xvEPA
United States
Environmental Protection
Agency
Effluent Guidelines Division
WH-552
Washington, DC 20460
EPA 440/1-79/102
July 1979
Water and Waste Management
Self-Monitoring Program
Analytical Methods Package
(Organic Chemicals)
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Self-Monitoring Program
Analytical Methods Package
U.S. Environmental Protection Agency
Effluent Guidelines Division
Organic Chemicals Branch
July 19/9
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Program Notice
At the time of printing of this package a list of the specific
priority pollutants discharged by each product/process for which
self-monitoring would be required was not available. However,
based on EPA's experience in the verification program, it can be
assumed that a maximum of 4 methods for organic pollutants would
have to be used. The methods would most likely be used for the
analysis of volatile organics (2 methods), phenols, polynuclear
aromatics. For the metals which have been found in organic chemicals
wastewaters, atomic absorption spectroscopy would be used.
The self-monitoring program is intended to provide long-term data
on only those priority pollutants present in wastewaters from the
product/processes or entering the treatment system. Continued
analysis for the entire spectrum of 129 priority pollutants throughout
the course of the 30 day sampling will not be required.
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Index
Chapter 1
Analytical Methods for the Verification
Phase of the BAT Review Edited by
(GC) William F. Cowen
Catalytic
Chapter 2
Quality Assurance Program for the
Organic Chemicals Industry (Self-Monitoring
Phase)
Chapter 3
Proposed Priority Pollutant
Test Procedures U.S. EPA
(GC:601-613) page 1 Environmental Monitoring &
(GC/MS:624-625) page 205 Support Laboratory
Cincinnati, Ohio 45268
Chapter 4
Quality Assurance and Quality Control
Procedures for Screening and Verification
of Industrial Effluents for Priority
Pollutants - EMSL
Chapter 5
Cost Estimate for Self-Monitoring
Program
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Chapter I
ANALYTICAL METHODS FOR THE
VERIFICATION PHASE
OF THE BAT REVIEW
M>v *-«^ »f» J» '-• §«••«:•» »••*»••» •«• •jr' <****i «r» "
PRELiMiNARi:
REVISED
March 21, 1979
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INDEX OF ANALYTICAL METHODS
Procedure Code No. Title
1 Direct Aqueous Injection Procedure for GC Analysis
of Acrolein and Acrylonitrile
2 Method for Benzidine and Its Salts in Wastewater
3 Method for Organochlorine Pesticides and Phthalate
Esters In Industrial Effluents
A Total Cyanide
5 A-26 Resin/GC-FID Method for Phenolr
6 Analysis of Nitrosamines
7 Microextraction Method for Organic Compounds in
Industrial Effluents
8 Purge and Trap Procedures for Analysis of Volatile
Organic Compounds in Effluents
9 Method for Polychlorinated Biphenyls (PCBs) in
Industrial Effluents
10 Analysis of Arsenic and Selenium in Industrial
Effluents by Flameless Atomic Absorption Spectrophotometry
11 Analysis of Silver, Antimony, and Thallium in Industrial
Effluents by Flameless Atomic Absorption
12 Analysis of Beryllium, Cadmium, Chromium, Copper, Nickel,
Lead, and Zinc in Industrial Effluents by Flame or
Flameless Atomic Absorption
13 Mercury in Water (Manual Cold Vapor-Atomic Absorption
Technique)
14 Pentane Extraction of Organics in Wastewaters for
GC Analysis
15 Acid Extraction Procedure for Phenols
16 Analysis of Nitroaromatics
17 Analysis of Polynuclear Aromatic Hydrocarbons in
Industrial Wastewater
4
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Procedure Code: 1
Variations: 1-3
REVISED
Title: Direct Aqueous Injection Procedure for GC Analysis ~;
of Acrolein and Acrylonitrile
1. Summary
This method is similar to that given by ASTM (1), method designation
D3371-74T, "Tentative Method of Test for Nitriles in Aqueous Solution by
Gas-Liquid Chromatography." The detection limit is approximately 1 rag/1
with this method. For increased sensitivity, the heated purge-and-trap
procedure (Procedure Code #8) is recommended.
2. Sampling and Analysis
No sample extraction or cleanup procedures are employed in this
analysis. Samples should be collected in Teflon-septa sealed-glass vials
headspace-free. A small volume (1-20 yl) of aqueous sample is injected
directly into the gas chromatograph for analysis on a porous polymer
column, which elutes the water in the sample ahead of the analyte peaks.
3. Quality Control
Duplicate and spiked sample analyses are recommended as quality
control checks. A standard of 1 mg/1 in water is injected (1-20 yl) ontc
the GC column to provide an external standard for quantification of
samples.
4. Variations to Procedure Code #1
Variation
No.
Effective
Date
June, 1977
Description
GC Column: Chromosorb 102, 60/80
mesh, in a 2 meter x 1/8" O.D.
stainless steel column.
Carrier: He at 30 ml/min.
Injector: 250°C
Detector: FID at 200°C
Temperature: 100 C isothermal
(Source: Envirodyne Engineers, Inc.
for Acrylonitrile)
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Variation Effective
No. Date). . Description
2 March, 1979 ~'GC Column: Chromosorb 101,"
60/80 mesh, in a 61 x 4 mm
ID glass column
Carrier: He at 60 nl/min. -'
Injector: 200°C
Detector: 150°C, FID
Temperature: 120°C isothermal
(Source: Southwest Research Institute,
for Acrolein)
3 June, 1977 GC Column: Chromosorb 101, in a
6' x 2imn ID glass column
Temperature: 135 isothermal
Injector: 225°C
Detector: FID
Carrier: He at 25 ml/min.
(Source: Envirodyne Engineers)
5. References
I. 1976 Annual Book of ASTM Standards (Water), Part 31, ASTM, Philadelphia
Pa., pp 534-536 (1976).
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Proposed pH7 Buffer for Acrylonitrile-Acrolein Samples
Design Buffer Index:^8 * 5X10 mole/1 (after dilution,
(dc \ _ a 1 ml buffer + 40 ml sample)
d pH / s«/
Calculated Buffer Concentration after dilution with sample: 0.091H,
Est. Ionic strength from buffer * 0.2
Est. /HPO* - 0.28
Est. H2PO~ - 0.73
pK°a2 - 7.198 for H3P04
Calculated species composition after dilution: 0.034M KH.PO ; FW » 136.1
0.057M Na2HP04; FW - 141.9
Preparation;
Weigh out 18.6g anhydrous KH PO and 32.2g anhydrous Na.HPO,. Pour the salts
Into a 100 ml volumetric flask and dilute to volume with organic-free distilled
water.
Use;
Add 1 ml of buffer to each clean 40 ml sample vial before taking the vial into
the field. At the plant site, pour the sample into the vial, cap the vial, and
invert for a short time to mix the buffer with the sample. Check the pH of the
sample at the time of analysis and record its value.
Bill Cowen
May 1, 1979
J--J
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Procedure Code: 2
Variations: 1
Title: Method for Benzidine and Its Salts in Wastewater*
1. Scope and Application
1.1 This method covers the determination of benzidine and its salts in
wastewaters. The method can be modified to apply also to the
determination of closely-related materials, as described under
Interferences (4.2)
1.2 The salts of benzidine, such as benzidine sulfate, are measured and
reported as benzidine.
1.3 The method detection limit is 0.2 yg/1 when analyzing 1 liter of
sample.
2. Summary
2.1 The water sample is made basic and the benzidine is extracted with
ethyl acetate. Cleanup is accomplished by extracting the benzidine
from the ethyl acetate with hydrochloric acid. Chloramine T is
added to the acid solution to oxidize the benzidine. The yellow
oxidation product is extracted with ethyl acetate and measured with
a scanning spectrophotometer. The spectrum from 510 nm to 370 nm is
used for qualitative identification.
3. Hazards
3.1 Benzidine is a known carcinogen. All manipulations of this method
should be carried out in a hood, with protection provided for the
hands and arms of the analyst. Consult OSHA regulations (1) before
working with benzidine.
4. Interferences
4.1 The multiple extractions effectively limit the interferences to
organic bases. The oxidation with Chloramine T to form a yellow
product is very selective and has been described in detail (2,3).
The use of the absorption spectrum for the identification of
benzidine results in a highly specific procedure.
4.2 Some compounds having a structure very similar to benzidine will
interfere with the quantification, if present. Examples of these
interfering compounds are dichlorobenzidine, o-tolidine, and
dianisidine.
*From EPA Environmental Monitoring and Support Laboratory, Cincinnati, OH
45268.
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4.3 A general yellow background color in the extract will limit the cell
pathlength that can be employed and thus limit the sensitivity of
the method.
5. Apparatus and Materials
5.1 Spectrophotometer-visible, scanning (510-370 nm).
5.2 Separatory Funnels - 125 ml, 2000 ml, 250 ml.
5.3 Cells - 1 to 5 cm pathlength, 20 ml volume maximum.
6. Reagents, Solvents and Standards
6.1 Ethyl acetate
6.2 Hydrochloric acid -IN. Add 83 ml cone, hydrochloric acid to
water, and dilute to one liter.
6.3 Chloramine T - 10% solution. Prepare fresh daily by dissolving
l.Og Chloramine T in 10 ml distilled water.
6.4 Stock standard - 0.2 pg/yl. Dissolve 100.0 mg purified benzidine
in about 30 ml 1 N HC1. Dilute to 500 ml with water.
7. Preparation of Calibration Curve
7.1 To a series of 125 ml separatory funnels, add 45 ml hydrochloric
acid and 10 ml ethyl acetate. Shake for one minute to saturate the
acid layers. Discard the solvent layers. Dose the series with
volumes from 1.0 to 20.0 Pi of stock standard, using syringes.
7.2 Treat standards according to the Procedure beginning with 8.5.
8. Procedure
8.1 Adjust the sample pH to 8.5 - 9.0 with dilute NaOH or HC1.
8.2 Transfer 1 liter of sample to a 2000 ml separatory funnel. Add
150 ml ethyl acetate and shake for two minutes. Allow the layers to
separate; then drain the water layer into a second 2-liter separatory
funnel. Drain the solvent layer into a 250 ml separatory funnel.
8.3 Repeat the extraction of the water layer twice (i.e., total of
three extractions) with 50-ml portions of ethyl acetate. Combine
all solvent layers; then discard the water layer.
J-
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8.4 Extract the solvent layer three times with 15-ml portions of hydro-
chloric acid by shaking 2 minutes and allowing the phases to separate.
Combine the acid layers in a glass-stoppered container for cold
storage until tine is available for analysis, or transfer the layers
directly into a 125 ml separatory funnel.
8.5 Prepare the spectrophotometer so that it is warmed and ready to use.
The'remaining steps of the procedure must be performed rapidly on
one sample at a time.
8.6 To the hydrochloric acid solution in a 125 ml separatory funnel, add
1.0 ml chloramine T solution, and mix. Add 25.0 ml ethyl acetate
with a pipet and shake for two minutes. Allow the layers to separate;
then discard the aqueous phase.
8.7 Filter the solvent layer through coarse filter paper, and fill a 5
cm
cell with the filtrate.
8.8 Scan the solvent from 510 nra to 370 nir.. Use ethyl acetate for
a blank with double beam instruments. Use shorter pathlength cells
in cases where absorbance exceeds 0.8.
9. Calculation of Results
9.1 Benzidine is identified by its absorbance maximum at 436 nm (figure
1). Dichlorobenzidine would appear to be similar, but has its
absorbance maximum at 445 nm.
9.2 Construct a baseline from the absorbance minimum at about 470 nm
to the minimum at 390 nm (or 420 nm minimum for samples with a high
background). Record the absorbance of the peak maximum and the
absorbance of the constructed baseline at the 436 nm. Treat samples
and standards in the same fashion.
9.3 Using the net absorbance values, prepare a calibration plot from
the standards. Determine the total micrograms in each sample from
this plot.
9.4 Divide the total micrograms by the sample volume, in liters, to
determine ug/1. Correct results for cell pathlength if necessary.
10. Reporting Results
10.1 Report results in micrograms per liter as benzidine, without correc-
tion for recovery data. When duplicate and spiked samples are
analyzed, all data obtained should be reported.
11. Accuracy and Precision
11.1 When 1-liter samples of Ohio River water were dosed with 1.80 up of
benzidine, an average of 1.24 ug was recovered. The standard
deviation was 0.092 yg/1 (n=8).
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12. References
(1) Federal Register. Volume 39, Page 3779, Paragraph 1910.93; (January
29, 1974).
(2) Classman, J. M., and Meigs, J. W., "Benzidine (A,4' - Diaminobiphenyl)
and Substituted Benzidines", Arch. Industr. Hye-. 4. 519, (1951).
(3) Butt, L. T. and Strafford, N., "Papilloma of the Bladder in the
Chemical Industry. Analytical Methods for the Determination of
Benzidine and B-Naphthylamine, Recommended by A. B. C. M. Sub-
Comittee", J. Appl. Chem.. £., 525 (1956).
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Procedure Code: 3
Variations: 1-3
Title: Method for Organochlorine Pesticides and Phthalate Esters in Industrial
Effluents
1. Summary
This method is the EPA (Federal Register, _3J3 No. 75, Pt. II) procedure
for the pesticides and metabolites on the priority pollutants list. The
method uses liquid-liquid extraction to extract and concentrate the
chlorinated pesticides and metabolites for gas chromatographic analysis.
There is an optional Florisil cleanup step to separate phthalate esters
(which are coextracted) from many of the pesticides. Hence, the method
can also be used to determine phthalate esters by selection of the proper
Florisil fraction.
The method variations in use are classified as follows:
Variation Effective
Code No. Date Description
1 June 1977 Midwest" Research Method
2 Feb. 1979 Southwest Research Method (EPA Method #606)
3 June 1977 Envirodyne Method, with GC conditions A-E
Each of these variations is described in Section 6, Procedures.
2. Interferences
2.1 Solvents, reagents, glassware, and other sample-processing hardware
must be demonstrated to be free from interfering compounds, which
contribute gas chromatographic peaks or high baselines. (See Refer-
ence (1) for suggestions on reducing interferences).
2.2 Care must be taken to ensure that the effect of a sample cleanup
procedure upon the concentration of each analyte is known, and that
the procedures for eliminating interferences with a cleanup step are
applied consistently from sample to sample and on the standards.
2.3 Polychlorinated Biphenyls (PCB's) are a potential source of inter-
ference in pesticide analysis. The presence of PCB's is indicated
by a large number of partially-resolved or unresolved peaks, which
may occur throughout the entire chromatogram. (See Procedure Code #9
for the determination of PCB's in the liquid-liquid extract from the
pesticide-phthalate methods).
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2.4 Organophosphorus pesticides that contain a nitro group (e.g.,
parathion) also respond to the electron-capture detector, and nay
interfere with the determination of the organochlorine pesticides. Such
compounds can be identified by their response to the flame photometric
detector (2). Elemental sulfur and other organics with conjugated carbonyl
groups, nitro groups, or halogens will also interfere with the electron-
capture response to pesticides or phthalates.
3. Apparatus and Materials
3.1 Gas Chromatograph - Equipped with glass-lined injection port.
3.2 Detector - For chlorinated pesticides, an electron-capture (EC)
detector is required. For phthalate esters, an EC or flame
ionization detector (FID) may be used. The FID detector might be
preferable to EC in the presence of a large quantity of EC -
responsive interferences, where the phthalate concentrations are
high enough to be measured accurately with FID.
3.3 Recorder - Potentiometric strip chart (10-inch) compatible with the
detector.
3.4 Gas Chromatographic Column Materials - See Procedure section.
3.5 Kuderna-Danish (K-D) Glassware (Kontes/
3.5.1 Snyder Column - three ball (macro) and two ball (micro)
3.5.2 Evaporative Flasks - 500 ml
3.5.3 Receiver ampules - 10 ml, graduated
3.5.4 Ampule Stoppers
3.6 Liquid Chromatography Column for Florisil - Chromaflex (400 mm long
by 19 mm ID) with a coarse fritted plate on the bottom and teflon
stopcock; the column has a 250 ml reservoir bulb at the top of the
column with flared-out funnel shape at the top of the bulb (Kontes
K-420540-9011).
3.7 Chromatographic Column - Pyrex (approximately 400 mm long by 20 mm
ID) with coarse fritted plate on bottom.
3.8 Blender - High-speed, glass or stainless steel cup.
Florisil - PR Grade (60/100 mesh); purchase activat
store in the dark in glass containers with glass stoppers or foil-
lined screw caps. Before use, ac
130 C in a foil-covered glass con
value as described in Section 11.
3.9 Florisil - PR Grade (60/100 mesh); purchase activated at 1250°F and
store in the dark in glass containers with glass stoppers or foj
lined screw caps. Before use, activate each batch overnight at
130 C in a foil-covered glass container; determine the lauric acid
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4. Reagents, Solvents and Standards
4.1 Ferrous Sulfate - (ACS) 30% solution in distilled water.
4.2 Potassium Iodide - (ACS) 10% solution in distilled water.
4.3 Sodium Chloride - (ACS) Saturated solution in distilled water (pre-
rinse NaCl with hexane).
4.4 Sodium Hydroxide - (ACS) 10 N in distilled water.
4.5 Sodium Sulfate - (ACS) Granular, anhydrous (conditioned at 400°C for
4 hours).
4.6 Sulfuric Acid - (ACS) Mix equal volumes of concentrated H.SO, with
distilled water. i *
4.7 Diethyl Ether - Nanograde, redistilled in glass, if necessary.
4.7.1 Must contain 2 percent alcohol and be free of peroxides by
following test: To 10 ml of ether in glass-stoppered cylinder,
previously rinsed with ether, add 1 ml of freshly-prepared
10 percent RI solution. Shake and let stand 1 minute. No
yellow color should be observed in either layer.
4.7.2 Decompose ether peroxides by adding 40 g of 30 percent ferrous
sulfate solution to each liter of solvent. CAUTION; Reaction
may be vigorous if the solvent contains a high concentration
of peroxides.
4.7.3 Distill deperoxidized ether in glass and add 2 percent ethanol.
4.8 Hexane, Methylene Chloride, Petroleum Ether (boiling range 30 to
60 C) - Nanograde, redistill in glass if necessary.
4.9 Phthalate or Pesticide - Compound being analyzed (99+Z purity).
5. Calibration and Quality Control
5.1 Gas chromatographic operating conditions are considered acceptable if
the response to dicapthon is at least 50 percent of full scale when
0.06 ng (or less) is injected for electron-capture detection. For
all quantitative measurements, the detector must be operated within
its linear response range, and the detector noise level should be
less than 2 percent of full scale.
5.2 Standards are injected frequently as a check on the stability of
operating conditions.
5.3 Duplicate and spiked sample analyses are used for quality control
checks. Each time a set of samples is extracted, a method blank is
determined on a volume of distilled water equal to that used to
dilute the sample.
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6. Procedures
6.1 Variation #1 (Midwest Research Phthalate Method):
Adjust the pH of a 1 liter sample to 7.0. Place the sample in a
heavier-than-water liquid-liquid extractor, and extract with methylene
chloride overnight (16-20 hr.). Pass the extract through a Na.SO,
drying column and evaporate to approximately 10 ml in a K-D macro
apparatus on a steam bath. Evaporate the samples and exchange with
hexane, to a small volume under a stream of dry nitrogen. Then
clean up the sample on a Florisil column according to Section 7, but
discard the 6 percent ether in petroleum ether fraction and elute
the column with a 50:50 (v:v) mixture of ethyl ether and petroleum
ether for phthalate recovery. The gas chromatographic conditions
are:
GC Column: 1% SP-2250 on 100/120 mesh Supelcoport in a
6 ft. x A mm ID (glass) column.
Temperature: 215°C Isothermal ,,
Detector: Electron capture, Ni at 300°C.
Carrier: Argon/methane at 60 ml/min.
6.2 Variation #2 (Southwest Research Phthalate Method; same as E?A
Method #606 (3)).
Measure 1-liter of sample into a graduated cylinder. Pour the
sample into a 2-liter separatory funnel and adjust the pH to 11 or
above with 6N NaOH, using pH paper indicator. Extract 3 times with
60 ml of methylene chloride, and combine the extracts in a 500 ml K-
D evaporator. Evaporate to about 5 ml on a steam bath, add 100 ml
of hexane, and evaporate to about 2 ml, using a nitrogen gas stream
for the final evaporation. To clean up the extract for phthalates,
take the following steps:
Prepare Florisil by heating to 400°C overnight. Then add 3% HJ)
(m/m) and mix thoroughly. Let stand for at least 2 hours. Use 10
mm ID column chromatography tube with glass frit and teflon stopcock.
(Kontes Catalog K420540-0213).
1. Add 10 g of Florisil (3% H20) to the 1 cm ID chromatography
tube and tap to settle.
2. Add 1 cm Na2SO, to top of column.
3. Add hexane to tube and pre-elute 40 ml. Discard.
4. Drain until solvent level is even with top of Na.SO,.
24
5. Add sample in 2 ml hexane to top of column.
6. Drain sample into column (to top of Na.SO,).
T- ii
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7. Wash sides of tube with = 2 ml hexane and drain to top of Na SO,.
8. Add hexane to top of column and eiute 60 ml of hexane.
Discard this.
9. Drain hexane to top of Na^SO, and add * 2 ml of 20% ether/hexane.
10. Drain new solvent to top of Na2SO,. Add more 20% ether hexane.
11. Place a 500 ml Kuderna-Danish evaporator below column.
12. Collect 100 ml of the 20% ether/hexane eluate in the K-D evaporator.
13. Evaporate the above eluate to =10 ml on a steam bath.
14. Transfer to a solvent rinsed 4-dram vial with teflon cap liner.
15. Adjust final volume of concentrate to 5 ml. Evaporate under
nitrogen if necessary.
16. For GC analysis a 1:20 to 1:100 dilution of this fraction may be
necessary.
The gas chromatographic conditions are:
GC Column: 6' x 1/4" 1.5% SP-2250/1.95% SP-2401 on 100/120
mesh Supelcoport.
Temperature: 180°C for 16 min, then heat rapidly to 220°C and
hold for rest of run (20 min).
Detector: Electron capture, 300 C.
Carrier: Argon/methane/60 cc/min
6.3 Variation #3 (Envirodyne Method for Pesticides and Phthalates):
Blend the sample if suspended matter is present, and adjust the pH to
6.5 to 7.5 with 50 percent sulfuric acid or 10 N sodium hydroxide.
Extract 1 liter of sample three times with 60 ml portions of 15
percent methylene chloride in hexane (v:v) in a 2-liter separatory
funnel, shaking for 2 min. each time. Collect the solvent layers
from the three extractions in a 500 ml K-D flask after drying the
extracts by passage through a column (3-4 inches) of anhydrous sodium
sulfate. Concentrate the extracts to 6-10 ml into the (10 ml) K-D
ampule, over a steam bath.
If Florisil cleanup is necessary for determining pesticides/phthalates
without excessive interference from each other or other compounds,
use the (EPA) Florisil procedure in Section 7.
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Gas chromatography is performed as follows:
A) GC Column:
Temperature:
Inj ector:
Detector:
Carrier:
3% SP-1000 on 100/120 mesh Supelcoport in
a 6* x 2 mm ID glass column.
100° to 250°C at 8°/min.
Ni
at 325°C
Argon/methane at 60 ml/min.
Note: (Specified for phthalate esters)
Other columns which have been used for phthalates or pesticides are:
B) GC Column:
Temperature:
Injector:
Detector:
Carrier:
C) GC Column:
Temperature:
Injector:
Detector:
Carrier:
D) GC Column:
Temperature:
Injector:
Detector:
Carrier:
3% QF-1 on Chromosorb WHP (00/100 mesh) in
a 6' x 2 mm ID glass column.
215°C Isothermal
Ni
at 325°C
Argon/methane at 60 ml/min.
3% SP-1000 on 100/120 mesh Supelcoport in
a 6* x 2 mm ID glass column.
220°C Isothermal
at 270C
Argon/methane at 60 ml/min.
3% SP-1000 on 100/120 mesh Supelcoport in a
6* x 2 mm ID glass column.
100°C to 250°C at 8 C/min.
Ni at 325C
Argon/methane at 60 ml/min.
E) GC Column:
Temperature:
Injector:
Detector:
Carrier:
F) GC Column:
Temperature:
Injector:
Detector:
Carrier:
7% OV-17 + 9% OV-210 on Chromosorb W in a
6* x 2 mm ID glass column.
190°C Isothermal
Ni"" at 325"C
Argon/methane at 60 ml/min.
1.5% SP-2250 -I- 1.95% SP-2401 on 100/120
mesh Supelcoport in a 6*
210°C Isothermal
x 2 mm glass column.
Ni at 325C
Argon/methane at 60 ml/min.
(Date: May 1979)
G) GC Column:
Temperature:
Injector:
Detctor:
Carrier:
1.5% SP-2250 + 1.95% SP-2401 on 100/120 mesh
Supelcoport in a 6' x 2 mm glass column.
190°C Isothermal
Ni
at 325°C
(Date: May 1979)
Argon/Methane at 40 ml/min.
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7. Florisil Column Adsorption Chromatography
7.1 Adjust the sample extract volume to 10 ml.
7.2 Place a charge of activated Florisil (weight determined by lauric-
acid value (see Section 10) in a Chromaflex column. After settling
the Florisil by tapping the column, add about one-half inch layer of
anhydrous granular sodium sulfate to the top.
7.3 Fre-elute the column, after cooling, with 50 to 60 ml of petroleum
ether. Discard the eluate and just prior to exposure of the sulfate
layer to air, quantitatively transfer the sample extract into the
column by decantation and subsequent petroleum ether washings.
Adjust the elution rate to about 5 ml per minute and, separately,
collect up to three eluates in 500 ml K-D flasks equipped with 10 ml
ampules. Perform the first elution with 200 ml of 6 volume percent
of peroxide-free ethyl ether (containing 2 percent ethanol) in
petroleum ether. Perform the second elution with 200 ml of 15
volume percent of peroxide-free ethyl ether (containing 2 percent
ethanol) in petroleum ether. Elute a third tine with 200 ml of 50
volume percent of peroxide-free ethyl ether (containing 2 percent
ethanol) in petroleum ether.
7.4 Eluate composition - By using an equivalent quantity of any batch of
Florisil as determined by Its lauric acid value, pesticides, phthalates
and PCB's will be separated into the eluates indicated in the following
lists. However, the distribution of the compounds of Interest
should be checked by performing the florisil clean-up with a mixed
standard of these compounds at a concentration which is readily
quantified on the GC.
62 Eluate
Aldrin Heptachlor Pentachloro-
BHC Heptachlor expoxide nitrobenzene
Chlordane Lindane Strobane
DDD Methoxychlor Toxaphene
DDE Mirex Trifluralin
DDT PCB's
15Z Eluate 50% Eluate
Endosulfan I Endosulfan II
Endrin Captan
Dieldrin
Dichloran
Phthalate esters
7.5 Concentrate the extracts to approximately 5 ml in the K-D evaporator
in a hot water bath and add a small amount of sodium sulfate to
assure a water-free sample.
T-/1
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7.6 Analyze by gas chromatography.
•
8. Calculation of Results
8.1 Determine the phthalate ester or pesticide concentration by using
the following absolute calibration procedure or the relative cali-
bration procedure described in Part 1, Section 3.4.2 of Reference (1).
(A) (B) (V )
Micrograms/liter »
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10.3 Reagents and Solvents
10.3.1 Alcohol, ethyl —USP or absolute, neutralized to phenol-
phthalein,
10.3.2 Hexane — Distilled from all glass apparatus.
10.3.3 Laurie acid -- Purified, CP.
10.3.4 Laurie acid solution — Transfer 10.000 g lauric acid to a
500 ml volumetric flask, dissolve in hexane, and dilute to
500 ml (1 ml = 20 mg).
10.3.5 Phenolphthalein Indicator — Dissolve 1 g in alcohol and
dilute to 100 ml.
10.3.6 Sodium hydroxide — Dissolve 20 g NaOH (pellets, reagent
grade) in water and dilute to 500 ml (1 BI). Dilute 25 ml
1 N NaOH to 500 ml with water (0.05 N). Standardize as
follows:
Weigh 100-200 mg lauric acid into 125 ml Erlenmeyer flask.
Add 50 ml neutralized ethyl alcohol and 3 drops phenol-
phthalein indicator; titrate to permanent end point. Cal-
culate mg lauric acid/ml 0.05 II NaOH (about 10 mg/ml).
10.4 Procedure
10.4.1 Transfer 2.000 g Florisil to 25 ml glass stoppered Erlen-
meyer flasks. Cover loosley with aluminum foil and heat
overnight at 130 C. Stopper, cool to room temperature,
add 20.0 ml lauric acid solution (400 mg), stopper, and
shake occasionally for 15 min. Let adsorbent settle and
pipet 10.0 ml of supernatant into 125 ml Erlenmeyer flask.
Avoid inclusion of any Florisil.
10.4.2 Add 50 ml neutral alcohol and 3 drops indicator solution;
titrate with 0.05 1$ NaOH to a permanent end point.
10.5 Calculation of Lauric Acid Value and Adjustment of Column Weight
10.5.1 Calculate amount of lauric acid adsorbed on Florisil as
follows:
Lauric Acid value » mg lauric acid/g Florisil » 200 - (ml
required for titration X mg lauric acid/ml 0.05 N NaOH).
10.5.2 To obtain an equivalent quantity of any batch of Florisil,
divide 110 by lauric acid value for that batch and multiply
by 20 g. Verify proper elution of pesticides and phthalates
by 13.6.
10.6 Test for Proper Elution Pattern and Recovery of Pesticides and
phthalates. Prepare a test mixture containing aldrin, heptachlor
epoxide, p,p' -DDE, phthalates, dieldrin, parathion and malathion.
Dieldrin and parathion and phthalates should eluta in the 15% eluate;
all but a trace of malathion in the 50% eluate and the others in
the 6% eluate.
-------
11. References
1. "Method for Organic Pesticides in Water and Wastewater", Environmental
Protection Agency, National Environmental Research Center, Cincinnati,
Ohio (1971).
2. "Method for Organopnosphorus Pesticides in Industrial Effluents",
Environmental Protection Agency, National Environmental Research
Center, Cincinnati, Ohio (1973).
3. Methods for Organic Compounds in Municipal and Industrial Wastewater.
U.S. EPA Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio, March, 1979.
4. "Pesticide Analytical Manual", U.S. Dept. of Health, Education and
Welfare, Food and Drug Administration, Washington, D.C., Volumes I
and II (1968).
5. Mills, P.A., "Variation of, Florisil Activity: Simple Method for
Measuring Adsorbent Capacity and its Use in Standardizing Florisil
Columns", Journal A.O.A.C.. 51, 29 (1968).
-------
Procedure Code: 4
Variations: 1-2
Title: Total Cyanide
1. Summary
The determination of simple cyanides plus complex cyanides in wastewaters
may be performed by the EPA (1) procedure, which uses an acid distillation
with cuprous chloride, or by the procedure in Standard Methods (2), which uses
an acid distillation with magnesium chloride and mercuric chloride. Recent
studies with coke plant effluents (3) showed a) that the use of cuprous
chloride gave lower results than the use of magnesium chloride, and b) the
presence of high thiocyanate in the samples led to sulfide in the cyanide
absorbing trap. This sulfide was removed by precipitation with cadmium
carbonate, followed by filtration, before final cyanide analysis.
In order to follow the method variations lor cyanide, the following table
should be used:
Variation Effective
Code No. date Method Source
1 June 1977 EPA (1)
2 June 1977 Standard Methods (2)
With either method, the cyanide can be determined by titration with
silver nitrate or colorimetry with pyridine-pyrazolone or pyridine-barbituric
acid reagents, as described in the methods referenced.
2. References
1. Methods for Chemical Analysis of Water and Wastes, U.S. EPA Office
of Technology Transfer, Washington, D.C. (1974).
2. Standard Methods for the Examination of Water and Wastewater,
14th Edition, American Public Health Assn., Washington, D.C. (1976).
3. Barton, P. J., Hammer, C.A., and Kennedy, D.C., "Analysis of Cyanides
in Coke Plant Wastewater Effluents, "JWPCF, 50, 234-239 (1978).
-------
Procedure Code: 5
Variations: 1 to 3
Title: A-26 Resin/GC-FID Method for Phenols
1. Summary
The method given here is that reported by Chriswell e_t al. (1).
phenols are trapped (as phenolate anions) on A-26 anion exchange resin by
passing alkaline sample through a column of resin in the hydroxyl form.
Any neutral organic compounds retained by the resin are washed off with
alkaline methanol. The column is then washed with aqueous hydrochloric
acid to convert the phenolate anions back to the acid fora and elute them
from the column. The elution is completed with an acetone-water solution
and all eluates extracted with methylene chloride for gas chromatography
with flame ionization detection (FID).
2. Interferences
2.1 Solvents, reagents, glassware, and other sample-processing hardware
may yield discrete artifacts and/or elevated baselines that could
cause misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the conditions
of the analysis Specific selection of reagents and purification of
solvents by distillation in all-glass systems may be required.
2.2 Common inorganic ions and many organic substances cause no inter-
ference, and neutral organics that are retained by the resin can be
removed by a methanol wash.
2.3 The interferences in industrial effluents are many and varied,
and often pose great difficulty in obtaining accurate and precise
measurement of phenols. Sample cleanup procedures are generally
required and may result in the loss of certain phenols. Therefore,
great care should be exercised in the selection and use of methods
for eliminating or minimizing interferences. It is not possible to
describe procedures for overcoming all the interferences that may be
encountered in industrial effluents.
3. Apparatus and Materials
3.1 Gas Chromatograph - equipped with dual flame ionization detectors.
3.2 Recorder - Potentiometric strip chart (10 inch) compatible with
the detectors.
3.3 Gas Chromatographic Column Materials - see method variations
-------
3.4 Detector Options
3.A.I Dual flame ionization
3.4.2 Electron capture - radioactive (tritium or nickel 63) - if
phenols of interest are halogenated.
3.5 Kuderna-Danish (K-D) Glassware
3.5.1 Snyder column - three ball (macro) and two ball (micro)
3.5.2 Evaporative flasks - 500 ml
3.5.3 Receiver ampules - 10 ml, graduated
3.5.4 Ampule stoppers
3.6 Chromatographic column - 1/2 in x 6 in glass chromatographic column
with A 1,000 ml reservoir and a coarse fritted plate on bottom,
prepared by pouring a water slurry of the cleaned A-26 resin into the
column until the resin bed is within about one-half inch of the top
of the column. The column is placed in the hydroxy form by passing
approximately 20 ml of 0.1 M sodium hydroxide solution through the
resin. Excess sodium hydroxide is washed from the resin bed with 50
ml of purified distilled water. Verify that there was excess hydrox-
ide on the column by testing the first portion of water wash with pH
paper (color should indicate pH^9).
