Stock standard solution -- A concentrated solution containing a single
certified standard that is a method analyte, or a concentrated solution
of a single analyte prepared in the laboratory with an assayed
reference compound. Stock standard solutions are used to prepare
primary dilution standards.
3.11 Primary dilution standard solution -- A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted as
needed to prepare calibration solutions and other needed analyte
solutions.
3.12 Calibration standard (CAL) -- a solution prepared from the primary
dilution standard solution and stock standard solutions of the internal
standards and surrogate analytes. The CAL solutions are used to
calibrate the instrument response with respect to analyte concentra-
tion.
3.13 Quality control sample (QCS) -- a sample matrix containing method
analytes or a solution of method analytes in a water miscible solvent
which is used to fortify reagent water or environmental samples. The
QCS is obtained from a source external to the laboratory, and is used
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to check laboratory performance with externally prepared test
materials.
4. INTERFERENCES
4.1 During analysis, major contaminant sources are volatile materials
in the laboratory and impurities in the inert purging gas and in
the sorbent trap. The use of non-polytetrafluoroethylene (PTFE)
plastic tubing, non-PTFE thread sealants, or flow controllers with
rubber components in the purging device should be avoided since
such materials out-gas organic compounds which will be concentrated
in the trap during the purge operation. Analyses of laboratory
reagent blanks provide information about the presence of contaminants.
When potential interfering peaks are noted in laboratory reagent
blanks, the analyst should change the purge gas source and regenerate
the molecular sieve purge gas filter. Subtracting blank values from
sample results is not permitted.
4.2 Interfering contamination may occur when a sample containing low
concentrations of volatile organic compounds is analyzed immediately
after a sample containing relatively high concentrations of volatile
organic compounds. A preventive technique is between-sample rinsing of
the purging apparatus and sample syringes with two portions of reagent
water. After analysis of a sample containing high concentrations of
volatile organic compounds, one or more laboratory reagent blanks
should be analyzed to check for cross contamination.
4.3 Special precautions must be taken to determine methylene chloride. The
analytical and sample storage area should be isolated from all
atmospheric sources of methylene chloride, otherwise random background
levels will result. Since methylene chloride will permeate through
PTFE tubing, all gas chromatography carrier gas lines and purge gas
plumbing should be constructed of stainless steel or copper tubing.
Laboratory worker's clothing should be cleaned frequently since
clothing previously exposed to methylene chloride fumes during common
liquid/liquid extraction procedures can contribute to sample contamina-
tion.
5. SAFETY
5.1 The toxicity or carcinogenicity of chemicals used in this method
has not been precisely defined; each chemical should be treated as
a potential health hazard, and exposure to these chemicals should
be minimized. Each laboratory is responsible for maintaining
awareness of OSHA regulations regarding safe handling of chemicals
used in this method. Additional references to laboratory safety
are available (4-6) for the information of the analyst.
5.2 The following pathod analytes have been tentatively classified as
known or susrjcted human or mammalian carcinogens: benzene, carbon
tetrachlorv-a, 1,4-dichlorobenzene, 1,2-dichlorethane, hexachloro-
butadiene 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, chloro-
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form, l,2-dibromoethane,tetrachloroethene, trichloroethene, and vinyl
chloride. Pure standard materials and stock standard solutions of
these compounds should be handled in a hood. A NIOSH/MESA approved
toxic gas respirator should be worn when the analyst handles high
concentrations of these toxic compounds.
6. APPARATUS AND EQUIPMENT
6.1 SAMPLE CONTAINERS -- 60-mL to 120-mL screw cap vials (Pierce #19832
or equivalent) each equipped with a PTFE-faced silicone septum
(Pierce #12718 or equivalent). Prior to use, wash vials and septa
with detergent and rinse with tap and distilled water. Allow the
vials and septa to air dry at room temperature, place in a 105°C
oven for 1 hr, then remove and allow to cool in an area known to be
free of organics.
6.2 PURGE AND TRAP SYSTEM -- The purge and trap system consists of three
separate pieces of equipment: purging device, trap, and desorber.
Systems are commercially available from several sources that meet all
of the following specifications.
6.2.1 The all glass purging device (Figure 1) should be designed to
accept 25-mL samples with a water column at least 5 cm deep. A
smaller (5-mL) purging device is recommended if the GC/MS system
has adequate sensitivity to obtain the method detection limits
required. Gaseous volumes above the sample must be kept to a
minimum (< 15 ml) to eliminate dead volume effects. A glass
frit should be installed at the base of the sample chamber so
the purge gas passes through the water column as finely divided
bubbles with a diameter of < 3 mm at the origin. Needle
spargers may be used, however, the purge gas must be introduced
at a point about 5 mm from the base of the water column.
6.2.2 The trap (Figure 2) must be at least 25 cm long and have an
inside diameter of at least 0.105 in. Starting from the inlet,
the trap should contain 1.0 cm of methyl silicone coated packing
and the following amounts of adsorbents: 1/3 of 2,6-diphenylene
oxide polymer, 1/3 of silica gel, and 1/3 of coconut charcoal.
If it is not necessary to determine dichlorodifluoromethane, the
charcoal can be eliminated and the polymer increased to fill 2/3
of the trap. Before initial use, the trap should be conditioned
overnight at 180°C by backflushing with an inert gas flow of at
least 20 mL/min. Vent the trap effluent to the room, not to the
analytical column. Prior to daily use, the trap should be
conditioned for 10 min at 180°C with backflushing. The trap may
be vented to the analytical column during daily conditioning;
however, the column must be run through the temperature program
prior to analysis of samples.
6.2.3 The use of the methyl silicone coated packing is recommended,
but not mandatory. The packing serves a dual purpose of
protecting the Tenax adsorbant from aerosols, and also of
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Insuring that the Tenax Is fully enclosed within the heated
zone of the trap thus eliminating potential cold spots.
Alternatively, silanized glass wool may be used as a spacer at
the trap inlet.
6.2.4 The desorber (Figure 2) must be capable of rapidly heating the
trap to 180°C either prior to or at the beginning of the flow of
desorption gas. The polymer section of the trap should not be
heated higher than 200°C or the life expectancy of the trap will
decrease. Trap failure is characterized by a pressure drop in
excess of 3 pounds per square inch across the trap during
purging or by poor bromoform sensitivities. The desorber
design illustrated in Fig. 2 meets these criteria.
6.3 GAS CHROMATOGRAPHY/MASS SPECTROMETER/DATA SYSTEM (GC/MS/DS)
6.3.1 The GC must be capable of temperature programming and should be
equipped with variable-constant differential flow controllers so
that the column flow rate will remain constant throughout
desorption and temperature program operation. The column oven
must be cooled to 10°C; therefore, a subambient oven controller
is required. If syringe injections of BFB will be used, a
split/splitless injection port is required.
6.3.2 Capillary Gas Chromatography Columns. Any gas chromatography
column that meets the performance specifications of this method
may be used. Separations of the calibration mixture must be
equivalent or better than those described in this method. Three
useful columns have been identified.
6.3.2.1 Column 1 -- 60 m x 0.75 mm ID VOCOL (Supelco, Inc.)
glass wide-bore capillary with a 1.5 im film thickness.
Column 2 -- 30 m x 0.53 mm ID DB-624 (J&W Scientific,
Inc.) fused silica capillary with a 3 /im film thick-
ness.
Column 3 -- 30 m x 0.32 mm ID DB-5 (J&W Scientific,
Inc.) fused silica capillary with a 1 (m film thick-
ness.
6.3.3 Interfaces between the GC and MS. The interface used depends on
the column selected and the gas flow rate.
6.3.3.1 The wide-bore columns 1 and 2 have the capacity to
accept the standard gas flows from the trap during
thermal desorption, and chromatography can begin with
the onset of thermal desorption. Depending on the
pumping capacity of the MS, an additional interface
between the end of the column and the MS may be
required. An open split interface (7), an all-glass
jet separator, or a cryogenic (Sect. 6.3.3.2) device
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are acceptable interfaces. Any interface can be used
if the performance specifications described in this
method can be achieved. The end of the transfer line
after the interface, or the end of the analytical
column if no interface is used, should be placed within
a few mm of the MS ion source.
6.3.3.2 The narrow bore column 3 cannot accept the thermal
desorption gas flow, and a cryogenic interface is
required. This interface (Tekmar Model 1000 or
equivalent) condenses the desorbed sample components at
liquid nitrogen temperature, and allows the helium gas
to pass through to an exit. The condensed components
are frozen in a narrow band on an uncoated fused silica
precolumn. When all components have been desorbed from
the trap, the interface is rapidly heated under a
stream of carrier gas to transfer the analytes to the
analytical column. The end of the analytical column
should be placed with a few mm of the MS ion source.
A potential problem with this interface is blockage of
the interface by frozen water from the trap. This
condition will result in a major loss in sensitivity
and chromatographic resolution.
6.3.4 The mass spectrometer must be capable of electron ionization at
a nominal electron energy of 70 eV. The spectrometer must be
capable of scanning from 35 to 260 amu with a complete scan
cycle time (including scan overhead) of 2 sec or less. (Scan
cycle time = Total MS data acquisition time in seconds divided
by number of scans in the chromatogram). The spectrometer must
produce a mass spectrum that meets all criteria in Table 3 when
25 ng or less of 4-bromofluorobenzene (BFB) is introduced into
the GC. An average spectrum across the BFB GC peak may be used
to test instrument performance.
6.3.5 An interfaced data system is required to acquire, store, reduce,
and output mass spectral data. The computer software should
have the capability of processing stored GC/MS data by recogniz-
ing a GC peak within any given retention time window, comparing
the mass spectra from the GC peak with spectral data in a
user-created data base, and generating a list of tentatively
identified compounds with their retention times and scan
numbers. The software must allow integration of the ion
abundance of any specific ion between specified time or scan
number limits. The software should also allow calculation of
response factors as defined in Sect. 9.2.6 (or construction of
a second or third order regression calibration curve), calcula-
tion of response factor statistics (mean and standard devia-
tion), and calculation of concentrations of analytes using
either the calibration curve or the equation in Sect. 12.
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6.4 SYRINGE AND SYRINGE VALVES
6.4.1 Two 5-mL or 25-mL glass hypodermic syringes with Luer-Lok tip
(depending on sample volume used).
6.4.2 Three 2-way syringe valves with Luer ends.
6.4.3 One 25-/U. micro syringe with a 2 1n x 0.006 in ID, 22° bevel
needle (Hamilton #702N or equivalent).
6.4.4 Micro syringes - 10, 100 /iL.
6.4.5 Syringes - 0.5, 1.0, and 5-mL, gas tight with shut-off valve.
6.5 MISCELLANEOUS
6.5.1 Standard solution storage containers -- 15-mL bottles with
PTFE-lined screw caps.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 TRAP PACKING MATERIALS
7.1.1 2,6-Diphenylene oxide polymer, 60/80 mesh, chromatographic
grade (Tenax GC or equivalent).
7.1.2 Methyl silicone packing (optional) -- OV-1 (3%) on Chromo-
sorb W, 60/80 mesh, or equivalent.
7.1.3 Silica gel -- 35/60 mesh, Davison, grade 15 or equivalent.
7.1.4 Coconut charcoal -- Prepare from Barnebey Cheney, CA-580-26
lot 0M-2649 by crushing through 26 mesh screen.
7.2 REAGENTS
7.2.1 Methanol -- Demonstrated to be free of analytes.
7.2.2 Reagent water -- Prepare reagent water by passing tap water
through a filter bed containing about 0.5 kg of activated
carbon, by using a water purification system, or by boiling
distilled water for 15 min followed by a 1-h purge with inert
gas while the water temperature is held at 90°C. Store in
clean, narrow-mouth bottles with PTFE-lined septa and screw
caps.
7.2.3 Hydrochloric acid (1+1) -- Carefully add measured volume of
cone. HC1 to equal volume of reagent water.
7.2.4 Vinyl chloride -- Certified mixtures of vinyl chloride in
nitrogen and pure vinyl chloride are available from several
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sources (for example, Matheson, Ideal Gas Products, and Scott
Gases).
7.2.5 Ascorbic acid -- ACS reagent grade, granular.
7.3 STOCK STANDARD SOLUTIONS -- These solutions may be purchased as
certified solutions or prepared from pure standard materials using the
following procedures. One of these solutions is required for every
analyte of concern, every surrogate, and the internal standard. A
useful working concentration is about 1-5 mg/mL.
7.3.1 Place about 9.8 ml of methanol into a 10-mL ground-glass
stoppered volumetric flask. Allow the flask to stand,
unstoppered, for about 10 min or until all alcohol-wetted
surfaces have dried and weigh to the nearest 0.1 mg.
7.3.2 If the analyte is a liquid at room temperature, use a 100-pL
syringe and immediately add two or more drops of reference
standard to the flask. Be sure that the reference standard
falls directly into the alcohol without contacting the neck
of the flask. If the analyte is a gas at room temperature,
fill a 5-mL valved gas-tight syringe with the standard to
the 5.0-mL mark, lower the needle to 5 mm above the methanol
meniscus, and slowly inject the standard into the neck area
of the flask. The gas will rapidly dissolve in the methanol.
7.3.3 Reweigh, dilute to volume, stopper, then mix by inverting
the flask several times. Calculate the concentration in
/xg//iL from the net gain in weight. When compound purity is
certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock standard.
7.3.4 Store stock standard solutions in 15-mL bottles equipped
with PTFE-lined screw caps. Methanol solutions prepared
from liquid analytes are stable for at least 4 weeks when
stored at 4°C. Methanol solutions prepared from gaseous
analytes are not stable for more than 1 week when stored
at <0°C; at room temperature, they must be discarded after
1 day.
7.4 PRIMARY DILUTION STANDARDS -- Use stock standard solutions to prepare
primary dilution standard solutions that contain all the analytes of
concern and the surrogates (but not the internal standard!) in
methanol. The primary dilution standards should be prepared at
concentrations that can be easily diluted to prepare aqueous calibra-
tion solutions that will bracket the working concentration range.
Store the primary dilution standard solutions with minimal headspace
and check frequently for signs of deterioration or evaporation,
especially just before preparing calibration solutions. Storage times
described for stock standard solutions in Sect. 7.4.4 also apply to
primary dilution standard solutions.
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7.5 FORTIFICATION SOLUTIONS FOR INTERNAL STANDARD AND SURROGATES
7.5.1 A solution containing the internal standard and the surrogates
is required to prepare laboratory reagent blanks (also used as a
laboratory performance check solution), and to fortify each
sample. Prepare a fortification solution containing fluoro-
benzene (internal standard), 1,2- dichlorobenzene-d4
(surrogate), and BFB (surrogate) in methanol at concentrations
of 5 /ig/mL of each. A 5-/iL aliquot of this solution added to a
25-mL water sample volume gives concentrations of 1 /jg/L of
each. A 5-/iL aliquot of this solution added to a 5-mL water
sample volume gives a concentration of 5 /zg/L of each).
Additional internal standrds and surrogate analytes are
optional.
7.5.2 A solution of the internal standard alone is required to prepare
calibration standards and laboratory fortified blanks. The
internal standard should be in methanol at a concentration of
5 /zg/mL.
7.6 PREPARATION OF LABORATORY REAGENT BLANK -- Fill a 25-mL (or 5-mL)
syringe with reagent water and adjust to the mark (no air bubbles).
Inject 10 /iL of the fortification solution containing the internal
standard and surrogates through the Luer Lok valve into the reagent
water. Transfer the LRB to the purging device. See Sect. 11.1.2.
7.7 PREPARATION OF LABORATORY FORTIFIED BLANK -- Prepare this exactly like
a calibration standard (Sect. 7.8). This is a calibration standard
that is treated as a sample.
7.8 PREPARATION OF CALIBRATION STANDARDS
7.8.1 The number of calibration solutions (CALs) needed depends on the
calibration range desired. A minimum of three CAL solutions is
required to calibrate a range of a factor of 20 in concentra-
tion. For a factor of 50, use at least four standards, and for
a factor of 100 at least five standards. One calibration
standard should contain each analyte of concern and each
surrogate at a concentration of 2-10 times the method detection
limit (Tables 4-6) for that compound. The other CAL standards
should contain each analyte of concern and each surrogate at
concentrations that define the range of the method. Every CAL
solution contains the internal standard at the same concentra-
tion (5 /zg/L suggested for a 5-mL sample; 1 /zg/L for a 25-mL
sample).
7.8.2 To prepare a calibration standard, add an appropriate volume of
a primary dilution standard (containing analytes and surrogates)
to an aliquot of reagent water in a volumetric flask. Use a
microsyringe and rapidly inject the methanol solutions into the
expanded area of the filled volumetric flask. Remove the needle
as quickly as possible after injection. Mix by inverting t
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flask three times only. Discard the contents contained in the
neck of the flask. Aqueous standards are not stable in a
volumetric flask and should be discarded after 1 hr unless
transferred to a sample bottle and sealed immediately.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE COLLECTION, DECHLORINATION, AND PRESERVATION
8.1.1 Collect all samples in duplicate. If samples contain residual
chlorine, and measurements of the concentrations of disinfection
by-products (trihalomethanes, etc.) at the time of sample
collection are desired, add about 25 mg of ascorbic acid to the
sample bottle before filling. Fill sample bottles to overflow-
ing, but take care not to flush out the rapidly dissolving
ascorbic acid. No air bubbles should pass through the sample as
the bottle is filled, or be trapped in the sample when the
bottle is sealed. Adjust the pH of the duplicate samples to <2
by carefully adding one drop of 1:1 HC1 for each 20 mL of sample
volume. Seal the sample bottles, PFTE-face down, and shake
vigorously for 1 min.
8.1.2 When sampling from a water tap, open the tap and allow the
system to flush until the water temperature has stabilized
(usually about 10 min). Adjust the flow to about 500 mL/min and
collect duplicate samples from the flowing stream.
8.1.3 When sampling from an open body of water, fill a 1-quart
wide-mouth bottle or 1-liter beaker with sample from a
representative area, and carefully fill duplicate sample
bottles from the 1-quart container.
8.1.4 The samples must be chilled, to 4°C on the day of collection and
maintained at that temperature until analysis. Field samples
that will not be received at the laboratory on the day of
collection must be packaged for shipment with sufficient ice to
ensure that they will be at 4°C on arrival at the laboratory.
8.2 SAMPLE STORAGE
8.2.1 Store samples at 4°C until analysis. The sample storage area
must be free of organic solvent vapors.
8.2.2 Analyze all samples within 14 days of collection. Samples
not analyzed within this period must be discarded and replaced.
8.3 FIELD REAGENT BLANKS
8.3.1 Duplicate field reagent blanks must be handled along with each
sample set, which is composed of the samples collected from the
same general sample site at approximately the same time. At
the laboratory, fill field blank sample bottles with reagent
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water, seal, and ship to the sampling site along with empty
sample bottles and back to the laboratory with filled sample
bottles. Wherever a set of samples is shipped and stored, it
is accompanied by appropriate blanks.
8.3.2 Use the same procedures used for samples to add ascorbic acid
and HC1 to blanks (Sect. 8.1.1).
9. CALIBRATION
9.1 Demonstration and documentation of acceptable initial calibration is
required before any samples are analyzed and is required intermittently
throughout sample analysis as dictated by results of continuing
calibration checks. After initial calibration is successful, a
continuing calibration check is required at the beginning of each 8 hr.
period during which analyses are performed. Additional periodic
calibration checks are good laboratory practice.
9.2 Initial calibration
9.2.1 Calibrate the mass and abundance scales of the MS with calibra-
tion compounds and procedures prescribed by the manufacturer
with any modifications necessary to meet the requirements in
Sect. 9.2.2.
9.2.2 Introduce into the GC (either by purging a laboratory reagent
blank or making a syringe injection) 25 ng of BFB and acquire
mass spectra for m/z 35-260 at 70 eV (nominal). Use the purging
procedure and/or GC conditions given in Sect. 11. If the
spectrum does not meet all criteria in Table 2, the MS must be
retuned and adjusted to meet all criteria before proceeding with
calibration. An average spectrum across the GC peak may be used
to evaluate the performance of the system.
9.2.3 Purge a medium CAL solution, for example 10-20 /zg/L, using the
procedure given in Sect. 11.
9.2.4 Performance criteria for the medium calibration. Examine the
stored GC/MS data with the data system software. Figure 3 shows
an acceptable total ion chromatogram.
9.2.4.1 GC performance. Good column performance will produce
symmetrical peaks with minimum tailing for most
compounds. If peaks are broad, or sensitivity poor,
see Sect. 9.3.6 for some possible remedial actions.
9.2.4.2 MS sensitivity. The GC/MS/DS peak identification
software should be able to recognize a GC peak in the
appropriate retention time window for each of the
compounds in calibration solution, and make correct
tentative identifications. If fewer than 99% of the
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compounds are recognized, system maintenance is
required. See Sect. 9.3.6.
9.2.5 If all performance criteria are met, purge an aliquot of each of
the other CAL solutions using the same GC/MS conditions.
9.2.6 Calculate a response factor (RF) for each analyte, surrogate,
and isomer pair for each CAL solution using the internal
standard fluorobenzene. Table 1 contains suggested quantitation
ions for all compounds. This calculation is supported in
acceptable GC/MS data system software (Sect. 6.3.4), and many
other software programs. RF is a unitless number, but units
used to express quantities of analyte and internal standard must
be equivalent.
RF= (Ax)(Qis)
(AlsHQx)
where:
Ais
Qx
Qis
integrated abundance of the quantitation ion
of the analyte.
integrated abundance of the quantitation ion
of the internal standard.
quantity of analyte purged in ng or
concentration units.
quantity of internal standard purged in ng
or concentration units.
9.2.6.1
9.2.7
For each analyte and surrogate, calculate the mean RF
from the analyses of the CAL solutions. Calculate the
standard deviation (SD) and the relative standard
deviation (RSD) from each mean: RSD = 100 (SD/M). If
the RSD of any analyte or surrogate mean RF exceeds
20%, either analyze additional aliquots of appropriate
CAL solutions to obtain an acceptable RSD of RFs over
the entire concentration range, or take action to
improve GC/MS performance. See Sect. 9.2.7.
As an alternative to calculating mean response factors and
applying the RSD test, use the GC/MS data system software or
other available software to generate a second or third order
regression calibration curve.
9.3 Continuing calibration check. Verify the MS tune and initial calibra-
tion at the beginning of each 8-hr work shift during which analyses
are performed using the following procedure.
9.3.1 Introduce into the GC (either by purging a laboratory reagent
blank or making a syringe injection) 25 ng of BFB and acquire a
mass spectrum that includes data for m/z 35-260. If the
spectrum does not meet all criteria (Table 2), the MS must be
299
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retimed and adjusted to meet all criteria before proceeding \
the continuing calibration check.
9.3.2 Purge a medium concentration CAL solution and analyze with the
same conditions used during the initial calibration.
9.3.3 Demonstrate acceptable performance for the criteria shown in
Sect. 9.2.4.
9.3.4 Determine that the absolute areas of the quantitation ions of
the internal standard and surrogates have not decreased by more
than 30% from the areas measured in the most recent continuing
calibration check, or by more than 50% from the areas measured
during initial calibration. If these areas have decreased by
more than these amounts, adjustments must be made to restore
system sensitivity. These adjustments may require cleaning of
the MS ion source, or other maintenance as indicated in Sect.
9.3.6, and recall bration. Control charts are useful aids in
documenting system sensitivity changes.
9.3.5 Calculate the RF for each analyte and surrogate from the data
measured in the continuing calibration check. The RF for each
analyte and surrogate must be within 30% of the mean value
measured in the initial calibration. Alternatively, if a second
or third order regression is used, the point from the continuing
calibration check for each analyte and surrogate must fall,
within the analyst's judgement, on the curve from the initial
calibration. If these conditions do not exist, remedial action
must be taken which may require re-initial calibration.
9.3.6 Some possible remedial actions. Major maintenance such as
cleaning an ion source, cleaning quadrupole rods, etc. require
returning to the initial calibration step.
9.3.6.1 Check and adjust GC and/or MS operating conditions;
check the MS resolution, and calibrate the mass scale.
9.3.6.2 Clean or replace the splitless injection liner;
silanize a new injection liner.
9.3.6.3 Flush the GC column with solvent according to manu-
facturer's instructions.
9.3.6.4 Break off a short portion (about 1 meter) of the column
from the end near the injector; or replace GC column.
This action will cause a change in retention times.
9.3.6.5 Prepare fresh CAL solutions, and repeat the initial
calibration step.
9.3.6.6 Clean the MS ion source and rods (if a quadrupole).
300
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9.3.6.7 Replace any components that allow analytes to come into
contact with hot metal surfaces.
9.3.6.8 Replace the MS electron multiplier, or any other faulty
components.
9.4 Optional calibration for vinyl chloride using a certified gaseous
mixture of vinyl chloride in nitrogen can be accomplished by the
following steps.
9.4.1 Fill the purging device with 25.0 ml (or 5-mL) of reagent water
or aqueous calibration standard.
9.4.2 Start to purge the aqueous mixture. Inject a known volume
(between 100 and 2000 0L) of the calibration gas (at room
temperature) directly Into the purging device with a gas tight
syringe. Slowly inject the gaseous sample through a septum seal
at the top of the purging device at 2000 /*L/m1n. If the
injection of the standard 1s made through the aqueous sample
Inlet port, flush the dead volume with several ml of room air or
carrier gas. Inject the gaseous standard before 5 m1n of the
Il-m1n purge time have elapsed.
9.4.3 Determine the aqueous equivalent concentration of vinyl chloride
standard, in /ig/L, injected with the equation:
S = 0.102 (C)(V)
where S = Aqueous equivalent concentration
of vinyl chloride standard in /ig/L;
C = Concentration of gaseous standard in ppm (v/v);
V = Volume of standard injected in milliliters.
10. QUALITY CONTROL
10.1 Quality control (QC) requirements are the initial demonstration of
laboratory capability followed by regular analyses of laboratory
reagent blanks, field reagent blanks, and laboratory fortified
blanks. The laboratory must maintain records to document the quality
of the data generated. Additional quality control practices are
recommended.
10.2 Initial demonstration of low system background. Before any samples
are analyzed, it must be demonstrated that a laboratory reagent blank
(LRB) is reasonably free of contamination that would prevent the
determination of any analyte of concern. Sources of background
contamination are glassware, purge gas, sorbants, and equipment.
Background contamination must be reduced to an acceptable level
before proceeding with the next section. In general, background from
method analytes should be below the method detection limit.
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10.3 Initial demonstration of laboratory accuracy and precision. Analy
five to seven replicates of a laboratory fortified blank containing
each analyte of concern at a concentration in the range of 0.2-5
(see regulations and maximum contaminant levels for guidance on
appropriate concentrations).
10.3.1 Prepare each replicate by adding an appropriate aliquot of a
quality control sample to reagent water. If a quality
control sample containing the method analytes is not
available, a primary dilution standard made from a source of
reagents different than those used to prepare the calibration
standards may be used. Also add the appropriate amounts of
internal standard and surrogates if they are being used.
Analyze each replicate according to the procedures described
in Section 11, and on a schedule that results in the analyses
of all replicates over a period of several days.
10.3.2 Calculate the measured concentration of each analyte in each
replicate, the mean concentration of each analyte in all
replicates, and mean accuracy (as mean percentage of true
value) for each analyte, and the precision (as relative
standard deviation, RSD) of the measurements for each
analyte. Calculate the MDL of each analyte using the
procedures described in Sect. 13.2 (2).
10.3.3 For each analyte and surrogate, the mean accuracy, expressed
as a percentage of the true value, should be 80-120% and the
RSD should be <20%. Some analytes, particularly the early
eluting gases and late eluting higher molecular weight
compounds, are measured with less accuracy and precision than
other analytes. The method detection limits must be
sufficient to detect analytes at the required levels. If
these criteria are not met for an analyte, take remedial
action and repeat the measurements for that analyte to
demonstrate acceptable performance before samples are
analyzed.
10.3.4 Develop and maintain a system of control charts to plot the
precision and accuracy of analyte and surrogate measurements
as a function of time. Charting of surrogate recoveries is
an especially valuable activity since these are present in
every sample and the analytical results will form a signi-
ficant record of data quality.
10.4 Monitor the integrated areas of the quantitation ions of the internal
standards and surrogates in continuing calibration checks. These
should remain reasonably constant over time. A drift of more than
50% in any area is indicative of a loss in sensitivity, and the
problem must be found and corrected. These integrated areas should
also be reasonably constant in laboratory fortified blanks and
samples.
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10.5 Laboratory reagent blanks. With each batch of samples processed as a
group within a work shift, analyze a laboratory reagent blank to
determine the background system contamination. A FRB (Sect. 10.7)
may be used in place of a LRB.
10.6 With each batch of samples processed as a group within a work shift,
analyze a single laboratory fortified blank (LFB) containing each
analyte of concern at a concentration as determined in 10.3. If more
than 20 samples are included in a batch, analyze one LFB for every 20
samples. Use the procedures described in 10.3.3 to evaluate the
accuracy of the measurements, and to estimate whether the method
detection limits can be obtained. If acceptable accuracy and method
detection limits cannot be achieved, the problem must be located and
corrected before further samples are analyzed. Add these results to
the on-going control charts to document data quality.
10.7 With each set of field samples a field reagent blank (FRB) should be
analyzed. The results of these analyses will help define contamina-
tion resulting from field sampling and transportation activities. If
the FRB shows unacceptable contamination, a LRB must be measured to
define the source of the impurities.
10.8 At least quarterly, replicates of laboratory fortified blanks should
be analyzed to determine the precision of the laboratory measure-
ments. Add these results to the on-going control charts to document
data quality.
10.9 At least quarterly, analyze a quality control sample (QCS) from an
external source. If measured analyte concentrations are not of
acceptable accuracy, check the entire analytical procedure to locate
and correct the problem source.
10.10 Sample matrix effects have not been observed when this method is used
with distilled water, reagent water, drinking water, and ground
water. Therefore, analysis of a laboratory fortified sample matrix
(LFM) is not required. It is recommended that sample matrix effects
be evaluated at least quarterly using the QCS described in 10.9.
10.11 Numerous other quality control measures are incorporated into other
parts of this procedure, and serve to alert the analyst to potential
problems.
11. PROCEDURE
11.1 SAMPLE INTRODUCTION AND PURGING
11.1.1 This method is designed for a 25-mL sample volume, but a
smaller (5 mL) sample volume is recommended if the GC/MS
system has adequate sensitivity to achieve the required
method detection limits. Adjust the purge gas (nitrogen or
helium) flow rate to 40 mL/min. Attach the trap inlet to the
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purging device and open the syringe valve on the purging
device.
11.1.2 Remove the plungers from two 25-mL (or 5-mL depending on
sample size) syringes and attach a closed syringe valve to
each. Uarm the sample to room temperature, open the sample
bottle, and carefully pour the sample into one of the syringe
barrels to just short of overflowing. Replace the syringe
plunger, invert the syringe, and compress the sample. Open
the syringe valve and vent any residual air while adjusting
the sample volume to 25.0-mL (or 5-mL). For samples and
blanks, add 5-jiL of the fortification solution containing the
internal standard and the surrogates to the sample through
the syringe valve. For calibration standards and laboratory
fortified blanks, add 5-jjL of the fortification solution
containing the internal standard only. Close the valve.
Fill the second syringe in an identical manner from the same
sample bottle. Reserve this second syringe for a reanalysis
if necessary.
11.1.3 Attach the sample syringe valve to the syringe valve on the
purging device. Be sure that the trap is cooler than 25°C,
then open the sample syringe valve and inject the sample into
the purging chamber. Close both valves and initiate purging.
Purge the sample for 11.0 min at ambient temperature.
11.2 SAMPLE DESORPTION
11.2.1 Non-cryogenic interface -- After the 11-min purge, place the
purge and trap system in the desorb mode and preheat the trap
to 180°C without a flow of desorption gas. Then simultan-
eously start the flow of desorption gas at 15-mL/min for
about 4 min, begin the temperature program of the gas
chromatograph, and start data acquisition.
11.2.2 Cryogenic interface -- After the 11-min purge, place the
purge and trap system in the desorb mode, make sure the
cryogenic interface is a -150°C or lower, and rapidly heat
the trap to 180°C while backflushing with an inert gas at
4 mL/min for about 5 min. At the end of the 5 min desorp-
tion cycle, rapidly heat the cryogenic trap to 250°C, and
simultaneously begin the temperature program of the gas
chromatograph, and start data acquisition.
11.2.3 While the trapped components are being introduced into the
gas chromatograph (or cryogenic interface), empty the purging
device using the sample syringe and wash the chamber with two
25-mL flushes of reagent water. After the purging device has
been emptied, leave syringe valve open to allow the purge gas
to vent through the sample introduction needle.
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11.3 GAS CHROMATOGRAPHY/MASS SPECTROMETRY -- Acquire and store data over
the mass range 35-260 with a total cycle time (including scan
overhead time) of 2 sec or less. Cycle time must be adjusted to
measure five or more spectra during the elution of each GC peak.
Several alternative temperature programs can be used.
11.3.1 Single ramp linear temperature program for wide bore columns
1 and 2 with a jet separator. Adjust the helium carrier gas
flow rate to about 15 mL/min. The column temperature is
reduced 10°C and held for 5 min from the beginning of
desorption, then programmed to 160°C at 6°C/min, and held
until all components have eluted.
11.3.2 Multi-ramp linear temperature program for wide bore column 2
with the open split interface. Adjust the helium carrier gas
flow rate to about 4.6 mL/min. The column temperature is
reduced 10°C and held for 6 min from the beginning of
desorption, then heated to 70°C at 10%nn, heated to 120°C
at 5°/min, heated to 180° at 8°/min, and held at 180° until
all compounds have eluted.
11.3.3 Single ramp linear temperature program for narrow bore column
3 with a cryogenic interface. Adjust the helium carrier gas
flow rate to about 4 mL/min. The column temperature is
reduced 10°C and held for 5 min from the beginning of
vaporization from the cryogenic trap, programmed at 6°C/min
for 10 min, then 15°C/min for 5 min to 145°C, and held until
all components have eluted.
11.4 TRAP RECONDITIONING -- After desorbing the sample for 4 min,
recondition the trap by returning the purge and trap system to the
purge mode. Wait 15 sec, then close the syringe valve on the
purging device to begin gas flow through the trap. Maintain the trap
temperature at 180°C. After approximately 7 min, turn off the trap
heater and open the syringe valve to stop the gas flow through the
trap. When the trap is cool, the next sample can be analyzed.
11.5 TERMINATION OF DATA ACQUISITION -- When all the sample components
have eluted from the GC, terminate MS data acquisition. Use
appropriate data output software to display full range mass spectra
and appropriate plots of ion abundance as a function of time. If any
ion abundance exceeds the system working range, dilute the sample
aliquot in the second syringe with reagent water and analyze the
diluted aliquot.
11.6 IDENTIFICATION OF ANALYTES -- Identify a sample component by
comparison of its mass spectrum (after background subtraction) to a
reference spectrum in the user-created data base. The GC retention
time of the sample component should be within three standard
deviations of the mean retention time of the compound in the
calibration mixture.
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11.6.1 In general, all ions that are present above 10% relative
abundance in the mass spectrum of the standard should be
present in the mass spectrum of the sample component and
should agree within absolute 20%. For example, if an ion has
a relative abundance of 30% in the standard spectrum, its
abundance in the sample spectrum should be in the range of 10
to 50%. Some ions, particularly the molecular ion, are of
special importance, and should be evaluated even if they are
below 10% relative abundance.
11.6.2 Identification requires expert judgement when sample
components are not resolved chromatographically and produce
mass spectra containing ions contributed by more than one
analyte. When GC peaks obviously represent more than one
sample component (i.e., broadened peak with shoulder(s) or
valley between two or more maxima), appropriate analyte
spectra and background spectra can be selected by examining
plots of characteristic ions for tentatively identified
components. When analytes coelute (i.e., only one GC peak is
apparent), the identification criteria can be met but each
analyte spectrum will contain extraneous ions contributed by
the coeluting compound. Because purgeable organic compounds
are relatively small molecules and produce comparatively
simple mass spectra, this is not a significant problem for
most method analytes.
11.6.3 Structural isomers that produce very similar mass spectra can
be explicitly identified only if they have sufficiently
different GC retention times. Acceptable resolution is
achieved if the height of the valley between two peaks is
less than 25% of the average height of the two peaks.
Otherwise, structural isomers are identified as isomeric
pairs. Two of the three isomeric xylenes and two of the
three dichlorobenzenes are examples of structural isomers
that may not be resolved on the capillary columns. If
unresolved, these groups of isomers must be reported as
isomeric pairs.
11.6.4 Methylene chloride and other background components appear in
variable quantities in laboratory and field reagent blanks,
and generally cannot be accurately measured. Subtraction of
the concentration in the blank from the concentration in the
sample is not acceptable because the concentration of the
background in the blank is highly variable.
12. CALCULATIONS
12.1 Complete chromatographic resolution is not necessary for accurate and
precise measurements of analyte concentrations if unique ions with
adequate intensities are available for quantitation.
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12.1.1 Calculate analyte and surrogate concentrations.
c (AX)(Q
-------
n = number of replicates
S = the standard deviation of the
replicate analyses.
14. REFERENCES
1. A. Alford-Stevens, J.W. Eichelberger, W.L. Budde, "Purgeable Organic
Compounds in Water by Gas Chromatography/Mass Spectrometry, Method
524." Environmental Monitoring and Support Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio, February 1983.
2. Madding, C., "Volatile Organic Compounds in Water by Purge and Trap
Capillary Column GC/MS", Proceedings of the Water Quality Technology
Conference, American Water Works Association, Denver, CO, December,
1984.
3. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde,
"Trace Analyses for Wastewaters, "Environ. Sci. Techno!.. 15, 1426,
1981.
4. "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.
5. "OSHA Safety and Health Standards, General Industry," (29CFR1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
6. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
7. Arrendale, R.F., R.F. Severson, and O.T. Chortyk, "Open Split Interface
for Capillary Gas Chromatography/Mass Spectrometry", Anal. Chem. 1984,
56, 1533.
8. Flesch, J.J., P.S. Fair, "The Analysis of Cyanogen Chloride in Drinking
Water," Proceedings of Water Quality Technology Conference, American
Water Works Association, St. Louis, MO., November 14-16, 1988.
308
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TABLE 1. MOLECULAR HEIGHTS AND QUANTITATION IONS FOR METHOD ANALYTES
Compound
MWa.