3.7 Filter - medium porosity, 150 ml sintered glass filter
3.8 Separatory funnels - 125 ml with Teflon stopcock
3.9 Graduated cylinders - 50 ml, 100 ml and 500 ml
3.10 Porous polymer resin - Rohm & Haas A-26 anion exchange resin screened
to remove beads smaller than 60 mesh. Clean new resin by placing
in a sintered glass filter attached to a suction flask, washing
successively with 2 M sodium hydroxide, purified distilled water,
4 M hydrochloric acid, purified distilled water and acetone.
Repeat sequence of washings until no color is apparent in the final
acetone wash (generally one to three times). Then extract with
acetone for 24 hours in a Soxhlet extractor.
4. Reagents, Solvents and Standards
4.1 Sodium hydroxide - (ACS)
4.1.1 2 M solution in distilled water
4.1.2 0.1 M solution in distilled water
-------
4.2 Hydrochloric acid - (ACS) 4 M solution in distilled water
4.3 Acetone, methylene chloride, methanol-nanograde, redistilled in
glass if necessary.
4.4 Hydroxylamine hydrochloride - ACS
4.5 Sodium hydrosulfite - ACS
4.6 Phenol - compound(s) being analyzed
5. Quality Control
5.1 Standards should be injected frequently, as a check on the stability
of the operating conditions.
5.2 Duplicate and spiked sample analyses are recommended as quality
control checks. When the routine occurrence of a phenol is being
observed, the use of quality control charts is recommended.
5.3 Each time a set of samples is extracted, a method blank is determined
on .a volume of distilled water equivalent to that used to dilute the
sample.
6. Sample Preparation
6.1 Take a 500 ml sample of water. If phenol standards are added to
water containing chlorine, add 15 to 25 mg of hydroxylamine hydro-
chloride and allow to stir for at least 5 minutes before proceeding.
Add 15 to 25 mg of sodium hydrosulfite to the sample, and adjust the
pH to between 12.0 and 12.5 with 2 M sodium hydroxide.
6.2 If a precipitate forms, coagulate it by allowing the sample to sit
for about 15 minutes. Decant the supernatant liquid through a
medium-porosity, 150 ml sintered-glass filter attached to a suction
flask. Wash the precipitate into the filter with a minimum amount
of water, then wash thoroughly with approximately 50 ml of distilled
water.
6.3 Pour the filtered water and washings into the reservoir of the
adsorption column and allow to flow through the resin column at a
rate of 10 to 15 ml/min.* When the liquid level reaches the top of
the resin bed, wash the column with 25 ml of basic methanol (2 ml of
2 M sodium hydroxide in 23 ml of methanol') and 25 ml of distilled
water.
6.4 Place a 125-ml separatory funnel under the column, and elute the
column with 25 ml of 4 M hydrochloric acid, then with 25 ml of
distilled water. (The first portion of water should test acidic to
pH paper). Extract the solution in the separatory funnel with 25 ml
of methylene chloride. Allow the phases to separate and become clear;
then drain the lower methylene chloride layer into a second 125-ml
separatory funnel and discard the aqueous layer.
*Note: Some workers have found better phenolate exchange when a slower flow
rate is used (about 3 ml/min.) in this step (see Variation #2).
-------
6.5 Elute the column with 60 ml of 5:1 (V:V) acetone'.water and 50 ml of
distilled water in sequence into the separatory funnel containing
the methylene chloride. Shake, allow the phases to separate and
clear; then drain the lower organic layer into a K-D evaporating
flask.
6.6 Add a small boiling chip to the flask, attach a bnyder column, and
evaporate the solvent over a steam bath until the volume is reduced
to approximately 0.5 ml. Remove the evaporating flask from the
steam, and immediately spray the outside with acetone to condense the
vapors inside. Adjust the volume to 'exactly 1.0 ml with acetone.
6.7 Inject 2 yl of the acetone solution into the gas chromatograph (see
GC conditions, below).
Note: If the acetone solvent peak interferes with some of the
individual phenol peaks, the acetone may be removed by
adding approximately 7 ml of pentane to the evaporated (1.0
ml) acetone extract. By evaporating and diluting to exactly
1.0 ml with pentane, the acetone is volatilized by azeotropic
distillation. Injection of a pentane solution permits
operation of the gas chromatograph at significantly lower
attenuation settings without interference from a tailing
edge of the solvent peak.
7. Calculation of Results
7.1 Determine the phenol concentration by comparing either peak heights
or areas against a previously-prepared calibration curve.
8. Reporting Results
8.1 Report results in micrograms per liter without correction for recovery
data. When duplicate and spiked samples are analyzed, all data
obtained must be reported on the analytical quality control report
sheets.
9. Variations to Procedure Code #5
Variation
Code No.
Effective
Date
June 1977
Description
GC Column:
Temperature:
Injector:
Detector:
1% SP-1240 DA on 100/120
mesh Supelcoport in a 4 ft
x 2 mm ID glass column.
80°C to 180°C at 10°/min.,
hold at 180°C for 15 min.
240°C
I-.23-
FID at 240 C
Carrier: He at 35 ml/min.
(Source: Envirodyne Engineers, Inc.)
-------
Variation
Code No.
Effective
Date
February 1979
June 1977
Description
Same as Variation 1, except Column
Temperature: 80 for 5 min.,
80° to 180° at 10°/min.t
hold at 180° for 15 min.
3 ml/min flow rate for initial phenolate
anion exchange (Step 6.3); regenerate
resin with 2M NaOH instead of 0.1M
NaOH.
GC column: 1% SP-1240 DA on Supelcoport
(100/120 mesh) in a 6' x 2mm ID glass
column.
Temperature: 4 min. at 85 C, then
10°/min to 200 C
Injector: 180°C
Detector: FID at 200°C
Carrier: N- at 30 ml/min
(Source: Midwest Research Institute)
10. References
1. Chriswell, C. D., Chang, R. C., and Fritz, J. S., "Chronatographic
Determination of Phenols in Water", Anal. Chem.. 47, 1325-1329
(1975).
-------
^ In I 71
Procedure Code: 5
Variations: 1 to 5
Title: A-26 Resin/GC-FID Method for Phenols
1. Summary
The method given here is that reported by Chrisvell et, al. .(1) •
Phenols are trapped (as phenolate anions) on A-26 anion exchange resin by
passing alkaline sample through a column of resin in the hydroxyl form.
Any neutral organic compounds retained by the resin are washed off with
alkaline methanol. The column is then washed with aqueous hydrochloric
acid to convert the phenolate anions back to the acid form and elute them
from the column. The elution is completed with an acetone-water solution
and all eluates extracted with methylene chloride for gas chromatography
with flame ionization detection (FID).
For other methods used for phenol analysis, see Procedure Codes #7
and 915.
2. Interferences
2.1 Solvents, reagents, glassware, and other sample-processing hardware
may yield discrete artifacts and/or elevated baselines that could
cause misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the conditions
of the analysis. Specific selection of reagents and purification of
solvents by distillation in all-glass systems may be required.
2.2 Common inorganic ions and many organic substances cause no inter-
ference, and neutral organics that are retained by the resin can be
removed by a methanol wash.
2.3 The interferences in industrial effluents are many and varied,
and often pose great difficulty in obtaining accurate and precise
measurement of phenols. Sample cleanup procedures are generally
required and may result in the loss of certain phenols. Therefore,
great care should be exercised in the selection and use of methods
for eliminating or minimizing interferences. It is not possible to
describe procedures for overcoming all the interferences that may be
encountered in industrial effluents.
3. Apparatus and Materials
3.1 Gas Chromatograph - equipped with dual flame ionization detectors.
3.2 Recorder - Potentiometric strip chart (10 inch) compatible with
the detectors.
3.3 Gas Chromatographic Column Materials - see method variations
>2 A
-------
3.4 Detector Options
3.4.1 Dual flame ionization
3.4.2 Electron capture - radioactive (tritium or nickel 63) - if
phenols of interest are halogenated.
3.5 Kuderna-Danish (K-D) Glassware
3.5.1 Snyder column - three ball (macro) and tvo ball (micro)
3.5.2 Evaporative flasks - 500 ml
3.5.3 Receiver ampules - 10 ml, graduated
3.5.4 Ampule stoppers
3.6 Chromatographic column - 1/2 in x 6 in glass chromatographic column
with a 1,000 ml reservoir and a coarse fritted plate on bottom,
prepared by pouring a water slurry of the cleaned A-26 resin into the
column until the resin bed is within about one-half inch of the top
of the column. The column is placed in the hydroxy form by passing
approximately 20 ml of 0.1 M sodium hydroxide solution through the
resin. Excess sodium hydroxide is washed from the resin bed with 50
ml of purified distilled water. Verify that there was excess hydrox-
ide on the column by testing the first portion of water wash with pH
paper (color should indicate pHj>9).
3.7 Filter - medium porosity, 150 ml sintered glass filter
3.8 Separatory funnels - 125 ml with Teflon stopcock
3.9 Graduated cylinders - 50 ml, 100 ml and 500 ml
3.10 Porous polymer resin - Rohm & Haas A-26 anion exchange resin screened
to remove beads smaller than 60 mesh. Clean new resin by placing
in a sintered glass filter attached to a suction flask, washing
successively with 2 M sodium hydroxide, purified distilled water,
4 M hydrochloric acid, purified distilled water and acetone.
Repeat sequence of washings until no color is apparent in the final
acetone wash (generally one to three times). Then extract with
acetone for 24 hours in a Soxhlet extractor.
4. Reagents, Solvents and Standards
4.1 Sodium hydroxide - (ACS)
4.1.1 2 M solution in distilled water
4.1.2 0.1 M solution in distilled water
-------
4.2 Hydrochloric acid - (ACS) 4 M solution in distilled water
4.3 Acetone, methylene chloride, methanol-nanograde, redistilled in
glass if necessary.
4.4 Hydroxylamine hydrochloride - ACS
4.5 Sodium hydrosulfite - ACS
4.6 Phenol - compound(s) being analyzed
5. Quality Control
5.1 Standards should be injected frequently, as a check on the stability
of the operating conditions.
5.2 Duplicate and spiked sample analyses are recommended as quality
control checks. When the routine occurrence of a phenol is being
observed, the use of quality control charts is recommended.
5.3 Each time a set of samples is extracted, a method blank is determined
on a volume of distilled water equivalent to that used to dilute the
sample.
6. Sample Preparation
6.1 Take a 500 ml sample of water. If phenol standards are added to
water containing chlorine, add 15 to 25 mg of hydroxylamine hydro-
chloride and allow to stir for at least 5 minutes before proceeding.
Add 15 to 25 mg of sodium hydrosulfite to the sample, and adjust the
pH to between 12.0 and 12.5 with 2 M sodium hydroxide.
6.2 If a precipitate forms, coagulate it by allowing the sample to sit
for about 15 minutes. Decant the supernatant liquid through a
medium-porosity, 150 ml sintered-glass filter attached to a suction
flask. Wash the precipitate into the filter with a minimum amount
of water, then wash thoroughly with approximately 50 ml of distilled
water.
6.3 Pour the filtered water and washings into the reservoir of the
adsorption column and allow to flow through the resin column at a
rate of 10 to 15 ml/min.* When the liquid level reaches the top of
the resin bed, wash the column with 25 ml of basic methanol (2 ml of
2 M sodium hydroxide in 23 ml of methanol) and 25 ml of distilled
water.
6.4 Place a 125-ral separatory funnel under the column, and elute the
column with 25 ml of 4 M hydrochloric acid, then with 25 ml of
distilled water. (The first portion of water should test acidic to
pH paper). Extract the solution in the separatory funnel with 25 ml
of methylene chloride. Allow the phases to separate and become clear;
then drain the lower methylene chloride layer into a second 125-ml
separatory funnel and discard the aqueous layer.
*Note: Some workers have found better phenolate exchange when a slower flow
rate is used (about 3 ml/min.) in this step (see Variation #2).
-------
6.5 Elute the column with 60 ml of 5:1 (V:V) acetonerwater and 50 ml of
distilled water in sequence into the separatory funnel containing
the methylene chloride. Shake, allow the phases to separate and
clear; then drain the lower organic layer into a K-D evaporating
flask.
6.6 Add a small boiling chip to the flask, attach a Snyder column, and
evaporate the solvent over a steam bath until the volume is reduced
to approximately 0.5 ml. Remove the evaporating flask from the
steam, and immediately spray the outside with acetone to condense the
vapors inside. Adjust the volume to exactly 1.0 ml with acetone.
6.7 Inject 2 yl of the acetone solution into the gas chromatograph (see
GC conditions, below).
Note: If the acetone solvent peak interferes with some of the
individual phenol peaks, the acetone may be removed by
adding approximately 7 ml of pentane to the evaporated (1.0
ml) acetone extract. By evaporating and diluting to exactly
1.0 ml with pentane, the acetone is volatilized by azeotropic
distillation. Injection of a pentane solution permits
operation of the gas chromatograph at significantly lower
attenuation settings without interference from a tailing
edge of the solvent peak.
7. Calculation of Results
7.1 Determine the phenol concentration by comparing either peak heights
or areas against a previously-prepared calibration curve.
8. Reporting Results
8.1 Report results in micrograms per liter without correction for recovery
data. When duplicate and spiked samples are analyzed, all data
obtained must be reported on the analytical quality control report
sheets.
9. Variations to Procedure Code #5
Variation Effective
Code No. Date Description
1 June 1977 GC Column: 1% SP-1240 DA on 100/120
mesh Supelcoport in a 4 ft
x 2 mm ID glass column.
Temperature: 80°C to 180°C at 10°/min.,
hold at 180°C for 15 min.
Injector: 240°C
Detector: FID at 240°C
Carrier: He at 35 ml/min.
(Source: Envirodyne Engineers, Inc.)
TOT-
-------
mVariation
Code No.
Effective
Date
February 1979
June 1977
A.
Description
Same as Variation 1, except Column
Temperature: 80 for 5 min.,
80° to 180° at 10°/min.,
hold at 180° for 15 min.
3 ml/min flow rate for initial phenolate
anion exchange (Step 6.3); regenerate
resin with 2M NaOH instead of 0.1M
NaOH.
May 1979
May 1979
B. GC column: 1% SP-1240 DA on Supelcoport
(100/120 mesh) in a 6' x 2mm ID glass
column.
Temperature: 4 min. at 85°C. then
10°/min to 200- C
Injector: 180°C
Detector: FID at 200°C
Carrier: N- at 30 ml/min
(Source: Midwest Research Institute)
Same as Variation 1, except as follows:
Column Temperature: 80°C for 10 min.,
increase to 180
at 5 /min., hold
at 180° for 30
minutes
Injector: 190°C
Detector: FID at 300°C
Carrier: He at 35 ml/min.
(Source: EEI)
Same as Variation 1, except as follows:
Column Temperature: 80°C for 13 min.,
increase to 180°C
at 10°/min.. and
hold at 180 C for
30 min.
10. References
1. Chriswell, C. D., Chang, R. C., and Fritz, J. S., "Chromatographic
Determination of Phenols in Water", Anal. Chem.. 47. 1325-1329
(1975).
-------
FV ':r*rr^
Procedure Code: 7 i V i tv 5/'~>/79
Variations: 1-6
Title: Microextraction Method for Organic Compounds
in Industrial Effluents
(Source: Southwest Research Institute)
1. Summary
The method given here is adapted from that of Henderson et al. (!)• (See
Procedure Code #14). The method is similar to that of Procedure Code #14,
except that the solvent/sample ratio is much smaller with the microextraction
method than with Procedure //14. In both methods a water sample is equili-
brated once with an organic solvent. In microextraction the water to
solvent ratio may be as high as 1000:1, but generally is in the range of
200:1 to 10:1. The water sample may or may not be saturated with salt as
required.
The method variations are classified as follows:
Variation Effective
Code No. Date Description
1 Feb. 1979 Benzene-Toluene-Ethyl Benzene procedure
2 March 1979 Nitrobenzene procedure
3 May 1979 Behzene-Toluerie-Ethyl Benzene procedure
4 May 1979 Phthalates procedure
5 May 1979 Phenols procedure
May 1979 Polynuclear Aromatic Hydrocarbon
procedure
2. Sample Collection
Samples to be analyzed for volatile organic compounds are collected in 40
ml serum vials. The vials are filled to overflowing so there is no head
space, then sealed with an aluminum crimped cap and a teflon-coated septum.
A layer of aluminum foil is placed between segtum and the cap to minimize
volatile losses. The samples are stored at 4 C until analysis. Altern-
atively, a 40 ml screw-cap vial may be used with a foil-covered teflon
disc to seal in the sample.
Water to be analyzed for phenols, phthalates or PAH Compounds Is collected
as part of the 24-hour composite sample or composite of grab samples used
for general non-volatile organics analyses.
-------
3. General Microextraction Procedure
3.1 Place "A" ml of sample In an appropriately sized extractor (see
Figure 1; Note 1) and sufficient salt is added to saturate the sample
if desired.
3.2 Add "B" microliters of solvent (pentane, hexane, diisopropylether,
etc.) to the extractor, which is then sealed.
3.3 Vigorously shake the extractor for 2 minutes to insure that partitioning
of compounds of interest between water sample and organic -solvent has
reached equilibrium.
3.4 Place or clamp the extractor in position so that solvent rises to the
top.
3.5 Using a micro syringe, withdraw a sample of the solvent layer directly
from the extractor for injection into the chromatograph or save the
solvent layer (3.6).
3.6 If desired, recover the solvent layer from the extractor into a small
screw cap vial for subsequent analysis (Note 2).
4. General Analytical Protocol
4.1 Estimate the concentration of compounds of interest by microextracting
a "blank" sample of wastewater, injecting 5-10 yl of extract and
calculating the apparent concentration with an external standard and
the following formula:
Formula I R01 x R03 x R04 ..
R02 x R05 x R06 " yg/1
Where: R01 « Standard ng injected
R02 - Area of injected standard
R03 - Area of sample Injected
R04 • Volume of extracting solvent (yl)
R05 - Volume of sample extract injected (yl)
R06 - Sample volume extracted (ml)
Note 1: Extractors can be of most any design or shape that allows for intimate
contact between water and solvent during shaking. It is desirable to
have a small diameter neck to simplify sample removal for injection or
storage.
Note 2: If an emulsion forms, forcing the emulsion through a glass wool plug
vill usually cause satisfactory separation and enable recovery of
extracting solvent.
-------
A. Silicone rubber septum
B. 2mm ID glass tubing
C. Semlball joint
D. Silicone rubber septum removed
when forcing solvent into
2"" tube
E. Glass wool plug
FIGURE 1. EXTRACTION VESSEL
(after Rhoades and Millar (2))
-------
For example: micro extract 40 ml ("A") of wastewater with 400 VI ("B")
of hexane as described. Then determine the "apparent" concentration by
injection of 5-10 yl of the extract and calculation of the "apparent"
concentration based on an external standard and assuming 400 VI of extract
(Formula I). Use the apparent concentration to plan the concentration of
spikes and internal standard used in quantitation Formula II, Section 4.2.
4.2 Measure two equal volumes of the same sample as the blank into each
of two extractors. Add an appropriate amount of IS (Internal Standard)
to each extractor and add an appropriate amount of "spike" (based on
the blank analysis in 4.1) to one of the extractors. The sample to
which only the IS has been added i& the "unspiked" sample. The
sample to which both the IS and the spike are added is the "spiked"
sample. Both samples are extracted and analyzed to collect peak area
data as in the analysis in 4.1. Formula II is used to calculate the
concentration in the wastewater:
Formula II Rll x ~ - R16
R16 x R15
(R13-R16) *
Where: Rll « area of unspiked sample
R12 » area of IS in unspiked sample
R13 «• area of spiked sample
R14 * area of IS in spiked sample
R.15 » ug/1 added for spike
5. Quality Control
Duplicate and spiked sample analyses (as described in Section 4) are
recommended as quality control. Each set of sample analyses is preceded
by a method blank using organic-free water.
6. Variations to Procedure Code #7
6.1 Variation #1 •- Benzene, Toluene, Ethyl Benzene (Feb. 1979).
Load 10 ml of sample into the 10-ml extraction vessel, using a syringe
to deliver the sample through the opening at the top (remove septum D,
Figure 1). Next, close the top with septum D. Carry out the entire
loading operation either in a cold room or with the extraction vessel
in an ice bucket. Add 500 vl of hexane UV (Burdick and Jackson) to
the extraction vessel through septum A. Internal standard (o-xylene)
and spiking solutions can also be added through septum A. After the
extractor has been prepared, follow the General Microextraction
Procedure in Sections 3.3-3.6. Analyse the extract by FID/GC using
the following conditions:
31
-------
GC Column: 15% Carbowax 1500 on 60/80 mesh Gas Chrotn Q
in a 6' x A mm ID glass column.
Carrier: He at 20 cc/min.
Injector: 150°C
Detector: 100°C FID
Temperature: 50°C (2 min), then 50°C to
70°C at l°C/min.
6.2 Variation 92 - Nitrobenzene (March. 1979)
Load 40 ml of sample into a 50-ml extraction vessel, using a syringe
to deliver the sample through the opening at the top (remove septum D,
Figure 1). Next, close the top with septum D. Carry out the entire
loading operation either in a cold room or with the extraction vessel
in an ice bucket. Add 500 yl of hexane UV (Burdick and Jackson) to
the extraction vessel through septum A. Internal Standard (nitro-
toluene) and spiking solutions can also be added through septum A.
After the extractor has been prepared, follow the General Micro-
extraction Procedure in Sections 3.3-3.6. Analyze the extract by
GC/AFID using the following conditions:
GC Column: 10% Carbowax 20 M + 22 KOH on Chromosorb W-
AW, 80/100 mesh in a 6' x A mm ID glass
column.
Temperature: 160°C Isothermal
Detector: AFID
Carrier: He at 60 ml/min.
6.3 Variation #3 - Benzene - Ethyl Benzene. Toluene (May. 1979)
Load 40 ml of sample into a 50-ml extraction vessel, using a syringe
to deliver the sample through the opening at the top (remove septum D,
Figure 1). Next, close the top with septum D. Carry out the entire
loading operation either in a cold room or with the extraction vessel
in an ice bucket. Add 500 yl of hexane UV (Burdick and Jackson) to
the extraction vessel through septum A. Internal Standard (o-xylene)
and spiking solutions can also be added through septum A. After the
extractor has been prepared, follow the General Microextraction
Procedure in Sections 3.3-3.6. Analyze the extract by FID/GC using
the following conditions:
33
-------
GC Column: 5% SP 1200/5% Bentone 34/10% Carbowax 1500
on 100/120 mesh Supelcoport, 6' x 4 mm ID
glass column.
Temperature: 50°C (4 min), then 50°C to 90°C at 4°C/min.,
hold 90°C for 20 min.
Detector: 200°C FID
Injector: 180°C
Carrier: He at 25 ml/min.
6.4 Variation #4 - Phthalates (May. 1979)
6.4.1 Place 100 ml of wastewater sample into a 100 ml pyrex
volumetric glass stoppered flask. If an internal standard
is to be used, it is added at this time. The compound
spike is also added at this time.
6.4.2 Add 1 ml of pesticide grade hexane.
6.4.3 Stopper and shake the flask vigorously for 2 minutes.
6.4.4 Allow the phases to separate sufficiently to permit a
portion of the hexane solvent to be withdrawn into a
microliter syringe for injection into the gas chromatograph.
6.4.5 Analyze by GC/EC with the following conditions:
GC Column: 1.5% SP 2250/1.95% SP 2401 on 100/120 mesh
Supelcoport, in a 1.8 m x 6.35 mm OD glass
column.
Temperature: 180°C (10 min), then 32°C/min to 220°C
Carrier: 5% Methane/Argon at 60 ml/min
Detector: Electron capture at 300°C
6.5 Variation #5 - Phenols (May. 1979)
6.5.1 Base - neutral extraction for clean-up
6.5.1.1 Place a 200 ml aliquot of sample into a 1L separatory
funnel equipped with a teflon stopcock.
6.5.1.2 Check the sample pH with indicator paper. If strongly
acidic, it may be necessary to add NaOH pellets to raise
the pH to 11 or above. If the sample is neutral, add 10 N
NaOH dropwise to reach a pH of 11 or above in the sample.
31
-------
6.5.1.3 Add 25 ml of methylene chloride and shake for 1 Bin.
Shake cautiously and vent frequently during the first 30
sec. of shaking.
6.5.1.4 Allow the organic solvent phase to separate. Drain
the solvent and any emulsion into a flask and discard.
6.5.1.5 Repeat steps 6.5.1.3 and 6.5.1.4.
6.5.1.6 After removal of the second methylene chloride extract,
add 60 ml of hexane and shake for 1 minute.
6.5.1.7 After 3-4 minutes of phase separation, rapidly drain about
50 ml of the aqueous phase into a flask and discard. This
step is necessary to remove any residual methylene chloride
solvent in the stopcock and delivery tube of the separately
funnel.
6.5.1.8 Drain 80 ml of sample into a 100 ml graduated cylinder.
6.5.2 Microextraction of Sample
6.5.2.1 Add 28 g NaCl to a 100 ml volumetric flask modified for
microextraction (Note 3).
6.5.2.2 Transfer 80 ml of sample to the flask and add 1:1 RJPO,:
H_0 until the pH of the sample is less than 2. Add internal
standard at this point if necessary.
6.5.2.3 Add 800 yl DIE (diisopropyl ether), stopper, and shake for
1 minute.
6.5.2.4 Allow phase separation prior to extract removal. To remove
DIE, inject organic-free water (Millipore Super Q or
equivalent) slowly into the flask with an inverted U-tube
on top. Collect the extract In a clean %-drain vial. A
small plug of glass wool In the U-tube will help break
emulsions if present.
Note 3: The 100 ml volumetric flask is modified by attaching a short length
of glass tubing to the lower portion of the flask to allow for
insertion of a rubber septum (as with "A" in Figure 1). An inverted
U-tube is made by attaching a U-shaped capillary bore glass tube to a
ground-glass joint which fits the taper of the 100 ml volumetric
flask.
-------
6.5.2.5 Analyze by the following GC/FID conditions:
GC Column: 1% SP-1240 DA on 100/120 mesh Supelcoport
in a 6' x 4 ram ID glass column with H.PO,
treated glass wool plugs.
Carrier: He at 100 ml/min
Detector: FID
Temperature: 120°C (2 min), then 20°C/min to 180°C, hold
20-35 min at 180°C (column can be taken to
190 C, to elute sample contaminants, for
short periods of time (<15 min).
Injector: 250°C
Detector: 250°C
6.6 Variation 06 - Polynuclear Aromatic Hydrocarbons (May, 1979).
Follow the General Microextraction Procedure for sample preparation,
Section 3. Analyze the hexane extract with the following GC conditions
GC Column: 15 meter x 0.25 mm ID SP 2100 WCOT (capillary)
Detector: FID at 300°C
Carrier: He at 0.75 ml/min after split
Split ratio: 50:1
Detector: 300°C
Calculate the results as shown in Section 4.
7. References
1. Henderson, J.E., Peyton, G.R., and Glaze, W.H., "A Convenient Liquid-
Liquid Extraction Method for the Determination of Halomethanes in
Water at the Parts-Per-Billion Level", in Identification and Analysis
of Organic Pollutants in Water, ed. L.H. Keith, Ann Arbor Science
Publishers, Inc., Ann Arbor, Michigan, pp 105-111 (1976).
2. Rhoades, J.W., and Millar, J.D., "Gas Chromatographic Method for
Comparative Analysis of Fruit Flowers", Agricultural and Food Chen..
13, 5-9 (1965).
-------
Procedure Code: 8
Variations: 1-5
Title: Purge and Trap Procedures for Analysis of Volatile
Organic Compounds in Effluents
1. Summary
All the procedures included in this code are derived from the method of
Bellar, Lichtenberg, and Eichelberger (1) and use gas stripping (purge)
with an inert gas to remove volatile, poorly-soluble organic compounds
from a water sample and deposit them on a suitable trapping column. The
trapping column is then heated and the compounds are back flushed onto
a gas chromatographic (GC) column held at a low temperature, to deposit
the compounds at the GC column inlet. The chromatograph is then rapidly
heated to its initial program temperature and the GC analysis is begun.
Procedural variations include: the trapping material; purge time,
temperature, and flow; trap desorption time, temperature, and flow; the
GC analysis parameters (column, temperature program, carrier flow); and
the type of GC detector chosen. Rather than assign a variation code to
all the possible sources of variation, each procedure used by an EPA
contractor or taken from the literature will be assigned a number, as
follows:
Variation Effective
Code No. Date Description
1 June 1977 Specific Method for Vinyl Chloride (2)
2 June 1977 Midwest Research Method
3a Jan. 1979 Southwest Research Method (a)
3b May 1979 Southwest Research Method (b)
4 Feb. 1979 Method for Acrylonitrile
5 May 1979 Method for Acrolein and Acrylonitrile
2. Sample Collection and Compositing
Samples are collected in 40 ml serum vials, filled to overflowing so
that there is no head space, then sealed with an aluminum crimp cap and
a teflon-coated septum. A layer of aluminum foil is placed between the
septum and the cap to minimize volatile losses. Alternatively, the
samples may be collected in 40 ml vials with screw caps, using teflon
discs covered with aluminum foil to seal the sample head-space free.
The samples are stored at 4 C until analysis. For compositing, the cold
samples are combined in a chilled flask (4 C); then an aliquot is
transferred to the purging device.
3. Vinyl Chloride Procedure
Variation #1
This method is described in detail in the paper by Bellar, Lichtenberg,
and Eichelberger (1). It uses a silica gel or carbosieve-B trap, designed
-------
to retain vinyl chloride. The method is hence compound-specific and should
be used in verification sampling when vinyl chloride is the only "purgeable"
compound present in the sample. ' If other purgeable compounds are present,
a liquid-liquid extraction method should be tried, or Variations //2 and #3
of the purge and trap procedure may be used if the sample matrix makes
liquid-liquid extraction ineffective.
Envirodyne Engineers, Inc., Parameters for Vinyl Chloride
Purge: 12 min. at 40 ml/min. He, ambient temperature, 5 ml sample.
»
Trap: 4.5 in X 2 mm ID, Carbosieve-B, 60/80 mesh.
Desorb: 4 min. at 180°C, 30 ml/min He.
GC Column: 8 ft. X 2 mm ID stainless steel with 0.2% Carbowax
1500 on Carbopack C, 60/80 mesh.
Temperature: Ambient during desorption, then heat to 60 C, hold
4 min., and program to 170 at 8 /min.
Detector: Hall electrolytic conductivity in halide mode or mass
spectrometer.
Carrier: He at 30 ml/min.
4. Variation 92 (Midwest Research)
Purge: 8 min. at 40 ml/min. N_, ambient temperature, 1-5 ml
sample.
Trap: 10 in. x 0.125 in. OD x 105 in. ID stainless steel, Tenax
GC, 60/80 mesh.
Desorb: 4 min. at 180°C
GC Column: 8 ft. x 0.125 in. OD stainless steel with 0.2% Carbowax
1500 on Carbopack C, 60/80 mesh.
GC Temperature: 4 min. at 40°C, 8°/min. to 160°C, hold 5 min.
Detector: FID and/or Hall electrolytic conductivity (halide mode)
Quantitation: Use area ratio to bromochloromethane as the internal
standard.
5. Variation #3(a) (Southwest Research)
Purge: 12 min. at 40 ml/min. He, ambient temperature, 5 ml sample.
Trap: 6 in. x 4 mm ID, Tenax GC, 60/80 mesh.
-------
Desorb: A min. at 180°C, 30 ml/min. He
GC Column: 8 ft. x 2 mm ID nickel, with 0.2% Carbowax 1500 on
Carbopack C, 60/80 mesh.
Temperature: Ambient during desorption: then heat to 60°C, hold 4 min. ,
and program to 170 C at 8 /min.
Detector: Hall electrolytic conductivity in halide mode.
Carrier: Helium at 30 ml/min
Quantitation: Concentrations of analytes are determined as follows:
*S " ^J " AAS
AAS
-££.. a
ngs
Tp
vp
Where:
AS • Area of peak in spiked run
A.. • Area of peak in unspiked run
A._ • Area of added epike
AS
o • Constant, area unita per ng
M.. • mass (ng) in unaplked peak
Vp • volume purged (ml)
C. » Concentration, ppb
Variation //3(b) (Southwest Research)
Same as Variation #3(a) except:
Purge Flow: 30 ml/min. He
Trap: 6 in. X 4 mm ID, with 5 in. of Tenax GC, 60/80 mesh at the
inlet, and 1 in. of silica gel 35/60 mesh, placed after the
Tenax in the trap flow direction.
Desorb Flow: 22 ml/min. He
GC Carrier Flow: 22 ml/min. He
-------
6. Variation #4 (Method for Acrylonitrile) (Source: EEI)*
Purge: 12 min. at 40 ml/min., at 60°C, with Helium; 25 ml sample
volume purged.
Trap: 6 in. x 4 mm ID stainless steel, Tenax GC, 60/80 mesh.
Desorb: 4 min. at 180°C, 30 ml/min.
GC Column: 6' x 1/4" OD glass column with Chromosorb 102, 60/80 mesh.
GC Temperature: 100 C isothermal.
Injector: 200°C.
Detector: FID at 200°C.
Carrier: Helium at 30 ml/min.
* See Procedure Code #1 for direct aqueous injection method for acrylonitrile.
7. Variation #5 (Method for Acrolein and Acrolonitrile) (Source: EEI)
Purge: 12 min. at 40 ml/min He, at 60°C, 25-40 ml sample. Composite
samples at 4 C, if necessary, before analysis.
Trap: 2 mm ID X 4.5 in. stainless steel, with 60/80 mesh Tenax GC.
Desorb: 7 min. x 200°C, 30 ml/min. He.
GC Column: Chromosorb 102 (60/80 mesh) in a 6' x 1/4" glass column.
Temperature: 110°C isothermal.
Detector: FID at 250°C.
Carrier: He at 35 ml/min.