Primary Secondary
Quantitation Quantitation
Ion Ions
Internal standard
Fluorobenzene 96
Surrogates
4-Bromofluorobenzene 174
l,2-Dichlorobenzene-d4 150
Target Analvtes
Benzene 78
Bromobenzene 156
Bromochloromethane 128
Bromodichloromethane 162
Bromoform 250
Bromomethane 94
n-Butylbenzene 134
sec-Butyl benzene 134
tert-Butylbenzene 134
Carbon tetrachloride 152
Chlorobenzene 112
Chloroethane 64
Chloroform 118
Chloromethane 50
2-Chlorotoluene 126
4-Chlorotoluene 126
Dibromochloromethane 206
l,2-Dibromo-3-Chloropropane 234
1,2-Dibromoethane 186
Dibromomethane 172
1,2-Dichlorobenzene 146
1,3-Dichlorobenzene 146
1,4-Dichlorobenzene 146
Dlchlorodifluoromethane 120
1,1-Dlchloroethane 98
1,2-Dichloroethane 98
1,1-Dichloroethene 96
cis-l,2-Dichloroethene 96
trans-l,2-Dichloroethene 96
1,2-Dichloropropane 112
1,3-Dichloropropane 112
2,2-Dichloropropane 112
1,1-Dichloropropene 110
96
95
152
78
156
128
83
173
94
91
105
119
117
112
64
83
50
91
91
129
75
107
93
146
146
146
85
63
62
96
96
96
63
76
77
75
77
174,176
115,150
77
77,158
49,130
85,127
175,252
96
134
134
91
119
77,114
66
85
52
126
126
127
155,157
109,188
95,174
111,148
111,148
111,148
87
65,83
98
61,63
61,98
61,98
112
78
97
110,77
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TABLE 1. (continued)
Compound
MWa
Primary Secondary
Quantitation Quantitation
Ion Ions
cis-l,3-dichloropropene
trans-l,3-dichloropropene
Ethyl benzene
Hexachl orobutadi ene
I sopropyl benzene
4-Isopropyltoluene
Methylene chloride
Naphthalene
n-Propyl benzene
Styrene
1,1,1 ,2-Tetrachl oroethane
1,1,2 , 2-Tetrachl oroethane
Tetrachloroethene
Toluene
1,2,3-Trichlorobenzene
1 , 2 , 4-Tr i chl orobenzene
1 , 1 , 1-Tri chl oroethane
1 ,1,2-Trichloroethane
Trichloroethene
Tri chl orof 1 uoromethane
1,2,3-Trichloropropane
1, 2, 4-Trimethyl benzene
1,3, 5-Trimethyl benzene
Vinyl Chloride
o-Xylene
m-Xylene
p-Xylene
110
110
106
258
120
134
84
128
120
104
166
166
164
92
180
180
132
132
130
136
146
120
120
62
106
106
106
75
75
91
225
105
119
84
128
91
104
131
83
166
92
180
180
97
83
95
101
75
105
105
62
106
106
106
110
110
106
260
120
134,91
86,49
120
78
133,119
131,85
168,129
91
182
182
99,61
97,85
130,132
103
77
120
120
64
91
91
91
aMonoisotopic molecular weight calculated from the atomic masses of the
isotopes with the smallest masses.
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TABLE 2. CHROMATOGRAPHIC RETENTION TIMES FOR METHOD ANALYTES
ON THREE COLUMNS WITH FOUR SETS OF CONDITIONS3
Retention kTime (min:sec)
Compound Column lb Column 2b Column 2C Column 3q
Internal standard
Fluorobenzene 8:49 6:27 14:06 8:03
Surrogates
4-Bromofluorobenzene 18:38 15:43 23:38
l,2-Dichlorobenzene-d4 22:16 19:08 27:25
Target Analvtes
Benzene 8:14 5:40 13:30 7:25
Bromobenzene 18:57 15:52 24:00 16:25
Bromochloromethane 6:44 4:23 12:22 5:38
Bromodichloromethane 10:35 8:29 15:48 9:20
Bromoform 17:56 14:53 22:46 15:42
Bromomethane 2:01 0:58 4:48 1:17
n-Butylbenzene 22:13 19:29 27:32 17:57
sec-Butyl benzene 20:47 18:05 26:08 17:28
tert-Butylbenzene 20:17 17:34 25:36 17:19
Carbon Tetrachloride 7:37 5:16 13:10 7:25
Chlorobenzene 15:46 13:01 20:40 14:20
Chloroethane 2:05 1:01 1:27
Chloroform 6:24 4:48 12:36 5:33
Chloromethane 1:38 0:44 3:24 0:58
2-Chlorotoluene 19:20 16:25 24:32 16:44
4-Chlorotoluene 19:30 16:43 24:46 16:49
Cyanogen chloride 1:03
Dibromochloromethane 14:23 11:51 19:12 12:48
l,2-Dibromo-3-Chloropropane 24:32 21:05 18:02
1,2-Dibromoethane 14:44 11:50 19:24 13:36
Dibromomethane 10:39 7:56 15:26 9:05
1,2-Dichlorobenzene 22:31 19:10 27:26 17:47
1,3-Dichlorobenzene 21:13 18:08 26:22 17:28
1,4-Dichlorobenzene 21:33 18:23 26:36 17:38
Dichlorodifluoromethane 1:33 0:42 3:08 0:53
1,1-Dichloroethane 4:51 2:56 10:48 4:02
1,2-Dichloroethane 8:24 5:50 13:38 7:00
1,1-Dichloroethene 2:53 1:34 7:50 2:20
cis-l,2-Dichloroethene 6:11 3:54 11:56 5:04
trans-l,2-Dichloroethene 3:59 2:22 9:54 3:32
1,2-Dichloropropane 10:05 7:40 15:12 8:56
1,3-Dichloropropane 14:02 11:19 18:42 12:29
2,2-Dichloropropane 6:01 3:48 11:52 5:19
1,1-Dichloropropene 7:49 5:17 13:06 7:10
311
-------
TABLE 2. (continued)
Compound
Retention ijinie (min:sec)
Column lb Column 2b Column 2C Column 3d
cis-l,3-dichloropropene
trans - 1 , 3-di chl oropropene
Ethyl benzene
Hexachlorobutadiene
I sopropyl benzene
4-Isopropyltoluene
Methyl ene Chloride
Naphthalene
n-Propyl benzene
Styrene
1,1,1 , 2-Tetrachl oroethane
1,1,2 , 2-Tetrachl oroethane
Tetrachloroethene
Toluene
1 , 2 , 3 -Tri chl orobenzene
1 , 2 , 4-Tr i chl orobenzene
1,1,1-Trichloroethane
1,1,2-Trichloroethane
Trichloroethene
Tri chl orof 1 uoromethane
1 , 2 , 3-Tr i chl oropropane
1 , 2 , 4-Tri methyl benzene
1,3, 5 -Tri methyl benzene
Vinyl chloride
o-Xylene
m-Xylene
p-Xylene
15:59
26:59
18:04
21:12
3:36
27:10
19:04
17:19
15:56
18:43
13:44
12:26
27:47
26:33
7:16
13:25
9:35
2:16
19:01
20:20
19:28
1:43
17:07
16:10
16:07
13:23
23:41
15:28
18:31
2:04
23:31
16:25
14:36
13:20
16:21
11:09
10:00
24:11
23:05
4:50
11:03
7:16
1:11
16:14
17:42
16:54
0:47
14:31
13:41
13:41
17:54
16:42
21:00
32:04
23:18
26:30
9:16
32:12
24:20
22:24
20:52
24:04
18:36
17:24
32:58
31:30
12:50
18:18
14:48
6:12
24:08
31:30
24:50
3:56
22:16
21:22
21:18
14:44
19:14
16:25
17:38
2:40
19:04
16:49
15:47
14:44
15:47
13:12
11:31
19:14
18:50
6:46
11:59
9:01
1:46
16:16
17:19
16:59
1:02
15:47
15:18
15:18
aColumns 1-3 are those given in Sect. 6.3.2.1; retention times were measured
from the beginning of thermal desorption from the trap (columns 1-2) or from
the beginning of thermal release from the cryogenic interface (column 3).
bGC conditions given in Sect. 11.3.1.
-------
TABLE 3. ION ABUNDANCE CRITERIA FOR 4-BROMOFLUOROBENZENE (BFB)
Mass
(M/z) Relative Abundance Criteria
50 15 to 40% of mass 95
75 30 to 80% of mass 95
95 Base Peak, 100% Relative Abundance
96 5 to 9% of mass 95
173 < 2% of mass 174
174 > 50% of mass 95
175 5 to 9% of mass 174
176 > 95% but < 101% of mass 174
177 5 to 9% of mass 176
313
-------
TABLE 4. ACCURACY AND PRECISION DATA FROM 16-31 DETERMINATIONS OF THE METHOC
ANALYTES IN REAGENT HATER USING HIDE BORE CAPILLARY COLUMN la
Compound
Benzene
Bromobenzene
Bromochl oromethane
Bromod i chl oromethane
Bromoform
Bromomethane
n-Butyl benzene
sec-Butyl benzene
tert-Butyl benzene
Carbon tetrachloride
Chl orobenzene
Chloroethane
Chloroform
Chl oromethane
2-Chlorotoluene
4-Chlorotoluene
Di bromochl oromethane
1 , 2-Di bromo-3-chl oropropane
1,2-Dibromoethane
Dibromomethane
1 , 2 -Di chl orobenzene
,3-Dichl orobenzene
,4-Dichl orobenzene
)i chl orodi f 1 uoromethane
,1-Di chloroethane
, 2-Di chloroethane
,1-Dichloroethene
cis-1,2 Dichloroethene
trans-l,2-Dichloroethene
1 , 2 -Di chl oropropane
1,3-Dichloropropane
2 , 2 -Di chl oropropane
1,1-Dichloropropene
ci s- 1 , 2-Di chl oropropene
trans-l,2-Dichloropropene
Ethyl benzene
Hexachl orobutad i ene
I sopropyl benzene
4- Isopropyl toluene
Methyl ene chloride
Naphthalene
n-Propyl benzene
Styrene
True
Cone.
Range
0.1-10
0.1-10
0.5-10
0.1-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.1-10
0.5-10
0.5-10
0.5-10
0.1-10
0.1-10
0.1-10
0.5-10
0.5-10
0.5-10
0.1-10
0.5-10
0.2-20
0.5-10
0.5-10
0.1-10
0.1-10
0.5-10
0.1-10
0.1-10
0.1-10
0.5-10
0.5-10
0.1-10
0.5-10
0.5-10
0.1-10
0.1-10
0.1-100
0.1-10
0.1-100
Mean
Accuracy
(% of True
Value)
97
100
90
95
101
95
100
100
102
84
98
89
90
93
90
99
92
83
102
100
93
99
103
90
96
95
94
101
93
97
96
86
98
99
100
101
99
95
104
100
102
Rel.
Std.
Dev.
m
5.7
5.5
6.4
6.1
6.3
8.2
7.6
7.6
7.3
8.8
5.9
9.0
6.1
8.9
6.2
8.3
7.0
19.9
3.9
5.6
6.2
6.9
6.4
7.7
5.3
5.4
6.7
6.7
5.6
6.1
6.0
16.9
8.9
8.6
6.8
7.6
6.7
5.3
8.2
5.8
7.2
Method
Det.
Limit
fua/L)
0.04
0.03
0.04
0.08
0.12
0.11
0.11
0.13
0.14
0.21
0.04
0.10
0.03
0.13
0.04
0.06
0.05
0.26
0.06
0.24
0.03
0.12
0.03
0.10
0.04
0.06
0.12
0.12
0.06
0.04
0.04
0.35
0.10
0.06
0.11
0.15
0.12
0.03
0.04
0.04
0.04
314
-------
TABLE 4. (Continued)
Compound
1,1,1 , 2-Tetrachl oroethane
1,1,2 , 2-Tetrachl oroethane
Tetrachloroethene
Toluene
1 , 2 , 3-Tri chl orobenzene
1,2, 4-Tri chl orobenzene
1,1, 1 -Tri chl oroethane
1 , 1 ,2-Trichloroethane
Trichloroethene
Tri chl orof 1 uoromethane
1,2, 3-Tri chl oropropane
1, 2, 4-Trimethyl benzene
1, 3, 5-Trimethyl benzene
Vinyl chloride
o-Xylene
m-Xylene
p-Xylene
True
Cone.
Range
(ua/L)
0.5-10
0.1-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.1-31
0.1-10
0.5-10
Mean
Accuracy
(% of True
Value)
90
91
89
102
109
108
98
104
90
89
108
99
92
98
103
97
104
Rel.
Std.
Dev.
m
6.8
6.3
6.8
8.0
8.6
8.3
8.1
7.3
7.3
8.1
14.4
8.1
7.4
6.7
7.2
6.5
7.7
Method
Det.
Limit
(jia/L)
0.05
0.04
0.14
0.11
0.03
0.04
0.08
0.10
0.19
0.08
0.32
0.13
0.05
0.17
0.11
0.05
0.13
aOata obtained by Robert W. Slater using column 1 with a jet separator
interface and a quadrupole mass spectrometer (Sect. 11.3.1) with analytes
divided among three solutions.
315
-------
TABLE 5. ACCURACY AND PRECISION DATA FROM SEVEN DETERMINATIONS OF THr
METHOD ANALYTES IN REAGENT WATER USING THE CRYOGENIC TRAFFIC
OPTION AND A NARROW BORE CAPILLARY COLUMN 3a
Compound
Benzene
Bromobenzene
Bromochl oromethane
Bromodi chl oromethane
Bromoform
Bromomethane
n-Butyl benzene
sec-Butyl benzene
tert-Butyl benzene
Carbon tetrachloride
Chlorobenzene
Chloroethane
Chloroform
Chl oromethane
2-Chlorotoluene
4-Chlorotoluene
Cyanogen chloride15
Di bromochl oromethane
1 , 2-Dibromo-3-chl oropropane
1,2-Oibromoethane
Dibromomethane
1,2-Dichlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
Di chl orodi f 1 uoromethane
1,1-Dichloroethane
1,2-Dichloroethane
1,1-Dichloroethene
cis-1,2 Dichloroethene
trans -1,2-Di chl oroethene
1, 2 -Di chl oropropane
1,3-Dichloropropane
2,2-Dichloropropane
1,1-Dichloropropene
cis-l,3-Dichloropropene
trans-l,3-Dichloropropene
Ethyl benzene
Hexachlorobutadiene
I sopropyl benzene
4-Isopropyltoluene
Methylene chloride
Naphthalene
True
Cone.
0.1
0.5
0.5
0.1
0.1
0.1
0.5
0.5
0.5
0.
0.
0.
0.
0.
0.
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.5
0.5
0.5
0.1
Mean
Accuracy
(% of True
Value)
99
97
97
100
99
99
94
90
90
92
91
100
95
99
99
96
92
99
92
97
93
97
99
93
99
98
100
95
100
98
96
99
99
98
99
100
98
87
97
98
Rel.
Std.
Dev.
6.2
7.4
5.8
4.6
5.4
7.1
6.0
7.1
2.5
6.8
5.8
5.8
3.2
4.7
4.6
7.0
10.6
5.6
10.0
5.6
6.9
3.5
6.0
5.7
8.8
6.2
6.3
9.0
3.7
7.2
6.0
5.8
4.9
7.4
5.2
6.7
6.4
13.0
13.0
7.2
Method
Dect.
Limit
(ua/L)
0.03
0.11
0.07
0.03
0.20
0.06
0.03
0.12
0.33
0.08
0.03
0.02
0.02
0.05
0.05
0.05
0.30
0.07
0.05
0.02
0.03
0.05
0.05
0.04
0.11
0.03
0.02
0.05
0.06
0.03
0.02
0.04
0.05
0.02
0.03
0.04
0.10
0.26
0.09
0.04
316
-------
TABLE 5. (Continued)
Comoound
n-Propyl benzene
Styrene
1,1,1 , 2-Tetrachl oroethane
1,1,2 , 2-Tetrachl oroethane
Tetrachloroethene
Toluene
1,2,3-Trlchlorobenzene
1 , 2 , 4-Tri chl orobenzene
1,1,1 -Trichl oroethane
1 , 1 , 2-Trichl oroethane
Trichloroethene
Trichlorofluoromethane
1 ,2,3-Trichloropropane
1 , 2 , 4-Tr imethyl benzene
1,3, 5-Tr Imethyl benzene
Vinyl chloride
o-Xylene
m-Xylene
p-Xylene
True
Cone.
(ua/L)
0.1
0.1
0.1
0.5
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.
0.
0.
0.
0.
Mean
Accuracy
(% of True
Value)
99
96
100
100
96
100
98
91
100
98
96
97
96
96
99
96
94
94
97
Rel.
Std.
Dev.
m
6.6
19.0
4.7
12.0
5.0
5.9
8.9
16.0
4.0
4.9
2.0
4.6
6.5
6.5
4.2
0.2
7.5
4.6
6.1
Method
Dect.
Limit
(ua/L)
0.06
0.06
0.04
0.20
0.05
0.08
0.04
0.20
0.04
0.03
0.02
0.07
0.03
0.04
0.02
0.04
0.06
0.03
0.06
aData obtained by Caroline A. Madding using column 3 with a cryogenic
interface and a quadrupole mass spectrometer (Sect 11.3.3).
Reference 8.
317
-------
TABLE 6. ACCURACY AND PRECISION DATA FROM SEVEN DETERMINATIONS
OF THE METHOD ANALYTES IN REAGENT WATER USING HIDE BORE
CAPILLARY COLUMN 2a
Compound
No.b
Mean Accuracy
(% of True
Value,
2 UQ/L Cone.)
RSO
(%)
Mean Accuracy
(% of True
Value,
0.2 ua/L Cone.)
RSD
(%)
Internal Standard
Fluorobenzene 1
Surrogates
4-Bromofluorobenzene 2 98
l,2-Dichlorobenzene-d4 3 97
Target Analvtes
Benzene
Bromobenzene
Bromochloromethane
Bromodichloromethane
Bromoform
Bromomethane
n-Butylbenzene
sec-Butyl benzene
tert-Butylbenzene
Carbon tetrachlorlde
Chlorobenzene
Chloroethanec
Chloroform
Chioromethane
2-Chlorotoluene
4-Chlorotoluene
Di bromochloromethane
1,2-Di bromo-3-chloropropanec
l,2-Dibromoethanec
Dibromomethane 13 99
1,2-Dichlorobenzene 45 93
1,3-Dichlorobenzene 46 100
1,4-Dichlorobenzene 47 98
Dichlorodifluoromethane 14 38
1,1-Dichloroethane 15 97
1,2-Dichloroethane 16 102
1,1-Dichloroethene 17 90
cis-l,2-Dichloroethene 18 100
trans-l,2-Dichloroethene 19 92
37
38
4
5
6
7
39
40
41
8
42
9
10
43
44
11
97
102
99
96
89
55
89
102
101
84
104
97
110
91
89
95
1.8
3.2
4.4
3.0
5.2
1.8
2.4
27.
4.8
3.5
4.5
3.2
3.1
2.0
5.0
2.4
2.0
2.7
2.1
2.7
4.0
4.1
25.
2.3
3.8
2.2
3.4
2.1
96
95
113
101
102
100
90
52
87
100
100
92
103
95
d
108
108
100
95
94
87
94
d
85
100
87
89
85
1.3
1.7
1.8
1.9
2.9
1.8
2.2
6.7
2.3
2.8
2.9
2.6
1.6
2.1
3.1
4.4
3.0
2.2
5.1
2.3
2.8
3.6
2.1
3.8
2.9
2.3
318
-------
TABLE 6. (Continued)
Comoound
1,2-Dichloropropane
1,3-Dichloropropane
2,2-Dichloropropanec
l,l-Dichloropropenec
ci s- 1 , 3-Di chl oropropenec
trans- 1,3-Dichloropropene
Ethyl benzene
Hexachl orobutadi ene
I sopropyl benzene
4-Isopropyltoluene
Methylene chloride
Naphthalene
n-Propyl benzene
Styrene
1,1,1, 2-Tetrachl oroethane
1,1,2,2-Tetrachloroethane
Tetrachloroethene
Toluene
1,2,3-Trichlorobenzene
1,2,4-Trichlorobenzene
1,1,1-Trichloroethane
1 , 1 ,2-Trichloroethane
Trichloroethene
Tri chl orof 1 uoromethane
1,2,3-Trichloropropane
1 , 2 , 4-Tri methyl benzene
1 ,3 , 5-Trimethyl benzene
Vinyl chloride
o-Xylene
m-Xylene
p-Xylene
Mean Accuracy
(% of True
Value, RSD
No.b 2 ua/L Conc.l (%)
20
21
25
48
26
49
50
27
51
52
53
28
29
30
54
55
56
31
32
33
34
35
57
58
36
59
60
61
102
92
96
96
91
103
95
e
93
102
95
99
101
97
105
90
92
94
107
99
81
97
93
88
104
97
f
98
2.2
3.7
1.7
9.1
5.3
3.2
3.6
7.6
4.9
4.4
2.7
4.6
4.5
2.8
5.7
5.2
3.9
3.4
2.9
4.6
3.9
3.1
2.4
3.5
1.8
2.3
Mean Accuracy
(% of True
Value, RSD
0.2 ua/L Cone.) (%)
103
93
99
100
88
101
95
e
78
97
104
95
84
92
126
78
83
94
109
106
48
91
106
97
115
98
f
103
2.9
3.2
2.1
4.0
2.4
2.1
3.1
8.3
2.1
3.1
3.8
3.6
3.3
1.7
2.9
5.9
2.5
2.8
2.5
13.