8. References
1. Bellar, T. A., Lichtenberg, J. J., and Eichelberger, J. W.,
"Determination of Vinyl Chloride at mg/1 Level in Water by Gas Chro-
ma to graphy," EnvJ^pjuS£i_:__andT^chnoL:_jL_10, 926-930 (1976).
-------
Procedure Code: 9
Title: Method for Polychlorinated Biphenyls (PCB's) in Industrial Effluents
1. Scope and Application
1.1 This method covers the determination of certain polychlorinated
biphenyl (PCB) mixtures, including: Aroclors 1221, 1232, 1242, 1248,
1254, 1260 and 1016.
1.2 The method is an extension of Procedure //3, the EPA method for
organochlorine pesticides and phthalate esters in industrial effluents
(1). It is designed so that determination of PCB's, organochlorine
pesticides, and phthalate esters may be made on the same sample.
1.3 The limit of detection is approximately I/ g/1 for each Aroclor
mixture.
2. Summary
2.1 The PCB's and the organochlorine pesticides are coextracted by
liquid-liquid extraction and, insofar as possible, the two classes
of separated from one another prior to gas chromatographic determination.
combination of the standard Florisil column cleanup procedure and a
gel microcolumn separation procedure (2,3) is employed. Identification
is made from gas chromatographic patterns obtained through the use
of two or more unlike columns. Detection and measurement is accomplished
by using an electron-capture detector. Techniques for confirming
qualitative identification are suggested.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines causing mis-
interpretation of gas chromatograms. All these materials must be
demonstrated to be free of interferences under the conditions of the
analysis. Specific selection of reagents and purification of solvents
by distillation in all-glass systems may be required. Refer to (4),
Part I. Sections 1.4 and 1.5.
3.2 The interferences in industrial effluents are high and varied, and
pose great-difficulty in obtaining accurate and precise measurement
of PCB's. Separation and cleanup procedures are generally required
to eliminate these interferences; however, such techniques may
result in the loss of certain organochlorine compounds. For this
reason, great care should be exercised in the selection and use of
methods for eliminating or minimizing interferences. It is not
possible to describe procedures for overcoming all the interferences
that may be encountered in industrial wastes.
-------
3.3 Phthalate esters, certain organophosphorus pesticides, and elemental
sulfur will interfere when using electron capture for detection.
Organochlorine pesticides and other halogenated compounds also
constitute interferences in the determination of PCB's. Most of
these are separated by the method described herein. However, certain
compounds, if present in the sample, will occur with the PCB's.
Included are: sulfur, Heptachlor, aldrin, DDE, technical chlordane,
mirex, and to some extent o,p'-DDT and p,p'-DDT.
A. Apparatus and Materials
A.I Gas Chromatograph - Equipped with glass-lined injection port.
4.2 Detector - Electron Capture
4.3 Recorder - Potentiometric strip chart (10 in.) compatible with
detector system.
4.4 Gas Chromatographic Column Materials:
4.4.1 Tubing - Pyrex (180 cm long x 4 mm ID/or 6' x 2 mm ID)
4.4.2 Glass Wool - Silanized
4.4.3 Solid Support* - Gas-Chrom Q (100/120 mesh)
4.4.4 Liquid Phases* - Expressed as weight percent coated on solid
Support:
4.4.4.1 SE-30 or OV-1, 3%
4.4.4.2 OV-17, 1.57. + QF-1, 1.95%
4.5 Kuderna-Danish (K-D) Glassware (Kontes)
4.5.1 Snyder Columns - Three ball (macro)
4.5.2 Evaporate Flasks - 500 ml
4.5.3 Receiver Ampuls - 10 ml graduated
4.5.4 Ampul stoppers
*See also Section 13.
-------
4.6 Chromatographic Column - Chromaflex (400 mm long x 19 mm ID) with
coarse fritted plate on bottom and Teflon stopcock; 250 ml reservoir
bulb at top of column with flared out funnel shape at top of bulb -
a special order (Kontes K-420540-9011).
4.7 Chromatographic Column - Pyrex (approximately 400 mm long x 20 nun ID)
with a coarse fritted plate on bottom.
4.8 Micro Column Pyrex - constructed according to Figure 1.
4.9 Capillary pipets disposable (5-3/4 in.) with rubber bulb. (Scientific
Products P5205-1).
4.10 Low-pressure regulator - 0 to 5 psig - with low-flow needle valve
(See Figure 1, Matheson Model 70).
4.11 Beaker - 100 ml.
4.12 Micro syringes - 10, 25, 50 and 100 jjl.
4.13 Separatory Funnels - 125 ml, 1000 ml, and 2000 ml with Teflon stopcocks,
4.14 Graduated Cylinders - 100 ml, 250 ml.
4.15 Blender - High speed, glass or stainless cup.
4.16 Florisil - PR Grade (60/100 mesh); purchase activated at 125°F
and store in the dark in glass containers with glass stoppers or
foil-lined screw caps. Before use, activate each batch overnight at
130° in foil-covered glass container. Determine lauric-acid value
(See Section 14).
4.17 Silica gel - Davison code 950-08-08-226 (60/200 mesh).
4.18 Glass Wool - Hexane extracted.
4.19 Centrifuge Tubes - Pyrex calibrated (15 ml).
5. Reagents, Solvents, and Standards
5.1 Ferrous Sulfate - (ACS) 30% solution in distilled water.
5.2 Potassium Iodide - (ACS) 10% solution in distilled water.
5.3 Sodium Chloride - (ACS) Saturated solution (pre-rinse NaCl with
hexane) in distilled water.
5.4 Sodium Hydroxide - (ACS) 10 N in distilled water.
5.5 Sodium Sulfate - (ACS) Granular, anhydrous, conditioned for 4 hours
at 400° C.
-------
5.6 Sulfuric Acid - (ACS) Mix equal volumes of cone. H?SO with distilled
water. 2 4
5.7 Diethyl Ether - Nanograde, redistilled in glass, if necessary.
5.7.1 Must contain 22 alcohol and be free of peroxides by
following test: to 10 ml of ether in glass-stoppered
cylinder previously rinsed with ether, add one ml of freshly
prepared 10% KI solution. Shake and let stand one minute.
No yellow color should be observed in either layer.
5.7.2 Decompose ether peroxides by adding 40g of 30% ferrous
sulfate solution to each liter of solvent. CAUTION: Reaction
may be vigorous if the solvent contains a high concentration
of peroxides.
5.7.3 Distill deperoxidized ether in glass, and add 2% ethanol.
5.8 n-Hexane - Pesticide quality (NOT MIXED HEXANES).
5.9 Acetonitrile, Hexane. Methanol, Methylene Chloride, Petroleum Ether
(Boiling range 30-60 C) - pesticide quality, redistilled in glass if
necessary.
5.10 Standards - Aroclors 1221, 1232, 1242, 1248, 1254, 1260, and 1016.
5.11 Anti-static Solution - STATNUL, Daystrom, Inc., Weston Instrument
Division, Newark, New Jersey 95212.
6. Calibration
6.1 Gas chromatographic operating conditions are considered acceptable
when the response to dicapthon is at least 50% of full scale when
0.06 ng or less is injected for electron-capture detection. For all
quantitative measurements, the detector must be operated within its
linear response range, and the detector noise level should be less
than 2% of full scale.
6.2 Standards are injected frequently as a check on the stability of
operating conditions, detector, and column. Example chromatograr.s,
shown in Figures 3 through 8, provide reference operating conditions.
'• Quality Control
7.1 Duplicate and spiked sample analyses are recommended as a quality
control check. When the routine occurrence of a pollution parameter
is observed, quality control charts are also recommended (5).
T-
-------
7.2 Each tine a set of samples is extracted, a method blank is determined
on a volume of distilled water equal to that used to dilute the sample.
8. Sample Preparat ion
8.1 Blend the sample if suspended matter is present, and adjust pH to near
neutral (pH 6.5-7.5) with 50% sulfuric acid or 10 N sodium hydroxide.
8.2 For sensitivity requirement of 1 yg/1, when using microcoulometric
or electrolytic conductivity methods for detection take 1000 ml of
sample for analysis. If interferences pose no problem, the sensitivity
of the electron-capture detector should permit a little as 100 ml of
sample to be used. Background information on the extent and nature
of interferences will assist the analyst in choosing the required sample
size and preferred detector.
8.3 Quantitatively transfer the proper aliquot into a two-liter separatory
funnel, and dilute to one liter.
9. Extraction
9.1 Add 60 ml of 15% methylene chloride in hexane (v:v) to the sample
in the separatory funnel, and shake vigorously for two minutes.
9.2 Allow the mixed solvent to separate from the sample;, then draw the
water into a one-liter Erlenmeyer flask. Pour the organic layer* into
a 100 ml beaker, and then pass it through a column containing 3-4
inches of anhydrous sodium sulfate and collect it in a 500 ml K-D
flask equipped with a 10 ml ampule. Return the water phase to the
separatory funnel. Rinse the Erlenmeyer flask with a second 60 ml
volume of solvent; add the solvent to the separatory funnel and
complete the extraction procedure a second time. Perform a third
extraction in the same manner.
9.3 Concentrate the extract to 6-10 ml in the K-D evaporator on a hot
water bath.
9.4 Qualitatively analyze the sample by gas chromatography with an
electron-capture detector. From the response obtained, decide:
a. If there are any organochlorine pesticides present.
b. If there are any PCB's present,
c. If there is a combination of a and b,
d. If elemental sulfur is present,
e. If the response is too complex to determine a,b, or c.
-------
f. If no response, concentrate to 1.0 ml or less, as required,
according to EPA Method (A), pg. 28, and repeat the analysis
looking for a, b, c, d, and e. Samples containing Aroclors
with a low percentage of chlorine, e.g., 1221 and 1232, may
require this concentration in order to achieve the detection
limit of 1 yg/1. Trace quantities of PCB's are often
masked by background which usually occur in the samples.
9.5 If condition a_ exists, quantitatively determine the organochlorine
pesticides according to Procedure #3, "Method for Organochlorine
Pesticides and Phthalate Esters in Industrial Effluents."
9.6 If condition b_ exists, PCB's only are present, and no further separa-
tion or cleanup is necessary. Quantitatively determine the PCB's in
accordance with Section 11.
9.7 If condition £ exists, compare peaks obtained from the sample to
those of standard Aroclors, and make a judgment as to which Aroclors
may be present. To separate the PCB's from the organochlorine
pesticides, continue as outlined in 10.4.
9.8 If condition ^ exists, separate the sulfur from the sample using
the method outlined in (10.3) followed by the method in (10.5).
9.9 If condition je exists, then the following macro cleanup and separation
procedures (10.2 and 10.3) should be employed and, if necessary,
followed by the micro separation procedures (10.4 and 10.5).
10. Cleanup and Separation Procedures
10.1 Interferences in the form of distinct peaks and/or high background
in the initial gas chromatographic analysis, as well as the physical
characteristics of the extract (color, cloudiness, viscosity) and
background knowledge of the sample, will indicate whether cleanup is
required. When these interfere with measurement of the pesticides or
affect column life or detector sensitivity, proceed as directed in
the following paragraphs.
10.2 Acetonitrile Partition - This procedure is used to remove fats and
oils from the sample extracts. It should be noted that not all
pesticides are quantitatively recovered by this procedure. The
analyst must be aware of this and demonstrate the efficiency of the
partitioning for the compounds of interest.
10.2.1 Quantitatively transfer the previously concentrated extract
to a 125 ml separatory funnel with enough hexane to bring the
final volume to 15 ml. Extract the sample four times by shaking
vigorously for one minute with 30-ml portions of hexane-saturated
acetonitrile.
-------
10.2.2 Combine and transfer the acetonitrile phases to a one-
liter separatory funnel, and add 650 ml of distilled water
and 40 ml of saturated sodium chloride solution. Mix
thoroughly for 30-35 seconds. Extract with two 100-ml
portions of hexane by vigorously shaking about 15 minutes.
10.2.3 Combine the hexane extracts in a one-liter separatory funnel,
and wash with two 100 ml portions of distilled water.
Discard the water layer and pour the hexane layer through a
3-A inch anhydrous sodium sulfate column into a 500 ml
-------
6Z Eluate
Aldrin DDT Pentachloro-
BHC Heptachlor nitrobenzene
Chlordane Heptachlor Epoxide Strobane
DDD Lindane Toxaphene
DDE Methoxychlor Trifluralin
Mirex PCB's
152 Eluate 50% Eluate
Endosulfan I Endosulfan II
Endrin Captan
Dieldrin
Dichloran
Phthalate esters
Certain thiophosphate pesticides will occur in each of the
foregoing fractions and in the 100% fraction. For additional
information regarding eluate composition, refer to the FDA
Pesticide Analytical Manual (6).
10.3.4 Concentrate the eluates to 6-10 ml in the K-D evaporator
in a hot-water bath.
10.3.5 Analyze by gas chromatography.
10.4 Silica Gel Microcolumn Separation Procedure (7)
10.4.1 Activation for Silica Gel
10.4.1.1 Place about 20 em of silica gel in a 100 ml beaker.
Activate at 180 C for approximately 16 hours. Transfer
the silica gel to a 100 ml glass stoppered bottle.
When cool, cover with about 35 ml of 0.50% diethy1
ether in benzene (volume:volume). Keep bottle well
sealed. If silica gel collects on the ground glass
surfaces, wash off with the solvent before resealing.
Always maintain an excess of the mixed solvent in
bottle (approximately 1/2 in. above silica gel).
Silica gel can be effectively stored in this manner
for several days.
10.4.2 Preparation of the Chromatographic Column
10.4.2.1 Pack the lower 2-mm ID section of the microcolumn
with glass wool. Permanently mark the column 120 mm
above the glass wool. Using a clean rubber bulb from
a disposable pipet, seal the lower end of the micro-
column. Fill the microcolumn with 0.50% ether in
benzene (v:v) to the bottom of the 10/30 Joint (Figure
1). Using a disposable capillary pipet, transfer
several aliquots of the silica gel slurry into the
microcolumn. After approximately 1 cm of silica gel
-------
collects in the bottom of the nlcrocolumn, remove the
rubber bulb seal, and tap the column to insure that
the silica gel settles uniformly. Carefully pack the
column until the silica gel reaches the 120 + 2 mm
mark. Be sure that there are no air bubbles in the
column. Add about 10 mm of sodium sulfate to the top
of the silica gel. Under low-humidity conditions, the
silica gel may coat the sides of the column and not
settle properly. This can be .minimized by wiping the
outside of the column with an anti-static solution.
10,.4.2.2 Deactivation of the Silica Gel
a. Pill the microcolumn to the base of the 10/30
joint with the 0.50% ether-benzene mixture,
assemble reservoir (using spring clamps), and
fill with approximately 15 ml of the 0.50% ether-
benzene mixture. Attach the air pressure device
(using spring clamps) and adjust the elution rate
to approximately 1 ml/tnin. with the air-pressure
control. 'Release the air pressure and detach the
reservoir just as the last of the solvent enters
the sodium sulfate. Fill the column with n-
hexane (not mixed hexanes) to the base of the
10/30 fitting. Evaporate all residual benzene
from the reservoir, assemble the reservoir section,
and fill with 5 ml of n-hexane. Apply air pressure
and adjust the flow to 1 ml/min. (The n-hexane
flows slightly faster than the benzene). Release
the air pressure, and remove the reservoir just
as the n-hexane enters the sodium sulfate. The
column is now ready for use.
b. Pipet a 1.0 ml aliquot of the concentrated sample
extract (previously reduced to a total volume of
2.0 ml) onto the column. As the last of the
sample passes into the sodium sulfate layer,
rinse down the internal wall of the column twice
with 0.25 ml of n-hexane. Then assemble the
upper section of the column. As the last of the
n-hexane rinse reaches the surface of the sodium
sulfate, add enough n-hexane (volume predetermined,
see 10.4.3) to just elute all the PCB's present
in the sample. Apply air pressure and adjust
until the flow is 1 ml/min. Collect the desired
volume of eluate (predetermined, see 10.4.3)
in an accurately calibrated ampule. As the last
of the n-hexane reaches the surface of the sodium
sulfate, release the air pressure and change the
collection ampule.
_L-
-------
c. Fill the column with 0.50% diethyl ether in
benzene, again apply air pressure, and adjust
flow to 1 ml/min. Collect the eluate until all
the organochlorlne pesticides of Interest have
been eluted (volume predetermined, tee 10.4.3).
d. Analyze the eluates by gas chromatography.
10.4.3 Determination of Elution Volumes
10.4.3.1 The elutlon volumes for the PCB's and the pesticides •
depend upon a number of factors which are difficult to
control. These include variation in:
a. Mesh size- of the silica gel
b. Adsorption properties of the silica gel
c. Polar contaminants present in the eluting solvent
d. Polar materials present in the sample and sample
solvent
e. The dimensions of the microcolumns
Therefore, the optimum elution volume must be experi-
mentally determined each time a factor is changed. To
determine the elution volumes, add standard mixtures
of Aroclors and pesticides to the column and serially
collect 1-ml elution volumes. Analyze the individual
eluates by gas chromatography and determine the cut-
off volume for n-hexane and for ether-benzene. Figure
2 shows the retention order of the various PCS compon-
ents and of the pesticides. Using this information,
prepare the mixtures required for calibration of the
microcolumn.
10.4.3.2 In determining the volume of hexane required to elute
the PCB's, the sample volume (1 ml) and the volume of
n-hexane used to rinse the column wall must be consid-
ered. Thus, if it is determined that a 10.0 ml elution
volume is required to elute the PCB's, the volume of
hexane to be added, in addition to the sample volume
but including the rinse volume, should be 9.5 ml.
10.4.3.3 Figure 2 shows that as the average chlorine content
of a PCB mixture decreases the solvent volume for
complete elution increases. Qualitative determination
(9.4) indicates which Aroclors are present and provides
the basis for selection of the ideal elution volume.
-------
This helps to minimize the quantity of organochlorine
pesticides which will elute along with the low-percent-
chlorine PCB's and insures the most efficient separations
possible for accurate analysis.
10.4.3.4 For critical analysis where the PCB's and pesticides
are not separated completely, the column should
be accurately calibrated (according to 10.4.3.1)
to determine the percent of material of interest that
elutes in each fraction. Then flush the column with
an additional IS ml of 0.50% ether in benzene followed
by 5 ml of n-hexane, and use this reconditioned column
for the sample separation. Using this technique one
can accurately predict the amount (%) of materials in
each microcolumn fraction.
10.5 Microcolumn Separation of Sulfur, PCB's, and Pesticides
10.5.1 See procedure for preparation and packing microcolumn
in PCB analysis section (10.4.1 and 10.4.2).
10.5.2 Microcolumn Calibration
10.5.2.1 Calibrate the microcolumn for sulfur and PCB separation
by collecting 1.0 ml fractions and analyzing them by
gas chromatography to determine the following:
a. The fraction with the first eluting PCB's (those
present in 1260)
b. The fraction with the last eluting PCB's
(those present in 1221)
c. The elution volume for sulfur
d. The elution volume for the pesticides of interest
in the 0.50% ether-benzene fraction
From these data, determine the following:
e. The eluting volume containing only sulfur
(Fraction I)
f. The eluting volume containing the last of
the sulfur and the early-eluting PCB's (Fraction
ID
g. The eluting volume containing the remaining
PCB's (Fraction 111)
h. The ether-benz.e*e. eluting volume containing
the pesticides oi interest (Fraction iv)
-------
10.5.3 Separation Procedure
10.5.3.1 Carefully concentrate the 6% eluate from the florisil
column to 2.0 ml in the graduated ampule on a warm
water bath.
10.5.3.2 Place 1.0 ml (50%) of the concentrate into the
microcolumn with a 1 ml pipet. Be careful not to get
any sulfur crystals into the pipet.
10.5.3.3 Collect Fractions I and II in calibrated centrifuge
tubes.
Collect Fractions III and IV in calibrated ground-
glass-stoppered ampules.
10.5.3.4 Sulfur Removal (9) - Add 1 to 2 drops of mercury
to Fraction II stopper and place on a wrist-action
shaker. A black precipitate indicates the presence of
sulfur. After approximately 20 minutes the mercury
may become entirely reacted or deactivated by the
precipitate. The sample should be quantitatively
transferred to a clean centrifuge tube and additional
mercury added. When crystals are present in the
sample, three treatments may be necessary to remove
all the sulfur. After all the sulfur has been removed
from Fraction II (check using gas chromatography)
combine Fractions II and III. Adjust the volume to 10
ml and analyze by gas chromatography. Be sure no
mercury is transferred to the combined Fractions II
and III, since it can react with certain pesticides.
By combining Fractions II and III (if PCB's are
present), it is possible to identify the Aroclor(s)
present, and a quantitative analysis can be performed
accordingly. Fraction I can be discarded since it
contains only the bulk of the sulfur. Analyze Fractions
III and IV for the PCB's and pesticides.
If DDT and its homologs (aldrin, heptachlor, and/or
technical chlordane) are present along with the PCB's,
an additional microcolumn separation can be performed;
this may help to further separate the PCB's from the
pesticides (See 10.4)
11. Quantitative Determination
11.1 Measure the volume of n-hexane eluate containing the PCB's, and
inject 1 to 5 pi into the gas chromatograph. If necessary, adjust
the volume of the eluate to give linear response to the electron-
capture detector.
-------
11.2 Calculations
11.2.1 When a single Aroclor IB present, compare quantitative
Aroclor reference standards (e.g., 1242, 1260) to the
unknown. Measure and sum the areas of the unknown and the
reference Aroclor, and calculate the result as follows:
(A) (B) (V )
Micrograra/liter - -rr-r rrr-r— X N
*V * s}
A m ng °^ Standard Injected m ng_
Z of Standard Peak Areas mm
2
B « £ of Sample Peak Areas * (mm )
V • Volume of sample injected (yl)
V « Volume of Extract (yl) from which sample
is injected into gas chromatograph
V » Volume of water sample extracted (ml)
N « 2 when microcolumn is used
1 when microcolumn is not used
Peak Area - Peak height (mm) x Peak Width at 1/2
height, or calculated by an electronic
integrator)
11.2.2 For complex situations, use the calibration method
described in the following paragraphs. Small variations in
conponents between different Aroclor batches make it
necessary to obtain samples of several specific Aroclors.
These are available from commercial chromatography supply
companies.
11.2.2.1 Using the OV-1 column, chromatograph a known
quantity of each Aroclor reference standard. Also
chromatograph a sample of p,p'-DDE. Suggested concen-
tration of each standard is 0.1 ng/1 for the Aroclors
and 0.02 ng/1 for the p,p'-DDE.
11.2.2.2 Determine the relative retention time (RRT) of
each PCB peak in the resulting chromatograms using
p,p'-DDE as 100. See Figures 3 through 5.
RT x 100
RRT -
RTDDE
RRT • Relative Retention Time
RT - Retention time of peak of interest
RT
DDE * Retention time of p,p'-DDE
-------
Retention time is measured as that distance in
nun between the first appearance of the solvent peak
and the maximum for the compound.
11.2.2.3 To calibrate the instrument for each PCS, measure
the area of each peak.
Area - Peak height (mm) x Peak width at 1/2 height.
Using Tables 1 through 6, obtain the proper mean
weight factor; then determine the response factor
g/mm
(ng.) (mean weight percent)
ng/mm -
ng. » ng of Aroclor Standard Injected
Mean weight percent » obtained from Tables 1
through 6.
11.2.2.4 Calculate the RRT value and the area for each PCB
peak in the sample chromatogram. Compare the sample
chromatogram to those obtained for each reference
Aroclor standard. If it is apparent that the PCB
peaks present are due to only one Aroclor, then
calculate the concentration of each PCB using the
following formula:
2
ng PCB - ng/mm x Area
2
Where: Area - Area (mm ) of sample peak
ng/mm « Response factor for that peak measured.
Then add the nanograms of PCB's present in the
injection to get the total number of nanograms of
PCB's present. Use the following formula to calculate
the concentration of PCB's in the sample:
(£ng) (V£)
micrograms/Liter » (v ) (v ) x N
V « volume of water extracted (ml)
V » volume of extract (ul)
V. • volume of sample injected (yl)
2ng - sum of all the PCB's in nanograms for that
Aroclor identified
N » 2 when mlcrocoluom is used
N • 1 when microcolunm is not used
-------
The value can then be reported as micrograms/liter
PCS's reported as the Aroclor. For samples containing
more than one Aroclor, use Figure 9 chronatogram
divisional flow chart to assign a proper response
factor to each peak and also identify the "most likely"
Aroclors present. Calculate the ng of each PCB isomer
present, and sum them according to the divisional flow
chart. Using the formula above, calculate the concen-
tration of the various Aroclors present in the sample.
12. Reporting Results
Report results in micrograms per liter without correction for recovery
data. When duplicate and spiked samples are analyzed, all data obtained
should be reported.
13. Variations to Procedure #9
Variation Effective
No. Date Description
1 May 1978 GC Column: 7% OV17 + 9% OV210 on
80/100 Chromosorb W in a
a 6 ft. x 2 mm ID glass
column
Temperature: 190 C isothermal
Injection: 27§ic
Detector: Ni at 325 C
Carrier: Ar/Me at 60 ml/min
14. Standardization of Florisil Column by Weight Adjustment Based on Adsorption
of Lauric Acid.
14.1 A rapid method for determining adsorptive capacity of Florisil is
based on adsorption of lauric acid from hexa'ne solution (6, 10). An
excess of lauric acid is used, and the amount not adsorbed is measured
by alkali titration. Weight of lauric acid adsorbed is used to
calculate, by simple proportion, equivalent quantities of florisil
for batches having different adsorptive capacities.
14.2 Apparatus
14.2.2 Buret. — 25 ml graduations.
14.2.2 Erlenmeyer flasks. — 125 ml narrow mouth and 25 ml,
glass stoppered.
14.2.3 Pipet. — 10 and 20 ml transfer.
14.2.4 Volumetric flasks. — 500 ml.
-------
14.3 Reagents and Solvents
14.3.1 Alcohol, ethyl. — USP or absolute, neutralized to
phenolphthalein.
14.3.2 Hexane, — Distilled from all-glass apparatus,
14.3.3 Laurie acid. — Purified, CP.
14.3.4 Laurie acid solution. — Transfer 10.000 g lauric acid to 500
ml volumetric flask, dissolve in benzene, and dilute to 500 ml
(ml * 20 mg).
14.3.5 Phenolphthalein Indicator. — Dissolve 1 g in alcohol and dilute
to 100 ml.
14.3.6 Sodium hydroxide. — Dissolve 20 g NaOH (pellets, reagent grade)
in water and dilute to 500 ml (IN). Dilute 25 ml IN NaOH to 500
ml with water (0.05N). Standardize as follows: Weigh 100-200
mg lauric acid into 125 ml Erlenmeyer flask. Add 50 ml neutral-
ized ethyl alcohol and 3 drops phenolphthalein indicator; titrate
to permanent end point. Calculate mg lauric acid/ml 0.05 JJ NaOH
(about 10 mg/ml).
14.4 Procedure
14.4.1 Transfer 2.000 g Florisil to 25 ml glass-stoppered
Erlenmeyer flasks. Cover loosely with aluminum foil and
heat overnight at 130 C. Stopper, and cool to room
temperature; add 20.0 ml lauric acid solution (400 mg),
stopper, and shake occasionally for 15 min. Let adsorbent
settle and pipet 10.0 ml of supernatant into 125 ml
Erlenmeyer flask. Avoid inclusion of any Florisil.
14.4.2 Add 50 ml neutral alcohol and 3 drops indicator solution;
titrate with 0.051? to a permanent end point.
14.5 Calculation of Lauric Acid Value and Adjustment of Column Weight
14.5.1 Calculate amount of lauric acid adsorbed on Florisil
as follows:
Lauric Acid value - mg lauric acid/g Florisil - 200 -
(ml required for titration x mg lauric acid/ml 0.05N NaOH).
14.5.2 To obtain an equivalent quantity of any batch of Florisil,
divide 110 by lauric acid value for that batch, and multiply
by 20 g. Verify proper elution of pesticides by 13.6.
14.6 Test for Proper Elution Pattern and Recovery of Pesticides.
Prepare a test mixture containing aldrin, heptachlor epoxide, p,p'-
DDE, dieldrin, Parathion, and malathion. Dieldrin and Parathion
should elute in the 15% eluate and all but a trace of malathion in
the 50% eluate and the others in the 6% eluate.
-------
15. References
(1) "Method for Organochlorine Pesticides in Industrial Effluents," U.S.
Environmental Protection Agency, National Environmental Research Center,
Analytical Quality Control Laboratory, Cincinnati, Ohio 45268, 1973.
(2) Leoni, V., "The Separation of Fifty Pesticides and Related Compounds
and Polychlorinated Biphenyls into Four Groups by Silica Gel Micro-column
Chromatography," Journal of Chromatography, 62. 63 (1971).
(3) McClure, V. E., "Precisely Deactivated Adsorbents Applied to the
Separation of Chlorinated Hydrocarbons," Journal of Chromatoeraphy,
20^168(1972).
(A) "Methods for Organic Pesticides in Water and Wastewater," U.S. Environ-
mental Protection Agency, National Environmental Research Center,
Analytical Quality Control Laboratory, Cincinnati, Ohio 45268, 1971.
(5) "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," Chapter 6, Section 6.4, U.S. Environmental Protection
Agency, National Environmental Research Center, Analytical Quality
Control Laboratory, Cincinnati, Ohio 45268, 1972.
(6) "Pesticide Analytical Manual," U.S. Dept. of Health, Education, and
Welfare, Food and Drug Administration, Washington, D.C.
(7) Bellar, T. A. and Lichtenberg, J.J., "Method for the Determination
of Polychlorinated Biphenyls in Water and Sediment," U.S. Environmental
Protection Agency, National Environmental Research Center, Analytical
Quality Control Laboratory, Cincinnati, Ohio 45268, 1973.
(8) Webb, R.G. and McCall, A. C., "Quantitative PCS Standards for Electron
Capture Gas Chromatography." Presented at the 164th National ACS
Meeting, New York, August 29, 1972. (Submitted to the Journal of
Chromatographic Science for publication).
(9) Goerlitz, D. F. and Law, L. M., "Note on Removal of Sulfur Interferences
from Sediment Extracts for Pesticide Analysis," Bulletin of Environmental
Contamination and Toxicology, 6, 9 (1971).
(10) Mills, P.A., "Variation of Florisil Activity: Sample Method for Measuring
Adsorbent Capacity and its Use in Standardizing Florisil Columns,"
Journal of the Association of Official Analytical Chemists. 51, 29 (1968).
-------
Table 1
Composition of Aroclor 1221
HPT8
11
14
16
19
21
28
32
P7
Uo
Total
Mean
Weight
Percent
31.8
19.3
10.1
2.8
20.8
5.4
1.4
1.7
93.3
Relative
6td. Dev.*>
IS. 8
9.1
9.7
9.7
9.3
13.9
30.1
48.8
Number of
Chlorines?
1
1
2
2
2
2] 65%
3J 15%
21 104
3J 90%
3
3
•Retention time relative to p,p*-DDE«100. Measured froc
first appearance of solvent. Overlapping peaXs that axe
quantitated as one peak are bracketed.
bstandard deviation of seventeen results as a percentage
of the mean of the results.
CFroxn GC-MS data. Peaks containing mixtures of isoroers
of different chlorine numbers are bracketed.
-------
Table 2
Composition of Aroclor 1232 C8>
HPT*
11
14
16
f20
Ul
28
32
37
40
47
54
58
70
78
Total
Mean
Weight
Percent
16.2
9.9
7.1
17.8
9.6
3.9
6.6
6.4
4.2
3.4
2.6
4.6
1.7
94.2
Relative
Std. Dev.b
3.4
2.5
6.6
2.4
3.4
4.7
2.5
2.7
4.1
3.4
3.7
3.1
7.5
Kunber of
Chlorinesc
1
1
2
2
2
21 40%
3J 60%
3
3
3
4
3] 331
4J 67%
4
4] 90%
5J 10%
4
•Retention tiroe relative to p,pf-DD£«100. Measured fror.
first appearance of solvent. Overlapping peaXs that are
quantitated as one peak are bracketed.
^Standard deviation of four results as a Dean of the
results.
CFron GC-MS data. Peaks containing mixtures of isomers
of different chlorine numbers.are bracketed.
-------
Table 3
Composition of Aroclor 1242
HKT8
11
16
21
28
32
37
40
47
54
58
70
78
64
98
104
125
146
Total
Mean
Weight
Percent
1.1
2.9
11.3
11.0
6.1
11.5
11.1
8.8
6.8
5.f
10.. 3
3.6
2.7
1.5
2.3
1.6
1.0
98.5
Relative.
Std. Dev.b
35.7
4.2
3.0
5.0
4.7
5.7
6.2
4.3
2.9
3.3
2.B
4.2
9.7
9.4
16.4
20.4
19.9
Nuraber of
Chlorines0
1
2
2
21 25%
3J 75%
3
3
3
4
31 33%
4J 67%
4
41 so;
5J 10%
4
5
5
5
51 85%
6J 151
51 75%
6J 25%
•Retention time relative to p,p*-DDE»100. Measured froa
first appearance of solvent.
^Standard deviation of six results as a percentage of
the »ean of the results.
cFron CC-MS data. PeaXs containing mixtares of iscsoers
Of different chlorine mu&ers are bracketed.
(,0
-------
Table 4
Composition of Aroclor 1248
*KT*
21
26
32
47
40
47
54
58
70
78
84
96
104
112
125
146
Total
Mean
Weight
Percent
1.2
5.2
3.2
8.3
8.3
15.6
9.7
9.3
19.0
6.6
4.9
3.2
3.3
1.2
2.6
1.5
103.1
Relative
Std. Dev.b
23.9
3.3
3.6
3.6
3.9
1.1
6.0
5.6
1.4
2 • "*
2.6
3.2
3.6
6.6
5.9
10.0
Number of
Chlorines0
2
3
3
3
3*1 85%
4] 15%
4
31 10%
4J 90%
4
41 80%
5J 20%
4
5
5
41 10%
5J 90%
5
51 90%
6J 10%
51 85%
6J 15%
•detention time relative to p,p'-DD£«100. Measured from
first appearance of solvent.
^Standard deviation of six results as a percentage of
the mean of the results.
cPron CC-MS data. Peaks containing mixtures of isomers
of different chlorine numbers are bracketed.