2.8
2.2
3.2
14.
1.7
1.4
aData obtained by James W. Eichelberger using column 2 with the open split
interface and an ion trap mass spectrometer (Sect. 11.3.2) with all method
analytes in the same reagent water solution.
"Designation in Figures 1 and 2.
cNot measured; authentic standards were not available.
dNot found at 0.2 /zg/L.
?Not measured; methylene chloride was in the laboratory reagent blank.
fm-xylene coelutes with and cannot be distinguished from its isomer p-xylene,
No 61.
319
-------
OPTIONAL
FOAM
TRAP
'/.IN.
0. 0. EXIT
-EXIT H IN.
0. D.
—14MM 0. D.
INLET % IN.
0.0.
SAMPLE INLET
—2-WAY SYRINGE VALVE
17CM. 20 GAUGE SYRINGE NEEDLE
6MM. 0. 0. RUBBER SEPTUM '
~10MM. 0. D. 1/16 IN. 0.0.
'STAINLESS ST
INLET
X IN. 0. D.
10MM GLASS FRIT
MEDIUM POROSITY
13X MOLECULAR
SIEVE PURGE
GAS FILTER
PURGE GAS
ROW
CONTROL
FIGURE 1. PURGING DEVICE
320
-------
PACKING PROCEDURE
CONSTRUCTION
en
*
GLASS
WOOL
ACTIVATED, „„.
CHARCOAL 7.7CH
GRADE 15
SIUCA
TENAX 7.7 C
3XOV-1
GLASS WOOL
10"
7 A/FOOT
RESISTANCE
WIRE WRAPPED
SOLID
(DOUBLE LAYER)
ns-
7^/FOOT.
RESISTANCE
WIRE WRAPPED
SOLID
(SINGLE LAYER)
8CMH
TRAP INLET
COMPRESSION
FITTING NUT
AND FERRULES
THERMOCOUPLE/
CONTROLLER
SENSOR
ELECTRONIC
TEMPERATURE
CONTROL
AND
.PYROMETER
v / TUBING 25CM
0.105 IN. I.D.
0.125 IN. O.D.
STAINLESS STEEL
FIGURE 2. TRAP PACKINGS AND CONSTRUCTION TO INCLUDE
DESORB CAPABILITY
321
-------
FIGURE 3. NORMALIZED TOTAL ION CURRENT CHROMATOGRAM FROM A VOLATILE COMPOUND CALIBRATION MIXTURE CONTAINING 25 ng
(5 ug/L) OF MOST COMPOUNDS. THE COMPOUND IDENTIFICATION NUMBERS ARE GIVEN IN TABLE 6.
10QX
CO
PO
I\J
TOT-
10
100
3:22
27
37
19 15 Vlli'.B
u,
209
6:42
300
10:02
13:22
16 33 13
ss
26:42 30:02 33:22
-------
IO.GURE 4. AMPLIFIED FIRST EIGHT MINUTES OF A TOTAL ION CURRENT CHROMATOGRAM FROM A VOLATILE COMPOUNL VIBRATION
MIXTURE CONTAINING 25 ng (5 pg/L) OF EACH COMPONENT. THE COMPOUND IDENTIFICATION NUMBERS ARE GIVEN. IN
TABLE 6.
CO
ro
co
my.
101-
50
i:42
._ J I/ \
34
108
3:22
I'
158
5182
17
\
288
6:42
CS,
250
8:22
-------
METHOD 525. DETERMINATION OF ORGANIC COMPOUNDS IN DRINKING WATER
BY LIQUID-SOLID EXTRACTION AND CAPILLARY COLUMN
GAS CHROMATOGRAPHY/MASS SPECTROMETRY
Revision 2.1
J. W. Eichelberger, T. D. Behymer, W. L. Budde - Method 525,
Revision 1.0, 2.0, 2.1 (1988)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
325
-------
METHOD 525
DETERMINATION OF ORGANIC COMPOUNDS IN DRINKING HATER
BY LIQUID-SOLID EXTRACTION AND CAPILLARY COLUMN
GAS CHROMATOGRAPHY/MASS SPECTROMETRY
1. SCOPE AND APPLICATION
1.1 This is a general purpose method that provides procedures for
determination of organic compounds in finished drinking water, raw
source water, or drinking water in any treatment stage. The method
is applicable to a wide range of organic compounds that are
efficiently partitioned from the water sample onto a CIQ organic
phase chemically bonded to a solid inorganic matrix, and sufficiently
volatile and thermally stable for gas chromatography. Particulate
bound organic matter will not be partitioned, and more than trace
levels of particulates in the water may disrupt the partitioning
process. Single-laboratory accuracy and precision data have been
determined at two concentrations with two instrument systems for the
following compounds:
Compound
Acenaphthylene
Alachlor
Aldrin
Anthracene
Atrazine
Benz[a]anthracene
Benzo[b]f1uoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
Benzo[g,h,i]perylene
Butyl benzylphthalate
Chlordane components
Alpha-chlordane
Gamma-chlordane
Trans nonachlor
2-Chlorobiphenyl
Chrysene
Dibenz[a,h]anthracene
Di-n-butylphthalate
2,3-Dichlorobiphenyl
Diethylphthalate
Oi(2-ethylhexylJadipate
Di(2-ethylhexyl)phthalate
Dimethylphthalate
Endrin
Fluorene
Heptachlor
Chemical Abstracts Service
MW* Registry Number
152 208-96-8
269 15972-60-8
362 309-00-2
178 120-12-7
215 1912-24-9
228 55-55-3
252 205-82-3
252 207-08-9
252 50-32-8
276 191-24-2
312 85-68-7
406 5103-71-9
406 5103-74-2
440 39765-80-5
188 2051-60-7
228 218-01-9
278 53-70-3
278 84-72-2
222 16605-91-7
222 84-66-2
370 103-23-1
390 117-81-7
194 131-11-3
378 72-20-8
166 86-73-7
370 76-44-8
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Heptachlor epoxide 386 1024-57-3
2,2',3,3',4,4',6-Heptachloro-
biphenyl 392 52663-71-5
Hexachlorobenzene 282 118-74-1
2,2',4,4',5,6'-Hexachloro-
biphenyl 358 60145-22-4
Hexachlorocyclopentadiene 270 77-47-4
Indeno[l,2,3,c,d]pyrene 276 193-39-5
Llndane 288 58-89-9
Methoxychlor 344 72-43-5
2,2',3>3',4,5',6,6'-Octa-
chlorobiphenyl 426 40186-71-8
2,2',3',4,6-Pentachloro-
biphenyl 324 60233-25-2
Pentachlorophenol 264 87-86-5
Phenanthrene 178 85-01-8
Pyrene 202 129-00-0
Simazine 201 122-34-9
2,2/,4,4'-Tetrachlorobiphenyl 290 2437-79-8
Toxaphene mixture 8001-35-2
2,4,5-Trichlorobiphenyl 256 15862-07-4
iMonoisotopic molecular weight calculated from the atomic masses of
the Isotopes with the smallest masses.
A laboratory may use this method to identify and measure additional
analytes after the laboratory obtains acceptable (defined in Sect.
10) accuracy and precision data for each added analyte.
1.2 Method detection limit (MDL) is defined as the statistically calcu-
lated minimum amount that can be measured with 99% confidence that the
reported value is greater than zero (1). The MDL is compound
dependent and is particularly dependent on extraction efficiency and
sample matrix. For the listed analytes, MDLs vary from 0.01 to
15 jig/L. The concentration calibration range of this method is
0.1 /ig/L to 10 pq/L.
2. SUMMARY OF METHOD
Organic compound analytes, internal standards, and surrogates are extracted
from a water sample by passing 1 liter of sample water through a cartridge
containing about 1 gram of a solid inorganic matrix coated with a chemical-
ly bonded Cjs organic phase (liquid-solid extraction, LSE). The organic
compounds are eluted from the LSE cartridge with a small quantity of
methylene chloride, and concentrated further by evaporation of some of the
solvent. The sample components are separated, identified, and measured by
injecting an aliquot of the concentrated methylene chloride extract into a
high resolution fused silica capillary column of a gas chromatography/mass
spectrometry (GC/MS) system. Compounds eluting from the GC column are
identified by comparing their measured mass spectra and retention times to
reference spectra and retention times in a data base. Reference spectra and
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retention times for analytes are obtained by the measurement of calibra
standards under the same conditions used for samples. The concentration of
each identified component is measured by relating the MS response of the
quantitation ion produced by that compound to the MS response of the
quantitation ion produced by a compound that is used as an internal
standard. Surrogate analytes, whose concentrations are known in every
sample, are measured with the same internal standard calibration procedure.
DEFINITIONS
3.1 Internal standard -- A pure analyte(s) added to a solution in known
amount(s) and used to measure the relative responses of other method
analytes and surrogates that are components of the same solution. The
internal standard must be an analyte that is not a sample component.
3.2 Surrogate analyte -- A pure analyte(s), which is extremely unlikely to
be found in any sample, and which is added to a sample aliquot in
known amount(s) before extraction and is measured with the same
procedures used to measure other sample components. The purpose of a
surrogate analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates (LD1 and LD2) -- Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample collection,
preservation, or storage procedures.
3.4 Field duplicates (FD1 and FD2) -- Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures. Analyses
of F01 and FD2 give a measure of the precision associated with sample
collection, preservation, and storage, as well as with laboratory
procedures.
3.5 Laboratory reagent blank (LRB) -- An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates that
are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.6 Field reagent blank (FRB) -- Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage, preservation,
and all analytical procedures. The purpose of the FRB is to determine
if method analytes or other interferences are present in the field
environment.
3.7 Laboratory performance check solution (LPC) -- A solution of method
analytes, surrogate compounds, and internal standards used to evaluate
the performance of the instrument system with respect to a defined f*
of method criteria.
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3.8 Laboratory fortified blank (LFB) -- An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.9 Laboratory fortified sample matrix (LFM) -- An aliquot of an environ-
mental sample to which known quantities of the method analytes are
added in the laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the
analytes in the sample matrix must be determined in a separate aliquot
and the measured values in the LFM corrected for background concentra-
tions.
3.10 Stock standard solution -- A concentrated solution containing a single
certified standard that is a method analyte, or a concentrated
solution of a single analyte prepared in the laboratory with an
assayed reference compound. Stock standard solutions are used to
prepare primary dilution standards.
3.11 Primary dilution standard solution -- A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
3.12 Calibration standard (CAL) -- a solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are used
to calibrate the instrument response with respect to analyte con-
centration.
3.13 Quality control sample (QCS) -- a sample matrix containing method
analytes or a solution of method analytes in a water miscible solvent
which is used to fortify reagent water or environmental samples. The
QCS is obtained from a source external to the laboratory, and is used
to check laboratory performance with externally prepared test
materials.
4. INTERFERENCES
4.1 During analysis, major contaminant sources are reagents and liquid-
solid extraction columns. Analyses of field and laboratory reagent
blanks provide information about the presence of contaminants.
4.2 Interfering contamination may occur when a sample containing low
concentrations of compounds is analyzed immediately after a sample
containing relatively high concentrations of compounds. Syringes and
splitless injection port liners must be cleaned carefully or replaced
as needed. After analysis of a sample containing high concentrations
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of compounds, a laboratory reagent blank should be analyzed to ensi
that accurate values are obtained for the next sample.
5.1 The toxicity or carcinogenicity of chemicals used in this method has
not been precisely defined; each chemical should be treated as a
potential health hazard, and exposure to these chemicals should be
minimized. Each laboratory is responsible for maintaining awareness
of OSHA regulations regarding safe handling of chemicals used in this
method. Additional references to laboratory safety are cited (2-4).
5.2 Some method analytes have been tentatively classified as known or
suspected human or mammalian carcinogens. Pure standard materials and
stock standard solutions of these compounds should be handled with
suitable protection to skin, eyes, etc.
6. Apparatus and Equipment
6.1 All glassware must be meticulously cleaned. This may be accomplished
by washing with detergent and water, rinsing with water, distilled
water, or solvents, air-drying, and heating (where appropriate) in an
oven. Volumetric glassware is never heated.
6.2 Sample containers. 1-liter or 1-quart amber glass bottles fitted
with a Teflon-lined screw cap. (Bottles in which high purity solvents
were received can be used as sample containers without additional
cleaning if they have been handled carefully to avoid contamination
during use and after use of original contents.)
6.3 Separatory funnels. 2-liter and 100-mL with a Teflon stopcock.
6.4 Liquid chromatography column reservoirs. Pear-shaped 100- or 125-mL
vessels without a stopcock but with a ground glass outlet joint sized
to fit the liquid-solid extraction column. (Lab Glass, Inc. part no.
ML-700-706S, with a 24/40 top outer joint and a 14/35 bottom inner
joint, or equivalent). A 14/35 outlet joint fits some commercial
cartridges.
6.5 Syringe needles. No. 18 or 20 stainless steel.
6.6 Vacuum flasks. 1- or 2-liter with solid rubber stoppers.
6.7 Volumetric flasks, various sizes.
6.8 Laboratory or aspirator vacuum system. Sufficient capacity to
maintain a slight vacuum of 13 cm (5 in.) of mercury in the vacuum
flask.
6.9 Micro syringes, various sizes.
6.10 Vials. Various sizes of amber vials with Teflon-lined screw caps.
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6.11 Drying column. Approximately 1.2 cm x 40 cm with 10 ml graduated
collection vial.
6.12 Analytical balance. Capable of weighing 0.0001 g accurately.
6.13 Fused silica capillary gas chromatography column. Any capillary
column that provides adequate resolution, capacity, accuracy, and
precision (Sect. 10) can be used. A 30 m X 0.25 mm id fused silica
capillary column coated with a 0.25 ion bonded film of polyphenyl-
methylsilicone is recommended (J&W DB-5 or equivalent).
6.14 Gas chromatograph/mass spectrometer/data system (GC/MS/DS)
6.14.1 The GC must be capable of temperature programming and be
equipped for splitless/split injection. The injection tube
liner should be quartz and about 3 mm in diameter. The
injection system must not allow the analytes to contact hot
stainless steel or other metal surfaces that promote decomposi-
tion.
6.14.2 The GC/MS interface should allow the capillary column or
transfer line exit to be placed within a few mm of the ion
source. Other interfaces, for example the open split inter-
face, are acceptable as long as the system has adequate
sensitivity (see Sect. 9 for calibration requirements).
6.14.3 The mass spectrometer must be capable of electron
ionization at a nominal electron energy of 70 eV. The
spectrometer must be capable of scanning from 45 to 450 amu
with a complete scan cycle time (including scan overhead) of
1.5 sec or less. (Scan cycle time = Total MS data acquisition
time in sec divided by number of scans in the chromatogram).
The spectrometer must produce a mass spectrum that meets all
criteria in Table 1 when 5 ng or less of DFTPP is introduced
into the GC. An average spectrum across the DFTPP GC peak may
be used to test instrument performance.
6.14.4 An interfaced data system is required to acquire, store,
reduce, and output mass spectral data. The computer software
must have the capability of processing stored GC/MS data by
recognizing a GC peak within any given retention time window,
comparing the mass spectra from the GC peak with spectral data
in a user-created data base, and generating a list of tenta-
tively identified compounds with their retention times and
scan numbers. The software must also allow integration of the
ion abundance of any specific ion between specified time or
scan number limits, calculation of response factors as defined
in Sect. 9.2.6 (or construction of a second or third order
regression calibration curve), calculation of response factor
statistics (mean and standard deviation), and calculation of
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concentrations of analytes using either the calibration cun
or the equation in Sect. 12.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Helium carrier gas, as contaminant free as possible.
7.2 Liquid-solid extraction (LSE) cartridges. Cartridges are inert non-
leaching plastic, for example polypropylene, or glass, and must not
contain plasticizers, such as phthalate esters or adipates, that leach
into methylene chloride. The cartridges are packed with about 1 gram
of silica, or other inert inorganic support, whose surface is modified
by chemically bonded octadecyl (Cjg) groups. The packing must have a
narrow size distribution and must not leach organic compounds into
methylene chloride. One liter of water should pass through the
cartridge in about 2 hrs with the assistance of a slight vacuum of
about 13 cm (5 in.} of mercury. Sect. 10 provides criteria for
acceptable LSE cartridges which are available from several commercial
suppliers.
7.3 Solvents
7.3.1 Methylene chloride, acetone, toluene and methanol. High
purity pesticide quality or equivalent.
7.3.2 Reagent water. Water in which an interferent is not observed
at the method detection limit of the compound of interest.
Prepare reagent water by passing tap water through a filter bed
containing about 0.5 kg of activated carbon or by using a water
purification system. Store in clean, narrow-mouth bottles with
Teflon-lined septa and screw caps.
7.4 Hydrochloric acid. 6N.
7.5 Sodium sulfate, anhydrous. (Soxhlet extracted with methylene chloride
for a minimum of 4 hrs.)
7.6 Stock standard solutions. Individual solutions of analytes, surro-
gates, and internal standards may be purchased as certified solutions
or prepared from pure materials. To prepare, add 10 mg (weighed on an
analytical balance to 0.1 mg) of the pure material to 1.9 ml of
methanol or acetone in a 2-mL volumetric flask, dilute to the mark,
and transfer the solution to an amber glass vial. If the analytical
standard is available only in quantities smaller than 10 mg, reduce
the volume of solvent accordingly. Some polycyclic aromatic hydro-
carbons are not soluble in methanol or acetone, and their stock
standard solutions are prepared in toluene. Methylene chloride should
be avoided as a solvent for standards because its high vapor pressure
leads to rapid evaporation and concentration changes. Methanol and
acetone are not as volatile as methylene chloride, but their solutions
must also be handled with care to avoid evaporation. Compounds 10, 11.
and 35 in Table 2 are soluble in acetone. Compounds 12, 13, and 20
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Table 2 are soluble In toluene. If compound purity Is certified by
the supplier at >96%, the weighed amount can be used without correc-
tion to calculate the concentration of the solution (5 /ig//zL). Store
the amber vials in a dark cool place.
7.7 Primary dilution standard solution. The stock standard solutions are
used to prepare a primary dilution standard solution that contains
multiple analytes. The recommended solvent for this dilution is
acetone. Aliquots of each of the stock standard solutions are
combined to produce the primary dilution in which the concentration of
the analytes is at least equal to the concentration of the most
concentrated calibration solution, that is, 10 ng/pL. Store the
primary dilution standard solution in an amber vial in a dark cool
place, and check frequently for signs of deterioration or evaporation,
especially just before preparing calibration solutions.