-------
Table 5
Composition of Aroclor 1254
**!•*
47
54
58
70
84
98
104
125
14 £
160
174
203
232
Total
Mean
Weight
Percent
6.2
2.9
1.4
13.2
17.3
7.5
13.6
15.0
10.4
1.3
8.4
1.8
1.0
100.0
Relativew
Std. Dev.b
3.7
2.6
2.6
2.7
1.9
5.3
3.6
2.4
2.7
8.4
5.5
18.6
26.1
Number of
Chlorines
4
4
4
41 25%
5J 75%
5
5
5
5"! 70%
6J 30%
5T 301
6J 70%
6
6
€
7
•Retention time relative to p,p'-DD£«100. Measured froir
first appearance.of solvent.
^Standard deviation of six results' as a percentage of the
nean of the results.
cFro» GC-MS data. Pea3cs containing mixtures of isomers
are bracketed.
-------
Table 6
Composition of Aroclor 1260 Ct)
^&p^*fl
*\*%*
70
84
r 98
Ll 04
117
125
146
160
174
203
T232
[244
280
332
372
448
528
Total
Mean
Weight
Percent
2.1
4!7
3.8
3.3
12.3
14.1
4.9
12.4
9.3
9.8
11.0
4.2
4.0
.6
1.5
98.6
Relative
Std. Dev.b .
6.3
1.6
3.5
6.7
3.3
3.6
2.2
2.7
4.0
3.4
2.4
5.0
8.6
25.3
10.2
Number of
Chlorines0
5
5
-Id
5 60%
6J 40%
6
51 15%
6J 85%
6
6*1 50%
7J 50%
6
61 10*
7j 90%
1«
6 10%
7J 90%
7
7
8
8
8
•Retention tine relative to p,p'-DDE«100. Measured froE
first appearance of solvent. Overlapping peaks that are
quantitated as one.peak arc bracketed.
.•^StAndard deviation of six results as a mean of the
re suits .-
^From CC-MS data. PeaXs containing mixtures of isoners
Of different chlorine nxunb&rs are bracketed. *
^Composition determined at the center of peak 104.
^Composition determined at the center of peak 232.
-------
COMPRESSED
AIR
SUPPLY
PRESSURE
'GAUGE
SHUT-OFF
VALVE
REGULATOR
NEEDLE o
VALVE w
I em
FLEXIBLE
TUBING
SILICA GEL
5 err
I em
I 10/30
. 15ml
RESERVOIR
§ 10/50
23 cm i 4.2 mm t.D.
2 cm • 2 mm I.D.
FIGURE I. WICROCOLUMN SYSTEM
X-H
-------
o
o
r
c
a.
*j
c
C\J
1V101 JO lN3DH3d
I- c^S"
-------
37
AROCLOR 1242
Pi£urc 3. Column: 3% OV-1, Carrier Gas: Nitrogen at 60 ml/min,
Column Temperature: 170 C, Detector: Electron Capture
-------
\
AROCIOR 1254
125
JJ2
Figure 4. Column: 3% OY-1, Carrier Gas: Nitrtgen it SO nl/nin,
Column Temperature: 170 C, Detector: Electron Capture.
-------
AROCLOR 1260
Fifure 5. Column: 3% OY-1. Carrier fits: Nitrogen it SO ul/nin,
Column Temperature: 170 C, Detector: Electron Capture
-------
AROCLOR 1242
j
I
I
1
I
I
i
24
i I 12 15 II 21
IEUKTIOK TIKE IK MIKCTES
S. Ctluno: 1.5% DM7 * M5% |F-1, Cirrfir gts:
it SO • I/lie, Column Timpintin: 200 C, Pitictir: Eij:trn (iptire,
-------
e
r
^ S
. if
55
w
- i
-------
*
1
r»
ii
I *
I
i
-------
} tRT it first M«fc«<7? J
TES/ \«D
Is tken i fistiict
ink with IRT 71?
III 47-51?
TES
/
TES
X
*
Use 1242 fir
14
Use 1242 fir
peikjl IRT 70
\ I/
Use 1254
fir fills
1 IRT 104
Is then i listiict
ftik witk IRT 117?
•se 1260 fir
all piiks
TES
RO
lite 12S4 fir ill
l IRT 174
• si 1260 fir
all itker pnks
Fi£urt 9. ChromatO£rim Division Flowchart [8].
-------
Procedure Code: 14
Variations: 1-11
Title: Pentane Extraction of Organics in Wastewaters for GC Analysis
1. Summary
The following procedure is adapted from that reported by Henderson e_t_
ajUl). It has been successfully applied to the analysis of benzene,
toluene, ethylbenzene, and halogenated volatile compounds, including
chloroalkyl ethers, in a wide variety of industrial wastewaters. A snail
volume of water sample is shaken with pentane in a headspace-free bottle.
The pentane layer is then injected, without concentration, into a gas
chromatograph equipped with electron-capture or Hall detector for halogen-
ated compounds, or with an FID detector for detection of non-halogenated
compounds. This method differs from the microextraction procedure (Pro-
cedure Code #7) in the volume of extracting solvent and to sample ratio;
this ratio is larger in Procedure #14 than in #7. Alternative procedures
for many of the compounds analyzed for by procedures #14 and #7 are given
in the purge and trap method Procedure Code #8.
2. Sample Collection
Samples are collected in 40-ral vials, which are filled to overflowing,
then capped with a teflon-coated rubber septum lined with aluminum foil.
The septum is held in place by a screw-cap retainer with a center hole.
The samples are held at 4 C until analysis.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample-processing hardware
may yield discrete artifacts and/or elevated baselines, which cause
misinterpretation of gas chromatograms. All these materials must be
shown to be free of interferences under the conditions of the analysis,
by running method blanks with each set of samples.
3.2 Because of the non-specificity of the extraction procedure, co-
extracted compounds other than those to be analyzed may interfere.
The use of GC/MS or other confirmatory techniques, such as second GC
columns, multiple-phase equilibration (2), or solvent-partition tests
(3), may be required to confirm the qualitative analysis.
4. Reagents, Solvents, and Standards
4.1 Sodium sulfate - (ACS) Granular, anhydrous (conditioned at 400 C for
4 hours)
4.2 Pentane-nanograde; redistill in glass if necessary.
4.3 Organic compounds standar oercent purity).
-------
5. Sample Preparation
5.1 Allow the sample to come to room temperature. (If compositing is
required, pour the required volumes together in a chilled (ice-bath)
flask while the samples are still cold from storage; then place the
composite in a vial, seal the vial, and warm to room temperature).
5.2 Add 5.0 ml of sodium sulfate saturated organic-free water directly to
the vial by piercing the septum with two 10-ml syringes (22-gauge
needles). One syringe should contain the water solution, and the
other should be empty. Insert the vial, inject the sodium sulfate
solution, and discard the displaced water.
5.3 Add 5.0 ml of nanograde pentane to the vial through the sane syringe
system described in 5.2.
5.4 Shake the vial for 15 minutes at 500 rpm with a gyratory platform
shaker or equivalent.
5.5 Withdraw portions of the pentane layer as required for GC analysis.
(See method variations in Section 8).
6. Quality Control
6.1 Duplicate and spiked sample analyses are recommended as quality
control checks.
6.2 With each set of samples, extract and analyze a method blank, using
organic-free water.
7. Calculation of Results
7.1 Determine concentrations by using the absolute calibration procedure
or by using a suitable internal standard calibration procedure.
Henderson e£ a_l (1) found that an internal standard could be conven-
iently added to the pentane used for extracting samples and standards.
(A) (B) (Vt)
micrograms/liter = TV~)—(V~~)
, . ng standard
where: A == —*—i—-.
standard area
B = Sample aliquot area
V. « Volume of extract injected (ul)
V » Volume of total extract (yl)
V = Volume of water extracted (ml)
-------
7.2 Report results in raicrograms per liter without correction for recovery
data. When duplicate and spiked samples are analyzed, all data
obtained .must be reported on Analytical Quality Control Report sheets.
8. Variations to Procedure Code //14:
Variation No.
Effective
Date
June 1977
June 1977
June 1977
Description
GC Column: 3% OF-1 on 80/100"mesh
Chromosorb WHP in a 6' x 2 mra ID glass
column.
Temperature: 35°C isothermal
Injector: 200°C
Detector: Ni63 at 300°C
Carrier: Argon/methane at 40 ml/min.
(Source: EEI)
GC Column: 20% OV-101 +0.1% Carbowax
1500 on 80/100 mesh Chromosorb W in a
6* x 2mm ID glass column.
Temperature: 65 C for 30 min; 65 C to
110 C at 10°/min.; hold at 110 C for
60 min. if dichlorobenzene is present.
at 325°C
Injector: 175 C
Detector: Ni
Carrier: Argon/methane at 40 ml/min.
(Source: EEI)
GC Column: 20% OV-101 + 0.1% Carbowax
1500 on 80/100 mesh Chromosorb W in a
6' x 2 mm ID glass column.
Temperature: 65 C isothermal
Injector: 175°C
Detector: Ni63 at 300°C
Carrier: Argon/methane at 40 ml/min.
(Source: EEI)
t-ts
-------
June 1977 GC Column: 20% OV-101 + 0.1% Carbowax
1500 on 80/100 mesh Chromosorb W in a
6* x 2 mm ID glass column.
Temperature: 65°C to 110°C at 10°C/min.
Injector: 175°C
Detector: Ni63 at 325°C
Carrier: Argon/methane at AO ml/min.
(Source: EEI)
June 1977 GC Column: 20% OV-101 +0.1% Carbowax
1500 on 80/100 Chromosorb W in a 6' x 2
mm ID glass column.
Temperature: 70 C for 16 min., then
10°C/min to 110°C; hold 15 min.
Injector: 150°C
Detector: Ni63 at 325°C
Carrier: Argon/methane at AO ml/nin.
(Source: EEI)
May 1978 GC Column: 20% OV-101 + 0.1% Carbowax
1500 on 80/100 Chromosorb W in a 6' x 2
mm ID glass column.
Temperature: 90 C isothermal
Injector: 175°C
Detector: Ni63 at 300°C
Carrier: He at 50 ml/min
(Source: EEI)
June 1977 GC Column: 20% SF-96 on 60/80 mesh
Chromosorb P-AW on a 6' x 1/8" OD
stainless steel column preceded by a 6'
x 1/8" OD stainless steel column packed
with Chromosorb 102.
Temperature: 75 C for 5 min.; program
to 150°C at A°C/min.
J
-------
10
11
Injector: 200 C
Detector: FID at 250°C
Carrier: He at 35 ml/min.
(Source: EE1)
June 1977 GC Column: 10% TCEP on 60/80 mesh
Chromosorb P in a 6' x 1/8" OD stainless
steel column.
Temperature: 90 isothermal
Injector: 145°C
Detector: FID at 190°C
Carrier: Ke at 35 ml/nin.
(Source: EEI)
June 1977 Same as (8), except Temperature = 70 C
isothermal
(Source: EEI)
May 1978 GC Column: 10% TCEP on 100/120 mesh
Chromosorb P-AW in an 8' x 1/8" OD
stainless steel column.
Temperature: 70 C isothermal
Injector: 175°C
Detector: FID
Carrier: He at 30 ml/min.
Jan. 1979 GC Column: 10% TCEP on 100/120 Chromo-
sorb P-AW in an 8' x 1/8" OD stainless
steel column.
Temperature: 75 C isothermal
Injector: 190°C
Detector: FID at 190°C
Carrier: He at 30 ml/min.
(Source: EEI)
-------
9. References
1. Henderson, J.E., Peyton, G.P., and Glaze W.H., "A Convenient Liquid-
Liquid Extraction Method for the Determination of Haloraethanes in
Water at the Parts-Per-Billion Level", in: Identification and Analysis
of Organic Pollutants in Water. Ed. L.H. Kieth, p 105, Ann Arbor
Science, Ann Arbor, MI (1977).
2. Beroza, M., and Bowman, M.C., "Identification of Pesticides at Nanogram
Level by Extraction p-Values", Anal. Chem.. .37, 291-292 (1965).
3. McAullife, C., "GC Determination of Solutes by Multiple Phase Equili-
bration", Chem. Tech., Jan., 46-51 (1971).
-------
Procedure Code: 14 ? / / 7 / 7 9
Variation 112 (Source: EEI) May, 1979
1. Allow the sample * to cone to room temperature, (If compositing
1> required pour the required volumes together in a chilled (ice-
bath) flask while the samples are still cold from storage; then
place the composite in a vial, seal the vial, and warm to room
temperature^-
* 40 ml vial or 125 ml vial, as required.
2. Remove 4.0 ml of sample with a 50-ml syringe. Then add 3.0 ml of
•odium sulfate saturated organic-free water using another 10 ml
syringe. This creates a 1.0 ml headspace in the vial.
3. Add 2.0 ml of nonograde pentane to the vial by piercing the septum
with two 10-ml syringes. One syringe should contain the pentane,
and the other should be empty. Insert the vial, inject the pentane,
and discard the displaced water.
4. Shake the vial for 15 minutes at 500 rpm with a gyratory platform
•baker or equivalent.
5. Withdraw portions of the pentane layer as required for GC analysis
6. GC Conditions (Non-Balogenated Compounds)
GC Column: 10Z 1, 2, 3-tricyanoethoxypropane
(TCEP) on 100/120 chromosorb PAW,
8* x 1/8" OD stainless steel column
Temperature: 75°C isothermal
Injector : 190°C
Detector : FID at 300°C
Carrier : H2 at 30 ml/mln
GC Conditions (Halogenated Compounds)
GC Column : 20Z OV-101 + 0,11 Carbowax 1500
on 80/100 Chromosorb VAW 6' x 2 mm
glass
Temperature: 60°C for 10 min. to 130°C at 15°/min
Injector s 130°C
Peteetor i Hi63 at 270°C
Carrier t N2 at 30 mil/mln
Variation 113
Same as Variation 112, except:
GC Temperature: 60 C Isothermal for halogenater compounds on 20Z
Q OV-101 + 0.1Z Carbowax 1500 Column.
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Procedure Code: 15
Title: Acid Extraction Procedures for Phenols
Variations: 1-2
1. Summary
The methods described here are similar to the EPA acid-extraction
procedure used during the screening of industrial effluents (1). Two
variations are presented. In Variation #1, the sample is extracted at
high pH (above pH 11) to remove base-neutral compounds, then is re-extracted
at pH<2. This extract can be analyzed for phenols with GC/FID, or it can
be back extracted into sodium hydroxide solution for cleanup by an A-26
anion exchange resin procedure similar to that of Chriswell et^ ai_. (2).
(See Procedure Code #5.) The final methylene chloride or hexane extract
is analyzed by GC/FID. In Variation #2, the sample is acidified and
extracted, as in the screening protocol (1), with methylene chloride.
GC/FID is then used instead of GC/MS for compound detection.
The following table gives the proper code numbers for each variation:
Variation Effective
No. Date Description
1 February 1979 Southwest Research
Method with A-26 resin
2 June 1977 Envirodyne/EPA Screening Method
2.0 Variation #1 (Southwest Research Method):
2.1 Measure a 1-liter water sample into a 1-liter graduated cylinder,
and pour into a 2-liter separatory funnel.
2.2 Adjust the pH to 11 or greater with 6N NaOH. Use multi-range pH
paper for measurement.
2.3 Extract 3 times with 60 ml of methylene chloride. (This extract
may be required for PAH (Procedure Code #17) or Pesticides-Phthalates
(Procedure Code #3); if so, do not discard.)
2.4 Adjust the pH of the water sample to 2 or less with 6N HC1, using
pH paper as indicator.
2.5 Extract 3 times with 60 ml of methylene chloride each time.
2.6 Combine the extracts in a 500-ml Kuderna-Danish evaporator, and
concentrate to about 5 ml on a steam bath.
-------
2.7 Solvent exchange to hexane (using 100 ml of hexane), and reconcentrate
to about 2 ml, using a stream of dry nitrogen for the final step.
2.8 Cleanup with A-26 Resin:
Since some neutral compounds may still be present in the hexane
extract and could cause artifact peaks during gas chromatography, the
following steps can be taken to remove neutral organics from the
phenols:
2.8.1 Pour the hexane extract into a 30-ml separatory funnel.
2.8.2 Extract 5 times with 5 ml of 0.IN NaOH, and combine the
NaOH extracts.
2.8.3 Prepare an Amberlyst A-26 column (10 on x 0.6 cm) from
resin that has been cleaned and sieved (2). Place the
column in the hydroxide form by washing the column with 20
ml of 0.1N NaOH, then with 20 ml of organic-free water.
Check the first portion of water with pH paper (pH should
be <9) to ensure that the resin is saturated with the base.
2.8.4 Flow the combined 0.1N NaOH extracts into the resin, down
to the top of the column.
2.8.5 Add 25 ml basic methanol (2 ml of 2N NaOH in 23 ml of
methanol), then 25 ml of organic-free water; discard the
eluate.
2.8.6 Place a 125-ml separatory funnel under the column, and
elute with 25 ml of 4N HC1 followed by 25 ml of organic-
free water.
2.8.7 Extract the eluate from step 2.8.6 with 25 ml of methylene
chloride, and drain the methylene chloride into a second
125-ml separatory funnel. Save the aqueous layer.
2.8.8 Elute the resin with 25 ml of acetone:water (5:1, v:v),
followed by elution with 25 ml of organic-free water,
into the second separatory funnel with the methylene
chloride.
2.8.9 Extract by shaking for two minutes, and drain the methylene
chloride layer into a 500 ml K-D evaporator. Save the
aqueous layer.
T -
-------
2.8.10 Repeat steps 2.8.7 and 2.8.9 twice more, with fresh 25-ml
portions of methylene chloride.*
2.8.11 Concentrate the combined organic extracts to about 5 ml
on a steam bath.
2.8.12 Solvent exchange the methylene chloride extract into
hexane (using 100 ml of hexane), and concentrate to 1 ml
for analysis, using a stream of dry nitrogen.
2.9 GC Conditions:
Column: 1% SP-1240DA on 100/120 mesh Supelcoport in a 6' x 2mm
ID glass column.
Temperature: 80°C for 2 min, 80° to 180° at 8°/min, hold at
180° for 15 min.
*This step differs from the Chriswell et al. (2) procedure, which does not
use the two additional liquid-liquid extractions of the aqueous layers.
Injector: 250°C
Detector: FID at 250°C
Carrier: He at 60 ml/min.
2.10 Quality Control and Quantification:
2.10.1 Duplicate and spiked sample analyses are recommended as
quality controls. Each set of sample analyses is preceded
by a method blank using organic-free water.
2.10.2 Calculations of phenol concentration are determined by
using the following calibration procedure, or by using
another suitable relative calibration procedure.
(A) (B) (Vt)
Micrograms/1 ~
(V±) (Vs)
yg Standard
Standard area
B = Sample aliquot area
V - Volume of extract injected (yl)
V - Volume of total extract (yl)
V « Volume cf water extracted (ml)
x-
-------
3.0 Variation #2 (Envirodyne/EPA Screening Method).
3.1 Transfer a 1000 ml sample to a separatory funnel, and adjust the
pH to 2 or less with 6N HC1.
3.2 Extract serially with 100 ml, 50 ml, and 50 ml of distilled-in-
glass methylene chloride.
3.3 Dry and filter the solvent extract by passing it through a short
column of sodium sulfate. (The sodium sulfate should be prewashed
in the column, using methylene chloride.) If necessary, the
sodium sulfate should be pretreated by heating at 500 C for 2
hours in an oven to remove interfering organics.
3.4 After passing the extract through the sodium sulfate, rinse the
column with methylene chloride and add the rinsings to the
extract.
3.5 Concentrate the extract to 5-10 ml in a 500 ml Kuderma-Danish
apparatus with a 3-ball macro Snyder column and 10 ml receiver
tube attached. Cool to room temperature, add fresh boiling
chips, and attach a micro Snyder column to the receiver tube.
Evaporate carefully to 1.0 ml or to the point when active
distillation ceases.
3.6 GC Conditions:
Column: 3% OV-17 on 80/100 mesh Chromosorb in a 6' x 1/8"
OD stainless steel column.
Temperature: 160° to 250°C at 8°/min.
Injector: 300°C
Detector: FID
Carrier: He at 35 ml/min.
3.7 Quantification is achieved by comparing peak areas of the sample
extracts to the peak area of a standard on the chromatograph.
Sample spikes are carried through the procedure to determine the
recovery of the various phenols.
-------
4.0 References
1. Sampling and Analysis Procedures for Screening of industrial
Effluents for Priority Pollutants, U.S. EPA Environmental
Monitoring and Support Laboratory, Cincinnati, OH, March
1977 Revised April, 1977.
2. Chriswell, C.D., Chang, R. C., and Fritz, J. S., "Chromato-
graphic Determination of Phenols in Water," Anal. Chem.,
47 1325-1329 (1975).
-------
Procedure Code: 16
Title: Analysis of Nitroaromatics
1. Summary
The method recommended for Nitroaromatics is that proposed by the
U.S. EPA, Method 609 (1). A 1-liter sample is extracted with methylene
chloride. The extract is dried and exchanged to toluene while being
concentrated to 1.0 ml. Nitrobenzene is measured by FID/GC; dinitro-
toluenes are measured by EC/GC. A florisil cleanup is provided to
eliminate interferences.
2. References
1. Methods for Organic Compounds in Municipal and Industrial Wastewater^
U.S. EPA Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio, March (1979).
-------
Procedure Code: 17
Variations: 1-5
REVISED
Title: Analysis of Polynuclear Aromatic Hydrocarbons (PAH) in Industrial
Wastevater
1. Summary
Four procedures for analysis of PAH compounds in industrial vastewaters,
•re described in this Procedure Code. In Variations 1 and 2, hexane is
used as an extraction solvent, followed by silica-gel cleanup. In
Variation 3, methylene chloride is used in a continuous extractor,
followed by a DHSO cleanup. Variation 4 also involves methylene chloride
extraction, with a separately funnel, followed by Florisil cleanup. The
following table lists the method variations:
Variation No. Effective Date Description
1 June, 1977 EEI Procedure a
2 January, 1979 EEI Procedure b
3 January, 1978 MRI Procedure
4 March, 1979 SwRI Procedure
5 May, 1979 EEI Procedure c
The SwRI extraction/cleanup procedure (Variation 14) is similar to the
extraction/cleanup procedure (Method 610) proposed by the U.S. EPA (1),
except for the use of a Florisil cleanup by SwRI instead of the silica-gel
cleanup in the EPA method.
Since the separation of some pairs of PAH compounds by conventional phase
packed-column gas chromatography is not possible, several options are
available to the analyst. With liquid chromatography and DV or fluorescence
detection (EPA Method 610) , all the priority pollutant PAH compounds can
be separated. Capillary-column GC has also been shown to separate them.
Liquid-crystal, packed-column GC has been shown to separate 2-ring to 6-
ring PAH compounds more effectively than conventional phase packed columns.
The difficult separations involving priority pollutants are the following
pairs:
a. benzo(a) pyrene from perylene
b. anthracene from phenanthrene
c. chrysene from benzo(a) anthracene
d. benzo(b) fluoranthene from benzo(k) fluoranthene
However, there may be other PAH compounds that interfere with the priority
pollutant PAH compounds in any of the analytical options presented here.
2. Variation tfl (EEI Procedure a)
2.1 Extract 500 ml of sample with one 100-ml portion and two 50-ml portions
of nanograde hexane. Concentrate the combined extracts to 1 ml using
a Kuderna-Danish evaporator followed by a stream of nitrogen.
-------
2.2 Transfer the extract to the top of a silica-gel column prepared in
a 4 1/2" "champagne" column (30 ml reservoir; Supelco> Inc.) with
about 4" of 60/80 mesh Davison Grade-12 silica gel. The silica gel
is activated by heating at 100 C for 20-30 minutes before use.
2.3 Elute the column with enough hexane to collect 5 ml of eluate; save
this eluate as Fraction #1. Elute with benzene and collect two 2-ml
fractions (#2 and #3). Finally, elute with enough benzene to collect
a 4-ml Fraction (#4).
2.4 Analyze benzene fractions #2 and #3 for PAH compounds by GC._ If
high concentrations are found in these fractions, analyze the last
benzene fraction (#4). Compute the volumes and concentrations in
each fraction, to determine the total weight of PAH recovered; then
compute the apparent concentration in the original sample, uncor-
rected for recovery. Report the results of duplicate and spiked
samples on quality-control sheets.
2.5 GC Conditions:
GC Column: 3Z OV-17 on 80/100 mesh Chromosorb W-AW in a 6'
x 1/8" OD stainless steel column.
Temperature: 140 C for 10 min., then programmed at 8 /min.
for 15 minutes, followed by 4°/min. to 280°C.
Injector: 280°C
Detector: FID at 300°C
Carrier: He at 50 ml/min.
Note: The four pairs of PAH compounds listed in Section 1
cannot be separated on this column.
3. Variation #2 (EEI Procedure b)
3.1 Extract 500 ml of sample with one 100-ml portion and two 50-ml
portions of nanograde hexane. Concentrate the combined extracts to
1 ml using a Kuderna-Danish evaporator followed by a stream of
nitrogen.
3.2 Transfer the extract to the top of a silica-gel column prepared in a
4 1/2" "Champagne" column (30 ml reservoir; Supelco, Inc.) with
about 4" of 60/80 mesh Davison Grade-12 silica gel. The silica gel
is activated by heating at 100°C for 20-30 minutes before use.
3.3 Elute the column with enough hexane to collect a 3-ml fraction (#1).
Next, collect a 2-ml fraction of hexane eluate (Fraction #2). Elute
with benzene and collect 2 ml of eluate; combine this 2-ml benzene
fraction with hexane Fraction #2. Continue to collect benzene
eluate until 7 ml of additional benzene eluate have been collected
in a separate fraction (Fraction
-------
3.4 Analyze Fraction #2 for an estimate of the quantity of PAH compounds
present in it. If the indicated amount is high, it may be necessary
to analyze Fraction $1 also. Otherwise, discard Fraction //I, and
analyze Fractions #2 and #3 as noted in 3.5.
3.5 Gas Chromatography Conditions
a. for Fraction #2 (and til if needed):
GC Column : 3% OV-17 on 80/100 Chromosorb W-AW in a 6' x 1/8"
stainless steel column.
Temperature: Hold at 110°C for 12 min., increase to 240°C at
10°/min., hold at 240°C for 5 min., and cool 5
minutes.
Injector: 280°C
Detector: FID at 300°C
Carrier: He at 35 ml/min.
b. for Fraction #3
GC Column: 2.5% BMBT liquid crystal on 100/120 mesh Chromosorb
WHP in a 4' x 1/8" stainless steel column (2).
Temperature: 185°C for 2 min., increase to 240°C at 4°/min.,
and hold for 10 minutes.
Injector: 265°C
Detector: FID at 265°C
Carrier: He at at 40 ml/minute.
4. Variation #3 (MRI Method) (3)
4.1 Measure and record the volume of sample. Adjust the pH to 11.0
or greater with 5N. NaOH. (If spiking is to be done, add an
aliquot of mixed PAH standard in acetone before pH adjustment).
4.2 Transfer the sample to a specially-designed liquid-liquid extractor
system (see Figure 1), which contains methylene chloride (Burdick and
Jackson, Muskegon, Michigan). Extract overnight (16-18 hours).
After extraction, drain off the methylene chloride and pass it
through a drying tube containing anhydrous sodium sulfate (ACS
grade, dried at 600°C and stored at 110°C) into a Kuderna-Danish (K-
D) evaporator with 10 ml of hexane in the receiver. Add boiling
chips and concentrate to about 10 ml.
-------
A.3 Transfer the hexane to a 125-ml separatory funnel, and extract
sequentially three times with 10 ml of dimethylsulfoxide (DMSO).
Combine the DMSO extracts, add 60 ml of deionized distilled water,
and extract three times sequentially with 90 ml of hexane in a 250-ml
separatory funnel.
4.4 Wash the hexane layer once, with an equal volume of water, to insure
removal of the DMSO; then pass the hexane solution of PAH compounds
through a drying tube of sodium sulfate into a K-D evaporator, and
concentrate to less than 5 ml. Further concentrate to 1.0 ml under a
gentle stream of prepurified nitrogen. This cleanup procedure is as
described in Natusch and Tomkins (3).
4.5 Analyze the extract of PAH compounds on a 30-m wall-coated open
tubular capillary column (WCOT) as follows:
GC Column: SE-30 WCOT capillary column, 30 in. long
(min: 2,500 effective plates/m) (Supelco, Inc.).
Temperature: 50°C for 1 min., then 8°C/min. to 260°C; hold
12 min. at 260°C.
Injector: 180°C
Detector: 250°C, FID
Inlet Split: 120:1 ratio
Carrier: 1.0 ml/min. N-; detector makeup flow 35 ml
min. N_.
Figure 2 shows a chromatogram from a 20 mg/1 standard (4 yl) injection.
5.0 Variation 14 (SwRI Method)
5.1 For extraction of PAH compounds, the EPA (1) procedure of Method is
used, as follows:
a. Measure a 1-liter water sample into a 1-liter graduated cylinder.
b. Pour into a 2-liter separatory funnel.
c. Adjust the pH to 11 or greater with 6N NaOH, using multirange pH
paper for measurement.
d. Extract 3 times with 60 ml of methylene chloride.
e. Combine extracts in a 500-ml Kuderna-Danish evaporator.
f. Evaporate to about 5 ml on a steam bath.
g. Solvent exchange to hexane, using 100 ml of hexane.
h. Concentrate to about 2 ml under a stream of dry nitrogen.
n
-------
5.2 Florisil preparation: Heat Florisil to 400 C overnight; add 3% HJO
(m/m) and .mix thoroughly; let stand for at least 2 hours. Use a
10mm ID column chromatography tube with a glass frit at the bottom
and a teflon stopcock (Kontes Catalog K420540-0213).
5.3 Cleanup of the hexane extract is performed as follows:
a. Add lOg of Florisil (32 H.O) to the column chromatography tube,
and tap to settle.
b. Add about 1cm of anhydrous sodium sulfate to the top of the
column.
c. Add hexane to the column and pre-elute 40 ml; discard the
eluate.
d. Drain until the solvent level is even with the top of the
sodium sulfate.
e. Add sample (in about 2 ml of hexane) to the top of the column.
f. Drain the sample into the column to the top of the sodium
sulfate.
g. Wash the sides of the tube with about 2 ml of hexane, and drain
to the top of the sodium sulfate.
h. Place a 500-ml Kuderna-Danish evaporator under the column.
i. Collect 20 ml of hexane eluate in th* K-D evaporator.
j. Evaporate the hexane eluate to less than 10 ml on a steam bath.
k. Transfer the concentrate to a solvent-rinsed 4-dram vial with a
teflon cap liner.
1. Adjust the final volume of concentrate to 5 ml. Evaporate
under nitrogen if necessary. (This is Fraction I, and contains
naphthalene).
m. Place another 500 ml K-D evaporator below the column.
n. Collect 40 ml of hexane eluate in the K-D evaporator.
o. Evaporate to less than 10 ml on a steam bath.
p. Transfer the concentrate to a solvent-rinsed 4-dram vial with a
teflon cap liner.
q. Adjust the final volume of concentrate to 5 ml. Evaporate
under nitrogen if necessary. (This is Fraction II, and contains
the other PAH compounds).
x-qc
-------
5.4 Gas chromatography is performed as follows for Fraction 1:
GC Column: 10.6 m x 0.25 mm ID SP2100 Wall-Coaced open
tubular capillary column.
Inlet Split Ratio: 100:1
300°C, FID
Detector:
Injector:
Carrier:
Temperature:
(used for naphthalene)
250°C
He at 0.75 ml/min. after split
95°C for 8 min., then 32°/min. to 270°C;
hold 15 min. at 270°C.
5.5 Gas chromatography for Fraction 2 is performed in the same manner as
for Fraction 1, with only a change in the temperature program:
Temperature: 110°C to 270 C at 4°/min.; hold at 270°C for
15 minutes, (used for fluoranthene, acenaphthene,
anthracene, fluorene, phenanthrene, pyrene.)
5.6 Gas chromatography for 1,2-Benzanthracene, 3,4-Benzopyrene,
3,4-Benzofluoranthene, 11,12-Benzofluoranthene, Chrysene, 1,2-
Benzoperylene, Indeno-(l,2,3-c,d) - pyrene:
GC Column: 1.5% SP301
(BPhBT liquid crystal) (A) on 100/120 mesh
Supelcoport in a 6' x 2 mm ID glass column
Temperature:
Detector:
Injector:
Carrier:
265 C Isothermal
300°C
300°C
He at 30 ml/min
Variation #5 (EEI Procedure c)
6.1 Extract 2 liters of sample with one 100-ml and two 50-ml portions of
nanograde hexane. Dry the combined extracts over sodium sulfate,
and concentrate to 2 ml using a Kuderna-Danish evaporator, followed
by a nitrogen stream.
6.2 Split the extract into two one-mi portions. Analyze one portion-at
the following GC conditions (used for naphthalene and acenanhthylene):
-------
GC Column: 3% OV-17 on 80/100 mesh Chromosorb WHP in a
6' x 1/8" OD stainless steel column.
Temperature: Hold at 80"C for 7 min.; increase to 120°C at
4°/min.; hold for 5 min. cool 5 min.
Injector: 185°C
Detector: FID at 300°C
Carrier: He at at 40 ml/min.
6.3 Transfer the other one-mi portion of hexane extract to the top of a
silica gel column prepared in a 4%". Champagne column (30-ml reservoir,
Supelco, Inc.) with about 4 in. of 60/80 mesh, Davison Grade-12
silica gel. (The silica gel is activated by heating at 100°C for
20-30 minutes before use.)
6.4 Elute the column with 5 ml of hexane, and discard the eluate. Then
elute with 50 ml of 40 percent methylene chloride in hexane.
Concentrate this eluate to 1 ml for GC analysis at the following
conditions (used for anthracene and phenanthrene):
GC Column:
Temperature:
Injector:
Detector:
Carrier:
2.5% BMBT liquid crystal on 100/120 mesh Chromosorb
WHP in a -4' x 1/8" O.D. stainless steel column
with a 7" precolumn of 2.5% BMBT
185°C isothermal
265°C
FID at 300°C
He at 40 ml/min.
7. Quality Control
Duplicate and spiked sample analyses are recommended as quality control.
Each set of sample analyses is preceded by a method blank using organic
free water.
-------
8. Calculations
The concentration will be determined by using the absolute calibration
procedure described below or a suitable relative calibration procedure.
(A) (B) (V2)
8/1 " 0^) (Vg)
. ng standard
standard area
B « Sample aliquot area
V. • Volume of extract injected ( 1)
Vt - Volume of total extract ( 1)
V » Volume of water extracted (ml)
9. References
1. Methods for Organic Compounds in Municipal and Industrial Wastewater.
U.S. EPA Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio, March, 1979.
2. Janini, G.M. Johnston, K., and Zielinski, W.L., "Use of a Nematic
Liquid Crystal for Gas-Liquid Chromatographic Separation of Polyaromatic
Hydrocarbons", Anal. Chem., 47_, 670-674 (1975).
3. Natusch, D.F.S., and Tonkins, B.A., Isolation of Polycyclic Organic
Compounds by Solvent Extraction with Dimethyl Sulfoxide", Anal.
Chem.. .50, 1429-1434 (1978).
4. Janini, G.M., Muschik, G.M., Schroer, J.A., and Zielinski, W.L., Jr.
"Gas-Liquid Chromatographic Evaluation and Gas-Chromatography/Mass
Spectrometric Application of New High-Temperature Liquid Crystal
Stationary Phases For Polycyclic Aromatic Hydrocarbon Separations,"
Anal. Chem.. .48, 1879-1883 (1976).
3
-------
6mm
Glom Red
TFE Reducing Union,
10-6mm ftomd Through et 6mm
25mm I.D.
Glass Drip Tip •
TFE Reducing Union, 10-omm,
lonxf Through at 6mm
$24/40 ».H
250ml
1*5/50
TFE Stopcock
TFE Reducing Union, 10-emnt
TFE Tubing, 6mm O.D.
Figure 1. MRI
Continuous Liquid-Liquid Extractor
for Use with Methylene Chloride
-------
«H
3
00
•
CO
> S1
*-. *o
i S
0
s
pt
o
00
M
I
Acenaphthcne
Acenaphthylene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Chryaene
Benzo(a)anthracene
I-95-
-------
Chapter II
Quality Assurance Program for the Organic Chemicals Industry
(Self Verification Phase)
Background
The approach that has been taken by the Organic Chemicals Branch
for the verification phase of the BAT review 1s unique among the
Industrial categories covered by the Effluent Guidelines Division.
To provide the most extensive data set possible on the chemical
Industry, a program has been devised to allow representative plants
to self-sample and analyze their own process streams. By collecting
samples for 30-60 consecutive days, the project officers will develop
a data base representing the variations 1n the waste streams over
a significant period of operating time. The success of this type
of program will depend, 1n a large part however, on the control
that the EPA maintains over the various Industrial laboratories.
Since 1t has become Increasingly obvious that the caliber of quality
assurance programs 1n commercial and Industrial labs ranges widely
1n terms of acceptability, 1t 1s desirable to Initiate a well-controlled
quality assurance program prior to the Initial samplings. This will
Insure that the data received at the conclusion of the program 1s
of known quality and should prevent any need for resampling as a
result of questionable analytical design.
Summary
To meet this need for a uniform quality assurance procedure, a
program has been developed to provide as much data on the precision
and accuracy of the analyses as 1s possible in the prescribed time
limits, without overburdening the Industrial laboratory. In doing so,
we have attempted to apply the EMSL guidelines of March, 1978.*
Although these were written for a long-term routine analysis program
and required modification for moderate length programs such as
this, they do Incorporate most facets of a basic QA procedure, and
as such have served as a guide for our use.
The program has assumed that a viable methodology has already
been developed and has been designed to Include a two to three week
period for the Initial work-up of the procedures. During this period
of time, the Instrument capabilities will be Investigated, validation
of the selected methods will be completed, and Initial precision and
accuracy data will be obtained. This preparation period 1s essential
to Insure that plant personnel are familiar with all procedures
and that data of known, and acceptable, quality can be compiled.
External standards are best utilized 1n this work to avoid
any possible Interference with the spectrum of the compounds of
Interest. External standards are defined as a known amount of a pure
compound of Interest that 1s measured externally from Identically
the same compound in a sample or sample extract. The con-
centration of the standard should be within a factor of two of the
sample concentration. The recommended method of use 1s to quantltate
Quality Assurance Program for the Analyses of Chemical
Constituents in Environmental Samples (March, 1978)
JT- 1
-------
•ach run with a standard run Immediately following 1t. In the
Interest of time and sample through-put however, 1t Is acceptable
to use a calibration curve for quantltatlon 1f 1t 1s checked and
updated between every three analyses. If slgnTf leant deviations
occur between standard runs, 1t must be Immediately corrected to
reflect the condition changes.
Time Requirements
The time required for such a program will be significant, but
adherence to the outlined procedures should allow problems to be
recognized when they first occur and thus avoid any repetition of
sampling or analysis at a later date. The actual percentages of time
for quality assurance are difficult to predict for any one plant without
knowledge of the number of sampling points, the nature of the sampling
points, and the number of data points gained from each method utilized.
The data gained from the Initial two weeks may allow several sampling
points to be grouped for obtaining QA data. Thus the number of spiked
samples and duplicates may well be reduced. The following data on
time needed 1s determined as 1f each point and each method were
unique.
Time Required for N data points (assuming 1 sample per sample point
per day for 30 days and 1 method of analysis)
N * number of data points gained from each analysis (e.g. N «
25 1f 25 priority pollutants are characterized by the same
method.)
30 (N) » number of data points (defined above) over 30 days.
Initial Analyses (Two week preparation period)
(1) 5 (X) analyses for determining linearity of GC where X «
number of detector/column combinations to be used.
(2) Distilled Water Method Validation
4 extractions
8 (X) analyses
3 (X) standard analyses for quantltatlon
(3) Sample Matrix Method Validation
2 extractions for Initial quantltatlon
4 (X) analyses for Initial quantltatlon
4 extractions for spiked analyses
8 (x) analyses for spiked analyses
3 (X) standard analyses for quantltatlon
for spiked analyses
Routine Analyses (30 day survey)
(1) Data Points
-------
30 extractions
30 (x) analyses
(2) Instrument checks (each shift)
Composite analytical standards of pollutants 1n question-
set of standards to check Instrument
and update standard curve
(3) Spiking/Recovery Studies
10 extractions (1 every 3 days)
10 (x) analyses
(4) Duplicate Analyses
20 extractions (1 every 3 days)
40 (x) analyses (Includes duplicates for both
samples and spiking studies)
for every 30 (x) sample points, 60 extractions
will be required.
for every 30 (x) sample points, 120 (x) analyses
will be required, plus additional runs to keep calibration
curve update or to quantltate ( 40-120 additional
analyses).
Recommendations
The following program of quality assurance should be released
to the participating Industries with the recommendation that 1t be
followed as closely as possible to Insure complete and comparable
data from all laboratories. Alternative plans may be drafted and
utilized by the Industrial firms 1f they are considered to be of
equal merit by the EPA, but these must be well-documented and be
approved prior to use. Industries may be warned that 1f their program
1s judged to.be Inadequate, or scientifically unsound, the data will
not be given full consideration 1n the evaluation of the verification
phase results.
Instrument Requirements and Calibration
The following equipment Is recommended:
1. One or more gas chromatographs equipped with
two different detector types, 1f possible, under a given
method.
2. Two distinct column types, preferably one polar
and one non-polar to allow Independent qualitative and
quantitative determinations. It 1s necessary, for truly
reliable characterization to have two Independent con-
firmations of the data.
3. An automated Injection system. The accuracy of
external standards depends upon the consistency of the
-------
Injection technique coupled with operator control. The
use of an automated system will standardize that source
of error and will allow fewer duplicate analyses during
the routine analysis program.
4. A data handling system. The volume of data that
will be generated 1n the relatively short period of time
will necessitate the use of a data handling program.
at the least, a peak Integrator.
Before any GC runs are made for quant1 tat1on purposes, the linearity
of the detectors must be determined. This 1s done by analyzing four or
more solutions of the priority pollutants of Interest. The concentrations
of the pollutants must vary, with two bracketing the range of expected
concentration and the remainder spaced at equal Intervals along the curve.
If the range 1s very narrow, four standards will suffice. Otherwise, five
or more must be used to sufficiently determine linearity. Once the
working range has been established, samples must be diluted 1f necessary,
to remain within the proper limits. These limits must be determined 1n
all the detectors, and must be rechecked 1f any major changes are made
(e.g. replacement of detectors).
Records should be kept on all Instruments during the period of
preparation and the 30 day sampling period. These records should Include
descriptions of all prepared columns and of all maintenance, routine or
emergency. In addition, the preparation of all standard solutions should
be documented.
Construction of Calibration Curve
Prior to analysis of samples, a standard curve that covers the
entire working range of the method must be constructed with at least
five standards, Including one near the upper limit of the concen-
tration range. The other standards should be equally spaced throughout
the operating concentration range. The data gained from the determina-
tion of linearity, described above, may be used for this purpose.
Each day, 1f operation 1s continuous, or prior to analyzing each
group of samples, 1f operation 1s non-continuous, analyze a minimum
of two standards to establish the validity of the original standard
curve. These standards should represent the range of the standard
curve. If these standards fall outside the established limits, a new
standard curve must be constructed. These limits should be established
by the analyst as a part of his ongoing quality control program.
Method Validation; I. Distilled Water Matrix
To Insure that the prescribed methods are viable for given water
samples, each must be tested through a series of distilled water spikes.
This will also Indicate the ability of the laboratory to follow the
procedures.
A large sample of distilled water of known purity 1s spiked, and
thoroughly mixed, with those priority pollutants of Interest. These
spikes should be within the ranges observed in the screening phase, or,
-------
If such data 1s not available, it ten times the limit of detection.
Four extractions (and/or purges or appropriate method of preparation)
should be performed according to the specified method, and each should
be analyzed 1n duplicate under each set of conditions (different -
columns, different detectors, etc,}.
The parameters of Interest must be quantltated using updated
calibration curves. For each parameter, use the resulting observed
values (01, 02, 03, and 04) to calculate the standard deviation
(s) of the replicates as follows:
)S
\J
v>«, *\**drr o^ f* r*' • f r^f
Using the same set of data, calculate the mean percent recovery
(P) of the method as follows:
where T « theoretical concentration that should have resulted from
the spike.
The precision and accuracy criteria should be recorded and sub-
mitted to the Environmental Protection Agency prior to the Initiation
of sample analysis. These Initial criteria are supplemented by, and
periodically revised, updated, and Improved using the data acquired
during the laboratory's routine analytical quality assurance program.
Method Validation: II. Sample Matrix
For this work, a large sample from each waste stream should be
composited from several smaller samples so that five extractions (or
purges) can be completed. One portion should be prepared and analyzed
with no added fortification so that the amount of each pollutant
already present can be determined. The remaining portions should
then be spiked at levels approximating that of the actual sample,
and four replicate extractions should be made. Each extract should
be analyzed 1n duplicate under each of Instrumental conditions. The
standard deviations of the observed values should be recorded and
preliminary quality control charts should be made as described below.
This data should be reviewed by EPA personnel before the actual
sampling begins.
Construction of Quality Control Charts
For each pollutant, a chart 1s made showing the standard deviation
of the observed values. The charts allow a quick review of the data
to Insure that the system 1s not out of control.
-------
When the results exceed the boundaries (1n this case 3 standard
deviation units) the system 1s considered to be out of control.
Further work should not be resumed until the problem(s) 1s Isolated
and resolved.
Procedure for 30 Day Analysis Program
Once the Initial data Indicates that the laboratory has adequate
control of the system, the routine sampling and analysis may proceed.
Quality control data will be charted as previously described and
any sign of loss of control must be Investigated. The analysis program
must Include the following elements:
(1) Method blanks
A method blank must be determined for each set of samples
analyzed and whenever a new source (new container) of reagent
or solvent 1s Introduced Into the analytical scheme. (Note:
the Individual solvents and reagents should be checked for
purity prior to use 1n determining the method blank or 1n
the analysis of samples.)
To determine the method blank, take a quantity of reagents
equivalent to that used 1n the analysis of the sample and carry
them through the entire analytical procedure Including all
glassware and other materials that may come Into contact with
the sample. Determine a method blank for each class of compounds
to be determined, I.e., pesticides, base-neutrals and acids,
metals, phenol1cs, cyanides, etc.
Reagents having background levels that Interfere with
the compounds to be determined must be purified and shown to
be acceptable or replaced with some that are acceptable prior
to proceeding with the analyses. Problems encountered and
corrective actions taken should be reported for Information and
possible resolution of problems encountered by other analysts.
(2) Field Blanks
A field blank must be analyzed with each set of samples
from a given source. This 1s particularly Important whenever
automatic samplers are used for collection of samples. The blanks
must be analyzed 1n the same manner as the sample. Field blanks
for purgeables are sent from the laboratory to the sampling site
and returned as a check on possible contamination of the sample
by permeation of volatHes through the septum seal.
When Interferences occur, the analytical results must be dis-
carded unless sufficient data from these blanks 1s available to
permit correction of the results.
-------
(3) Spiked Samples
The sample from each sampling point (or group of sampling
points) 1s used In spiking and recovery studies every third '
day.Determine the recovery of the method for the analysis of
environmental samples by adding a spike (T;, true value)
sufficient to approximately double the background concentration
level (X-J of the sample selected. If the original concentration
1s higher than the midpoint of the standard curve (range of the
method), then the concentration of the spike should be
approximately one-half the original concentration. If the con-.
centratlon of the original sample was not detectable, the
concentration of the spike should be five to fifteen times the
lower limit of detections The volume of standard added 1n
aqueous solution should not dilute the sample by more than
ten percent. The volume of standard added In an organic solvent
solution should be kept small (100 ul/1 or less), so that the
solubility of the standard 1n the water will not be affected.
Analyze the sample, calculate the observed value (Oj
and then calculate the recovery for the spike as follows:
where PJ. 1s the percent recovery. If the sample was diluted due
to the addition of the spike, adjust X-t accordingly.
After the Initial analyses (See above) the first five
recovery data points should be combined with those determined
on the Initial method validation work and the mean percent
recovery (P) and standard deviation (3f) of the recovery should
be calculated: .-
t»\
where n « the number of percent recovery values available.
Subsequent results should be compared to this data. If the
percent recovery of the spike 1s not within the Interval of
P + 3S? , the system 1s out of control, and the source of this
systematic error should be Identified and resolved before
continuing with routine analysis. Quality control charts, as
previously described should be maintained.
(4) Replicate Analyses
Every third sample should be extracted 1n duplicate and
each extract should be analyzed 1n duplicate. After two weeks,
IT-7
-------
use the obtained results to calculate the range (R;) of these
results as follows:
where R;1s the difference between the results of the pair
(x^and xlt) from sample 1 « 1 through n. The concentration
of each sample 1s represented by the mean:
'
a.
- .
where Xi 1s the average of the results of the replicate pair.
A preliminary estimate of the critical difference (R^) between
replicate analyses for any specific concentration level (C) can
be calculated as:
From these data develop a table of such Revalues for various
C values that span the concentration range of Interest.
These preliminary drtlcal difference values may be used
to Judge the acceptability of the succeeding replicate results.
To do this, calculate the mean(x) and difference (R) between
the replicate results. Referring to the table of critical range
values developed above, find the nearest to 3 and use Its R^
to evaluate the acceptability of R. If the R 1s greater than
R*, the system precision 1s out of control and the source of
this unusual variability should be Identified and resolved before
continuing with routine analyses.
(5) Check Samples
Check samples should be prepared from standards of known
purity (e.g. compounds received from National Bureau of
Standards, or other recognized primary standards). The check
standards should be approximately equal to the concentration
found 1n routine samples. Alternately, one standard above and
one standard below the midpoint of the range of the method should
be used. Analyze the standard and calculate Oc (the observed
value). The percent recovery (P;) 1s then calculated as follows:
,* \ooCoi)
V «—••-•—•
where T^ * the true value.
After determining the Pj, for approximately 15 check standards,
calculate the mean (P) and standard deviation (S^) of the percentages
as follows:
- 8
-------
?
and:
V J^iU.**- (£,p)i/lx
where n « the number of results available.
If the percent recovery for succeeding check standards 1s
not within the Interval of P *_2 Sp, the system should be checked
for problems. If problems exist, they must be resolved before
continuing with routine analysis.
Evaluation of Quality Assurance Data
After the Initial two weeks of sampling, the precision and accuracy
data should be compiled to represent the Initial five samples.
Standards of performance should be defined based on these results,
and the remaining analyses should fit the defined criteria. Procedures
and equations for obtaining this Information have been given under
the appropriate section. Problems must be resolved before the analysis
1s continued.
At the end of the program all quality assurance data should be
tabulated and returned to EPA. All data sheets, Including charts
and raw data should be Included 1n the final report.
jr-9
-------
Chapter III
PROPOSED PRIORITY POLLUTANT
TEST PROCEDURES
Parameter (ug/1)
Method No.
STORET No,
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
acenaphthene
acrolein
aery Ion itrile
benzene
benzidine
carbon tetrachloride
chlorobenzene
1 ,2,4-trichlorobenzene
hexachlorobenzene
1,2-dichloroethane
1 ,1 ,1-trichloroethane
hexachloroethane
1,1-dichloroethane
1 , 1 ,2-trichloroethane
1 , 1 ,2,2-tetrachloroethane
chloroethane
b is (chloromethyl) ether
bis(2-chloroethyl)ether
2-chloroethyl vinyl ether
610,
603,
603,
602,
605,
601,
601,
612,
612,
601,
601,
612,
601,
601,
601,
601,
625
624
624
624
625*
624*
602, 624*
625*
625
624
624
625
624
624*
624*
624
No method
611,
601,
625
624
34205
34210
32415
34030
39120
32102
34301
34551
39700
34531
34506
34396
34496
34511
34516
34311
34268
34273
34576
-------
20. 2-chloronaphthalene
21. 2,4,6-trichlorophenol
22. 4-chloro-3-methyl phenol
23. chloroform
24. 2-chlorophenol
25. 1,2-dichlorobenzene
26. 1,3-dichlorobenzene
27. 1,4-dichlorobenzene
28. 3,3'-dichlorobenzidine
29. 1,1-dichloroethene
30. trans-l,2-dichloroethene
31. 2,4-dichlorophenol
32. 1,2-dichloropropane
33. trans-l,3-dichloropropene
34. cis-l,3-dichloropropene
35. 2,4-dimethylphenol
36. 2,4-dinitrotoluene
37. 2,6-dinitrotoluene
38. 1,2-diphenylhydrazine
39. ethylbenzene
40. fluoranthene
41. 4-chlorophenyl phenyl ether
42. 4-bromophenyl phenyl ether
43. bis(2-chloroisopropyl)ether
612, 625 34581
604, 625 34621
604, 625 34452
601, 624* 32106
604, 625 34586
601, 602, 612, 625 34536
601, 602, 612, 625 34566
601, 602, 612, 625 34571
605, 625 34631
601, 624 34501
601, 624 34546
604, 625 34601
601, 624 34541
601, 624 34561
601, 624 34561
604, 625 34606
609, 625 34611
609, 625 34626
625 34346
602, 624 34371
610, 625 34376
611, 625 34641
611, 625 34636
611, 625 34283
11
-------
44. bis(2-chloroethoxy)methane
45. methylene chloride
46. chloromethane
47. bromomethane
48. bromoform
49. bromodichloromethane
50. fluorotrichloromethane
51. dichlorodifluoromethane
52. chlorodibromomethane
53. hexachlorobutadiene
54. hexachlorocyclopentadiene
55. isophorone
56. naphthalene
57. nitrobenzene
58. 2-nitrophenol
59. 4-nitrophenol
60. 2,4-dinitrophenol
61. 4,6-dinitro-2-methylphenol
62. N-nitrosodimethylamine
63. N-nitrosodiphenylamina
64. N-nitrosodipropylamine
65. pentachlorophenol
66. phenol
67. bis(2-ethylhexyl)phthalate
611, 625
601, 624*
601, 624
601, 624
601, 624
601, 524
601, 624
601
601, 624
612, 625
612, 625
609, 625
610, 625
609, 625
604, 625
604, 625
604, 625
604, 625
607, 625
607, 625
607, 625
604, 625*
604, 625
606, 625
34278
34423
34418
34413
32104
32101
34488
34668
32105
34391
34386
34408
34696
34447
34591
34646
34616
34657
34438
34433
34428
39094
34694
39100
-------
68. benzyl butyl phthalate
69. di-n-butyl phthalate
70. di-n-octyl phthalate
71. diethyl phthalate
72. dimethyl phthalate
73. benzo(a)anthracene
74. benzo(a)pyrene
75. benzo (b) fluoranthene
76. benzo(k)fluoranthene
77. chrysene
78. acenaphthylene
79. anthracene
80. benzo(ghi)perylene
81. fluorene
82. phenanthrene
83. dibenzo(ah)anthracene
84. indeno(l,2,3-cd)pyrene
85. pyrene
86. tetrachloroethene
87. toluene
88. trichloroethene
89. vinyl chloride
90. 2,3,7,8-tetrachlorodibenzo
-p-dioxin
606, 625
606, 625
606, 625
606, 625
606, 625
610, 625
610, 625
610, 625
610, 625
610, 625
610, 625
610, 625
610, 625
610, 625
610, 625
610, 625
610, 625
610, 625
601, 624
602, 624
601
601
613
34292
34110
34596
34336
34341
34526
34247
34230
34242
34320
34200
34220
34521
34381
34461
34556
34403
34469
34475
34010
39180
39175
34675
-------
91. aldrin
92. dieldrin
93. chlordane
94. 4,4'-DDT
95. 4,4'-OOE
96. 4,4'-ODD
97. endosulfan I
98. endosulfan II
99. endosulfan sulfate
100. endrin
101. endrin aldehyde
102. heptachlor
103. heptachlor epoxide
104. a-BHC
105. b-BHC
106. d-BHC
107. g-BHC
108. toxaphene
109. PCB-1016
110. PCB-1221
111. PCB-1232
112. PCB-1242
113. PCB-1248
114. PCB-1254
115. PCB-1260
608, 625*
608, 625*
608, 625*
608, 625*
608, 625*
608, 625*
608, 625*
608, 625*
608, 625
608, 625*
608, 625
608, 625*
608, 625*
608, 625*
608, 625
608, 625
608, 625*
608, 625*
608, 625*
608, 625*
608, 625*
608, 625*
608, 625*
608, 625*
608, 625*
39330
39380
39350
39300
39320
39310
34361
34356
34351
39390
34366
39410
39420
39337
39338
39259
39340
39400
34671
39488
39492
39496
39500
39504
39508
*Approved EPA procedures are already available for these parameters
(Federal Register, December 1, 1976).
-------
PURGEABLE HALOCARBONS
METHOD 601
1. Scope and Application
1.1 This method covers the determination of 29 purgeable
halocarbons. The following parameters may be deter-
mined by this method:
Parameter STORET No.
Bromoform 32104
Bromodichloromethane 32101
Bromomethane 34413
Carbon tetrachloride 32102
Chlorobenzene 34301
Chloroethane 34311
2-Chloroethylvinyl ether 34576
Chloroform 32106
Chloromethane 34418
Dibromochloromethane 34105
1,2-Dichlorobenzene 34536
1,3-Dichlorobenzene 34566
1,4-Dichlorobenzene 34571
Dichlorodifluoromethane 34668
1,1-Dichloroethane 34496
1,2-Dichloroethane 34531
1,1-Dichloroethene 34501
trans-l,2-0ichloroethene 34546
771 I
-------
Parameter Storet No.
1,2-Dichloropropane 34541
cis-l,3-0ichloropropene 34561
trans-l,3-Dich1oropropene 34561
Methylene chloride 34423
1,1,2,2-Tetrachloroethane 34516
Tetrachloroethene 34475
1,1,1-Trichloroethane 34506
1,1,/2-Trichloroethane 34511
Trichloroethene 39180
Trichlorofluoromethane 34488
Vinyl chloride 39175
1.2 This method is applicable to the determination of these
compounds in municipal and industrial discharges. It
is designed to be used to meet the monitoring require-
ments of the National Pollutant Discharge Elimination
System (NPDES). As such, it presupposes a high expec-
tation of finding the specific compounds of interest.
If the user is attempting to screen samples for any or
all of the compounds above, he must develop independent
protocols for the verification of identity.
1.3 The sensitivity of this method is usually dependent
upon the level of interferences rather than instrumen-
tal limitations. The limits of detection listed in
Table 1 represent sensitivities that can be achieved in
wastewaters under optimum operating conditions.
-------
1.4 This method is recommended for use only by experienced
residue analysts or under the close supervision of such
qualified persons.
2. Summary of Method
2.1 An inert gas is bubbled through a 5 ml water sample
contained in a specially-designed purging chamber. The
halocarbons are efficiently transferred from the
aqueous phase to the vapor phase. The vapor is swept
through a short sorbent tube where the halocarbons are
trapped. After the purge is completed, the trap is
heated and backflushed with gas to desorb the halo-
carbons into a gas chromatographic system. A temper-
ature program is used in the GC system to separate the
halocarbons before detection with a halide-specific
detector.
2.2 If interferences are encountered, the method provides
an optional gas chromatographic column that may be
helpful in resolving the compounds of interest from the
interferences.
3. Interferences
3.1 Impurities in the purge gas and organic compounds
out-gasing from the plumbing ahead of the trap account
for the majority of contamination problems. The analy-
tical system must be demonstrated to be free from
contamination under the conditions of the analysis by
3
-------
running method blanks. Method blanks are run by charg-
ing the purging device with organic-free water and
analyzing it in a normal manner. The use of non-TFE
plastic tubing, non-TFE thread sealants, or flow
controllers with rubber components in the purging
device should be avoided.
3.2 Samples can be contaminated by diffusion of "volatile
organics (particularly freons and methylene chloride)
through the septum seal into the sample during shipment
and storage. A sample blank prepared from organic-free
water and carried through the sampling and handling
protocol can serve as a check on such contamination.
3.3 Cross contamination can occur whenever high level and
low level samples are sequentially analyzed. To reduce
the likelihood of this, the purging device and sample
syringe should be rinsed out twice between samples with
organic-free water. Whenever an unusually concentrated
sample is encountered, it should be followed by an
analysis of organic-free water to check for cross
contamination. For samples containing large amounts of
water-soluble materials, suspended solids, high boiling
compounds or high organohalide levels, it may be
necessary to wash out the purging device with a soap
solution, rinse with distilled water, and then dry in a
105°C oven between analyses.
-------
4. Apparatus and Materials
4.1 Sampling equipment, for discrete sampling.
4.1.1 Vial, with cap - 40 ml capacity screw cap
(Pierce f13075 or equivalent). Detergent wash
and dry at 105°C before use.
4.1.2 Septum - Teflon - faced silicone (Pierce #12722
or equivalent). Detergent wash, rinse with tap
and distilled water, and dry at 105°C for one
hour before use.
4.2 Purge and trap device - The purge and trap equipment
consists of three separate pieces of apparatus: the
purging device, trap, and desorber. Several complete
devices are now available commercially. The device
must meet the following specifications: the unit must
be completely compatible with the gas chromatographic
system; the purging chamber must be designed for a 5 ml
volume and be modeled after Figure 1; the dimensions
for the sorbant portion of the trap must meet or exceed
those in Figure 2. Figures 3 and 4 illustrate the
complete system in the purge and the desorb mode.
4.3 Gas chromatograph - Analytical system complete with
programmable gas chromatograph suitable fcr on-column
injection and all required accessories Including
halide-specific detector, column supplier, reorder,
and gases. A data system for measuring - areas is
recommended.
-------
4.4 Syringes - 5-rol glass hypodermic with luerlok tip (2
each).
4.5 Micro syringes - 10, 25, 100 ul.
4.6 2-way syringe valve with Luer ends (3 each).
4.7 Syringe - 5-ml gas-tight with shut-off valve.
4.8 Bottle - 15-ml screw-cap, with Teflon cap liner.
5. Reagents
5.1 Sodium thiosulfate - (ACS) Granular.
5.2 Trap Materials
5.2.1 Porous polymer packing 60/80 mesh chroma-
tographic grade Tenax SC (2,6-diphenylene oxide).
5.2.2 Three percent OV-1 on Chromosorfa-W 60/80 mesh.
5.2.3 Silica gel-(35/60 mesh) - Oavison, grade-15 or
equivalent.
5.2.4 Coconut- charcoal 6/10 mesh Bamafay Chaney,
CA-580-26 lot # M-2649 or equivalent.
5.3 Activated carbon - Filtrasorb-200 '(Calgon Corp.) or
equivalent.
5.4 Organic-free water
5.4.1 Organic-free water is defined as water free of
interference when employed in the purge and trap
procedure described herein. It is generated by
passing tap water through a carbon filter bed
containing about 1 Ib. of
-------
activated carbon.
5.4.2 A water purification system (Millipore Super-Q
or equivalent) may be used to generate
organic-free deionized water.
5.4.3 Organic-free water may also be prepared by
boiling water for 15 minutes. Subsequently,
while maintaining the temperature at 90°C,
bubble a contaminant-free inert gas through the
water for one hour. While still hot, transfer
the water to a narrow mouth screw cap bottle and
seal with a Teflon lined septum and cap.
5.5 Stock standards - Prepare stock standard solutions in
methyl alcohol using assayed liquids or gas cylinders
as appropriate. Because of the toxicity of some of the
organohalides, primary dilutions of these materials
should be prepared in a hood. A NIOSH/MESA approved
toxic gas respirator should be used when the analyst
handles high concentrations of such materials.
5.5.1 Place about 9.8 ml of methyl alcohol into a 10
ml ground glass stoppered volumetric flask.
Allow the flask to stand, unstop- pered, for
about 10 minutes or until all alcohol wetted
surfaces have dried. Weigh the flask to the
nearest 0.1 mg.
nr-
-------
5.5.2 Add the assayed reference material:
5.5.2.1 Liquids - Using a 100 ul syringe, imme-
diately add 2 drops of assayed reference
material to the flask, then reweigh. Be
sure that the 2 drops fall directly into
the alcohol without contacting the neck of
the flask.
5.5.2.2 Gases - To prepare standards for any of
the six halocarbons that boil below 30°C
(bromomethane, chloroethane,
ch1oromethane, di ch1orodif1uoromethane,
trichlorofluoromethane, vinyl chloride),
fill a 5 ml valved gas-tight syringe with
the reference standard to the 5.0-ml
mark. Lower the needle to 5 rran above the
methyl alcohol menicus. Slowly inject the
reference standard above the surface of
the liquid (the heavy gas will rapidly
dissolve into the methyl alcohol).
5.5.3 Reweigh, dilute to volume, stopper, then mix by
inverting the flask several times. Transfer the
standard solution to a 15 ml screw-cap bottle
with a Teflon cap liner.
5.5.4 Calculate the concentration in micrograms per
microliter from the net gain in weight.
UT-
-------
5.5.5 Store stock standards at 4°c. Prepare fresh
standards weekly for the six gases and
2-chloroethylvinyl ether. All other stan- dards
must be replaced with fresh standard each month.
6. Calibration
6.1 Using stock standards, prepare secondary dilution stan-
dards in methyl alcohol that contain the compounds of
interest, either singly or mixed together. The
standards should be prepared at concentrations such
that the aqueous standards prepared in 6.2 will com-
pletely bracket the working range of the analytical
system.
6.2 Using secondary dilution standards, prepare calibration
standards by carefully adding 20.0 ul of standard in
methyl alcohol to 100, 500, or 1000 ml of organic-free
water. A 25 ul syringe (Hamilton 702N or equivalent)
should be used for this operation. These aqueous
standards must be prepared fresh daily.
6.3 Assemble the necessary gas chromatographic apparatus
and establish operating parameters equivalent to those
indicated in Table 1. By injecting secondary dilution
standards, establish the sensitivity limit and the
linear range of the analytical system for each compound.
6.4 Assemble the necessary purge and trap device. The trap
must meet the minimum specifications as shown in
-------
Figure 2 to achieve satisfactory results. Condition
the trap overnight at 180°C by backflushing with an
inert gas flow of at least 20 ml/nrin. Prior to use,
daily condition traps 10 minutes while backflushing at
180°C. Analyze aqueous calibration standards (6.2)
according to the purge and trap procedure in Section
8. Compare the responses to those obtained by injec-
tion of standards (6.3); to determine purging effici-
ency and also calculate analytical precision. The
purging efficiencies and analytical precision of the
analysis of aqueous standards must be comparable to
data presented by Bellar and Lichtenberg (1978) before
reliable sample analysis may begin.
6.5 By analyzing calibration standards, establish the sen-
sitivity limit and linear range of the entire analyti-
cal system for each compound.
7. Quality Control
7.1 Before processing any samples, the %alyst should daily
demonstrate through the analysis of an organic-free
water method blank that the entire analytical system is
i nterference-free.
7.2 Standard quality assurance practices should be used
with this method. Field replicates should be collected
to validate the precision of the sampling technique.
Laboratory replicates should be analyzed to validate
the precision of the analysis. Fortified samples
or 10
-------
should be analyzed to validate the accuracy of the
analysis. Where doubt exists over the identification
of a peak on the gas chromatogram, confirmatory tech-
niques such as mass spectroscopy should be used.
7.3 The analyst should maintain constant surveillance of
both the performance of the analytical system and the
effectiveness of the method in dealing with each sample
matrix by spiking each sample, standard and blank with
surrogate halocarbons. A combination of bromo-
chloromethane, 2-bromo-l-chloropropane, and
1,4-dichlorobutane is recommended to encompass the
boiling range covered by this method. From stock
standard solutions prepared as above, add a volume to
give 1000 ug of each surrogate to 45 ml of organic-free
water contained in a 50-ml volumetric flask, mix and
dilute to volume (20 ng/ul). Dose 5.0 ul of this
surrogate spiking solution directly into the 5 ml
syringe with every sample and reference standard
analyzed. Prepare a fresh surrogate spiking solution
on a weekly basis.
8. Sample Collection, Preservation, and Handling
8..1 Grab samples must be collected in glass containers
having a total volume in excess of 40 ml. Fill the
sample bottles in such a manner that no air bubbles
pass through the sample as the bottle is being filled.
Seal the bottle so that no air bubbles are entrapped
-------
in it. Maintain the hermetic seal on the sample bottle
until time of analysis.
8.2 The samples must be iced or refrigerated from the time
of collection until extraction. If the sample contains
free or combined chlorine, add sodium thiosulfate
preservative (10 mg/40 ml will suffice for up to 5 ppm
Cip) to the empty sample bottles just prior to ship-
ping to the sampling site, fill with sample just to
overflowing, seal the bottle, and shake vigorously for
1 minute.