7.8 Fortification solution of internal standards and surrogates. Prepare
a solution of acenaphthene-DjQ, phenanthrene-Djp, chrysene-D^, and
perylene-Di2 in methanol or acetone at a concentration of 500 /ig/mL of
each. This solution is used 1n the preparation of the calibration
solutions. Dilute a portion of this solution by 10 to 50 fig/ml and
use this solution to fortify the actual water samples (see Sect.
11.2). Other surrogates, for example, caffe1ne-l5N? and pyrene-Din
may be Included in this solution as needed (a 100-/JL aliquot of this
50 /ig/mL solution added to 1 liter of water gives a concentration of
5 /ig/L of each internal standard or surrogate). Store this solution in
an amber vial in a dark cool place.
7.9 MS performance check solution. Prepare a 5 ng//iL solution of DFTPP
in methylene chloride. Store this solution in an amber vial in a dark
cool place.
7.10 Calibration solutions (CAL1 through CAL6). Prepare a series of six
concentration calibration solutions in acetone which contain all
analytes except pentachlorophenol and toxaphene at concentrations of
10, 5, 2, 1, 0.5, and 0.1 ng//iL, with a constant concentration of
5 ng//iL of each internal standard and surrogate in each CAL solution.
CAL1 through CAL6 are prepared by combining appropriate aliquots of
the primary dilution standard solution (7.7) and the fortification
solution (500 /ig/mL) of internal standards and surrogates (7.8).
Pentachlorophenol is included in this solution at a concentration four
times the other analytes. Toxaphene CAL solutions should be prepared
as separate solutions at concentrations of 250, 200, 100, 50, 25, and
10 ng//iL. Store these solutions in amber vials in a dark cool place.
Check these solutions regularly for signs of deterioration, for
example, the appearance of anthraquinone from the oxidation of
anthracene.
7.11 Reducing agents. Sodium sulfite or sodium arsenite. Sodium thio-
sulfate is not recommended as it may produce a residue of elemental
sulfur that can interfere with some analytes.
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7.12 Fortification solution for optional recovery standard. Prepare a
solution of terphenyl-Du in methylene chloride at a concentration ,
500 /ig/mL. An aliquot or this solution may be added (optional) to the
extract of the LSE cartridge to check on the recovery of the internal
standards in the extraction process.
8. SAMPLE COLLECTION. PRESERVATION. AND HANDLING
8.1 Sample collection. When sampling from a water tap, open the tap and
allow the system to flush until the water temperature has stabilized
(usually about 2-5 min). Adjust the flow to about 500 mL/min and
collect samples from the flowing stream. Keep samples sealed from
collection time until analysis. When sampling from an open body of
water, fill the sample container with water from a representative
area. Sampling equipment, including automatic samplers, must be free
of plastic tubing, gaskets, and other parts that may leach analytes
into water. Automatic samplers that composite samples over time must
use refrigerated glass sample containers.
8.2 Sample dechlorination and preservation. All samples should be iced or
refrigerated at 4°C from the time of collection until extraction.
Residual chlorine should be reduced at the sampling site by addition
of a reducing agent. Add 40-50 mg of sodium sulfite or sodium
arsenite (these may be added as solids with stirring until dissolved)
to each liter of water. Hydrochloric acid should be used at the
sampling site to retard the microbiological degradation of some
analytes in unchlorinated water. The sample pH is adjusted to <2 with
6 N hydrochloric acid. This is the same pH used in the extraction,
and is required to support the recovery of pentachlorophenol.
8.3 Holding time. Samples must be extracted within 7 days and the
extracts analyzed within 30 days of sample collection.
8.4 Field blanks.
8.4.1 Processing of a field reagent blank (FRB) is recommended along
with each sample set, which is composed of the samples
collected from the same general sample site at approximately
the same time. At the laboratory, fill a sample container with
reagent water, seal, and ship to the sampling site along with
the empty sample containers. Return the FRB to the laboratory
with filled sample bottles.
8.4.2 When hydrochloric acid is added to samples, use the same
procedures to add the same amount to the FRB.
9. CALIBRATION
9.1 Demonstration and documentation of acceptable initial calibration is
required before any samples are analyzed and is required intermittent-
ly throughout sample analysis as dictated by results of continuing
calibration checks. After initial calibration is successful, a
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continuing calibration check is required at the beginning of each 8
hr. period during which analyses are performed. Additional periodic
calibration checks are good laboratory practice.
9.2 Initial calibration
9.2.1 Calibrate the mass and abundance scales of the MS with
calibration compounds and procedures prescribed by the
manufacturer with any modifications necessary to meet the
requirements in Sect. 9.2.2.
9.2.2 Inject into the GC a 1-pL aliquot of the 5 ng/nl DFTPP solution
and acquire a mass spectrum that includes data for m/z 45-450.
Use GC conditions that produce a narrow (at least five scans
per peak) symmetrical peak. If the spectrum does not meet all
criteria (Table 1), the MS must be retuned and adjusted to meet
all criteria before proceeding with calibration. An average
spectrum across the GC peak may be used to evaluate the
performance of the system.
9.2.3 Inject a l-/zL aliquot of a medium concentration calibration
solution, for example 0.5-2 /ig/L, and acquire and store data
from m/z 45-450 with a total cycle time (including scan
overhead time) of 1.5 sec or less. Cycle time should be
adjusted to measure at least five or more spectra during the
elution of each GC peak.
9.2.3.1 Multi-ramp temperature program GC conditions. Adjust
the helium carrier gas flow rate to about 33 cm/sec.
Inject at 45°C and hold in splitless mode for 1 min.
Heat rapidly to 130°C. At 3 min start the temperature
program: 130-180°C at 12°/min; 180-240°C at 7°/nrin;
240-320°C at 12°/min. Start data acquisition at 5 min.
9.2.3.2 Single ramp linear temperature program. Adjust the
helium carrier gas flow rate to about 33 cm/sec.
Inject at 40°C and hold in splitless mode for 1 min.
Heat rapidly to 160°C. At 3 min start the temperature
program: 160-320°C at 6%iin; hold at 320° for 2 min.
Start data acquisition at 3 min.
9.2.4 Performance criteria for the medium calibration. Examine the
stored GC/MS data with the data system software. Figure 1
shows an acceptable total ion chromatogram.
9.2.4.1 GC performance. Anthracene and phenanthrene should be
separated by baseline. Benz[a]anthracene and chrysene
should be separated by a valley whose height is less
than 25% of the average peak height of these two
compounds. If the valley between benz[a]anthracene and
chrysene exceeds 25%, the GC column requires main-
tenance. See Sect. 9.3.6.
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9.2.4.2 MS sensitivity. The GC/MS/DS peak identification
software should be able to recognize a GC peak in the
appropriate retention time window for each of the
compounds in calibration solution, and make correct
tentative identifications. If fewer than 99% of the
compounds are recognized, system maintenance is
required. See Sect. 9.3.6.
9.2.4.3 Lack of degradation of endrin. Examine a plot of the
abundance of m/z 67 in the region of 1.05-1.3 of the
retention time of endrin. This 1s the region of
elutlon of endrin aldehyde, a product of the thermal
isomerizatlon of endrin. Confirm that the abundance of
m/z 67 at the retention time of endrin aldehyde 1s <10%
of the abundance of m/z 67 produced by endrin. If more
than 10% endrin aldehyde 1s observed, system main-
tenance is required to correct the problem. See Sect.
9.3.6.
9.2,5 If all performance criteria are met, inject a 1-pL aliquot of
each of the other CAL solutions using the same GC/MS condi-
tions.
9.2.6 Calculate a response factor (RF) for each analyte and surrogate
for each CAL solution using the internal standard whose
retention time is nearest the retention time of the analyte or
surrogate. Table 2 contains suggested internal standards for
each analyte and surrogate, and quantitation ions for all
compounds. This calculation is supported in acceptable GC/MS
data system software (Sect. 6.14.4), and many other software
programs. RF is a unitless number, but units used to express
quantities of analyte and internal standard must be equivalent.
DC (Ax)(Qis)
Kr —
(Als)Wx)
where:
Ax = integrated abundance of the quantitation ion
of the analyte.
Ais = integrated abundance of the quantitation ion
internal standard.
Qx = quantity of analyte injected in ng or
concentration units.
Qis = quantity of internal standard injected in ng
or concentration units.
9.2.6.1 For each analyte and surrogate, calculate the mean RF
from the analyses of the six CAL solutions. Calculate
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the standard deviation (SD) and the relative standard
deviation (RSD) from each mean: RSD = 100 (SD/M). If
the RSD of any analyte or surrogate mean RF exceeds
30%, either analyze additional aliquots of appropriate
CAL solutions to obtain an acceptable RSD of RFs over
the entire concentration range, or take action to
improve GC/MS performance. See Sect. 9.2.7.
9.2.7 As an alternative to calculating mean response factors and
applying the RSD test, use the GC/MS data system software or
other available software to generate a linear, second, or third
order regression calibration curve.
9.3 Continuing calibration check. Verify the MS tune and initial
calibration at the beginning of each 8 hr. work shift during which
analyses are performed using the following procedure.
9.3.1 Inject a l-/tL aliquot of the 5ng//zL DFTPP solution and acquire
a mass spectrum that includes data for m/z 45-450. If the
spectrum does not meet all criteria (Table 1), the MS must be
retuned and adjusted to meet all criteria before proceeding
with the continuing calibration check.
9.3.2 Inject a l-/zL aliquot of a medium concentration calibration
solution and analyze with the same conditions used during the
initial calibration.
9.3.3 Demonstrate acceptable performance for the criteria shown in
Sect. 9.2.4.
9.3.4 Determine that the absolute areas of the quantitation ions of
the internal standards and surrogate(s) have not decreased by
more than 30% from the areas measured in the most recent
continuing calibration check, or by more than 50% from the
areas measured during initial calibration. If these areas have
decreased by more than these amounts, adjustments must be made
to restore system sensitivity. These adjustments may require
cleaning of the MS ion source, or other maintenance as
indicated in Sect. 9.3.6, and recalibration. Control charts
are useful aids in documenting system sensitivity changes.
9.3.5 Calculate the RF for each analyte and surrogate from the data
measured in the continuing calibration check. The RF for each
analyte and surrogate must be within 30% of the mean value
measured in the initial calibration. Alternatively, if a
second or third order regression is used, the point from the
continuing calibration check for each analyte and surrogate
must fall, within the analyst's judgement, on the curve from
the initial calibration. If these conditions do not exist,
remedial action must be taken which may require reinitial
calibration.
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9.3.6 Some possible remedial actions. Major maintenance such as
cleaning an Ion source, cleaning quadrupole rods, etc. requi...
returning to the Initial calibration step.
9.3.6.1 Check and adjust GC and/or MS operating conditions;
check the MS resolution, and calibrate the mass
scale.
9.3.6.2 Clean or replace the splitless injection liner;
silanize a new injection liner.
9.3.6.3 Flush the GC column with solvent according to
manufacturer's instructions.
9.3.6.4 Break off a short portion (about 1 meter) of the column
from the end near the injector; or replace GC column.
This action will cause a change in retention times.
9.3.6.5 Prepare fresh CAL solutions, and repeat the initial
calibration step.
9.3.6.6 Clean the MS ion source and rods (if a quadrupole).
9.3.6.7 Replace any components that allow analytes to come into
contact with hot metal surfaces.
9.3.6.8 Replace the MS electron multiplier, or any other faulty
components.
10. QUALITY CONTROL
10.1 Quality control (QC) requirements are the initial demonstration of
laboratory capability followed by regular analyses of laboratory
reagent blanks, laboratory fortified blanks, and laboratory fortified
matrix samples. The laboratory must maintain records to document the
quality of the data generated. Additional quality control practices
are recommended.
10.2 Initial demonstration of low system background and acceptable particle
size and packing. Before any samples are analyzed, or any time a new
supply of cartridges is received from a supplier, it must be demon-
strated that a laboratory reagent blank (LRB) is reasonably free of
contamination that would prevent the determination of any analyte of
concern. In this same experiment, it must be demonstrated that the
particle size and packing of the LSE cartridge are acceptable.
Consistent flow rate is an indication of acceptable particle size
distribution and packing.
10.2.1 A major source of potential contamination is the liquid-solid
extraction (LSE) cartridges which very likely will contain
phthalate esters, silicon compounds, and other contaminants
that could prevent the determination of method analytes (5).
338
-------
Generally, phthalate esters will be leached from the cartridges
into methylene chloride and produce a variable background that
is equivalent to <2 /ig/L in the water sample. If the back-
ground contamination is sufficient to prevent accurate and
precise analyses, the condition must be corrected before
proceeding with the initial demonstration. Figure 2 shows
unacceptable background contamination from a poor quality
commercial LSE cartridge. The background contamination is the
large broad peak, and the small peaks are method analytes
present at a concentration equivalent to 2 /ig/L. Several
sources of LSE cartridges may be evaluated before an acceptable
supply is identified.
10.2.2 Other sources of background contamination are solvents,
reagents, and glassware. Background contamination must be
reduced to an acceptable level before proceeding with the next
section. In general, background from method analytes should be
below the method detection limit.
10.2.3 One liter of water should pass through the cartridge in about
2 hrs with a partial vacuum of about 13 cm (5 in.) of mercury.
The extraction time should not vary unreasonably among LSE
cartridges.
10.3 Initial demonstration of laboratory accuracy and precision. Analyze
four to seven replicates of a laboratory fortified blank containing
each analyte of concern at a concentration 'in the range of 2-5 jig/L
(see regulations and maximum contaminant levels for guidance on
appropriate concentrations).
10.3.1 Prepare each replicate by adding an appropriate aliquot of the
primary dilution standard solution, or another certified
quality control sample, to reagent water. Analyze each
replicate according to the procedures described in Sect. 11
and on a schedule that results in the analyses of all repli-
cates over a period of several days.
10.3.2 Calculate the measured concentration of each analyte in each
replicate, the mean concentration of each analyte in all
replicates, and mean accuracy (as mean percentage of true
value) for each analyte, and the precision (as relative
standard deviation, RSD) of the measurements for each analyte.
Calculate the MDL of each analyte using the procedures
described in Sect. 13.1.2 (1).
10.3.3 For each analyte and surrogate, the mean accuracy, expressed as
a percentage of the true value, should be 70-130% and the RSD
should be <30%. Some analytes, particularly the polycyclic
aromatic hydrocarbons with molecular weights >250, are measured
at concentrations below 2 /ig/L, with a mean accuracy of 35-130%
of true value. The MDLs should be sufficient to detect
analytes at the regulatory levels. If these criteria are not
339
-------
met for an analyte, take remedial action and repeat the
measurements for that analyte to demonstrate acceptable
performance before samples are analyzed.
10.3.4 Develop and maintain a system of control charts to plot the
precision and accuracy of analyte and surrogate measurements as
a function of time. Charting of surrogate recoveries is an
especially valuable activity since these are present in every
sample and the analytical results will form a significant
record of data quality.
10.4 Monitor the integrated areas of the quantitation ions of the internal
standards and surrogates in continuing calibration checks (see Sect.
9.3.4). In laboratory fortified blanks or samples, the integrated
areas of internal standards and surrogates will not be constant
because the volume of the extract will vary (and is difficult to keep
constant). But the ratios of the areas should be reasonably constant
in laboratory fortified blanks and samples. The addition of 10 /il_ of
the recovery standard, terphenyl-Di4 (500 jig/mL), to the extract is
optional, and may be used to monitor the recovery of internal
standards and surrogates in laboratory fortified blanks and samples.
Internal standard recovery should be in excess of 70%.
10.5 Laboratory reagent blanks. With each batch of samples processed as a
group within a work shift, analyze a laboratory reagent blank to
determine the background system contamination. Any time a new batch
of LSE cartridges is received, or new supplies of other reagents are
used, repeat the demonstration of low background described in 10.2.
10.6 With each batch of samples processed as a group within a work shift,
analyze a single laboratory fortified blank (LFB) containing each
analyte of concern at a concentration as determined in 10.3. If more
than 20 samples are included in a batch, analyze a LFB for every 20
samples. Use the procedures described in 10.3.3 to evaluate the
accuracy of the measurements, and to estimate whether the method
detection limits can be obtained. If acceptable accuracy and method
detection limits cannot be achieved, the problem must be located and
corrected before further samples are analyzed. Add these results to
the on-going control charts to document data quality.
10.7 Determine that the sample matrix does not contain materials that
adversely affect method performance. This is accomplished by
analyzing replicates of laboratory fortified matrix samples and
ascertaining that the precision, accuracy, and method detection limits
of analytes are in the same range as obtained with laboratory
fortified blanks. If a variety of different sample matrices are
analyzed regularly, for example, drinking water from groundwater and
surface water sources, matrix independence should be established for
each. A laboratory fortified sample matrix should be analyzed for
every 20 samples processed in the same batch.
340
-------
10.8 With each set of field samples a field reagent blank (FRB) should be
analyzed. The results of these analyses will help define contamina-
tion resulting from field sampling and transportation activities.
10.9 At least quarterly, replicates of laboratory fortified blanks should
be analyzed to determine the precision of the laboratory measurements.
Add these results to the on-going control charts to document data
quality (as in Sect. 10.3).
10.10 At least quarterly, analyze a quality control sample from an external
source. If measured analyte concentrations are not of acceptable
accuracy (Sect. 10.3.3), check the entire analytical procedure to
locate and correct the problem source.
10.11 Numerous other quality control measures are incorporated into other
parts of this procedure, and serve to alert the analyst to potential
problems.
11. PROCEDURE
11.1 Setup the extraction apparatus shown in Figure 3A. The reservoir is
not required, but recommended for convenient operation. Water drains
from the reservoir through the LSE cartridge and into a syringe needle
which is inserted through a rubber stopper into the suction flask. A
slight vacuum of 13 cm (5 in.) of mercury is used during all opera-
tions with the apparatus. The pressure used is critical as a vacuum >
than 13 cm may result in poor precision. About 2 hrs is required to
draw a liter of water through the system.
11.2 Pour the water sample into the 2-L separatory funnel with the stopcock
closed. Residual chlorine should not be present as a reducing agent
should have been added at the time of sampling. Also the pH of the
sample should be about 2. If residual chlorine is present and/or the
pH is >2, the sample may be invalid. Add a 100-jtL aliquot of the
fortification solution (50 /ig/mL) for internal standards and surro-
gates, and mix immediately until homogeneous. The concentration of
these compounds in the water should be 5 pg/L.
11.3 Flush each cartridge with two 10 ml aliquots of methylene chloride,
followed by two 10 ml aliquots of methanol, letting the cartridge
drain dry after each flush. These solvent flushes may be accomplished
by adding the solvents directly to the solvent reservoir in Figure 3A.
Add 10 ml of reagent water to the solvent reservoir, but before the
reagent water level drops below the top edge of the packing in the LSE
cartridge, open the stopcock of the separatory funnel and begin adding
sample water to the solvent reservoir. Close the stopcock when an
adequate amount of sample is in the reservoir.
11.4 Periodically open the stopcock and drain a portion of the sample water
into the solvent reservoir. The water sample will drain into the
cartridge, and from the exit into the suction flask. Maintain the
packing material in the cartridge immersed in water at all times.
341
-------
After all of the sample has passed through the LSE cartridge, wash
separatory funnel and cartridge with 10 ml of reagent water, and draw
air through the cartridge for 10 mln.
11.5 Transfer the 125-mL solvent reservoir and LSE cartridge (from Figure
3A) to the elutlon apparatus (Figure 3B). The same 125-mL solvent
reservoir 1s used for both apparatus. Wash the 2-liter separatory
funnel with 5 mL of methylene chloride and collect the washings.