8.3 All samples must be analyzed within 14 days of collec-
tion.
9. Sample Extraction and Gas Chromatography
9.1 Adjust the purge gas (nitrogen or helium) flow rate to
40 ml/min. Attach the trap inlet to the purging de-
vice, and set the device to purge. Open the syringe
valve located on the purging device sample introduction
needle.
9.2 Remove the plunger from a 5 ml syringe and attach a
closed syringe valve. Open the sample bottle (or stan-
dard) and carefully pour the water into the syringe
barrel until it overflows. Replace the syringe plunger
and compress the sample. Open the syringe valve and
vent any residual air while adjusting the sample volume
to 5.0 ml. Since this process of taking an aliquot de-
stroys the validity :" the sample for future analysis,
-------
the analyst should fill a second syringe at this time
to protect against possible loss of data. Add 5.0 ul
of the surrogate spiking solution (7.3) through the
valve bore, then close the valve.
9.3 Attach the syringe-syringe valve assembly to the
syringe valve on the purging device. Open the syringe
valves and inject the sample into the purging chamber.
9.4 Close both valves and purge the sample for 11.0 + .05
minutes.
9.5 After the 11 minute purge time, attach the trap to the
chromatograph, and adjust the device to the desorb
mode. Introduce the trapped materials to the GC column
by rapidly heating the trap to 180°C while back-
flushing the trap with an inert gas between 20 and 60
ml/min for 4 minutes. If rapid heating cannot be
achieved, the gas chromatographic column must be used
as a secondary trap by cooling it to 30°C (or sub-
ambient, if problems persist) instead of the initial
program temperature of 45°C.
9.6 While the trap is being desorbed into the gas chroma-
tograph, empty the purging chamber using the sample
introduction syringe. Wash the chamber with two 5 ml
flushes of organic-free water.
9.7 After desorbing the sample for approximately four
minutes recondition the trap by returning the purge and
trap device to the purge mode. Wait 15 seconds
Jt • 13
-------
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 seven
minutes turn off the trap heater and open the syringe
valve to stop the gas flow through the trap. When cool.
the trap is ready for the next sample.
9.8 Table 1 summarizes some recommended gas chromatographic
column materials and operating.conditions for the
instrument. Included in this table are estimated
retention times and sensitivities that should be
achieved by this method. An example of the separation
achieved by column 1 is shown in Figure 5. Calibrate
the system daily by analysis of a minimum of three
concentration levels of calibration standards.
10. Calculations
10.1 Determine the concentration of individual compounds
directly from calibrations plots of concentration
(ug/1) vs. peak height or area units.
10.2 Report results in micrograms per liter. When duplicate
and spiked samples are analyzed, all data obtained
should be reported.
11. Accuracy and Precision
The U.S. EPA Environmental Monitoring and Support Laboratory
in Cincinnati is in the process of conducting an inter-
laboratory method study to determine the accuracy and pre-
cision of this test procedure.
-------
BIBLIOGRAPHY
1. Bellar, T. A., and J. J. Lichtenberg, Journal American Water
Works Association, Vol. 66, No. 12, Dec. 1974, pp. 739-744.
2. Bellar, T. A., and J. J. Lichtenberg, "Semi-Automated
Headspace Analysis of Drinking Waters and Industrial Waters
for Purgeable Volatile Organic Compounds," Proceeding from
ASTM Symposium on Measurement of Organic Pollutants in Water
and Wastewater, June 1978 (In Press).
3. "Development and Application of Test Procedures for Specific
Organic Toxic Substances in Wastewaters. Category 11 -
Purgeables and Category 12 - Acrolein, Acrylonitrile, and
Dichlorodifluoromethane." Report for EPA Contract
68-03-2635 (In preparation).
jzr
-------
TABLE 1
Organohalides Tested Using Purge and Trap Method
Retention Time Detection Limitt
Compound (min.) ug/1
Column 1 Column 2
Chloromethane
Bromomethane
Dichlorodifluoromethane
Vinyl chloride
Chloroethane
Methylene chloride
Trichlorofluoromethane
1,1-Dichloroethene
1,1-Dichloroethane
trans-l,2-Dichloroethene
Chloroform
1,2-Dichloroethane
1,1,1 -Tr i ch 1 oroet hane
Carbon tetrachloride
Bromodi chloromethane
1 ,2-Dichloropropane
trans-l,3,-Dichloropropene
Trichloroethene
Di bromoch 1 oromet hane
1,1, 2-Tri chl oroethane
Cis-l,3-dichloropropene
2-Chloroethylvinyl ether
1.50
2.17
2.62
2.67
3.33
5.25
7.18
7.93
9.30
10.1
10.7
11.4
12.6
13.0
13.7
14.9
15.2
15.8
16.5
16.5
16.5
18.0
5.28
7.05
nd
5.28
8.68
10.1
nd
7.72
12.6
9.38
12.1
15.4
13.1
14.4
14.6
16.6
16.6
13.1
16.6
18.1
18.0
nd
0.009
0.03
0.03
0.01
0.01
0.01
0.01
0.006
0.004
0.006
0.006
0.006
0.005
0.007
0.006
0.004
0.006
0.005
0.01
0.006
0.008
0.06
-------
TABLE 1 (continued)
Organohalides Tested Using Purge and Trap Method
Retention Time Detection Limit*
Compound (min.) ug/1
Column 1 Column 2
Bromoform
1,1,2,2-Tetrachloroethane
Tetrachloroethene
Chlorobenzene
1 , 3-Dichlorobenzene
1,2-Di Chlorobenzene
1,4-Dichlorobenzene
19.2
21.6
21.7
24.2
34.0
34.9
35.4
19.2
nd
15.0
18.8
22.4
23.5
22.3
0.02
0.006
0.007
0.03
0.04
0.04
0.04
nd = not determined
Column 1 conditions: Carbopack B 60/80 mesh coated with 1£
SP-1000 packed in an 8 ft x 0.1 in ID stainless steel or
glass column with helium carrier gas at 40 ml/min flow
rate. Column temperature held at 45°C for 3 min. then
programmed at 8°C/min. to 220° then held for 15 min.
Column 2 conditions: Porisil-C 100/120 mesh coated with
n-octane packed in a 6 ft x 0.1 in ID stainless steel or
glass column with helium carrier gas at 40 ml/min flow
rate. Column temperature held at 50°C for 3 min then
programmed at 6 C/min to 170 then held for 4 min.
* Detection limit is calculated from the minimum detectable GC
response being equal to five times the GC background noise,
using a Hall Model 700-A Detector.
3D"- 1*7
-------
OPTIONAL
FOAM
TRAP
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0. D. EXIT
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0. D.
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INLET tt IN.
0. D.
SAMPLE INLET
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-^SMM. 0. D. RUBBER SEPTUM
. 0. D.
INLET
1/4 IN. 0. D.
1/16 IN. O.D.
' STAINLESS STEEL
10MM GLASS FRIT
MEDIUM POROSITY
13X MOLECULAR
SIEVE PURGE
GAS FILTER
PURGE GAS
FLOW
CONTROL
Figure 1. Purging device
izr-M-7
-------
PACKING PROCEDURE
CONSTRUCTION
GLASS
WOOL
ACTIVATED., ,„.,.
CHARCOAL 7.7CM[
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RESISTANCE
WIRE WRAPPED
SOLID<
(DOUBLE LAYER)
15CM
7^/FOOT.
RESISTANCE
WIRE WRAPPED
SOLID
(SINGLE LAYER)
8CM-
TRAP INLET
COMPRESSION
"FITTING NUT
AND FERRULES
THERMOCOUPLE/
CONTROLLER
SENSOR
ELECTRONIC
TEMPERATURE
CONTROL
AND
PYROMETER
TUBING 25CM
0.105 IN. I.D.
0.125 IN. O.D.
STAINLESS STEEL
Figure 2. Trap packings and construction to include
desorb capability
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-------
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CARRIER GAS
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PRESSURE
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LIQUID INJECTION PORTS
PURGE GAS
FLOW CONTROL
13X MOLECULAR^
SIEVE FILTER ^
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OPTIONAL 4-PORT COLUMN
SELECTION VALVE
,16-PORT TRAP INLET
RESISTANCE WIRE
COLUMN OVEN
CONFIRMATORY COLUMN
i DETECTOR
•ANALYTICAL COLUMN
i VALVE
TRAP
180°C
ON
HEATER
CONTROL
_y
PURGING
DEVICE
Note:
ALL LINES BETWEEN
TRAP AND GC
SHOULD BE HEATED
TO 80°C.
Figure 4. Schematic of purge and trap device - desorb mode
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-------
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-------
PURGEABLE AROMATICS
METHOD 602
1. Scope and Application
1.1 This method covers the determination of various purge-
able aromatics. The following parameters may be deter-
mined by this method:
Parameter STORET No.
Benzene 34030
Chlorobenzene 34301
1,2-Dichlorobenzene 34536
1,3-DiChlorobenzene 34566
1,4-Dichlorobenzene 34571
Ethylbenzene 34371
Toluene 34010
1.2 This method is applicable to the determination of these
compounds in municipal and industrial discharges. It
is designed to be used to meet the monitoring require-
ments of the National Pollutant Discharge Elimination
System (NPDES). As such, it presupposes a high expec-
tation of finding the specific compounds of interest.
If the user is attempting to screen samples for any or
all of the compounds above, he must develop independent
protocols for the verification of identity.
1.3 The sensitivity of this method is usually dependent
upon the level of interferences rather than instru-
-------
mental limitations. The limits of detection listed in
Table 1 represent sensitivities that can be achieved in
wastewaters under optimum operating conditions.
1.4 This method is recommended for use only by experienced
residue analysts or under the close supervision of such
qualified persons.
2. Summary of Method
2.1 An inert gas-is bubbled through a 5 ml water sample
contained in a specially-designed purging chamber. The
aromatics are efficiently transferred from the aqueous
phase to the vapor phase. The vapor is swept through a
short sorbent tube where the aromatics are trapped.
After the purge is completed, the trap is heated and
backflusned with gas to desorb the aromatic compounds
into a gas chromatographic system. A temperature
program is used in the GC system to separate the
aromatics before detection with a photoionization
detector.
3. Interferences
3.1 Impurities in the purge gas and organic compounds
out-gasing from the plumbing ahead of the trap account
for the majority of contamination problems. The ana-
lytical system must be demonstrated to be free from
interferences under the conditions of the analysis by
running method blanks. Method blanks are run by
charging the purging device with organic-free water and
Tf
-------
analyzing it in a normal manner. The use of non-TFE
plastic tubing, non-TFE thread sealants or flow con-
trollers with rubber components in the purging device
should be avoided.
3.2 Samples can be contaminated by diffusion of volatile
organics through the septum seal into the sample during
shipment and storage. A sample blank prepared from
organic-free water and carried through the sampling and
handling protocol can serve as a check on such contam-
ination.
3.3 Cross contamination can occur whenever high level and
low level samples are sequentially analyzed. To reduce
the likelihood of this, the purging device and sample
syringe should be rinsed out twice between samples with
organic-free water. Whenever an unusually concentrated
sample is encountered, it should be followed by an
analysis of organic-free water to check for cross
contamination. For samples containing large amounts of
water soluble materials, suspended solids, high boiling
compounds or high levels of aromatics, it may be
necessary to wash out the purging device with a soap
solution, rinse with distilled water, and then dry in a
105°C oven between analyses.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete sampling.
4.1.1 Vial, with cap - 40 ml capacity screw cap
-BT- z-o
-------
(Pierce 113075 or equivalent). Detergent wash and dry
at 105°C before use.
4.1.2 Septum - Teflon - faced silicone (Pierce #12722
or equivalent). Detergent wash, rinse with tap
and distilled water, and dry at 105°C for one
hour before use.
4.2 Purge and trap device - The purge and trap equipment
consists of three separate pieces of apparatus: the
purging device, trap, and desorber. Several complete
devices are available commercially. The device must
meet the following specifications: the unit must be
completely compatible with the gas chromatographic
system; the purging chamber must be designed for a 5 ml
volume and be modeled after Figure 1; the dimensions
for the sorbant portion of the trap must meet or exceed
those in Figure 2. Figures 3 and 4 illustrate the
complete system in the purge and the desorb mode.
4.3 Gas chromatograph - Analytical system complete with
programmable gas chromatograph suitable for on-column
injection and all required accessories including Model
PI-51-02 photoiom'zation detector (h-nu Systems, Inc.),
column supplies, recorder, and gases. A data system
for measuring peak areas is recommended.
4.4 Syringes - 5-ml glass hypodermic with luerlok tip (2
each).
4.5 Micro syringes - 10, 25, 100 ul.
-------
4.6 2-way syringe valve with Luer ends (3 each).
4.7 Bottle - Ib-ml screw-cap, with Teflon cap liner.
5. Reagents
5.1 Sodium thiosulfate - (ACS) Granular.
5.2 Trap Materials
5.2.1 Porous polymer packing 60/80 mesh chromato-
graphic grade Tenax GC (2,6-diphenylene
oxide).
5.2.2 Three percent OV-1 on Chromosorb-W 60/80 mesh.
5.3 Activated carbon - Filtrasorb-200 (Calgon Corp.) or
equivalent.
5.4 Organic-free water
5.4.1 Organic-free water is defined as water free
of interference when employed in the purge
and trap procedure described herein. It is
generated by passing tap water through a
carbon filter bed containing about 1 Ib. of
activated carbon.
5.4.2 A water purification system (Millipore
Super-Q or equivalent) may be used to
generate organic-free deionized water.
5.4.3 Organic-free water may also be prepared by
boiling water for 15 minutes. Subsequently,
while maintaining the temperature at 90°C,
bubble a contaminant-free inert gas through
the water for one hour. While still hot,
IDT - 27.
-------
transfer the water to a narrow mouth screw
cap bottle and seal with a Teflon lined
septum and cap.
5.5 Stock standards - Prepare stock standard solutions in
methyl alcohol using assayed liquids. Because benzene
and 1,4-dichlorobenzene are suspected carcinogens,
primary dilutions of these compound should be prepared
in a hood.
5.5.1 Place about 9.8 ml of methyl alcohol into a
10 ml ground glass stoppered volumetric
flask. Allow the flask to stand, unstop-
pered, for about 10 minutes or until all
alcohol wetted surfaces have dried. Weigh
the flask to the nearest 0.1 mg.
5.5.2 Using a 100 ul syringe, immediately add 2
drops of assayed reference material to the
flask, then reweigh. Be sure that the 2
drops fall directly into the alcohol without
contacting the neck of the flask.
5.5.3 Dilute to volume, stopper, then mix by in-
verting the flask several times. Transfer
the standard solution to a 15 ml screw-cap
bottle with a Teflon cap liner.
5.5.4 Calculate the concentration in micrograms per
microliter from the net gain in weight.
.TIT- 2-3
-------
5.5.5 Store stock standards at 4°C. All stan-
dards must be replaced with fresh standard
each month.
6. Calibration
6.1 Using stock standards, prepare secondary dilution
standards in methyl alcohol that contain the compounds
of interest, either singly or mixed together. The
standards should be prepared at concentrations such
that the aqueous standards prepared in 6.2 will com-
pletely bracket the working range of the analytical
system.
6.2 Using secondary dilution standards,,prepare calibration
standards by carefully adding 20.0 ul of standard in
methyl alcohol to 100, 500, or 1000 ml of organic-free
water. A 25 ul syringe (Hamilton 702N or equivalent)
should be used for this operation. These aqueous
standards must be prepared fresh daily.
6.3 Assemble the necessary gas chromatographic apparatus
and establish operating parameters equivalent to those
indicated in Table 1. By injecting secondary dilution
standards, establish the sensitivity limit and the
linear range of the analytical system for each compound.
6.4 Assemble the necessary purge and trap device. The trap
must meet the minimum specifications shown in Figure 2
to achieve satisfactory results. Condition the trap
overnight at 180°C by backflushing with an inert gas
-------
flow of at least 20 ml/min. Prior to use, daily con-
dition traps 10 minutes while backflushing at 180°C.
Analyze aqueous calibration standards (6.2) according
to the purge and trap procedure in Section 8. Compare
the responses to those obtained by injection of stan-
dards (6.3), to determine purging efficiency and also
to calculate analytical precision. The purging effici-
encies and analytical precision of the analysis of
aqueous standards must be comparable to data presented
by Bellar and Lichtenberg (1978) before reliable sample
analysis may begin.
6.5 By analyzing calibration standards, .establish the
sensitivity limit and linear range of the entire
analytical system for each compound.
7. Quality Control
7.1 Before processing any samples, the analyst should
demonstrate daily through the analysis of an
organic-free water method blank that the entire
analytical system is interference-free.
7.2 Standard quality assurance practices should be used
with this method. Field replicates should be collected
to validate the precision of the sampling technique.
Laboratory replicates should be analyzed to validate
the precision of the analysis. Fortified samples
should be analyzed to validate the accuracy of the
analysis. Where doubt exists over the identification
77T
-------
of a peak on the gas chromatogram, confirmatory tech-
niques such as mass spectroscopy should be used.
7.3 The analyst should maintain constant surveillance of
both the performance of the analytical system and the
effectiveness of the method in dealing with each sample
matrix by spiking each sample, standard and blank with
surrogate compounds.
8. Sample Collection, Preservation, and Handling
8.1 Collect about 500 ml sample in a clean container.
Adjust the pH of the sample to about 2 by adding 1:1
diluted HC1 while stirring vigorously. If the sample
contains free or combined chlorine, add 35 mg of sodium
thiosulfate per part per million of free chlorine per
liter of sample. Fill a 40 ml sample bottle in such a
manner that no air bubbles pass through the sample as
the bottle is being filled. Seal the bottle so that no
air bubbles are entrapped in it. Maintain the hermetic
seal on the sample bottle until time of analysis.
8.2 The samples must be iced or refrigerated from the time
of collection until extraction.
8.3 All samples must be analyzed within 7 days of collec-
tion.
9. Sample Extraction and Gas Chromatography
9.1 Adjust the purge gas (nitrogen or helium) flow rate to
40 ml/min. Attach the trap inlet to the purging
device, and set the device to purge. Open the syringe
3ZT-
-------
valve located on the purging device sample introduction
needle.
9.2 Remove the plunger from a 5 ml syringe and attach a
closed syringe valve. Open the sample bottle (or
standard) and carefully pour the water into the syringe
barrel until it overflows. Replace the syringe plunger
and compress the sample. Open the syringe valve and
vent any residual air while adjusting the sample volume
to 5.0 ml. Since this process of taking an aliquot
destroys the validity of the sample for future analy-
sis, the analyst should fill a second syringe at this
time to protect against possible loss of data. Add the
surrogate spiking solution (7.3) through the valve
bore, then close the valve.
9.3 Attach the syringe-syringe valve assembly to the
syringe valve on the purging device. Open the syringe
valves and inject the sample into the purging chamber.
9.4 Close both valves and purge the sample for 12.0 +; .05
minutes.
9.5 After the 12 minute purge time, disconnect the purge
chamber from the trap. Dry the trap by maintaining a
flow rate of 40 cc/min dry purge gas for 6 min. Attach
the trap to the chromatograph, and adjust the device to
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 between
2-7
-------
20 and 60 ml/min for 4 minutes. If rapid heating can-
not be achieved, the gas chromatographic column must be
used as a secondary trap by cooling it.to 30°C (or
subambient, if problems persist) instead of the initial
program temperature of 50°C.
9.6 While the trap is being desorbed into the gas chroma-
tograph, empty the purging chamber using the sample
introduction syringe. Wash the chamber with two 5 ml
flushes of organic-free water.
9.7 After desorbing the sample for approximately four
minutes recondition the trap by returning the purge and
trap device to the purge mode. Wait 15 seconds then
close the syringe valve on the purging device to begin
gas flow through the trap. Maintain the trap temper-
ature at 180°C. After approximately seven minutes
turn off the trap heater and open the syringe valve to
stop the gas flow through the trap. When cool the trap
is ready for the next sample.
9.8 Table 1 summarizes the recommended gas chromatographic
column material and operating conditions for the
instrument. Included in this table are estimated
retention times and sensitivities that should be
achieved by this method. An example of the separation
achieved by this column is shown in Figure 5. Cali-
brate the system daily by analysis of a minimum of
three concentration levels of calibration standards.
jr-
-------
10. Calculations
10.1 Determine the concentration of Individual compounds
directly from calibrations plots of concentration
(ug/1) vs. peak height or area units.
10.2 Report results 1n micrograms per liter. When duplicate
and spiked samples are analyzed, all data obtained
should be reported.
11. Accuracy and Precision
The U.S. EPA Environmental Monitoring and Support Laboratory
in Cincinnati is in the process of conducting an interlabor-
atory method study to determine the accuracy and precision
of this test procedure.
TOT
-------
BIBLIOGRAPHY
1. Bellar, T. A., and 0. J. Lichtenberg, Journal American Water
Works Association, Vol. 66, No. 12, Dec. 1974, pp. 739-744.
2. Bellar, T. A., and 0. J. Lichtenberg, "Semi-Automated
Headspace Analysis of Drinking Waters and Industrial Waters
for Purgeable Volatile Organic Compounds," Proceeding from
ASTM Symposium on Measurement of Organic Pollutants in Water
and Wastewater, June 1978 (In Press).
3. Bellar, T. A., and J. J. Lichtenberg, "The Determination of
Purgeable Aromatic Compounds in Drinking Waters and
Industrial Wastes," (In preparation).
4. "Development and Application of Test Procedures for Specific
Organic Toxic Substances in Wastewaters. Category 11 -
Purgeables and Category 12 - Acrolein, Acrylonitrile, and
Dichlorodifluoromethane." Report for EPA Contract
68-03-2635 (In preparation).
or- 50
-------
TABLE 1
Chromatography of Aromatics Using Purge and Trap Method
Retention Time (min.) Detection Limit
Compound ug/1
Column 1
Benzene
Toluene
Ethyl benzene
Chlorobenzene
1 , 4-Di ch 1 orobenzene
1 , 3-Di ch 1 orobenzene
1,2-Oi Chlorobenzene
3.33
5.75
8.25
9.17
16.8
18.2
25.9
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
nd = not determined
Column 1 conditions: Supelcoport 100/120 mesh coated with 5%
SP-2100 and 1.75% Bentone-34 packed in a 6 ft. x 0.085 in ID
stainless steel column with helium carrier gas at 36 cc/min
flow rate. Column temperature held at 50°C for 2 min.
then programmed at 6 C/min. to 90 C for a final hold.
Detection limit is calculated from the minimum detectable GC
response being equal to five times the GC background noise,
using a h-nu Model PI-51-02 photoionization detector with a
10.2 ev lamp.
Jfl-
-------
[OPTIONAL
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TRAP
EXIT '/4 IN.
0. D.
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INLET V4 IN.
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2-WAY SYRINGE VALVE
17OA. 20 GAUGE SYRINGE NEEDLE
6MM. 0. D. RUBBER SEPTUM
10MM. 0. D. 1/16 IN. O.D.
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IN. 0. D.
13X MOLECULAR
SIEVE PURGE
GAS FILTER
10MM GLASS FRIT
MEDIUM POROSITY
PURGE GAS
FLOW
CONTROL
Figure 1. Purging device
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PACKING PROCEDURE
GLASS
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TENAX 23CM
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CONSTRUCTION
COMPRESSION FITTING
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TUBING 25CM.
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STAINLESS STEEL
TRAP INLET
Figure 2. Trap packings and construction to include
desorb capability
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13X MOLECULAR ^
S.EVE FILTER
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CONFIRMATORY COLUMN
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FLOW 180°C
PURGING
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Note:
ALL LINES BETWEEN
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Figure 4. Schematic of purge and trap device - desorb mode
-------
LU
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LU
2
LU
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cc
9
LU
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RETENTION TIME-MINUTES
Figure 5. Gas chromatogram of purgeable aromatics
-------
ACROLEIN AND ACRYLONITRILE
METHOD 603
1. Scope and Application
1.1 This method covers the determination of acrolein and
acrylonitrile. The following parameters may be deter-
mined by this method:
Parameter STORET No.
Acrolein 34210
Acrylonitrile 32415
1.2 This method is applicable to the determination of these
compounds in municipal and industrial discharges. It
is designed to be used to meet the monitoring require-
ments of the National Pollutant Discharge Elimination
System (NPDES). As such, it presupposes a high expec-
tation of finding the specific compounds of interest.
If the user is attempting to screen samples for any or
all of the compounds above, he must develop independent
protocols for the verification of identity.
1.3 The sensitivity of this method is usually dependent
upon the level of interferences rather than instru-
mental limitations. The limits of detection listed in
Table 1 represent sensitivities that can be achieved in
wastewaters under optimum operating conditions.
1.4 This method is recommended for use only by experienced
3z-
-------
residue analysts or under the close supervision of such
qualified persons.
2. Summary of Method
2.1 An inert gas is bubbled through a 5 ml water sample
contained in a specially-designed heated purging
chamber. Acrolein and acrylonitrile are transferred
from the vapor phase to the gaseous phase. The vapor
is passed through a short sorbent tube where the com-
pounds are trapped. After the extraction is completed,
the trap is heated and backflushed with gas to desorb
the compounds into a gas chromatographic system. A
temperature program is used in the GC system to
separate the compounds before detection with a flame
ioni2ation detector.
3. Interferences
3.1 Impurities in the purge gas and organic compounds
out-gasing from the plumbing ahead of the trap account
for the majority of contamination problems. The
analytical system must be demonstrated to be free from
interferences under the conditions of the analysis by
running method blanks. Method blanks are run by charg-
ing the purging device with organic-free water and
analyzing it in a normal manner. The use of non-TFE
plastic tubing, non-TFE thread sealants, or flow con-
trollers with rubber components in the purging device
should be avoided.
-------
3.2 Samples can be contaminated by diffusion of volatile
organics (particularly methylene chloride) through the
septum seal into the sample during shipment and
storage. A sample blank prepared from organic-free
water and carried through the sampling and handling
protocol can serve as a check on such contamination.
3.3 Cross contamination can occur whenever high-level and
low level samples are sequentially analyzed. To reduce
the likelihood of this, the purging device and sample
syringe should be rinsed out twice between samples with
organic-free water. Whenever an unusually concentrated
sample is encountered, it should be followed by an
analysis of organic-free water to check for
cross-contamination. For samples containing large
amounts of water soluble materials, suspended solids,
high boiling compounds or high organohalide levels it
may be necessary to wash out the purging device with a
soap solution, rinse with distilled water, and then dry
in a 105°C oven between analyses.
3.4 Interferences are sometimes reduced or eliminated by
first purging the water sample for 5 minutes at room
temperature in 9.4. Then the purge device is rapidly
heated to 85°C and purged as in 9.4. With such a
modification, approximately 5 to 10% of the acryloni-
trile and a trace of the acrolein in the sample will be
-------
lost. Therefore, calibration must be established for
the compounds under the conditions of this modified
procedure.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete sampling.
4.1.1 Vial, with cap - 40 ml capacity screw cap
(Pierce #13075 or equivalent). Detergent
wash and dry at 105°C before use.
4.1.2 Septum - Teflon - faced silicone (Pierce
#12722 or equivalent). Detergent wash, rinse
with tap and distilled water, and dry at
105°C for one hour before use.
4.2 Purge and trap device - The purge and trap equipment
consists of three separate pieces of apparatus: the
purging device, trap, and desorber. The purging device
should be equipped for heating in the same manner as
the trap (electrically) or with a circulating water
jacket. If electrical heating is used the electrical
parts must be protected so that water will not drip on
the conductors, causing dangerous electrical shock or
shorts. All temperature parameters must be carefully
controlled. Several complete devices are available
commercially although most are not equipped to heat the
purging chamber. The device must meet the following
specifications: the unit must be completely compatible
with the gas chromatographic system; the purging
ITT"
-------
chamber must be designed for a 5 ml volume and be
modeled after Figure 1; the dimensions for the sorbant
portion of the trap must meet or exceed those in figure
2. Figures 3 and 4 illustrate the complete system in
the purge and the desorb mode.
4.3 Gas chromatograph - Analytical system complete with
programmable gas chromatograph suitable for on-column
injection, equipped with matched columns for dual
column analysis and a differential flame ionization
detector. A nitrogen specific detector (thermionic or
Hall) may be used if only acrylonitrile is to be
detected. Required accessories include: column
supplies, recorder, and gases. A data system for
measuring peak areas is recommended.
4.4 Syringes - 5-ml glass hypodermic with luerlok tip (2
each).
4.5 Micro syringes - 10, 25, 100 ul.
4.6 2-way syringe valve with Luer ends (3 each).
4.7 Bottle - 15-ml screw-cap, with Teflon cap liner.
5. Reagents
5.1 Preservatives
5.1.1 Sodium hydroxide - (ACS) 10 N in distilled
water.
5.1.2 Sulfuric acid - (ACS). Mix equal volumes of
cone. H-SO, with distilled water.
5.1.3 Sodium thiosulfate - (ACS) Granular.
5.2 Trap absorbent - Porous polymer packing, 50/80 mesh
2T-
-------
chromatographic grade Porapak N.
5.3 Activated carbon - Filtrasorb-200 (Calgon Corp.) or
equivalent.
5.4 Organic-free water
5.4.1 Organic-free water is defined as water free
of interference when employed in the purge
and trap procedure described herein. It is
generated by passing tap water through a
carbon filter bed containing about 1 Ib. of
activated carbon.
5.4.2 A water purification system (Millipore
Super-Q or equivalent) may be used to
generate organic-free deionized water.
5.4.3 Organic-free water may also be prepared by
boiling water for 15 minutes. Subsequently,
while maintaining the temperature at 90°C,
bubble a contaminant-free inert gas through
the water for one hour. While still hot,
transfer the water to a narrow mouth screw
cap bottle and sealwith a Teflon lined septum
and cap.
5.5 Stock standards - Prepare stock standard solutions
daily in water using assayed standards. Because of
toxicity, primary dilutions of these materials should
be prepared in a hood. A NIOSH/MESA approved toxic gas
respirator should be used when the analyst handles high
-------
concentrations of the materials.
5.5.1 Place about 9.8 ml of water (pH 6.5 to 7.5)
into a 10 ml ground glass stoppered volu-
metric flask. Allow the flask to stand,
unstoppered, for about 10 minutes or until
all water wetted surfaces have dried. Weigh
the flask to the nearest 0.1 mg.
5.5.2 Using a 100 ul syringe, immediately add 2
drops of assayed reference material to the
flask, then reweigh. Be sure that the 2
drops fall directly into the water without
contacting the neck of the flask.
5.5.3 Dilute to volume, stopper, then mix by in-
verting the flask several times. Transfer
the standard solution to a 15 ml screw-cap
bottle with a Teflon cap liner.
5.5.4 Calculate the concentration in micrograms per
microliter from the net gain in weight.
6. Calibration
6.1 Using stock standards, prepare secondary dilution stan-
dards in water. The standards should be prepared at
concentrations such that the aqueous standards prepared
in 6.2 will completely bracket the working range of the
chromatographic system.
6.2 Using secondary dilution standards, prepare calibration
standards by carefully adding ul of stock standard
-£-
-------
to 100, 500, or 1000 ml of organic-free water.
6.3 Assemble the necessary gas chromatographic apparatus
and establish operating parameters equivalent to those
indicated in Table 1. By injecting secondary dilution
standards, establish the sensitivity limit and the
linear range of the analytical system for each compound.
6.4 Assemble the necessary purge and trap device. The trap
must meet the minimum specifications as shown in Figure
2 to achieve satisfactory results. Condition the trap
overnight at 180°C by backflushing with an inert gas
flow of at least 20 ml/min. Prior to use, daily
condition traps 10 minutes while backflushing at
180°C. Analyze aqueous calibration standards (6.2)
according to the purge and trap procedure in Section
9. Compare the responses to those obtained by injec-
tion of standards (6.3), to determine purging effici-
ency and also to calculate analytical precision. The
purging efficiencies and analytical precision of the
analysis of aqueous standards should be 85 + 5% for
acrolein and 98% + 5% for acrylonitrile.
6.5 By analyzing calibration standards, establish the sen-
sitivity limit and linear range of the entire analy-
tical system for each compound.
7. Quality Control
7.1 Before processing any samples, the analyst should
demonstrate daily through the analysis of an
-------
organic-free water method blank that the entire analy-
tical system is interference-free.
7.2 Standard quality assurance practices should be used
with this method. Field replicates should be collected
to validate the precision of the sampling technique.
Laboratory replicates should be analyzed to validate
the precision of the analysis. Fortified samples
should be analyzed to validate the" accuracy of the
analysis
Where doubt exists over the identification of a peak on
the gas chromatogram, confirmatory techniques such as
mass spectroscopy should be used.
7.3 The analyst should maintain constant surveillance of
both the performance of the analytical system and the
effectiveness of the method in dealing with each sample
matrix by spiking each sample, standard and blank with
surrogate compounds.
8. Sample Collection, Preservation, and Handling
8.1 Collect about 500 ml sample in a clean container.
Adjust the pH of the sample to 6^5 to 7.5 by adding 1:1
diluted hUSO^ or NaOH while stirring vigorously.
If the sample contains residual chlorine, add 35 mg of
sodium thiosulfate per part per million of free
chlorine per liter of sample. Fill a 40 ml sample
bottle and seal the bottle so that no air bubbles are
entrapped in it. Maintain the hermetic seal on the
jr
-------
sample bottle until time of analysis.
8.2 The samples must be'iced or refrigerated at 4°C from
the time of collection until extraction.
8.3 All samples must be analyzed within 3 days of collec-
tion.
9. Sample Extraction and Gas Chromatography
9.1 Adjust the helium purge gas flow rate to 2(H1 ml/min
and the temperature of the purge device to 85°C.
Attach the trap inlet to the purging device, and set
the device to purge. Open the syringe valve located on
the purging device sample introduction needle.
9.2 Remove the plunger from a 5 ml syringe and attach a
closed syringe valve. Open the sample bottle (or
standard) and carefully pour the water into the syringe
barrel until it overflows. Replace the syringe plunger
and compress the sample. Open the syringe valve and
vent any residual air while adjusting the sample volume
to 5.0 ml.
9.3 Attach the syringe-syringe valve assembly to the
syringe valve on the purging device. Open the syringe
valves and inject the sample into the purging chamber.
9.4 Close both valves and purge the sample for 30.0 + 0.1
minutes. Monitor and control the temperature of the
purge device to obtain 85+l°C.
9.5 After the 30-minute purge time, attach the trap to tN
chromatograph, and adjust the device to the desorb
-------
mode. Introduce the trapped materials to the GC column
by rapidly heating the trap to 170°C while back-
flushing the trap with helium at 45 ml/min for 5
minutes. The backflushing time and gas flow rate must
be carefully reproduced from sample to sample. During
backflushing the chromatographic column is held at
100°C. Record GC retention time from the beginning
of desorption.
9.6 While the trap is being desorbed into the gas chroma-
tograph, empty the purging chamber using the sample
introduction syringe. Wash the chamber with two 5 ml
flushes of organic-free water.
9.7 After desorbing the sample for 5 minutes recondition
the trap by returning the purge and trap device to the
purge mode and begin the GC program. Wait 15 seconds
then close the syringe valve on the purging device to
begin gas flow through the trap. Maintain the trap
temperature at 170°C. After approximately seven
minutes turn off the trap heater and open the syringe
valve to stop the gas flow through the trap. When cool
the trap is ready for the next sample.
9.8 Table 1 summarizes some recommended gas chromatographic
column materials and operating conditions for the
instrument. Included in this table are estimated
retention times and sensitivities that should be
achieved by this method. An example of the separation
_ZT-
-------
achieved by this column is shown in Figure 5. Cali-
brate the system daily by analysis of a minimum of
three concentrations levels of calibration standards.
10. Calculations
10.1 Determine the concentration of individual compounds
directly from calibrations plots of concentration
(ug/1) vs. peak height or area units.
10.2 Report results in micrograms per liter. When duplicate
and spiked samples are analyzed, all data obtained
should be reported.
11. Accuracy and Precision
The U.S. EPA Environmental Monitoring and Support Laboratory
in Cincinnati is in the process of conducting an interlaboratory
method study to determine the accuracy and precision of this
test procedure.
BIBLIOGRAPHY
1. Bellar, T. A., and J. J. Lichtenberg, Journal American Water
Works Association, Vol. 66, No. 12, Dec. 1974, pp. 739-744.
2. Bellar, T. A., and J. J. Lichtenberg, "Semi-Automated
Headspace Analysis of Drinking Waters and Industrial Waters
for Purgeable Volatile Organic Compounds," Proceeding from
ASTM Symposium on Measurement of Organic Pollutants in Water
and Wastewater, June 1978 (In Press).
-------
3. "Development and Application of Test Procedures for Specific
Organic Toxic Substances in Wastewaters. Category 11 -
Purgeables and Category 12 - Acrolein, Acrylonitrile, and
Dichlorodlfluoromethane." Report for EPA Contract
68-03-2635 (In preparation).
4. Going, John, et al., "Environmental Monitoring Near
Industrial Sites-Acrylonltrile," EPA Report No.
560/6-79-003, 1979.
-------
TABLE 1
Gas Chromatography by Heated Purge and Trap
Detection Limit
Compound Retention Time (min.) ug/1
Acrolein 7.6 2
Acrylonitrile 8.9 1
Column conditions: Chromosorb 101 80/100 mesh packed 1n a
6' x 1/8" O.D. stainless steel column with helium carrier
gas at 45 ml/min flow rate. Column temperature is held at
100°C for 5 minutes during trap desorption, then
programmed at 10°C/min to 140°C and held for 5 minutes.
Detection limit is estimated, based upon the use of a flame
ionization detector.
-------
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PACKING PROCEDURE
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CARRIER GAS
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Figure 4. Schematic of purge and trap device - desorb mode
-------
:OLUMN: CHROMOSORB 101
ROGRAM: 80eC-5 MINUTES.
8°C< MINUTE TO 1505C
DETECTOR: FLAME IONIZATION
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RETENTION TIME-MINU":
Figured. Gas chromatogram -
ein and acrylonitrile
-------
PHENOLS
METHOD 604
1. Scope and Application
1.1 This method covers the determination of various
•
phenolic compounds. The following parameters may be
determined by this method:
Parameter STORET No.
4-Chloro-3-methylphenol 34452
2-Chlorophenol 34586
2,4-Dichlorophenol 34601
2,4-Dimethylphenol 34606
2,4-Dinitrophenol 34616
2-Methyl-4,6-dinitrophenol 34657
2-Nitrophenol 34591
4-Nitrophenol 34646
Pentachlorophenol 39094
Phenol 34694
2,4,6-Trichlorophenol 34621
1.2 This method is applicable to the determination of these
compounds in municipal and industrial discharges. It
is designed to be used to meet the monitoring
requirements of the National Pollutant Discharge
Elimination System (NPDES). As such, it presupposes a
nr-'K
-------
high expectation of finding the specific compounds of
interest. If the user is attempting to screen samples
for any or all of the compounds above, he must develop
independent protocols for the verification of identity.
1.3 The sensitivity of this method is usually dependent
upon the level of interferences rather than
instrumental limitations. The limits of detection
listed in Table I represent sensitivfties that can be
achieved in wastewaters with a flame ionization
detector in the absence of interferences. If the
derivatization cleanup is required, the sensitivity of
the method is 10 ug/1. This concentration represents
the minimum amount proven to date to give reproducible
and linear response during derivatization.
1.4 This method is recommended for use only by experienced
residue analysts or under the close supervision of such
qualified persons.
2. Summary of Method
2.1 A 1-liter sample of wastewater is acidified and
extracted with methylene chloride using separatory
funnel techniques. The extract is dried and
concentrated to a volume of 10 ml or less. Flame
ionization gas chromatographic conditions are described
which allow for the measurement of the compounds in the
extract.
2.2 The method also provides for the preparation of
•nr-47
-------
pentafluorobenzylbromide derivatives for electron
capture gas chromatography with additional cleanup
procedures to aid the analyst in the elimination of
interferences.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample
processing hardware may yield discrete artifacts and/or
elevated baselines causing misinterpretation of gas
chromatograms. All of these materials must be
demonstrated to be free from interferences under the
conditions of the analysis by running method blanks.
Specific selection of reagents and purification of
solvents by distillation in all-glass systems may be
required.
3.2 Interferences coextracted from the samples will vary
considerably from source to source, depending upon the
diversity of the industrial complex or municipality
being sampled. While general cleanup techniques are
provided as part of this method, unique samples may
require additional cleanup approaches to achieve the
sensitivities stated in Table I.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete or composite sampling.
4.1.1 Grab sample bottle - amber glass, 1-liter or
1-quart volume. French or Boston Round design
is recommended. The container must be washed
-------
and solvent rinsed before use to minimize
interferences.
4.1.2 Bottle caps - Threaded to screw on to the sample
bottles. Caps must be lined with Teflon.
4.1.3 Compositing equipment - Automatic or manual
compositing system. Must incorporate glass
sample containers for the collection of a
minimum of 250 ml. Sample containers must be
kept refrigerated during sampling. No tygon or
rubber tubing may be used in this system.
4.2 Separatory funnel - 2000 ml, with Teflon stopcock.
4.3 Drying column - 20 mm ID Pyrex chromatograhic column
with coarse frit.
4.4 Kuderna-Danish (K-D) Apparatus
4.4.1 Concentrator tube - 10 ml, graduated (Kontes
K-570050-1025 or equivalent). Calibration must
be checked. Ground glass stopper (size 19/22
joint) is used to prevent evaporation of
extracts.
4.4.2 Evaporative flask - 500 ml (Kontes K-57001-0500
or equivalent). Attach to concentrator tube
with springs. (Kontes K-662750-0012).
4.4.3 Snyder column - three-ball macro (Kontes
K-503000-0121 or equivalent).
4.4.4 Snyder column - two-ball micro (Kontes
K-569001-0219 or equivalent).
ft
-------
4.4.5 Boiling chips - solvent extracted, approximately
10/40 mesh.
4.5 Water bath - Heated, with concentric ring cover,
capable of temperature control (+ 2°C). The bath
should be used in a hood.
4.6 Gas chromatograph - Analytical system complete with gas
chromatograph suitable for on-column injection and all
required accessories including flame ionization and
electron capture detector, column supplies, recorder,
gases, syringes. A data system for measuring peak
areas is recommended.
4.7 Chromatographic column - 10 mm ID by 100 mm length,
with Teflon stopcock.
4.8 Reaction vial.
5. Reagents
5.1 Preservatives:
5.1.1 Sodium hydroxide - (ACS) 10 N in distilled water.
5.1.2 Sulfuric acid - (1+1) Mix equal volumes of cone.
H2S04 (ACS) with distilled water.
5.1.3 Sodium thiosulfate - (ACS) Granular.
5.2 Methylene chloride, acetone, 2-propanol, hexane,
toluene - Pesticide quality or equivalent.
5.3 Sodium sulfate - (ACS) Granular, anhydrous (purified by
heating at 400°c for 4 hrs. in a shallow tray).
5.4 Stock standards - Prepare stock standard solutions at a
concentration of 1.00 ug/ul by dissolving 0.100 grams
so
-------
of assayed reference material in pesticide quality
2-propanol and diluting to volume in a 100 ml ground
glass stoppered volumetric flask. The stock solution
is transferred to ground glass stoppered reagent
bottles, stored in a refrigerator, and checked
frequently for signs of degradation or evaporation,
especially just prior to preparing working standards
from them.
5.5 Sulfuric acid - (ACS) 1 N in distilled water.
5.6 ' Potassium carbonate - (ACS) powdered.
5.7 Pentafluorobenzyl bromide (a-Bromopentaf luorotoluene) -
97% minimum purity.
5.8 1,4,7,10,13,16 - Hexaoxacyclooctadecane (18 crown 6) -
98% minimum purity.
5.9 Derivatization reagent - Add 1 ml pentafluorobenzyl
bromide and 1 gram 18 crown 6 to a 50 ml volumetric
flask and dilute to volume with 2-propanol. Prepare
fresh weekly.
5.10 Silica gel - (ACS) 100/200 mesh, grade 923; activated
at 130°C and stored in a desiccator.
Calibration
6.1 Prepare calibration standards for the flame ionization
detector that contain the compounds of interest, either
singly or mixed together. The standards should be
prepared at concentrations covering two or more orders
of magnitude that will completely bracket the working
.TIT
-------
range of the chromatographic system. If the
sensitivity of the detection system can be calculated
from Table I as 100 ug/1 in the final extract, for
example, prepare standards at 10 ug/1, 50 ug/1, 100
ug/1, 500 ug/1, etc. so that injections of 1-5 ul of
each calibration standard will define the linearity of
the detector in the working range.
6.2 Assemble the necessary gas chromatogrphic apparatus and
establish operating parameters equivalent to those
indicated in Table I. By injecting calibration
standards, establish the sensitivity limit of* the
detector and the linear range of the analytical system
for each compound.
6.3 Before using the derivatization cleanup procedure, the
analyst must process a series of calibration standards
through the procedure to validate the precision of the
derivatization and the absence of interferences from
the reagents.
7. Quality Control
7.1 Before processing any samples, the analyst should
demonstrate through the analysis of a distilled water
method blank, that all glassware and reagents are
interference-free. Each time a set of samples is
extracted or there is a -nange in reagents, a method
blank should be process = - a safeguard against
chronic laboratory cc~ "ion.
.52
-------
7.2 Standard quality assurance practices should be used
with this method. Field replicates should be collected
to validate the precision of the sampling technique.
Laboratory replicates should be analyzed to validate
the precision of the analysis. Fortified samples
should be analyzed to validate the accuracy of the
analysis. Where doubt exists over the identification
of a peak on the chromatogram, confirmatory techniques
such as mass spectroscopy should be used.
8. Sample Collection, Preservation, and Handling
8.1 Grab samples must be collected in glass containers.
Conventional sampling practices should be followed,
except that the bottle must not be prewashed with
sample before collection. Composite samples should be
collected in refrigerated glass containers in
accordance with the requirements of the program.
Automatic sampling equipment must be free of tygon and
other potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time
of collection until extraction. At the sampling
location fill the glass container with sample. Add 35
mg of sodium thiosulfate per part per million free
chlorine per liter. Adjust the sample pH to
approximately 2, as measured by pH paper, using
appropriate sulfuric acid solution or 10 N sodium
hydroxide. Record the volume of acid used on the
-------
sample identification tag so the sample volume can be
corrected later.
8.3 All samples must be extracted within 7 days and
completely analyzed within 30 days of collection.
9. Sample Extraction
9.1 Mark the water meniscus on the side of the sample
bottle for later determination of sample volume. Pour
the entire sample into a two-liter separatory funnel.
Adjust the sample pH to 12 with sodium hydroxide.
9.2 Add 60 ml methylene chloride to the sample bottle,
seal, and shake 30 seconds to rinse the inner walls.
Transfer the solvent into the separatory funnel, and
extract the sample by shaking the funnel for one minute
with periodic venting to release vapor pressure. Allow
the organic layer to separate from the water phase for
a minimum of ten minutes. If the emulsion interface
between layers is more than one-third the size of the
solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The
optimum technique depends upon the sample, but may
include stirring, filtration of the emulsion through
glass wool, or centrifugation. Discard the methylene
chloride layer, and wash the sarrple with an additional
two 60 ml portions of methylene chloride in similar
fashion.
9.3 Adjust the aqueous layer to a p- sf 1-2 with sulfuric
acid.
-------
9.4 Add 60 ml of methylene chloride to the sample and shake
for two minutes. Allow the solvent to separate from
the sample and collect the methylene chloride in a 250
ml Erlenmeyer flask.
9.5 Add a second 60 ml volume of methylene chloride to the
sample bottle and complete the extraction procedure a
second time, combining the extracts in the Erlenmeyer
flask.
9.6 Perform a third extraction in the same manner. Pour
the combined extract through a drying column containing
3-4 inches of anhydrous sodium sulfate, and collect it
in a 500-ml Kuderna-Danish (K-D) flask equipped with a
10 ml concentrator tube. Rinse the Erlenmeyer flask
and column with 20-30 ml methylene chloride to complete
the quantitative transfer.
9.7 Add 1-2 clean boiling chips to the flask and attach a
three-ball Snyder column. Prewet the Snyder column by
adding about 1 ml methylene chloride to the top. Place
the K-D apparatus on a hot water bath (60-65°C) so
that the concentrator tube is partially immersed in the
hot water, and the entire lower rounded surface of the
flask is bathed in vapor. Adjust the vertical position
of the apparatus and the water temperature as required
to complete the concentration in 15-20 minutes. At the
proper rate of distillation the balls of the column
1JL- ^^
-------
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 for at least 10
minutes while cooling.
9.8 Increase the temperature of the hot water bath to
95-100°C. Remove the Snyder column and rinse the
flask and its lower joint into the concentrator tube
with 1-2 ml of 2-propanol. A 5-ml syringe is
recommended for this operation. Attach a micro-Snyder
column to the concentrator tube and prewet the column
by adding about 0.5 ml 2-propanol 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 5-10 minutes. At the proper rate of
distillation, the balls of the column will actively
chatter but the chambers will not flood. When the
apparent volume of the liquid reaches 2.5 ml, remove
the K-D apparatus and allow it to drain for at least 10
minutes while cooling. Add an additional 2 ml of
2-propanol through the top of the micro-Snyder column
and resume concentrating as before. When the apparent
volume of liquid reaches 0.5 ml, remove the K-D
apparatus and allow it to drain for at least 1C vnutes
while cooling. Remove the micro-Snyder colurn-
-------
rinse its lower joint into the concentrator tube with a
minimum of 2-propanol. Adjust the extract volume to
1.0 ml. Stopper the concentrator tube and store in
refrigerator, if further processing will not be
performed immediately. If the sample extract requires
no further cleanup, proceed with flame ionization gas
chromatographic analysis. If the sample requires
cleanup, proceed to Section 11.
9.9 Determine the original sample volume by refilling the
sample bottle to the mark and transferring the liquid
to a 1000 ml graduated cylinder. After correction for
sulfuric acid preservative, record the sample volume to
the nearest 5 ml.
10. Gas Chromatography - Flame Ionization Detector
10.1 Table I summarizes some recommended gas chromatographic
column materials and operating conditions for the
instrument. Included in this table are estimated
retention times and sensitivities that should be
achieved by this method. An example of the separation
achieved by one of these columns is shown in Figure 1.
Calibrate the gas chromatographic system daily with a
minimum of three injections of calibration standards.
10.2 Inject 2-5 ul of the sample extract using the
solvent-flush technique. Smaller (1.0 ul) volumes can
be injected if automatic devices are employed. Record
the volume injected to the nearest 0.05 ul, and the
Iff- -
-------
resulting peak size, 1n area units.
10.3 If the peak area exceeds the linear range of the
system, dilute the extract and reanalyze.
10.4 If the peak area measurement 1s prevented by the
presence of Interferences, the phenols must be
derlvatlzed and analyzed by electron capture gas
chromatography.
11. Derivat1zation and Electron Capture Gas Chromatography
11.1 P1pet a 1.0 ml aliquot of the 2-propanol solution of
standard or sample extract Into a glass reaction vial.
Add 1.0ml der1vat1zat1on reagent. This 1s a
sufficient anount of reagent to derlvatlze a solution
whose total phenalic content does not exceed 0.3 mg/ml.
11.2 Add about 3 mg of potassium carbonate to the solution
and shake gently.
11.3 Cap the mixture and heat 1t for 4 hours at SO°C 1n a
hot water bath.
11.4 Remove the solution from the hot water bath and allow
1t to cool
11.5 Add 10 ml hexane to the reaction vial and shake
vigorously for one nrinute. Add 3.0 ml of distilled,
delonlzed water to the reaction vial and shake for two
minutes.
11.6 Decant organic layer into a concentrator tube and cap
with a glass stopper.
11.7 Pack a 10 mm ID chromatographic column with 4.0 grams
s-8
-------
of activated silica gel. After settling the silica gel
by tapping the column, add about two grams of anhydrous
sodium sulfate to the top.
11.8 Pre-elute the column with 6 ml hexane. Discard the
eluate and just prior, to exposure of the sulfate layer
to air, pipet onto the column 2.0 ml of the hexane
solution (11.6) that contains the derivatized sample or
standard. Elute the column with 10.0 ml of hexane
(Fraction 1) and discard this fraction. Elute the
column, in order, with: 10.0 ml 15% toluene in hexane
(Fraction 2); 10.0 ml 40% toluene in hexane (Fraction
3); 10.0 ml 75% toluene in hexane (Fraction 4); and
10.0 ml 15% 2-propanol in toluene (Fraction 5).
Elution patterns for the phenolic derivatives are shown
in Table II. Fractions may be combined as desired,
depending upon the specific phenols of interest or
level of interferences.
11.9 Analyze the fractions by electron capture gas
chromatography. Table II summarizes some recommended
gas chromatographic column materials and operating
conditions for the instrument. Included in this table
are estimated retention times that should be achieved
by this method. Examples of the separation achieved by
this column is shown in Figure 2. Calibrate the system
daily with a minimum of three aliquots of calibration
standards, containing each of the phenols of interest
-------
that are derlvatlzed according to the procedure.
11.10 Inject 2-5 ul of the column fractions using the
solvent-flush technique. Smaller (1.0 ul} volumes can
be Injected 1f automatic devices are employed. Record
the volume Injected to the nearest 0.05 ul, and the
resulting peak size, In area units. If the peak area
exceeds the linear range of the system, dilute the
extract and reanalyze.
12. Calculations
12.1 Determine the concentration of Individual compounds
measured by the flame 1on1zat1on procedure (without
der1vat1zat1on) according to the formula:
Concentration, ug/1 « JA) (B) (Vt)
where A » Calibration factor for chromatographlc
system, 1n nanograms material per area unit.
B » Peak size in Injection of sample extract, 1n area
units
Vj » volume of extract Injected (ul)
Vt » Volume of total extract (ul)
V » Volume of water extracted (ml)
12.2 Determine the concentration of Individual compounds
measured by the der1vat1zat1on and electron capture
procedure according to the following procedure:
12.2.1 From the concentration of the calibration
standards that were derlvatlzed with the
-------
samples, calculate the amounts, 1n nano grains,
of underivatized phenols that were added as
2-propanol solution (11.1) From the size of
the Injection Into the electron capture gas
chromatograph, determine the nano grams of
material (calculated as the underivatized
phenol) Injected onto the column. Compare the
detector responses obtained to develop a
calibration factor for the chromatographlc
system, 1n nanograms of material per area unit.
12.2.2 Determine the concentration of Individual
compounds according to the formula:
Concentration, ug/1 » (A) (B) (Vt)(10) (D)
where A » Calibration factor for chromatographic
system, in nanograms material per area
unit, calculated as underivatized
phenol .
B * Peak size 1n Injection of sample
extract, in area units.
V1 » Volume of eluate injected (ul)
Vt » Total volume of column eluate (ul)
•
V * Volume of water extracted (ml)
C * Volume of hexane sample solution added
to cleanup column, in ml.
0 * Total volume of 2-propanol extract
JJL
-------
after concentration.
E = Volume of 2-propanol extract used for
derivatization.
12.3 Report results in micrograms per liter without
correction for recovery data. When duplicate and
spiked samples are analyzed, all data obtained should
be reported.
13. Accuracy and Precision
13.1 The U.S. EPA Environmental Monitoring and Support
Laboratory in Cincinnati is in the process of
conducting an interlaboratory method study to determine
the accuracy and precision of this test procedure.
BIBLIOGRAPHY
"Development and Application of Test Procedures for Specific
Organic Toxic Substances in Wastewaters. Category 3 -
Chlorinated Hydrocarbons and Category 8 - Phenols." Report for
EPA Contract 68-03-2625 (In preparation).
-------
TABLE I
Flame lonization Gas Chromatography of Phenols
Compound
2-Chlorophenol
2-Nitrophenol
Phenol
2,4-Dimethylphenol
2,4-Dichlorophenol
2,4,6-Trichlorophenol
4-Ch1oro-3-methyIpheno1
2,4-Dinitrophenol
2-Methyl-4,6-dinitrophenol
Pentachlorophenol
4-Nitrophenol
Retention Time
Detection
Limit (ug/L)
1.70
2.00
3.01
4.03
4.30
6.05
7.50
10.00
10.24
12.42
24.25
2.0
2.5
1.4
1.7
2.1
5.0
8.3
7.0
10.0
10.0
10.0
Column conditions: Supelcoport 80/100 mesh coated with 1%
SP-1240 DA in 6 ft long x 2 mm ID glass column with nitrogen
carrier gas at 30 ml/min flow rate. Column temperature is
80°C at injection, programmed immediately at 8°C/min to
150°c final temperature.
- 63
-------
Detection limit is calculated from the minimum detectable GC
response being equal to five times the GC background noise,
assuming a 10 ml final extract volume of the 1 liter sample
extract, and assuming a GC injection of 5 micro!iters.
-------
TABLE II
Electron Capture Gas Chromatography
of PFBB Derivatives
Retention Recovery(%) by Fraction*
Parent Compound Time (min) J 2 3 4 5
2-chlorophenol 3.3 - 90 <1
2-nitrophenol 9.1 - - 9 90
Phenol 1.8 - 90 10
2,4-Dimethyphenol 2.9 - 95 7
2,4-Dichlorophenol 5.8 - 95 <1
2,4,6-Trichlorophenol 7.0 50 50
4-Chloro-2-methyphenol 4.8 - 84 14
Pentachlorophenol 28.8 75 20
4-Nitrophenol 14.0 - - - <1 90
(2,4-Dinitrophenol) 46.9**
(2-Methyl-4,6-dinitrophenol) 36.6**
Column conditions: Chromosorb W-AW-DMCS 80/100 mesh coated with
5% OV-17 packed in a 1.8 m long x 2.0 mm ID glass column with 5%
methane/95% argon carrier gas at 30 ml/min flow r»te. Column
temperature is 200°C.
-------
*From: "Development and Application of Test Procedures for
Specific Organic Toxic Substances in Wastewaters. Categories
3-Chlorinated Hydrocarbons and Category 8-Phenols."
**Retention times included for qualitative information only.
The lack of accuracy and precision of the derivatization
reaction precludes the use of this approach for quantitative
purposes.
'til
-------
o
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o- I
O £
q_i s COLUMN: 1% SP-1240DA ON SUPELCOPORT
~'~ 5 PROGRAM: 80°C.-0 MINUTES 8° /MINUTE TO 15Q°C.
DETECTOR: FLAME IONIZATION
o
•2.
LU
O
SS
/
8 12 16 20 2A
RETENTION TIME-MINUTES
28
Figure 1. Gas chromatogram of ph*
IT
-------
COLUMN: 5% OV-17 ON CHROMOSORB W-AW
TEMPERATURE: 200°C.
DETECTOR: ELECTRON CAPTURE
a.
O
cc
O
8 12 16 20 24
RETENTION TIME-MINUTES
28
32
Figure 2. Gas chromatogram of PFB derivatives of phenols
71!
-------
BENZIDINES
METHOD 605
1. Scope and Application
1.1 This method covers the determination of benzidine and selected
derivatives. The following parameters may be determined by this
method:
Parameter STORET No.
Benzidine 39120
3,3'-Dichlorobenzidine 34631
1.2 This method is applicable to the measurement of these compounds
in municipal and industrial discharges. It is designed to be
used to meet the monitoring requirements of the National
Pollutant Discharge Elimination System (NPDES). As such, it
presupposes a high expectation of finding the specific compounds
of interest. If the user is attempting to screen samples for any
or all of the compounds above, he must develop independent
protocols for the verification of identifications implied with
the use of these techniques.
1.3 This sensitivity of this method is usually dependent upon the
level of interferences rather than instrumental limitations. The
limits of detection listed in Table I represent sensitivities
that can be achieved in wastewaters in the absence of inter-
ferences.
1.4 This method is recommended for use only by experienced residue
analysts or under the close supervision of such qualified persons.
- lol
-------
2. Summary of Method
2.1 Benzidine and 3,3-dichlorobenzidine (DCB) are extracted from the
sample at pH 7-8 using chloroform. The extract is then back
extracted into acid, re-extracted into chloroform at neutral pH,
and concentrated. The benzidines are determined in the final
extract using high performance liquid chromatography (HPLC) with
electrochemical detection.
2.2 If interferences are encountered with the measurement of benzi-
dine, the method provides additional detector settings to
increase the selectivity of the analytical system.
3. Interferences
3.1 Although the detection system is highly selective, solvents,
reagents, glassware, and other sample processing hardware may
yield discrete artifacts and/or elevated baselines causing
misinterpretation of chromatograms. All of these materials must
be demonstrated to be free from interferences under the
conditions of the analysis. Specific selection of reagents and
purification of solvents by distillation in all-glass systems may
be required.
3.2 Interferences coextracted from the samples will vary considerably
from source to source, depending upon the diversity of the
industrial complex or municipality being sampled. While general
cleanup techniques are provided as part of this method, unique
samples may require additional cleanup approaches to achieve the
sensitivities stated in Table I.
3.3 Some dye plant effluents contain large amounts of components with
-------
retention times in the vicinity of benzidine. In these cases,-it
has been found useful to reduce the electrode potential in order
to eliminate the interferences but still detect the benzidine.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete or composite sampling.
4.1.1 Grab sample bottle - amber glass, 1-pint or 1-quart
volume. Quart bottles should be only half filled in the
field. French or Boston Round design is recommended. The
container should be washed and solvent rinsed before use
to minimize interferences.
4.1.2 Bottle caps - Threaded to screw on to the sample bottles.
Caps must be lined with Teflon. Foil may be substituted
if the sample is not corrosive.
4.1.3 Compositing equipment - Automatic or manual compositing
system. Must incorporate glass sample containers for the
collection of a minimum of 250 ml. Sample containers must
be kept refrigerated during sampling. No tygon or rubber
tubing or fittings may be used in the system.
4.2 Separatory funnel - 1000 ml and 250 ml, with Teflon stopcock.
4.3 Water bath - Heated, with concentric ring cover, capable of
temperature control within 2°C. The bath should be used in a
hood.
4.4 Rotating evaporator.
4.5 Flasks - round bottom, 100 ml, with 24/40 joints.
4.6 Centrifuge tubes - conical, screw capped, graduated, with Teflon
lined caps.
-------
4.7 Pipettes - Pasteur, with bulbs.
4.8 High performance liquid chromatograph - Analytical system
complete with column supplies, recorder, syringes, and the
following components:
4.8.1 Solvent delivery system - with pulse damper (Altex 110A,
or equivalent).
4.8.2 Injection valve Waters Model U6K (or equivalent).
4.8.3 Electrochemical detector Bioanalytical Systems LC-2A with
glassy carbon electrode, (or equivalent).
4.8.4 Electrode polishing kit Princeton Applied Research Model
9320 (or equivalent).
5. Reagents
5.1 Preservatives:
5.1.1 Sodium hydroxide - (ACS) 10 N in distilled water.
5.1.2 Sulfuric acid - (ACS) Mix equal volumes of c'onc. H-S04
with distilled water.
5.1.3 Sodium thiosulfate - (ACS) granular.
5.2 Sulfuric acid (1M) - (ACS) 58 ml/liter in distilled water.
5.3 Sodium hydroxide (20%) - (ACS) 20 grams/100 ml in distilled water.
5.4 Sodium hydroxide (1M) - (ACS) 40 grams/liter in distilled water.
5.5 Sodium tribasic phosphate (0.4M) - (ACS) 160 grams
Na3P04(12H20) in 1 liter of distilled water.
5.6 Acetate buffer 0.1M, pH 4.7; 5.8 mis glacial acetic acid (ACS)
and 13.6 grams of sodium acetate trihydrate (ACS) per liter in
distilled water.
5.7 ' Acetonitrile - Pesticide quality or equivalent.
W~ 7O
-------
5.8 Methyl alcohol - Pesticide quality or equivalent.
5.9 Chloroform (preserved with 1% ETOH) - Burdick and Jackson (or
equivalent).
5.10 Water - Purified (e.g. from Millipore RO-4 System or equivalent).
5.11 HPLC mobile phase - Place 1 liter of filtered (through Millipore
type FH filter, or equivalent) acetonitrile and 1 liter of
filtered (through Millipore type GS filter, or equivalent)
acetate buffer in a narrow mouth, glass, one gallon jug and mix
thoroughly. Prepare fresh weekly.
5.12 Stock standards - Prepare stock standard solutions at a concen-
tration of 0.100 ug/ul by dissolving 0.0100 grams of assayed
reference material in pesticide quality methyl alcohol and
diluting to volume in a 100 ml ground glass stoppered volumetric
flask. The stock solution is transfered to ground glass
stoppered reagent bottles, stored in a refrigerator, and checked
frequently for signs of degradation or evaporation, especially
just prior to preparing working standards from them.
6. Calibration
6.1 Prepare calibration standards that contain the compounds of
interest, either singly or mixed together. The standards should
be prepared from the stock standards at the following concen-
trations that will bracket the working range of the
chromatographic system:
-------
Solution Cone, (nq/ul) Sensitivity fnA Full Scale)
0.05 10
0.10 10
0.50 50
1.0 100
5.0 500
6.2 Assemble the necessary liquid chromatographic apparatus and
establish operating parameters equivalent to those Indicated 1n
Table I.
6.3 A constant Injection volume of 25 microliters should be employed
for all subsequent measurements.
6.4 In order to determine the precision of the HPLC system, a series
of 6 replicate Injections of a 1 ng/ul solution of benzldlne and
3,3'HJlchlorobenz1d1ne (DCS) should be made on the 50nA full
scale setting. A precision of +43J should be achieved for the
peak heights of both benzldlne and DCS. This measurement should
be made every few weeks or whenever Instrument related problems
are apparent. A sample chromatogram is shown 1n Figure 1.
6.5 Retention times should remain relatively constant (within +5S day
to day) with benzldlne being 6.1 minutes and DCS-being 12.1
minutes under the specified conditions. These values should be
checked daily when the calibration injections are made.
6.6 If serious loss of response occurs, it may be necessary to polish
the surface of the carbon electrode (according to the instruc-
tions supplied with the polishing kit). In this case, it will be
necessary to recalibrate the system.
-------
6.7 When leaving the Instrument Idle, It is advisable to maintain a
flow of 0.1 inl/m1n of mobile phase through the HPLC column in
order to prolong column life.
7. Quality Control
7.1 Before processing any samples, the analyst should demonstrate
through the analysis of a distilled water blank, that all
glassware and reagents are interference-
free. Each time a set of samples is extracted or there is a
change in reagents, a method blank should be processed as a
safeguard against laboratory contamination.
7.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to validate the
precision of the sanpling technique. Laboratory replicates
should be analyzed to validate the precision of the analysis.
Fortified samples should be analyzed to validate the accuracy of
the analysis. Where doubt exists over the identification of a
peak on the chromatogram, confirmatory techniques such as mass
spectroscopy should be used.
8. Sample Collection. Preservation, and Handling
3.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle
must not be prewashed with sample before collection. Composite
samples should be collected in refrigerated glass containers in
accordance with the requirements of the program. Automatic
sampling equipment must be free of tygon and other potential
n 7
'
-------
sources of contamination.
8.2 The samples must be iced or refrigerated from the time of
collection to extraction. Benzidine and dichlorobenzidine are
easily oxidized by materials such as free chlorine. For
chlorinated wastes, immediately add 35 mg sodium thiosulfate per
part per million of free chlorine per liter.
8.3 If 1,2-diphenylhydrazine is likely to be present, adjust the pH
of the sample to 4+0.2 units to prevent rearrangement to
benzidine. Otherwise, if the samples will not be extracted
within 48 hours of collection, the sample pH should be adjusted
to 2-3 with sodium hydroxide or sulfuric acid.
8.4 All samples must be extracted within 7 days and completely
analyzed within 30 days of collection.
9. Sample Extraction
9.1 Mark the water meniscus on the side of the sample bottle for
later determination of sample volume. Quantitatively pour the
entire sample into a one-liter separatory funnel. Check the pH
of the sample with wide-range pH paper and adjust to within the
range of 6.5 - 7.5 with sodium hydroxide or sulfuric acid.
9.2 Add 100 ml chloroform to the sample bottle, seal, and shake 30
seconds to rinse the inner walls. Transfer the solvent into the
separatory funnel, and extract the sample by shaking the funnel
for two minutes. Allow the organic layer to separate from the
sample a minimum of ten minutes, then collect the chloroform
extract in a 250-ml separatory funnel.
9.3 Add 50 ml chloroform to the sample bottle and complete the
-------
extraction procedure a second time, combining the extracts in the
separatory funnel. Perform a third extraction in the same manner
with an additional 50 ml volume.
9.4 Discard any aqueous layer from the 250 ml funnel containing the
combined organic layers. Add 25 ml of 1 M sulfuric acid and
extract the sample by shaking the funnel for two minutes.