Close the stopcock on the 100-ml separatory funnel of the elutlon
apparatus, add the washings to the reservoir and enough additional
methylene chloride to bring the volume back up to 5 mL and elute the
LSE cartridge. Elute the LSE cartridge with an additional 5 mL of
methylene chloride (10-mL total). A small amount of nitrogen positive
pressure may be used to elute the cartridge. Small amounts of
residual water from the LSE cartridge will form an immiscible layer
with the methylene chloride in the 100-ml separatory funnel. Open the
stopcock and allow the methylene chloride to pass through the drying
column packed with anhydrous sodium sulfate (1-in) and into the
collection vial. Do not allow the water layer to enter the drying
column. Remove the 100 mL separatory funnel and wash the drying
column with 2 mL of methylene chloride. Add this to the extract.
Concentrate the extract to 1 mL under a gentle stream of nitrogen. If
desired, gently warm the extract in a water bath to evaporate to
between 0.5 - 1.0 mL (without gas flow). Do not concentrate the
extract to less than 0.5 mL (or dryness) as this will result in losses
of analytes. If desired, add an aliquot of the recovery standard to
the concentrated extract to check the recovery of the internal
standards (see Sect. 10.4).
11.6 Analyze a 1-2 /iL aliquot with the GC/MS system under the same
conditions used for the initial and continuing calibrations (Sect.
9.2.3).
11.7 At the conclusion of data acquisition, use the same software that was
used in the calibration procedure to tentatively identify peaks in
retention time windows of interest. Use the data system software to
examine the ion abundances of components of the chromatogram. If any
ion abundance exceeds the system working range, dilute the sample
aliquot and analyze the diluted aliquot.
11.8 Identification of analytes. Identify a sample component by comparison
of its mass spectrum (after background subtraction) to a reference
spectrum in the user-created data base. The GC retention time of the
sample component should be within 10 sec of the time observed for
that same compound when a calibration solution was analyzed.
11.8.1 In general, all ions that are present above 10% relative
abundance in the mass spectrum of the standard should be
present in the mass spectrum of the sample component and should
agree within absolute 20%. For example, if an ion has a
relative abundance of 30% in the standard spectrum, its
abundance in the sample spectrum should be in the range of 1
342
-------
to 50%. Some ions, particularly the molecular ion, are of
special importance, and should be evaluated even if they are
below 10% relative abundance.
11.8.2 Identification is hampered when sample components are not
resolved chromatographically and produce mass spectra contain-
ing Ions contributed by more than one analyte. When GC peaks
obviously represent more than one sample component (i.e.,
broadened peak with shoulder(s) or valley between two or more
maxima), appropriate analyte spectra and background spectra can
be selected by examining plots of characteristic ions for
tentatively identified components. When analytes coelute
(i.e., only one GC peak is apparent), the identification
criteria can be met but each analyte spectrum will contain
extraneous ions contributed by the coeluting compound.
11.8.3 Structural isomers that produce very similar mass spectra can
be explicitly identified only if they have sufficiently
different GC retention times. See Sect. 9.2.4.1. Acceptable
resolution is achieved if the height of the valley between two
isomer peaks is less than 25% of the average height of the two
peak heights. Otherwise, structural isomers are identified as
isomeric pairs. Benzo[b] and benzo[k]fluoranthene are measured
as an isomeric pair.
11.8.4 Phthalate esters and other background components appear in
variable quantities in laboratory and field reagent blanks, and
generally cannot be accurately measured at levels below about
2 /ig/L. Subtraction of the concentration in the blank from the
concentration in the sample at or below the 2 /ig/L level is not
recommended because the concentration of the background in the
blank is highly variable.
12. CALCULATIONS
12.1 Complete chromatographic resolution is not necessary for accurate and
precise measurements of analyte concentrations if unique ions with
adequate intensities are available for quantitation. For example,
although two listed analytes, dibenz[a,h]anthracene and indeno-
[l,2,3,c,d]pyrene, were not resolved with the GC conditions used, and
produced mass spectra containing common ions, concentrations (Tables
3-6) were calculated by measuring appropriate characteristic ions.
12.1.1 Calculate analyte and surrogate concentrations.
(AxMQis)
(Ais) RF V
where:
Cx = concentration of analyte or surrogate in /ig/L in
the water sample.
343
-------
Ax = Integrated abundance of the quantitation ion
of the analyte in the sample.
Ais = integrated abundance of the quantitation ion
of the internal standard in the sample.
Qjs = total quantity (in micrograms) of internal
standard added to the water sample.
V = original water sample volume in liters.
RF = mean response factor of analyte from the
initial calibration.
12.1.2 Alternatively, use the GC/MS system software or other
available proven software to compute the concentrations of
the analytes and surrogates from the second or third order
regression curves.
12.1.3 Calculations should utilize all available digits of precision,
but final reported concentrations should be rounded to an
appropriate number of significant figures (one digit of
uncertainity). Experience indicates that three significant
figures may be used for concentrations above 99 /ig/L, two
significant figures for concentrations between 1-99 /jg/L, and
one significant figure for lower concentrations.
13. METHOD PERFORMANCE
13.1 Single laboratory accuracy and precision data (Tables 3-7} for each
listed analyte was obtained at two concentrations with the same
extracts analyzed on two different instrument systems. Seven 1-liter
aliquots of reagent water containing 2 pg/L of each analyte, and five
to seven 1-liter aliquots of reagent water containing 0.2 jig/L of
each analyte were analyzed with this procedure.
13.1.2 With these data, method detection limits (MDL) were calculated
using the formula:
MDL = S t(n.1>1.aipha = 0.99)
where:
tfn-l.l-alpha = 0.99} " Student's t value for the 99%
confidence level with n-1 degrees of freedom
n = number of replicates
S = standard deviation of replicate analyses.
13.2 Problem compounds
13.2.1 The common phthalate and adipate esters (compounds 14, 21, and
23-26}, which are widely used commercially, appear in variable
quantities in laboratory and field reagent blanks, and
generally cannot be accurately or precisely measured at levels
below about 2 M9/L- Subtraction of the concentration in the
blank from the concentration in the sample at or below the
344
-------
2 /ig/L level is not recommended because the concentrations of
the background in blanks is highly variable.
13.2.2 Some polycyclic aromatic hydrocarbons are rapidly oxidized
and/or chlorinated in water containing residual chlorine.
Therefore residual chlorine must be reduced before analysis.
13.2.3 In water free of residual chlorine, some polycyclic aromatic
hydrocarbons (for example, compounds 9, 12, 13, 20, and 35) are
not accurately measured because of low recoveries in the
extraction process.
13.2.4 Pentachlorophenol No. 40 and hexachlorocyclopentadiene No. 34
may not be accurately measured. Pentachlorophenol is a strong
acid and elutes as a broad weak peak. Hexachlorocyclo-
pentadiene is susceptible to photochemical and thermal
decomposition.
14. REFERENCES
1. Glaser, J. A., D. L. Foerst, G. D. McKee, S. A. Quave, and W. L.
Budde, "Trace Analyses for Wastewaters," Environ. Sci. Techno!. 1981
15, 1426-1435.
2. "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, Aug. 1977.
3. "OSHA Safety and Health Standards, General Industry," (29CFR1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
5. Junk, G.A., M. J. Avery, J. J. Richard, "Interferences in Solid-Phase
Extraction Using C-18 Bonded Porous Silica Cartridges," Anal. Chem.
1988, 60, 1347.
345
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TABLE 1. ION ABUNDANCE CRITERIA FOR BIS(PERFLUOROPHENYL)PHENYL
PHOSPHINE (DECAFLUOROTRIPHENYLPHOSPHINE, DFTPP)
Mass Relative Abundance
(M/z) Criteria
Purpose of Checkpoint*
51 10-80% of the base peak
68 <2% of mass 69
70 <2% of mass 69
127 10-80% of the base peak
197 <2% of mass 198
198 base peak or >50% of 442
199 5-9% of mass 198
275 10-60% of the base peak
365 >1% of the base peak
441 Present and < mass 443
442 base peak or >50% of 198
443 15-24% of mass 442
low mass sensitivity
low mass resolution
low mass resolution
low-mid mass sensitivity
mid-mass resolution
mid-mass resolution and sensitivity
mid-mass resolution and isotope ratio
mid-high mass sensitivity
baseline threshold
high mass resolution
high mass resolution and sensitivity
high mass resolution and isotope ratio
IAH ions are used primarily to check the mass measuring accuracy of the mass
spectrometer and data system, and this is the most important part of the
performance test. The three resolution checks, which include natural abundance
isotope ratios, constitute the next most important part of the performance
test. The correct setting of the baseline threshold, as indicated by the
presence of low intensity ions, is the next most important part of the
performance test. Finally, the ion abundance ranges are designed to encourage
some standardization to fragmentation patterns.
346
-------
TABLE 2. RETENTION TIME DATA, QUANTITATION IONS, AND INTERNAL
STANDARD REFERENCES FOR METHOD ANALYTES.
Comoound
Compound
Number
Retention
Time(min:sec)
Aa
Internal
Quantitation Standard
Ion (m/z) Reference
Internal standards
acenaphthene-Djo 1
phenanthrene-Djo 2
chrysene-Dj2 3
4:49 7:45
8:26 11:08
18:14 19:20
164
188
240
Surrogate
perylene-Dj2
23:37 22:55
264
Target analvtes
acenaphthylene
aldrin
anthracene
atrazine
benz[a]anthracene
benzo[b]fluoranthene
benzo[k]fluoranthene
benzo[a]pyrene
benzo[g,h,i]perylene
butyl benzylphthalate
chlordane components
alpha-chlordane
gamma-chlordane
trans nonachlor
2-chlorobiphenyl
chrysene
dibenz[a,h]anthracene
di-n-butylphthalate
2,3-dichlorobiphenyl
diethylphthalate
di(2-ethylhexyl)
phthalate
di(2-ethylhexyl)adipate 25
dimethylphthalate
endrin
fluorene
heptachlor
heptachlor epoxide
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
4:37
11:21
8:44
7:56
18:06
22:23
22:28
23:28
27:56
16:40
13:44
13:16
13:54
4:56
18:24
27:15
10:58
7:20
5:52
19:19
17:17
4:26
15:52
6:00
10:20
12:33
7:25
13:36
11:20
10:42
19:14
22:07
22:07
22:47
26:44
18:09
15:42
15:18
15:50
7:55
19:23
25:57
13:20
10:12
8:50
20:01
18:33
7:21
16:53
8:53
12:45
14:40
152
66
178
200/215
228
252
252
252
276
149
375
375
409
188
228
278
149
222
149
149
129
163
81
166
100/160
81/353
1
2
2
1/2
3
3
3
3
3
2/3
2/3
2/3
2/3
1
3
3
2
1
1
2/3
2/3
1
2/3
1
2
2
347
-------
TABLE 2. (Continued)
Compound
Compound Retention
Number Time(min:sec)
Internal
Quantitation Standard
Ion (m/z) Reference
2,2',3,3',4,4',6-hepta-
chlorobiphenyl 31
hexachlorobenzene 32
2,2',4,4')5,6'-hexa-
chlorobiphenyl 33
hexachlorocyclo-
18:25
7:37
19:25
10:20
14:34 16:30
394/396
284/286
360
3
1/2
pentadlene 34
1ndeno[l,2,3,c,d]pyrene 35
llndane 36
methoxychlor 37
2, 2', 3, 3', 4, 5', 6,6'-
octachloroblphenyl 38
2,2',3',4,6-penta
chiorobiphenyl 39
pentacnlorophenol 40
phenanthrene 41
pyrene 42
simazlne 43
2,2',4,4'-tetrachloro-
biphenyl 44
toxaphene 45
2,4,5-trichlorobiphenyl 46
alachlor 47
3:36
27:09
8:17
18:34
18:38
12:50
8:11
8:35
13:30
7:47
11:01
11:30-23:30
9:23
~ ™
6:15
25:50
10:57
19:30
19:33
15:00
10:51
11:13
15:29
10:35
13:25
13:00-21:30
11:59
13:19
237
276
181/183
227
430
326
266
178
202
201
292
159
256
160
1
3
1/2
3
3
2
2
2
2/3
1/2
2
2
2
2
^Single ramp linear temperature program conditions (Sect. 9.2.3.2).
''Multi-ramp linear temperature program conditions (Sect. 9.Z.3.1).
348
-------
TABLE 3. ACCURACY AND PRECISION DATA FROM SEVEN DETERMINATIONS OF
THE METHOD ANALYTES AT 2 nG/l WITH LIQUID-SOLID EXTRACTION
AND THE ION TRAP MASS SPECTROMETER
Compound
Number
(Table 2)
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
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46U
Meanb
"ee Table
True
Cone.
(ua/L)
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
8
2
2
2
2
25
2
2
Mean
Observed
Cone.
(UQ/L)
5.0
1.9
1.6
1.7
2.2
1.8
Rel
Std. Std
Dev. Oev
(uo/L) (%)
0.3 6.0
0.2 11.
0.2 13.
0.1 5.9
0.3 14.
0.2 11.
not separated from No.
4.2
0.8
0.7
2.0
2.0
2.2
2.7
1.9
2.2
0.3
2.2
2.3
2.0
1.9
1.6
1.9
1.8
2.2
2.2
2.3
1.4
1.7
1.6
1.1
0.4
2.1
1.8
1.8
1.9
8.2
2.4
1.9
2.1
1.5
28.
1.7
1.8
4. DCompounds 4, 40,
0.3 7.1
0.2 25.
0.1 14.
0.3 15.
0.2 10.
0.3 14.
1.0 37.
0.1 5.2
0.1 4.5
0.3 100.
0.3 14.
0.1 4.3
0.3 15.
0.2 11.
0.3 19.
0.2 11.
0.1 5.5
0.2 9.1
0.3 14.
0.2 8.7
0.2 14.
0.2 12.
0.4 25.
0.1 9.1
0.2 50.
0.2 9.5
0.2 11.
0.2 11.
0.1 5.3
1.2 15.
0.1 4.2
0.1 5.3
0.2 9.5
0.1 6.7
4.7 17.
0.1 5.9
0.2 15.
and 45 excl
349
Mean Method
Accuracy
(% of True
Cone.)
100
95
80
85
110
90
11; measured with
105
40
35
100
100
110
135
95
110
15
110
115
100
95
80
95
90
110
110
115
70
85
80
55
20
105
90
90
95
102
120
95
105
75
112
85
91
Method
Detection
Limit (MDL)
(UQ/L)
a
a
a
a
a
a
No. 11
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
15.
a
0.6
uded from the means.
-------
TABLE 4. ACCURACY AND PRECISION DATA FROM FIVE TO SEVEN DETERMINATIONS
OF THE HETHOD ANALYTES AT 0.2
-------
TABLE 5. ACCURACY AND PRECISION DATA FROM SEVEN DETERMINATIONS
OF THE METHOD ANALYTES AT 2 uG/L WITH LIQUID-SOLID EXTRACTION
AND A MAGNETIC SECTOR MASS SPECTROMETER
Compound
Number
(Table 2)
4
5
6
7
3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46U
Meanb
aSee Table
True
Cone.
(ua/L)
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
8
2
2
2
2
25
2
2
Mean
Observed
Cone.
(ua/L)
5.7
1.9
1.6
2.2
2.4
2.2
Rel. Mean Method Method
Std. Std. Accuracy Detection
Dev. Dev. (% of True Limit (MDL)
(ua/L) (%) Cone.) (ua/L)
0.34 6.0
0.22 12.
0.18 11.
0.67 30.
0.46 19.
0.87 40
not separated from No.
4.0
0.85
0.69
2.0
2.2
2.1
1.9
2.0
2.1
0.75
2.5
2.0
3.5
2.0
1.4
2.9
1.7
2.6
1.2
2.6
1.5
1.5
1.9
0.89
0.83
2.2
2.0
1.5
1.6
12.
2.3
2.0
2.5
1.6
28.
1.9
1.8
6. Compounds 4, 40,
0.37 9.3
0.15 18.
0.12 17.
0.20 10.
0.41 19.
0.38 18.
0.10 5.2
0.29 14.
0.32 15.
0.18 24.
0.32 13.
0.23 12.
1.8 51.
0.28 14.
0.16 11.
0.70 24.
0.45 26.
1.0 38.
0.10 8.3
0.42 16.
0.19 13.
0.35 23.
0.17 8.9
0.11 12.
0.072 8.7
0.10 4.5
0.88 44.
0.11 7.3
0.14 8.8
2.6 22.
0.18 7.8
0.26 13.
0.34 14.
0.17 11.
2.7 10.
0.073 3.8
0.32 16.
and 45 excl
114
95
80
110
120
110
11; measured
100
43
35
100
110
105
95
100
105
38
125
100
175
100
70
145
85
130
60
130
75
75
95
45
42
110
100
75
80
150
115
100
125
80
112
95
88
uded from the
a
a
a
a
a
a
with No. 11
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
9.
a
1.
means.
351
-------
TABLE 6. ACCURACY AND PRECISION DATA FROM SIX OR SEVEN DETERMINATIONS
OF THE METHOD ANALYTES AT 0.2 uG/L WITH LIQUID-SOLID EXTRACTION AND
A MAGNETIC SECTOR MASS SPECTROMETER.
Compound
Number
(Table 2)
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
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Meana
Compounds
True
Cone.
(ua/L)
0.5
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.8
0.2
0.2
0.2
0.2
Mean
Observed
Cone.
(ua/L)
0.67
0.11
0.11
0.14
0.26
0.24
Rel . Mean Method Method
Std. Std. Accuracy Detection
Dev. Dev. (% of True Limit (MDL)
(ua/L) (%) Cone.) (ua/L)
0.07 9.4
0.03 24.
0.02 21.
0.02 17.
0.08 31.
0.06 26.
not separated from No. 11;
0.40
0.08
0.07
0.33
0.19
0.17
0.19
0.17
0.27
0.09
1.1
0.18
0.29
0.42
0.32
0.20
0.53
0.18
0.11
0.33
0.17
0.11
0.17
0.05
0.08
0.27
0.24
0.15
0.13
1.8
0.21
0.19
0.27
0.13
0.10 25.
0.02 27.
0.01 22.
0.16 48.
0.02 13.
0.03 45.
0.04 18.
0.02 13.
0.08 28.
0.01 15.
1.2 109.
0.05 30,
0.17 59.
0.23 55.
0.16 50.
0.09 47.
0.30 57.
0.03 15.
0.05 42.
0.08 26.
0.01 7.1
0.04 40.
0.03 15.
0.02 35.
0.06 8.1
0.03 11.
0.09 39.
0.02 12.
0.02 13.
0.82 46.
0.07 33.
0.04 23.
0.07 27.
0.03 22.
134
55
56
70
130
120
measured with
100
38
33
160
95
85
95
85
135
46
550
90
145
210
160
100
265
90
55
165
85
55
85
24
40
135
120
75
65
225
105
95
135
65
0.2
0.1
0.1
0.1
0.3
0.2
No. 11
0.3
0.1
0.1
0.5
0.1
0.3
0.1
0.1
0.3
0.1
4.
0.2
0.6
0.8
0.5
0.3
1.
0.1
0.2
0.3
0.04
0.2
0.1
0.1
0.02
0.1
0.3
0.1
0.1
3.
0.2
0.1
0.2
0.1
not measured at this level
0.2
0.2
4, 40,
0.16
0.21
0.04 23.
0.09 28.
80
102
0.12
0.3
and 45 excluded from the means.
352
-------
TABLE 7. ACCURACY AND PRECISION DATA FROM SEVEN
DETERMINATIONS AT 2 uG/L WITH LIQUID-SOLID EXTRACTION
AND A QUADRUPOLE MASS SPECTROMETER
Compound
Number
(Table 2)
True
Cone.
(ua/L)
Mean
Observed
Cone.
(ua/L)
Std.
Dev.
(ua/L)
Rel.
Std.
Dev.
m
Mean Method
Accuracy
(% of True
Cone.)
Method
Detection
Limit (MDL)
(ua/L)
47 2 2.4 0.4 16. 122 1.0
353
-------
FIGURE 1. TOTAL ION CHROMATOCRAM OF TWO MANOCRAMS OF ANALYTES
AND KIVE NANOCRAMS OF SURROGATES AND INTERNAL STANDARD
100
90
80 j
70
60
50
40
30
20
10
0
Scan
R.T.
TIC
5
34 .5
J jl
1
2
18
^ J
100
4:55
3
2
I
28
if 37 Ul *6 4A
1 j 36 fr 7 i ji
niRlilf 1 7 Q n £.
IlkfJJli ft IL lU
200 300
7:20 9:46
8925696
39i6 42 15 j3
400 500
12:12 14:38
CO
tn
100
90
80
70
60
50
40
30
20
10
0
TIC
38 24
10 11
M »
^J^^^^/^J >Vw^.. .*J
2513216
13
35.20
Scan
R.T.
600
17:04
700
19:29
21:55
90®
24:21
1000
26:47
-------
FIGURE 2. TOTAL ION CHROMATOGRAM FROM A LABORATORY BLANK
WITH AN UNACCEPTABLY HIGH BACKGROUND
to
01
en
METHOD ANALYTES AT 2ufl/L
-------
n
2 Liter
separator/
funnel
125 ml
solvent
reservoir
ground glass T 14/35
LSE cartridge
rubber stopper
No. 18-2O luer-lok
syringe needle
1 liter
vacuum flask
125 ml
solvent
reservoir
ground glass
114/35
LSE cartridge
100ml
separator/
funnel
drying
column
1.2 cm x 4O cm
1O ml
graduated
vial
A. Extraction apparatus
FIGURE 3
356
B. Elution apparatus
-------
METHOD 531.1. MEASUREMENT OF N-METHYLCARBAMOYLOXIMES
AND N-METHYLCARBAMATES IN HATER BY DIRECT AQUEOUS INJECTION HPLC
WITH POST COLUMN DERIVATIZATION
Revision 3.0
D. L. Foerst - Method 531, Revision 1.0 (1985)
T. Engels (Battelle Columbus Laboratories) - National Pesticide Survey
Method 5, Revision 2.0 (1987)
R. L. Graves - Method 531.1, Revision 3.0 (1989)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
357
-------
METHOD 531.1
MEASUREMENT OF N-METHYLCARBAMOYLOXIMES
AND N-METHYLCARBAMATES IN HATER BY DIRECT AQUEOUS INJECTION HPLC
WITH POST COLUMN DERIVATIZATION
1. SCOPE AND APPLICATION
1.1 This is a high performance liquid chromatographic (HPLC) method
applicable to the determinations of certain N-methylcarbamoyloximes
and N-methylcarbamates in ground water and finished drinking water(l).
The following compounds can be determined using this method:
Chemical Abstract Services
Analvte Registry Number
Aldicarb 116-06-3
Aldicarb sulfone 1646-88-4
Aldicarb sulfoxide 1646-87-3
Baygon 114-26-1
Carbaryl 63-25-2
Carbofuran 1563-66-2
3-Hydroxycarbofuran 16655-82-6
Methiocarb 2032-65-7
Methomyl 16752-77-5
Oxamyl 23135-22-0
1.2 This method has been validated in a single laboratory and estimated
detection limits (EDLs) have been determined for the analytes above
(Sect.12). Observed detection limits may vary between ground waters,
depending upon the nature of interferences in the sample matrix and
the specific instrumentation used.
1.3 This method is restricted to use by or under the supervision of
analysts experienced in the use of liquid chromatography and in the
interpretation of liquid chromatograms. Each analyst must demonstrate
the ability to generate acceptable results with this method using the
procedure described in Sect. 10.3.
1.4 When this method is used to analyze unfamiliar samples for any or all
of the analytes above, analyte identifications should be confirmed by
at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 The water sample is filtered and a 400-0L aliquot is injected into a
reverse phase HPLC column. Separation of the analytes is achieved
using gradient elution chromatography. After elution from the HPLC
column, the analytes are hydrolyzed with 0.05 N sodium hydroxide
(NaOH) at 95"C. The methyl amine formed during hydrolysis is reacted
with o-phthalaldehyde (OPA) and 2-mercaptoethanol to form a highly
358
-------
fluorescent derivative which is detected by a fluorescence detector
(2).
3. DEFINITIONS
3.1 Internal standard -- A pure analyte(s) added to a solution in known
amount(s) and used to measure the relative responses of other method
analytes and surrogates that are components of the same solution. The
internal standard must be an analyte that is not a sample component.
3.2 Surrogate analyte -- A pure analyte(s), which is extremely unlikely to
be found in any sample, and which is added to a sample aliquot in
known amount(s) before extraction and is measured with the same
procedures used to measure other sample components. The purpose of a
surrogate analyte is to monitor method performance with each sample.
3.3 Laboratory duplicates (LD1 and LD2) -- Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample collection,
preservation, or storage procedures.
3.4 Field duplicates (FD1 and FD2) -- Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures. Analyses
of FD1 and FD2 give a measure of the precision associated with sample
collection, preservation and storage, as well as with laboratory
procedures.
3.5 Laboratory reagent blank (LRB) -- An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates that
are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.6 Field reagent blank (FRB) -- Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage, preservation
and all analytical procedures. The purpose of the FRB is to determine
if method analytes or other interferences are present in the field
environment.
3.7 Laboratory performance check solution (LPC) -- A solution of method
analytes, surrogate compounds, and internal standards used to evaluate
the performance of the instrument system with respect to a defined set
of method criteria.
3.8 Laboratory fortified blank (LFB) -- An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
359
-------
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.9 Laboratory fortified sample matrix (LFM) -- An aliquot of an
environmental sample to which known quantities of the method analytes
are added in the laboratory. The LFM is analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be determined
in a separate aliquot and the measured values in the LFM corrected for
background concentrations.
3.10 Stock standard solution -- A concentrated solution containing a single
certified standard that is a method analyte, or a concentrated
solution of a single analyte prepared in the laboratory with an
assayed reference compound. Stock standard solutions are used to
prepare primary dilution standards.
3.11 Primary dilution standard solution -- A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
3.12 Calibration standard (CAL) -- a solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are used
to calibrate the instrument response with respect to analyte
concentration.
3.13 Quality control sample (QCS) -- a sample matrix containing method
analytes or a solution of method analytes in a water miscible solvent
which is used to fortify reagent water or environmental samples. The
QCS is obtained from a source external to the laboratory, and is used
to check laboratory performance with externally prepared test
materials.
INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware and other sample processing apparatus that lead to
discrete artifacts or elevated baselines in liquid chromatograms.
Specific sources of contamination have not been identified. All
reagents and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running
laboratory reagent blanks as described in Sect. 10.2.
4.1.1 Glassware must be scrupulously cleaned.(2) Clean all
glassware as soon as possible after use by thoroughly rinsing
with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap and reagent
water. Drain dry, and heat in an oven or muffle furnace at
450°C for 1 hour. Do not heat volumetric ware. Thermally
360
-------
stable materials might not be eliminated by this treatment.
Thorough rinsing with acetone may be substituted for the heat-
ing. After drying and cooling, seal and store glassware in a
clean environment to prevent any accumulation of dust or other
contaminants. Store inverted or capped with aluminum foil.
4.1.2 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. WARNING:
When a solvent is purified, stabilizers added by the
manufacturer are removed, thus potentially making the solvent
hazardous. Also, when a solvent is purified, preservatives
added by the mamufacturer are removed, thus potentially
reducing the shelf-life.
4.2 Interfering contamination may occur when a sample containing low
concentrations of analytes is analyzed immediately following a sample
containing relatively high concentrations of analytes. A preventive
technique is between-sample rinsing of the sample syringe and filter
holder with two portions of reagent water. After analysis of a sample
containing high concentrations of analytes, one or more laboratory
method blanks should be analyzed.
4.3 Matrix interference may be caused by contaminants that are present in
the sample. The extent of matrix interference will vary considerably
from source to source, depending upon the water sampled. Positive
identifications must be confirmed.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound must
be treated as a potential health hazard. Accordingly, exposure to
these chemicals must be reduced to the lowest possible level. 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 and have been identified (4-6) for the information of the
analyst.
5.2 WARNING: When a solvent is purified, stabilizers added by the
manufacturer are removed, thus potentially making the solvent
hazardous.
6. APPARATUS AND EQUIPMENT (All specifications are suggested. Catalog numbers
are included for illustration only.)
6.1 SAMPLING EQUIPMENT
361
-------
6.1.1 Grab sample bottle -- 60-mL screw cap vials (Pierce No. 1307
or equivalent) and caps equipped with a PTFE-faced silicone
septa (Pierce No. 12722 or equivalent). Prior to use, wash
vials and septa as described in Sect. 3.1.1.
6.2 BALANCE -- Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.3 FILTRATION APPARATUS
6.3.1 Macrofiltration -- to filter derivatization solutions and
mobile phases used in HPLC. Recommend using 47 mm filters
(Millipore Type HA, 0.45 pm for water and Millipore Type FH,
0.5 im for organics or equivalent).
6.3.2 Microfiltration -- to filter samples prior to HPLC analysis.
Use 13 mm filter holder (Millipore stainless steel XX300/200
or equivalent), and 13 mm diameter 0.2 /un polyester filters
(Nuclepore 180406 or equivalent).
6.4 SYRINGES AND SYRINGE VALVES
6.4.1 Hypodermic syringe -- 10-mL glass, with Luer-Lok tip.
6.4.2 Syringe valve -- 3-way (Hamilton HV3-3 or equivalent).
6.4.3 Syringe needle -- 7 to 10-cm long, 17-gauge, blunt tip.
6.4.4 Micro syringes -- various sizes.
6.5 MISCELLANEOUS
6.5.1 Solution storage bottles -- Amber glass, 10- to 15-mL capacity
with TFE-fluorocarbon-lined screw cap.
6.5.2 Helium, for degassing solutions and solvents.
6.6 HIGH PERFORMANCE LIQUID CHROMATOGRAPH (HPLC)
6.6.1 HPLC system capable of injecting 200- to 400-/U. aliquots, and
performing binary linear gradients at a constant flow rate. A
data system is recommended for measuring peak areas. Table 1
lists retention times observed for method analytes using the
columns and analytical conditions described below.
6.6.2 Column 1 (Primary column) -- 150 mm long x 3.9 mm I.D.
stainless steel packed with 4 ion NovaPak CIS. Mobil Phase is
establirned at 10:90 methanol:water, hold 2 min., then
program as a linear gradient to 80:20 methanol:water in 25
min. Alternative columns may be used in accordance with the
provisions described in Sect. 10.4.
362
-------
6.6.3 Column 2 (Alternative column)* -- 250 mm long x 4.6 mm I.D.
stainless steel packed with 5 /MI Beckman Ultrasphere ODS.
Mobile phase Is established at 1.0 mL/min as a linear gradient
from 15:85 methanol:water to methanol 1n 32 mln. Data
presented In this method were obtained using this column.
* Newer manufactured columns have not been able to resolve
aldlcarb sulfone from oxamyl.
6.6.4 Column 3 (Alternative column) -- 250 mm long x 4.6 mm I.D.
stainless steel packed with 5 fun Supelco LC-1. Mobile phase
Is established at 1.0 mL/min as a linear gradient from 15:85
methanol:water to methanol In 32 mln.
6.6.5 Post column reactor -- Capable of mixing reagents Into the
mobile phase. Reactor should be constructed using PTFE tubing
and equipped with pumps to deliver 0.1 to 1.0 mL/min of each
reagent; mixing tees; and two 1.0-mL delay coils, one thermo-
stated at 95°C (ABI URS 051 and URA 100 or equivalent).
6.6.6 Fluorescence detector -- Capable of excitation at 230 nm and
detection of emission energies greater than 418 nm. A
Schoffel Model 970 fluorescence detector was used to generate
the validation data presented in this method.
REAGENTS AND CONSUMABLE MATERIALS -- WARNING: When a solvent is purified,
stabilizers added by the manufacturer are removed, thus potentially making
the solvent hazardous. Also, when a solvent is purified, preservatives
added by the manufacturer are removed, thus potentially reducing the shelf-
life.
7.1 REAGENT WATER -- Reagent water is defined as water that is reasonably
free of contamination that would prevent the determination of any
analyte of interest. Reagent water used to generate the validation
data in this method was distilled water obtained from the Magnetic
Springs Water Co., 1801 Lone Eagle St., Columbus, Ohio 43228.
7.2 METHANOL -- Distilled-in-glass quality or equivalent.
7.3 HPLC MOBILE PHASE
7.3.1 Water -- HPLC grade (available from Burdick and Jackson).
7.3.2 Methanol -- HPLC grade. Filter and degas with helium before
use.
7.4 POST COLUMN DERIVATIZATION SOLUTIONS
7.4.1 Sodium hydroxide, 0.05 N -- Dissolve 2.0 g of sodium hydroxide
(NaOH) in reagent water. Dilute to 1.0 L with reagent water.
Filter and degas with helium just before use.
363
-------
7.4.2 2-Mercaptoethanol (1+1) -- Mix 10.0 ml of 2-mercapto-ethar
and 10.0 ml of acetonitrile. Cap. Store in hood (CAUTION
stench).
7.4.3 Sodium borate (0.05 N) -- Dissolve 19.1 g of sodium borate
(NagB^y • 10K20) in reagent water. Dilute to 1.0 L with
reagent water. The sodium borate will completely dissolve at
room temperature if prepared a day before use.
7.4.4 OPA reaction solution -- Dissolve 100 ± 10 mg of o-phthal-
aldehyde (mp 55-58'C) in 10 ml of methanol. Add to 1.0 L of
0.05 N sodium borate. Mix, filter, and degas with helium.
Add 100 pL of 2-mercaptoethanol (HI) and mix. Make up fresh
solution daily.
7.5 MONOCHLOROACET1C ACID BUFFER (pH3) -- Prepare by mixing 156 ml of 2.5
M monochloroacetic acid and 100 ml 2.5 M potassium acetate.
7.6 4-BROMO-3.5-DIMETHYLPHENYL N-METHYLCARBAMATE (BDMC) -- 98% purity, for
use as internal standard (available from Aldrich Chemical Co.).
7.7 STOCK STANDARD SOLUTIONS (1.00 ng/nl) -- Stock standard solutions may
be purchased as certified solutions or prepared from pure standard
materials using the following procedure:
7.7.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material in HPLC quality methanol and dilute to volume in a
10-mL volumetric flask. Larger volumes may be used at the
convenience of the analyst. If compound purity is certified
at 96% 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.
7.7.2 Transfer the stock standard solutions into TFE-fluoro-
carbon- sealed screw cap vials. Store at room temperature and
protect from light.
7.7.3 Stock standard solutions should be replaced after two months
or sooner if comparison with laboratory fortified blanks, or
QC samples indicate a problem.
7.8 INTERNAL STANDARD SOLUTION -- Prepare an internal standard
fortification solution by accurately weighing approximately 0.0010 g
of pure BDMC. Dissolve the BDMC in pesticide-quality methanol and
dilute to volume in a 10-mL volumetric flask. Transfer the internal
standard fortification solution to a TFE-fluorocarbon-sealed screw cap
bottle and store at room temperature. Addition of 5 pL of the
internal standard fortification solution to 50 mL of sample results in
a final internal standard concentration of 10 pg/L. Solution shoul '
364
-------
be replaced when ongoing QC (Sect. 10) indicates a problem. Note:
BDMC has been shown to be an effective internal standard for the
method analytes (1), but other compounds may be used, if the quality
control requirements in Sect. 9 are met.
7.9 LABORATORY PERFORMANCE CHECK SOLUTION -- Prepare concentrate by
adding 20 /iL of the 3-hydroxycarbofuran stock standard solution,
1.0 mL of the aldicarb sulfoxide stock standard solution, 200 /iL of
the methiocarb stock standard solution, and 1 mL of the internal
standard fortification solution to a 10-mL volumetric flask. Dilute
to volume with methanol. Thoroughly mix concentrate. Prepare check
solution by placing 100 /iL of the concentrate solution into a 100-mL
volumetric flask. Dilute to volume with buffered reagent water.
Transfer to a TFE-fluorocarbon-sealed screw cap bottle and store at
room temperature. Solution should be replaced when ongoing QC
(Sect. 10) indicates a problem.
8. SAMPLE COLLECTION. PRESERVATION AND HANDLING
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices (8) should be followed; however, the bottle must
not be prerinsed with sample before collection.
8.2 SAMPLE PRESERVATION/PH ADJUSTMENT -- Oxamyl, 3-hydroxycarbofuran,
aldicarb sulfoxide, and carbaryl can all degrade quickly in neutral
and basic waters held at room temperature.(6,7) This short term
degradation is of concern during the time samples are being shipped
and the time processed samples are held at room temperature in
autosampler trays. Samples targeted for the analysis of these three
analytes must be preserved at pH 3. The pH adjustment also minimizes
analyte biodegradation.
8.2.1 Add 1.8 mL of monochloroacetic acid buffer to the 60-mL sample
bottle. Add buffer to the sample bottle at the sampling site
or in the laboratory before shipping to the sampling site.
8.2.2 If residual chlorine is present, add 80 mg of sodium thio-
sulfate per liter of sample to the sample bottle prior to
collecting the sample.
8.2.3 After sample is collected in bottle containing buffer, seal
the sample bottle and shake vigorously for 1 min.
8.2.4 Samples must be iced or refrigerated at 4°C from the time of
collection until storage. Samples must be stored at -10°C
until analyzed. Preservation study results indicate that
method analytes are stable in water samples for at least 28
days when adjusted to pH 3 and stored at -10°C. However,
analyte stability may be effected by the matrix; therefore,
the analyst should verify that the preservation technique is
applicable to the samples under study.
365
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9. CALIBRATION
9.1 Establish HPLC operating parameters equivalent to those Indicated In
Sect. 6.6. The HPLC system may be calibrated using either the
Internal standard technique (Sect. 9.2) or the external standard
technique (Sect. 9.3).
9.2 INTERNAL STANDARD CALIBRATION PROCEDURE. The analyst must select one
or more Internal standards similar 1n analytical behavior to the
analytes of Interest. The analyst must further demonstrate that the
measurement of the Internal standard Is not affected by method or
matrix Interferences. BDMC has been Identified as a suitable Internal
standard.
9.2.1. Prepare calibration standards at a minimum of three (recommend
five) concentration levels for each analyte of Interest by
adding volumes of one or of the more stock standards to a
volumetric flask. To each calibration standard, add a known
constant amount of one or more Internal standards, and dilute
to volume with buffered reagent water. To prepare buffered
reagent water, add 10 mL of 1.0 M monochloroacetic acid buffer
to 1 L of reagent water. The lowest standard should repre-
sent analyte concentrations near, but above, their respective
EDLs. The remaining standards should bracket the analyte
concentrations expected in the sample extracts, or should
define the working range of the detector.
9.2.2 Analyze each calibration standard according to the procedure
(Sect. 11.2). Tabulate peak height or area responses against
concentration for each compound and Internal standard.
Calculate response factors (RF) for each analyte, surrogate
and internal standard using Equation 1.
RF = (AsHcis) Equation 1
(A1s)(Cs)
where:
As = Response for the analyte to be measured.
AJS = Response for the Internal standard.
Cis = Concentration of the internal standard pg/L).
Cs - Concentration of the analyte to be measured 0g/L).
9.2.3 If the RF value over the working range is constant (20% RSD or
less) the average RF can be used for calculations.
Alternatively, the results can be used to plot a calibration
curve of response ratios (As/AjS) vs. Cs.
9.2.4 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any analyte varies from the
predicted response by more than ±20%, the test must be
366
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repeated using a fresh calibration standard. If the
repetition also fails, a new calibration curve must be
generated for that analyte using freshly prepared standards.
9.2.5 Single point calibration is a viable alternative to a
calibration curve. Prepare single point standards from the
secondary dilution standards. The single point standards
should be prepared at a concentration that deviates from the
sample extract response by no more than 20%.
9.2.6 Verify calibration standards periodically, recommend at least
quarterly, by analyzing a standard prepared from reference
material obtained from an independent source. Results from
these analyses must be within the limits used to routinely
check calibration.
9.3 EXTERNAL STANDARD CALIBRATION PROCEDURE
9.3.1 Prepare calibration standards at a minimum of three (recommend
five) concentration levels for each analyte of interest by
adding volumes of one or more stock standards to a volumetric
flask. Dilute to volume with buffered reagent water. The
lowest standard should represent analyte concentrations near,
but above, the respective EDLs. The remaining standards
should bracket the analyte concentrations expected in the
sample extracts, or should define the working range of the
detector.
9.3.2 Starting with the standard of lowest concentration, analyze
each calibration standard according to Sect. 11.2 and tabulate
responses (peak height or area) versus the concentration in
the standard. The results can be used to prepare a
calibration curve for each compound. Alternatively, if the
ratio of response to concentration (calibration factor) is a
constant over the working range <20% relative standard
deviation), linearity through the origin can be assumed and
the average ratio or calibration factor can be used in place
of a calibration curve.
9.3.3 The working calibration curve or calibration factor must be
verified on each working day by the measurement of a minimum
of two calibration check standards, one at the beginning and
one at the end of the analysis day. These check standards
should be at two different concentration levels to verify the
concentration curve. For extended periods of analysis
(greater than 8 hr), it is strongly recommended that check
standards be interspersed with samples at regular intervals
during the course of the analyses. If the response for any
analyte varies from the predicted response by more than ±20%,
the test must be repeated using a fresh calibration standard.
If the results still do not agree, generate a new calibration
367
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curve or use a single point calibration standard as descr
in Sect. 9.3.3.
9.3.4 Single point calibration is a viable alternative to a
calibration curve. Prepare single point standards from the
secondary dilution standards. The single point standards
should be prepared at a concentration that deviates from the
sample extract response by no more than 20%.
9.3.5 Verify calibration standards periodically, recommend at least
quarterly, by analyzing a standard prepared from reference
material obtained from an independent source. Results from
these analyses must be within the limits used to routinely
check calibration.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration of
laboratory capability, monitoring internal standard peak area or
height in each sample and blank (when internal standard calibration
procedures are being employed), analysis of laboratory reagent blanks,
laboratory fortified samples, laboratory fortified blanks and QC
samples.
10.2 LABORATORY REAGENT BLANKS. Before processing any samples, the analyst
must demonstrate that all glassware and reagent interferences are
under control. Each time a set of samples is extracted or reagents
are changed, a laboratory reagent blank (LRB) must be analyzed. If
within the retention time window of any analyte of interest the LRB
produces a peak that would prevent the determination of that analyte,
determine the source of contamination and eliminate the interference
before processing samples.
10.3 INITIAL DEMONSTRATION OF CAPABILITY.
10.3.1 Select a representative concentration (about 10 times EDL) for
each analyte. Prepare a sample concentrate (in methanol)
containing each analyte at 1000 times selected concentration.
With a syringe, add 50 /iL of the concentrate to each of at
least four 50-mL aliquots of reagent water, and analyze each
aliquot according to procedures beginning in Sect. 11.
10.3.2 For each analyte the recovery value for all four of these
samples must fall in the range of R ± 30% (or within R ± 3$R
if broader) using the values for R and SR for reagent water in
Table 2. For those compounds that meet the acceptance
criteria, performance is judged acceptable and sample analysis
may begin. For those compounds that fail these criteria,
this procedure must be repeated using four fresh samples until
satisfactory performance has been demonstrated.
368
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10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples via a
new, unfamiliar method prior to obtaining some experience with
it. It is expected that as laboratory personnel gain
experience with this method the quality of data will improve
beyond those required here.
10.4 The analyst is permitted to modify HPLC columns, HPLC conditions,
internal standards or detectors to improve separations or lower
analytical costs. Each time such method modifications are made, the
analyst must repeat the procedures in Sect. 10.3.
10.5 ASSESSING THE INTERNAL STANDARD
10.5.1 When using the internal standard calibration procedure, the
analyst is expected to monitor the IS response (peak area or
peak height) of all samples during each analysis day. The IS
response for any sample chromatogram should not deviate from
the daily calibration check standard's IS response by more
than 30%.
10.5.2 If >30% deviation occurs with an individual extract, optimize
instrument performance and inject a second aliquot.
10.5.2.1 If the reinjected aliquot produces an acceptable
internal standard response, report results for that
aliquot.
10.5.2.2 If a deviation of greater than 30% is obtained for
the reinjected extract, analysis of the sample
should be repeated beginning with Sect. 11, provided
the samples is still available. Otherwise, report
results obtained from the reinjected extract, but
annotate as suspect.
10.5.3 If consecutive samples fail the IS response acceptance
criterion, immediately analyze a calibration check standard.
10.5.3.1 If the check standard provides a response factor
(RF) within 20% of the predicted value, then follow
procedures itemized in Sect. 10.5.2 for each sample
failing the IS response criterion.
10.5.3.2 If the check standard provides a response factor
which deviates more than 20% of the predicted value,
then the analyst must recalibrate, as specified in
Sect. 9.
10.6 ASSESSING LABORATORY PERFORMANCE - LABORATORY FORTIFIED BLANKS
10.6.1 The laboratory must analyze at least one laboratory fortified
blank (LFB) sample with every 20 samples or one per sample set
369
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(all samples analyzed within a 24-h period) whichever Is
greater. The fortification concentration of each analyte i
the LFB should be 10 times EDL or the MCL, whichever is less.
Calculate accuracy as percent recovery (Xj). If the recovery
of any analyte falls outside the control limits (see Sect.
10.7.2), that analyte is judged out of control, and the source
of the problem must be identified and resolved before
continuing analyses.
10.6.2 Until sufficient data become available from within their own
laboratory, usually a minimum of results from 20 to 30
analyses, the laboratory should assess laboratory performance
against the control limits in Sect. 10.3.2 that are derived
from the data in Table 2. When sufficient internal performance
data becomes available, develop control limits from the mean
percent recovery (X) and standard deviation (S) of the percent
recovery. These data are used to establish upper and lower
control limits as follows:
UPPER CONTROL LIMIT = X + 3S
LOWER CONTROL LIMIT = X - 3S
After each five to ten new recovery measurements, new control
limits should be calculated using only the most recent 20-30
data points. These calculated control limits should never
exceed those established in Sect. 10.3.2.
10.6.3 It is recommended that the laboratory periodically determine
and document its detection limit capabilities for analytes of
interest.
10.6.4 At least quarterly, analyze a QC sample from an outside source.
10.6.5 Laboratories are encouraged to participate in external
performance evaluation studies such as the laboratory
certification programs offered by many states or the studies
conducted by USEPA. Performance evaluation studies serve as
independent checks on the analyst's performance.
10.7 ASSESSING ANALYTE RECOVERY - LABORATORY FORTIFIED SAMPLE MATRIX
10.7.1 The laboratory must add a known concentration to a minimum of
5% of the routine samples or one sample concentration per set,
whichever is greater. The concentration should not be less
then the background concentration of the sample selected for
fortification. Ideally, the concentration should be the same
as that used for the laboratory fortified blank (Sect. 10.6).
Over time, samples from all routine sample sources should be
fortified.
10.7.2 Calculate the percent recovery, P of the concentration for each
analyte, after correcting the analytical result, X, from the
370
-------
fortified sample for the background concentration, b, measured
in the unfortified sample, i.e.,:
P = 100 (X - b) / fortifying concentration,
and compare these values to control limits appropriate for
reagent water data collected in the same fashion. If the
analyzed unfortified sample is found to contain NO background
concentrations, and the added concentrations are those
specified in Sect. 10.7, then the appropriate control limits
would be the acceptance limits in Sect. 10.7. If, on the other
hand, the analyzed unfortified sample is found to contain
background concentration, b, estimate the standard deviation at
the background concentration, sj,, using regressions or
comparable background data and, similarly, estimate the mean,
Xa and standard deviation, sa, of analytical results at the
total concentration after fortifying. Then the appropriate
percentage control limits would be P ± 3sp , where:
P = 100 X / (b + fortifying concentration)
2 2 !/2
and sp = 100 (sa + sb ) /fortifying concentration
For example, if the background concentration for Analyte A was
found to be 1 /ig/L and the added amount was also 1 /jg/L, and
upon analysis the laboratory fortified sample measured 1.6 /i/L,
then the calculated P for this sample would be (1.6 /ig/L minus
1.0 /ig/L)/l /ig/L or 60%. This calculated P is compared to
control limits derived from prior reagent water data. Assume
it is known that analysis of an interference free sample at 1
Mg/L yields an s of 0.12 /tg/L and similar analysis at 2.0 /zg/L
yields X and s of 2.01 /ig/L and 0.20 /ig/L, respectively. The
appropriate limits to judge the reasonableness of the percent
recovery, 60%, obtained on the fortified matrix sample is
computed as follows:
[100 (2.01 /ig/L) / 2.0 /ig/L]
_ 1/2
± 3 (100) [(0.12 /ig/L)2 + (0.20 /ig/L)2] / 1.0 /ig/L =
100.5% ± 300 (0.233) =
100.5% ± 70% or 30% to 170% recovery of the added analyte.
10.7.3 If the recovery of any such analyte falls outside the
designated range, and the laboratory performance for that
analyte is shown to be in control (Sect. 10.6), the recovery
problem encountered with the dosed sample is judged to be
matrix related, not system related. The result for that
analyte in the unfortified sample is labeled suspect/matrix to
inform the data user that the results are suspect due to matrix
effects.
371
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10.8 ASSESSING INSTRUMENT SYSTEM - LABORATORY PERFORMANCE CHECK SAMPLu -
Instrument performance should be monitored on a daily basis by
analysis of the LPC sample. The LPC sample contains compounds
designed to indicate appropriate instrument sensitivity, column
performance (primary column) and chromatographic performance. LPC
sample components and performance criteria are listed in Table 3.
Inability to demonstrate acceptable instrument performance indicates
the need for reevaluation of the instrument system. The sensitivity
requirements are set based on the EDLs published in this method. If
laboratory EDLs differ from those listed in this method,
concentrations of the instrument QC standard compounds must be
adjusted to be compatible with the laboratory EDLs.
10.9 The laboratory may adopt additional quality control practices for use
with this method. The specific practices that are most productive
depend upon the needs of the laboratory and the nature of the
samples. For example, field or laboratory duplicates may be
analyzed to assess the precision of the environmental measurements or
field reagent blanks may be used to assess contamination of samples
under site conditions, transportation and storage.
11. PROCEDURE
11.1 PH ADJUSTMENT AND FILTRATION
11.1.1 Add preservative to any samples not previously preserved (Sect.
8). Adjust the pH of the sample or standard to pH 3 ± 0.2 by
adding 1.5 mL of 2.5 M monochloroacetic acid buffer to each 50
mL of sample. This step should not be necessary if sample pH
was adjusted during sample collection as a preservation
precaution. Fill a 50-mL volumetric flask to the mark with the
sample. Add 5 ftl of the internal standard fortification
solution (if the internal standard calibration procedure is
being employed) and mix by inverting the flask several times.
11.1.2 Affix the three-way valve to a 10-mL syringe. Place a clean
filter in the filter holder and affix the filter holder and the
7- to 10-cm syringe needle to the syringe valve. Rinse the
needle and syringe with reagent water. Prewet the filter by
passing 5 mL of reagent water through the filter. Empty the
syringe and check for leaks. Draw 10 mL of sample into the
syringe and expel through the filter. Draw another 10 mL of
sample into the syringe, expel through the filter, and collect
the last 5 mL for analysis. Rinse the syringe with reagent
water. Discard the filter.
11.2 LIQUID CHROMATOGRAPHY
11.2.1 Sect. 6.6 summarizes the recommended operating conditions for
the liquid chromatograph. Table 1 lists retention times
observed using this method. Other HPLC columns, chromato-
372
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graphic conditions, or detectors may be used if the
requirements of Sect. 10.4 are met.
11.2.2 Calibrate the system daily as described in Sect. 10. The
standards and samples must be in pH 3 buffered water.
11.2.3 Inject 400 0L of the sample. Record the volume injected and
the resulting peak size in area units.
11.2.4 If the response for the peak exceeds the working range of the
system, dilute the sample with pH 3 buffered reagent water and
reanalyze.
11.3 IDENTIFICATION OF ANALYTES
11.3.1 Identify a sample component by comparison of Us retention time
to the retention time of a reference chromatogram. If the
retention time of an unknown compound corresponds, within
limits, to the retention time of a standard compound, then
Identification is considered positive.
11.3.2 The width of the retention time window used to make
Identifications should be based upon 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.
11.3.3 Identification requires expert judgement when sample
components are not resolved chromatographically. When peaks
obviously represent more than one sample component (i.e.,
broadened peak with shoulder(s) or valley between two or more
maxima), or any time doubt exists over the identification of a
peak on a chromatogram, appropriate alternate techniques, to
help confirm peak identification, need to be employed. For
example, more positive identification may be made by the use of
an alternative detector which operates on a chemical/physical
principle different from that originally used; e.g., mass
spectrometry, or the use of a second chromatography column. A
suggested alternative column is described in Sect. 6.6.3.
12. CALCULATIONS
Determine the concentration of individual compounds in the sample using
the following equation:
r - Av • °-c
cx - _J L_
As . RF
373
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where Cx = analyte concentration in micrograms per liter;
Ax = response of the sample analyte;
As = response of the standard (either internal or
external), in units consistent with those used
for the analyte response;
RF = response factor (with an external standard, RF = 1, because
the standard is the same compound as the measured analyte);
Qs = concentration of internal standard present or concentration
of external standard that produced As, in micrograms per
liter.
13. PRECISION AND ACCURACY
13.1 In a single laboratory, analyte recoveries from reagent water were
determined at five concentration levels. Results were used to
determine analyte EDLs and demonstrate method range.(1) Analyte
recoveries and standard deviation about the percent recoveries at one
concentration are given in Table 2.
13.2 In a single laboratory, analyte recoveries from two standard
synthetic ground waters were determined at one concentration level.
Results were used to demonstrate applicability of the method to
different ground water matrices.(1) Analyte recoveries from the two*
synthetic matrices are given in Table 2.
14. REFERENCES
1. National Pesticide Survey Method No. 5.,"Measurement of N-
Methylcarbamoyloximes and N-Methylcarbamates in Groundwater by HPL with
Post Column Derivatization."
2. Moye, H.A., S.J. Sherrer, and P.A. St. John, "Dynamic Labeling of
Pesticides for High Performance Liquid Chromatography: Detection of
N-Methylcarbamates and o-Phthalaldehyde," Anal. Lett. 10, 1049, 1977.
3. ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard
Practice for Preparation of Sample Containers and for Preservation",
American Society for Testing and Materials, Philadelphia, PA, p. 86, 1986.
4. "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, Aug.
1977.
5. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
6. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
374
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7. Foerst, D.L. and H.A. Moye, "Aldicarb in Drinking Water via Direct Aqueous
Injection HPLC with Post Column Derivatization," Proceedings of the 12th
annual AWWA Water Quality Technology conference, 1984.
8. Hill, K.M., R.H. Hollowell, and L.A. DalCortevo, "Determination of
N-Methylcarbamate Pesticides in Well Water by Liquid Chromatography and
Post Column Fluorescence Derivatization," Anal. Chem. 56, 2465 (1984).
9. ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82, "Standard
Practice for Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 130, 1986.
375
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TABLE 1. RETENTION TIMES FOR METHOD ANALYTES
Analvte
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl
3-Hydroxycarbofuran
Aldicarb
Baygon
Carbofuran
Carbaryl
Methiocarb
BDMC
Primary^)
80
77
8.20
8.94
13.65
16.35
18.86
19.17
20.29
24.74
25.28
Retention Time(a)
Alternative*** A1ternative(3)
15
15
17
18
23
27
29
29
30.8
34.9
35.5
17.5
12.2
14.6
14.8
19
21.4
24,
23,
25,
28.6
(a) Columns and analytical conditions are described in Sect. 6.6.2 and 6.6.3.
(1) Waters NovaPak C18
(2) Beckman Ultrasphere ODS
(3) Supelco LC-1
376
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TABLE 2. SINGLE LABORATORY ACCURACY, PRECISION AND ESTIMATED DETECTION LIMITS (EDLSJ
FOR ANALYTES FROM REAGENT WATER AND SYNTHETIC GROUND WATERS(A)
Water 2f
Analvte
EDLh
UQ/Lb
Concentration Level
UQ/L Rc
Reagent Water
Synthetic Water le
SR R_
Synthetic
SR_
—i
—j
Aldicarb
Aldicarb sulfone
Aldicarb sulfoxide
Baygon
Carbaryl
Carbofuran
3-Hydroxycarbofuran
Methiocarb
Methomyl
Oxamyl
a Data corrected for
b EDL = Estimated del
1.0
2.0
2.0
1.0
2.0
1.5
2.0
4.0
0.5
2.0
amount detected
lection limit: d<
5
10
10
5
10
7.5
10
20
2.5
10
in blank
jfined as
115
101
97
106
97
102
102
94
105
100
and represent
either MDL (Ai
3.5
4.0
4.9
3.2
5.8
5.1
4.1
1.9
4.2
4.0
the mean
joendix B
106
98
105
96
94
102
98
102
98
97
of 7-8 samples
to 40 CFR Part
3.2
3.9
4.2
4.8
4.7
3.1
4.9
4.1
3.9
2.9
.
136 - De
102
95
94
97
104
100
101
112
105
102
finition and Pr
8.2
9.5
10.3
5.8
10.4
7.0
10.1
3.4
9.5
10.2
-ocedure fo
the Determination of the Method Detection Limit - Revision 1.11) or a level of compound in a sample yielding a peak
in the final extract with signal-to-noise ratio of approximately 5, whichever value is higher. The concentration
level used in determining the EDL is not the same as the concentration level presented in this table.
c R = Average percent recovery.
d SR = Standard deviation of the percent recovery.
e Corrected for amount found in blank; Absopure Nature Artesian Spring Water Obtained from the Absopure Water Company
in Plymouth, Michigan.
f Corrected for amount found in blank; reagent water fortified with fulvic acid at the 1 mg/L concentration level. A
well-characterized fulvic acid, available from the International Hunnc Substances Society (associated with the
United States Geological Survey in Denver, Colorado) was used.
-------
TABLE 3. LABORATORY PERFORMANCE CHECK SOLUTION
OJ
^J
oo
Test
Sensitivity
Chromatographic performance
Column performance
Anal vte
3 -Hydroxycarbof uran
Aldicarb sulfoxide
Methiocarb
4-Bromo-3 , 5-di methyl phenyl
N-methylcarbamate (IS)
Cone,
ua/tnL
2
100
20
10
Requirements
Detection of analyte; S/N > 3
0.90 1.0C
a PGF = peak Gaussian factor. Calculated using the equation:
PGF = 1.83 x WH/2)
where W(l/2) is the peak width at half height and W(l/10) is the peak width at tenth height.
b Resolution between the two peaks as defined by the equation:
R = _t_
W
where t is the difference in elution times between the two peaks and M is the average peak width, at the
baseline, of the two peaks.
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