Transfer the aqueous layer to a 250 ml beaker. Extract with two
additional 25 ml portions of 1 M sulfuric acid and combine the
acid extracts in the beaker.
9.5 Place a stirbar in the 250 ml beaker and stir the acid extract
while carefully adding 5 ml of a .4M sodium tribasic phosphate.
With the aid of a pH meter neutralize the extract to pH 6-7 by
dropwise addition of 20 percent NaOH while stirring the solution
vigorously. Approximately 25-30 ml of 20 percent NaOH will be
required and it should be added over at least a 2-minute period.
Do not allow the sample pH ever to exceed pH 8.
9.6 Pour the neutralized extract into a 250 ml separatory funnel.
Add 30 ml of chloroform and shake the funnel for 2 minutes.
Allow phases to separate, and transfer the organic layer in a
second 250 ml separatory funnel.
9.7 Extract the aqueous layer with two additional 20 ml aliquots of
chloroform as before. Combine the extracts in the 250 ml
separatory funnel.
9.8 Add 20 ml of distilled water to the combined organic layers,
shake for 30 seconds, and discard aqueous layer.
9.9 Transfer the organic extract into a 100 ml round bottom flask.
T/T- T5"
-------
Add 20 ml of methyl alcohol and concentrate to 5 ml on a
rotating evaporator at 35°C.
9.10 Using a 9-inch Pasteur pipette, transfer the extract to a 15 ml
conical screw-capped centrifuge tube. Rinse the flask, including
the entire side wall, with two ml portions of methyl alcohol and
combine with the original extract.
9.11 Carefully concentrate the extract to 0.5 ml using a gentle stream
of nitrogen and a 30°C water bath, dilute to 2 ml with methyl
alcohol, reconcentrate to 1 ml, and dilute to 5 ml with acetate
buffer. Mix extract thoroughly. Stopper the ampul and store
refrigerated if further processing will not be performed
immediately.
9.12 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the liquid to a 1000 ml
graduated cylinder. Record the sample volume to the nearest 5 ml.
10. Liquid Chromatography fHPLC)
10.1 Table I summarizes the recommended liquid chromatographic column
material and operating conditions for the instrument. Included
in this table are estimated retention times and sensitivities
that should be achieved by this method. An example of the
separation achieved by this column is shown in Figure 1.
Calibrate the system daily with a minimum of three injections of
calibration standards.
10.2 Inject 25 ul of the sample extract. If the peak area exceeds the
linear range of the system, dilute the extract and reanalyze.
10.3 If the peak area measurement for benzidine is prevented by the
-------
presence of interferences, reduce the electrode potential to 0.7
V and reanalyze.
11. Calculations
11.1 Determine the concentration of individual compounds according to
the formula:
Concentration, ug/1 = (A) (B) (Vt)
(V^ (Vs)
where A = Calibration factor for chromatographic
system, in nanograms material per unit peak area.
B = Peak size in injection of sample extract, in
peak area units
V.. = volume of extract injected (ul)
Vt = Volume of total extract (ul)
V = Volume of water extracted (ml)
11.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
all data obtained should be reported.
12. Accuracy and Precision
12.1 The U.S. EPA Environmental Monitoring and Support Laboratory in
Cincinnati is in the process of conducting an interlaboratory
method study to determine the accuracy and - - 'on of this test
procedure.
-------
BIBLIOGRAPHY
"Development and Application of Test Procedures for Specific
Organic Toxic Substances in Wastewaters Category 7 -
Benzidines," Report for EPA Contract 68-03-2624 (In preparation)
71-
-------
TABLE I
Liquid Chromatography of Benzidines
Retention Detection
Compound Time (min.) Limit fug/1)
Benzidine 6.1 0.05
3,3'-dich1orobenzidine 12.1 0.1
Column conditions: Lichrosorb RP-2, 5 micron particle diameter,
packed in a 25 cm x 4.6 mm ID stainless steel column with
0.8 ml/min flow rate of mobile phase (50% acetonitrile: 50%
0.1 M pH 4.7 acetate buffer).
Detection limit is calculated from the minimum detectable
response of the electrochemical detector being equal to five
times the background noise, assuming a 5 ml final extract
volume of the 500 ml sample, and assuming an injection
volume of 25 micro!iters.
sr-7?
-------
uu
Q
INI
Z
LU
03
CO
O
ee
O
O
PO
0 4 8 12
RETEMTION TINIE-WINUTES
Figure 1. Liquid chromatogram of benzidines
nt
-------
PHTHALATE ESTERS
METHOD 606
1. Scope and Application
1.1 This method covers the determination of certain
phthalate esters. The following parameters may be
determined by this method:
Parameter STORET No.
Benzyl butyl phthalate 34292
B1s(2-ethy1hexy1) phthalate 39100
01-n-butyl phthalate 34110
01-n-octyl phthalate 34596
01ethyl phthalate 34336
Dimethyl phthalate 34341
1.2 This method 1s applicable to the determination of these
compounds In municipal and industrial discharges. It
1s designed to be used to meet the monitoring require-
ments of the National Pollutant Discharge Elimination
System (NPOES). As such, it presupposes a high expec-
tation of finding the specific compounds of interest.
If the user is attempting to screen samples for any or
all of the compounds above, he must develop independent
protocols for the verification of identity.
1.3 The sensitivity of this method is usually dependent
upon the level of interferences rather than instru-
mental limitations. The limits of detection listed in
-------
Table I represent sensitivities that can be achieved in
wastewaters in the absence of interferences.
1.4 This method is recommended for use only by experienced
residue analysts or under the close supervision of such
qualified persons.
2. Summary of Method
2.1 A 1-liter sample of wastewater is extracted with
methylene chloride using separatory funnel techniques.
The extract is dried and concentrated to a volume of 10
ml or less. Chromatographic conditions are described
which allow for the accurate measurement of the com-
pounds in the extract.
2.2 If interferences are encountered, the method provides
selected general purpose cleanup procedures to aid the
analyst in their elimination.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample pro-
cessing hardware may yield discrete artifacts and/or
elevated baselines causing misinterpretation of gas
chromatograms. All of these materials must be
demonstrated to be free from interferences under the
conditions of the analysis by running method blanks.
Specific selection of reagents and purification of
solvents by distillation in all-glass systems may be
required.
3.2 Interferences coextracted from the samples will vary
-------
considerably from source to source, depending upon the
diversity of the industrial complex or municipality
being sampled. While general cleanup techniques are
provided as part of this method, unique samples may
require additional cleanup approaches to achieve the
sensitivities stated in Table I.
3.3 Phthalate esters contaminate many types of products
commonly found in the laboratory. The analyst must
demonstrate that no phthalate residues contaminate the
sample or solvent extract under the conditions of the
analysis. Of particular importance is the avoidance of
plastics because phthalates are commonly used as
plasticizers and are easily extracted from plastic
materials. Serious, phthalate contamination may result
at any time if consistent quality control is not
practiced.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete or composite sampling.
4.1.1 Grab sample bottle - amber glass, 1-liter or
1-quart volume. French or Boston Round
design is recommended. The container must be
washed and solvent rinsed before use to
minimize interferences.
4.1.2 Bottle caps - Threaded to screw on to the
sample bottles. Caps must be lined with
Teflon. Foil may be substituted if sample is
-------
not corrosive.
4.1.3 Compositing equipment - Automatic or manual
compositing system. Must incorporate glass
sample containers for the collection of a
minimum of 250 ml. Sample containers must be
kept refrigerated during sampling. No tygon
or rubber tubing may be used in the system.
4.2 Separatory funnel - 2000 ml, with Teflon stopcock.
4.3 Drying column - 20 mm ID pyrex chromatographic column
with coarse frit.
4.4 Kuderna-Danish (K-D) Apparatus
4.4.1 Concentrator tube - 10 ml, graduated (Kontes
K-570050-1025 or equivalent). Calibration
must be checked. Ground glass stopper (size
19/22 joint) is used to prevent evaporation
of extracts.
4.4.2 Evaporative flask - 500 ml (Kontes
K-57001-0500 or equivalent). Attach to
concentrator tube with springs. (Kontes
K-662750-0012).
4.4.3 Snyder column - three-ball macro (Kontes
K503000-0121 or equivalent).
4.4.4 Snyder column - two-ball micro (Kontes
K-569001-0219 or equivalent).
4.4.5 Boiling chips - solvent extracted, approxi-
mately 10/40 mesh.
-------
4.5 Water bath - Heated, with concentric ring cover,
capable of temperature control (+ 2°C). The bath
should be used in a hood.
4.6 Gas chromatograph - Analytical system complete with gas
chromatograph suitable for on-column injection and all
required accessories including electron capture or
flame ionization detector, column supplies, recorder,
gases, syringes. A data system for measuring peak
areas is recommended.
4.7 Chromatography column - 300 mm long x 10 mm ID with
coarse fritted disc at bottom and Teflon stopcock
(Kontes K-420540-0213 or equivalent).
5. Reagents
5.1 Preservatives:
5.1.1 Sodium hydroxide - (ACS) 10 N in distilled
water.
5.1.2 Sulfuric acid - (ACS) Mix equal volumes of
cone. H2S04 with distilled water.
5.2 Methylene chloride - Pesticide quality or equivalent.
5.3 Sodium Sulfate - (ACS) Granular, anhydrous (purified by
heating at 400°C for 4 hrs. in a shallow tray).
5.4 Stock standards - Prepare stock standard solutions at a
concentration of 1.00 ug/ul by dissolving 0.100 grams
of assayed reference material in pesticide quality
isooctane or other appropriate solvent and diluting to
volume in a 100 ml ground glass stoppered volumetric
ITT
-------
flask. The stock solution is transferred to ground
glass stoppered reagent bottles, stored in a refriger-
ator, and checked frequently for signs of degradation
or evaporation, especially just prior to preparing
working standards from them.
5.5 Diethyl Ether - Nanograde, redistilled in glass if
necessary.
5.5.1 Must be free of peroxides as indicated by EM
Quant test strips. (Test strips are avail-
able from EM Laboratories, Inc., 500
Executive Blvd., Elmsford, N.Y. 10523.)
5.5.2 Procedures recommended for removal of
peroxides are provided with the test strips.
After cleanup, 20 ml ethyl alcohol preserva-
tive must be added to each liter of ether.
5.6 Florisil - PR grade (60/100 mesh); purchase activated
at 1250°F and store in dark in glass container with
ground glass stoppers or foil-lined screw caps.
5.7 Alumina - Activity Super I, Neutral, W200 series, (ICN
Life Sciences Group, No. 404583).
5.8 Hexane - Pesticide quality.
6. Calibration
6.1 Prepare calibration standards that contain the com-
pounds of interest, either singly or mixed together.
The standards should be prepared at concentrations
covering two or more orders of magnitude that will
72T-
-------
completely bracket the working range of the
chromatographic system. If the sensitivity of the
detection system can be calculated from Table I as 100
ug/1 in the final extract, for example, prepare stan-
dards at 10 ug/1, 50 ug/1, 100 ug/1, 500 ug/1, etc. so
that injections of 1-5 ul of each calibration standard
will define the linearity of the detector in the work-
ing range.
6.2 Assemble the necessary gas chromatographic apparatus
and establish operating parameters equivalent to those
indicated in Table I. By injecting calibration stan-
dards, establish the sensitivity limit of the detector
and the linear range of the analytical system for each
compound.
6.3 Before using any cleanup procedure, the analyst must
process a series of calibration standards through the
procedure to validate elution patterns and the absence
of interferences from the reagents.
7. Quality Control
7.1 Before processing any samples, the analyst should
demonstrate through the analysis of a distilled water
method blank, that all glassware and reagents are
interference-free. Each time a set of samples is
extracted or there is a change in reagents, a method
blank should be processed as a safeguard against
chronic laboratory contamination.
JT-ffc
-------
7.2 Standard quality assurance practices should be used
with this method. Field replicates should be collected
to validate the precision of the sampling technique.
Laboratory replicates should be analyzed to validate
the precision of the analysis. Fortified samples
should be analyzed to validate the accuracy of the
analysis. Where doubt exists over the identification
of a peak on the chromatogram, confirmatory techniques
such as mass spectroscopy should be used.
8. Sample Collection, Preservation, and Handling
8.1 Grab samples must be collected in glass containers.
Conventional sampling practices should be followed,
except that the bottle must not be prewashed with
sample before collection. Composite samples should be
collected in refrigerated glass containers in accor-
dance with the requirements of the program. Automatic
sampling equipment must be free of tygon and other
potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time
of collection until extraction. Chemical preservatives
should not be used in the field unless more than 24
hours will elapse before delivery to the laboratory.
If the samples will not be extracted within 48 hours of
collection, the sample should be adjusted to a pH range
of 6.0-8.0 with sodium' hydroxide or sulfuric acid.
8.3 All samples must be extracted within 7 days and
777-
-------
completely analyzed within 30 days of collection.
9. Sample Extraction
9.1 Mark the water meniscus on the side of the sample
bottle for later determination of sample volume. Pour
the entire sample into a two-liter separatory funnel.
Check the pH of the sample pH with wide-range paper and
adjust to within the range of 5-9 with sodium hydroxide
or sulfuric acid.
9.2 Add 60 ml methylene chloride to the sample bottle,
seal, and shake 30 seconds to rinse the inner walls.
Transfer the solvent into the separatory funnel, and
extract the sample by shaking the funnel for two
minutes with periodic venting to release vapor pres-
sure. Allow the organic layer to separate from the
water phase for a minimum of ten minutes. If the emul-
sion interface between layers is more than one-third
the size of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separa-
tion. The optimum technique depends upon the sample,
but may include stirring, filtration of the emulsion
through glass wool, or centrifugation. Collect the
methylene chloride extract in a 250-ml Erlenmeyer flask,
9.3 Add a second 60-ml volume of methylene chloride to the
sample bottle and complete the extraction procedure a
second time, combining the extracts in the Erlenmeyer
flask.
TTt--7
-------
9.4 Perform a third extraction in the same manner. Pour
the combined extract through a drying column containing
3-4 inches of anhydrous sodium sulfate, and collect it
in a 500-ml Kuderna-Danish (K-D) flask equipped with a
10 ml concentrator tube. Rinse the Erlenmeyer flask
and column with 20-30 ml methylene chloride to complete
the quantitative transfer.
9.5 Add 1-2 clean boiling chips to the flask and attach a
three-ball Snyder column. Prewet the Snyder column by
adding about 1 ml methylene chloride to the top. Place
the K-D apparatus on a hot water bath (60-65°C) so
that the concentrator tube is partially immersed in the
hot water, and the entire lower rounded surface of the
flask is bathed in vapor. Adjust the vertical position
of the apparatus and the water temperature as required
to complete the concentration in 15-20 minutes. At the
proper rate of distillation the balls of the column
will actively chatter but the chambers will not flood.
When the apparent volume of liquid reaches 1 ml, remove
the K-D apparatus and allow it to drain for at least 10
minutes while cooling.
9.6 Increase the temperature of the hot water bath to about
80°C. Momentarily remove the Snyder column, add 50
ml of hexane and a new boiling chip and reattach the
Snyder column. Pour about 1 ml of hexane into the top
of the Snyder column and concentrate the solvent
-------
extract as before. Elapsed time of concentration
should be 5 to 10 minutes. When the apparent volume of
liquid reaches 1 ml, remove the K-D apparatus and allow
it to drain at least 10 minutes while cooling. Remove
the Snyder column and rinse the flask and its lower
joint into the concentrator tube with 1-2 ml of hexane,
and adjust the volume to 10 ml. A 5-ml syringe is
recommended for this operation. Stopper the concen-
trator tube and store refrigerated if further process-
ing will not be performed immediately. If the sample
extract requires no further cleanup, proceed with gas
chromatographic analysis. If the sample requires
cleanup, proceed to Section 10.
9.7 Determine the original sample volume by refilling the
sample bottle to the mark and transferring the liquid
to a 1000 ml graduated cylinder. Record the sample
volume to the nearest 5 ml.
10. Cleanup and Separation
10.1 If the entire extract is to be cleaned up by one of the
following two procedures, it must be concentrated to
about 2 ml . To the concentrator tube in 9.6, add a
clean boiling chip and attach a two-ball micro-Snyder
column. Prewet the column by adding about 0.5 ml
hexane through the top. Place the K-D apparatus on a
hot water bath (80°C) so that the concentrator tube
is partially immersed in the hot water. Adjust the
is
-------
vertical position of the apparatus and the water
temperature as required to complete the concentration
in 5-10 minutes. At the proper rate of distillation
the balls of the column will actively chatter but the
chambers will not flood. When the apparent volume of
liquid reaches about 0.5 ml, remove the K-D apparatus
and allow it to drain for at least 10 minutes while
cooling. Remove the micro-Snyder column and rinse its
lower joint into the concentrator tube with 0.2 ml of
hexane. Proceed with one of the following clean-up
procedures.
10.2 Florisil Column Cleanup for Phthalate Esters
10.2.1 Place lOOg of Florisil into a 500 ml beaker and
heat for approximately 16 hours at 400°C.
After heating transfer to a 500 ml reagent
bottle. Tightly seal and cool to room temper-
ature. When cool add 3 ml of distilled water
which is free of phthalates and interferences.
Mix thoroughly by shaking or rolling for 10
minutes and let it stand for at least 2 hours.
Keep the bottle sealed tightly.
10.2.2 Place lOg of this Florisil preparation into a
10 mm ID chromatography column and tap the
column to settle the Florisil. Add 1 cm of
anhydrous sodium sulfate to the top of the
Florisil.
70
-------
10.2.3 Preelute the column with 40 ml of hexane. Dis-
card this eluate and just prior to exposure of
the sodium sulfate layer to the air transfer the
2 ml sample extract onto the column, using an
additional 2 ml of hexane complete the transfer.
10.2.4 Just prior to exposure of the sodium sulfate
layer to the air add 40 ml hexane and continue
the elution of the column. Discard this hexane
eluate.
10.2.5 Next elute the phthalate esters with 100 ml of
20% ethyl ether /80% hexane (V/V) into a 500 ml
K-D flask equipped with a 10 ml concentrator
tube. Elute the column at a rate of about 2 ml
per minute for all fractions. Concentrate the
collected fraction by standard K-D technique.
No solvent exchange is necessary. After
concentration and cooling, adjust the volume of
the cleaned up extract to 10 ml in the concen-
trator tube and analyze by gas chromatography.
10.3 Alumina Column Cleanup for Phthalate Esters
10.3.1 Place 100 g of alumina into a 500 ml beaker and
heat for approximately 16 hours at 400°C.
After heating transfer to a 500 ml reagent
bottle. Tightly seal and cool to room
temperature. When cool add 3 ml of distilled
water which is free from phthalates and
2-
-------
interferences. Mix thoroughly by shaking or
rolling for 10 minutes and let it stand for at
least 2 hours. Keep the bottle sealed tightly.
10.3.2 Place lOg of this alumina preparation into a 10
mm ID chromatography column and tap the column
to settle the alumina. Add 1 cm of anhydrous
sodium sulfate to the top of the alumina.
10.3.3 Preelute the column with 40 ml of hexane. Dis-
card this eluate and just prior to exposure of
the sodium sulfate layer to the air, transfer
the 2 ml sample extract onto the column, using
an additional 2 ml of hexane to complete the
transfer.
10.3.4 Just prior to exposure of the sodium sulfate
layer to the air add 35 ml hexane and continue
to elution of the column. Discard this hexane
eluate.
10.3.5 Next elute the column with 140 ml of 20% ethyl
ether/80% hexane (V/V) into a 500 ml K-D flask
equipped with a 10 ml concentrator tube. Elute
the column at a rate of about 2 ml per minute
for all fractions. Concentrate the collected
fraction by standard K-D technique. No solvent
exchange is necessary. After concentration and
cooling adjust the volume of the cleaned up
extract to 10 ml in the concentrator tube and
-13
-------
analyze by gas chromatography.
11. Gas Chromatography
11.1 Table I summarizes some recommended gas chromatographic
column materials and operating conditions for the
instrument. Included in this table are estimated
retention times and sensitivities that should be
achieved by this method. Examples of the separations
achieved by the primary column are shown in Figures 1
and 2. Calibrate the system daily with a minimum of
three injections of calibration standards.
11.2 Inject 2-5 ul of the sample extract using the
solvent-flush technique. Smaller (1.0 ul) volumes can
be injected if automatic devices are employed. Record
the volume injected to the nearest 0.05 ul, and the
resulting peak size, in area units.
11.3 If the peak area exceeds the linear range of the
system, dilute the extract and reanalyze.
11.4 If the peak area measurement is prevented by the
presence of interferences, further cleanup is required.
12. Calculations
12.1 Determine the concentration of individual compounds
according to the formula:
Concentration, ug/1 = (A) (B) (Vt)
(Vi) (Vs)
where A = Calibration factor for chromatographic
system, in nancgrams material per area unit.
\ll-~
-------
B « Peak size in injection of sample extract, in
area units
V. = volume of extract injected (ul)
Vt = Volume of total extract (ul)
V$ = Volume of water extracted (ml)
12.2 Report results in micrograms per liter without correc-
tion for recovery data. When duplicate and spiked
samples are analyzed, all data obtained should be
reported.
13. Accuracy and Precision
13.1 The U.S. EPA Environmental Monitoring and Support
Laboratory in Cincinnati is in the process of conduct-
ing a interlaboratory method study to determine the
accuracy and precision of this test procedure.
BIBLIOGRAPHY
"Development and Application of Test Procedures for Specific
Organic Toxic Substances in Wastewaters. Category
1-Phthalates." Report for EPA Contract 68-03-2606 (In
preparation).
¥• 9*"
-------
TABLE I
GAS CHROMATOGRAPHY OF PHTHALATE ESTERS
Retention Time (min.) Detection Limit (ug/1)
Compound Column 1 Column 2 EC FID
Dimethyl phthalate
Diethyl phthalate
Di-n-butyl phthalate
Butyl benzyl
phthalate
Bis(2-ethylhexyl)
phthalate
Di-n-octyl
phthalate
2.03
2.82
8.65
6.94*
8.92*
16.2*
0.95
1.27
3.50
5.11**
10.5**
18.0**
0.11
0.13
0.02
0.02
0.04
0.11
19
31
14
15
20
31
Column 1 conditions: Supelcoport ToO/120 mesh coated with 1.5%
SP-2250/1.95% SP-2401 packed in a 180 cm long x 4 mm ID
glass column with carrier gas at 60 ml/min flow rate.
Column temperature is 180°C except where * indicates
220°C. Under these conditions R.T. of Aldrin is 5.49 min.
at 180°C and 1.84 min at 220°C.
Column 2 conditions: Supelcoport 100/120 mesh coated with 3%
OV-1 in a 180 cm long x 4 mm ID glass column with carrier
gas at 60 ml/min flow rate. Column temperature is 200°C
except where ** indicates 220°C. Under these conditions
R.T. of Aldrin is 3.18 min. at 200°C and 1.46 min. at
220°C.
Detection limit is calculated from the minimum detectable GC
response being equal to five times the GC background noise,
assuming a 10 ml final volume of the 1 liter sample extract,
and assuming a GC injection of 5 micro!iters.
-------
I COLUMN: 1.5% SP-2250 +
1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 180°C.
DETECTOR; ELECTRON CAPTURE
<
_i
<
x
a.
oa
a
0 2 4 6 8 10 12
RETENTION TIME-MINUTES
Figure 1. Gas chromatogram of phthalates
•a.
-------
COLUMN: 1.5% SP-2250 +
1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 180°C.
DETECTOR: ELECTRON CAPTURE
X
a.
X X
a. t—
_a ^
*" d
LU oa
ca
X
a.
O
c
0 4 8 12 16 :
RETENTION TIME-MINUTES
Figure 2. Gas chromatogram c* phthalates
-------
NITROSAMINES
METHOD 607
1. Scope and Application
1.1 This method covers the determination of certain nitrosamines. The
following parameters may be determined by this method:
Parameter STORET No.
N-nitrosodimethylamine 34438
N-nitrosodiphenylamine 34433
N-nitrosodi-n-propylamine 34428
1.2 This method is applicable to the determination of these compounds
in municipal and industrial discharges. It is designed to be used
to meet the monitoring requirements of the National Pollutant
/
Discharge Elimination System (NPDES). As such, it presupposes a
high expectation of finding the specific compounds of interest. If
the user is attempting to screen samples for any or all of the
compounds above, he must develop independent protocols for the
verification of identity.
1.3 The sensitivity of this method is usually dependent upon the level
of interferences rather than instrumental limitations. The limits
of detection listed in Table I represent sensitivities that can be
achieved in wastewaters in the absence of interferences.
1.4 This method is recommended for use only by experienced residue
analysts or under the close supervision of such qualified persons.
1.5 The analyst must understand that nitrosamines are known carcino-
gens. Utmost care must be exercised in the handling of materials
-------
which are known or believed to contain nitrosanrines.
2. Summary of Method
2.1 A 1-liter sample of wastewater is extracted with methylene chloride
using separatory funnel techniques. The extract is dried and
concentrated to a volume of 10 ml or less. Depending upon the
nitrosamines being measured, a column cleanup procedure may be
required. Chromatographic conditions are described which allow for
the accurate measurement of the compounds in the extract.
2.2 If interferences are encountered, the method provides selected
general purpose cleanup procedures to aid the analyst in their
elimination.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines causing
misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the
conditions of the analysis by running method blanks. Specific
selection of reagents and purification of solvents by distillation
in all-glass systems may be required.
3.2 Interferences coextracted from the samples will vary considerably
from source to source, depending upon the diversity of the indus-
trial complex or municipality being sampled. While general
clean-up techniques are provided as part of this method, unique
samples may require additional cleanup approaches to achieve the
sensitivities stated in Table 1.
3.3 It is necessary to remove diphenylamine frr- tr.e sample extract
-------
prior to gas chromatography because it will interfere with the
determination of N-nitrosodiphenylamine. Removal is achieved if
the sample is processed completely through one of the clean-up
procedures detailed in the method.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete or composite sampling.
4.1.1 Grab sample bottle - amber glass, 1-liter or 1-quart
volume. French or Boston Round design is recommended.
The container must be washed and solvent rinsed before
use to minimize interferences.
4.1.2 Bottle caps - Threaded to screw on to the sample
bottles. Caps must be lined with Teflon. Foil may be
substituted if sample is not corrosive.
4.1.3 Compositing equipment - Automatic or manual compositing
system. Must incorporate glass sample containers for the
collection of a minimum of 250 ml. Sample containers
must be kept refrigerated during sampling. No tygon or
rubber tubing may be used in the system.
4.2 Separatory funnels - 2000 ml and 250 ml, with Teflon stopcock.
4.3 Drying column - 20 mm ID pyrex chromatographic column with coarse
frit.
4.4 Kuderna-Danish (K-D) Apparatus
4.4.1 Concentrator tube - 10 ml, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be
checked. Grc-^d glass stopper (size 19/22 joint) is used
to prevent ev;~?ration of extracts.
-------
4.4.2 Evaporative flask - 500 ml (Kontes K-57001-0500 or equiva-
lent). Attach to concentrator tube with springs. (Kontes
K-662750-0012).
4.4.3 Snyder column - three-ball macro (Kontes K503000-0121 or
equivalent).
4.4.4- Snyder column - two-ball micro (Kontes K-569001-0219 or
equivalent).
4.4.5 Boiling chips - solvent extracted, approximately 10/40 mesh.
4.5 Water bath - Heated, with concentric ring cover, capable of temper-
ature control ( + 2°C). The bath should be used in a hood.
4.6 Gas chromatograph - Analytical system complete with gas chromato-
graph suitable for on-column injection and all required accessories
including nitrogen-phosphorus or reductive Hall detector, column
supplies, recorder, gases, syringes. A data system for measuring
peak areas is recommended.
4.7 Chromatographic column - Pyrex (approximately 300 mm long x 10 mm
ID) with coarse fritted disc at bottom and Teflon stopcock (Kontes
K-420540-0213 or equivalent).
4.8 Chromatographic column - Pyrex (approximately 400 mm long x 22 mm
ID) with coarse fritted disc at bottom and Teflon stopcock (Kontes
\
K-420540-0234 or equivalent).
5. Reagents
5.1 Preservatives:
5.1.1 Sodium hydroxide - (ACS) 10 N in distilled water.
5.1.2 Sulfuric acid - (ACS) Mix equal volumes of cone. H2S04
with distilled water.
'00
-------
5.1.3 Sodium thiosulfate - (ACS) Granular.
5.2 Methylene chloride - Pesticide quality or equivalent.
5.3 Sodium Sulfate - (ACS) Granular, anhydrous (purified by heating at
400°C for 4 hrs. in a shallow tray).
5.4 Stock standards - Prepare stock standard solutions at a concentra-
tion of 1.00 ug/ul by dissolving 0.100 grams of assayed reference
material in pesticide quality isooctane or other appropriate
solvent and diluting to volume in a 100 ml ground glass stoppered
volumetric flask. The stock solution is transferred to ground
glass stoppered reagent bottles, stored in a refrigerator, and
checked frequently for signs of degradation or evaporation,
especially just prior to preparing working standards from them.
5.5 Methyl alcohol, pentane, acetone - Pesticide quality or equivalent.
5.6 Diethyl Ether - Nanograde, redistilled in glass if necessary.
5.6.1 Must be free of peroxides as indicated by EM Quant test
strips. (Test strips are available from EM Laboratories,
Inc., 500 Executive Blvd., Elmsford, N.Y. 10523.)
5.6.2 Procedures recommended for removal of peroxides are provided
with the test strips. After cleanup, 20 ml ethyl alcohol
preservative must be added to each liter of ether.
5.7 Florisil - PR grade (60/100 mesh); purchase rjtivated at 1250°F
and store in dark in glass containers witr r' — s stoppers or
foil-lined screw caps. Before use, activate ^;n batch at least 16
hours at 130°C in a foil covered glass ccr'j'-~*r.
5.8 Alumina - Activity Super I, Basic, W20G ser.'er ^ICN Life Sciences
Group, No. 404571).
TIT-
-------
5.9 Hydrochloric acid, 10%-(ACS) Add one volume of cone. HC1 to nine
volumes distilled water.
6. Calibration
6.1 Prepare calibration standards that contain the compounds of
interest, either singly or mixed together. The standards should be
»
prepared at concentrations covering two or more orders of magnitude
that will completely bracket the working range of the chromato-
graphic system. If the sensitivity of the detection system can be
calculated from Table I as 100 ug/1 in the final extract, for
example, prepare standards at 10 ug/1, 50 ug/1, 100 ug/1, 500 ug/1,
etc. so that injections of 1-5 ul of each calibration standard will
define the linearity of the detector in the working range.
6.2 Assemble the necessary gas chromatographic apparatus and establish
operating parameters equivalent to those indicated in Table I. By
injecting calibration standards, establish the sensitivity limit of
the detector and the linear range of the analytical system for each
compound.
6.3 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from the reagents.
7. Quality Control
7.1 Before processing any samples, the analyst should demonstrate
through the analysis of a distilled water method blank, that all
glassware and reagents are interference-free. Each time a set of
samples is extracted or there is a change in reagents, a method
-------
blank should be processed as a safeguard against chronic laboratory
contamination.
7.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to validate the
precision of the sampling technique. Laboratory replicates should
be analyzed to validate the precision of the analysis. Fortified
samples should be analyzed to validate the accuracy of the
analysis. Where doubt exists over the identification of a peak on
the chromatogram, confirmatory techniques such as mass spectroscopy
should be used.
8. Sample Collection, Preservation, and Handling
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be prewashed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be free of tygon and other potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time of collec-
tion until extraction. Chemical preservatives should not be used
in the field unless more than 24 hours will elapse before delivery
to the.laboratory. If the samples will not be extracted within 48
hours of collection, they must be preserved as follows:
8.2.1 Add 35 mg of sodium thiosulfate per part per million of free
chlorine per liter of sample.
8.2.2 Adjust the pH of the water sample to pH 7 to 10 using sodium
hydroxide or sulfuric acid. Record the volume of acid or
base added.
-------
8.3 All samples must be extracted within 7 days and completely analyzed
within 30 days of collection.
9. Sample Extraction
9.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
two-liter separatory funnel. Check the pH of the sample with
wide-range pH paper and adjust to within the range of 7 to 10 with
sodium hydroxide or sulfuric acid.
9.2 Add 60 ml methylene chloride to the sample bottle, seal, and shake
30 seconds to rinse the inner walls. Transfer the solvent into the
separatory funnel, and extract the sample by shaking the funnel for
two minutes with periodic venting to release vapor pressure. Allow
the organic layer to separate from the water phase for a minimum of
ten minutes. If the emulsion interface between layers is more than
one-third the size of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool, or
centrifugation. Collect the methylene chloride extract in a 250-ml
separatory funnel.
9.3 Add a second 60-ml volume of methylene chloride to the sample
bottle and complete the extraction procedure a second time,
combining the extracts in the 250-ml separatory funnel.
9.4 Perform a third extraction in the same manner. Add 10 ml of 10%
HC1 solution to the combined extracts and shake for 2 minutes.
Allow the layers to separate. Drain the methylene chloride layer
-------
through a drying column containing 3-4 inches of anhydrous sodium
sulfate, and collect it in a 500-ml Kuderna-Danish (K-D) flask
equipped with a 10 ml concentrator tube. Rinse the column with
20-30 ml methylene chloride to complete the quantitative transfer.
9.5 Add 1-2 clean boiling chips to the flask and attach a three-ball
Snyder column. Prewet the Snyder column by adding about 1 ml
methylene chloride to the top. Place the K-D apparatus on a hot
water bath (60-65°C) so that the concentrator tube is partially
immersed in the hot water, and the entire lower rounded surface of
the flask is bathed in vapor. Adjust the vertical position of the
apparatus and the water temperature as required to complete the
concentration in 15-20 minutes. At the proper rate of distillation
the balls of the column will actively chatter but the chambers will
not flood. Because of the volatility of N-nitrosodimethyl amine,
K-D concentration must be carefully carried out. When the apparent
volume of liquid reaches 1 ml, remove the K-D apparatus and allow
it to drain for at least 10 minutes while cooling. Remove the
Snyder column and rinse the flask and its lower joint into the
concentrator tube with 1-2 ml of methylene chloride. A 5-ml
syringe is recommended for this operation. Unless the entire
extract will be subjected to a cleanup operation (Section 10),
adjust the extract volume to 10.0 ml with methylene chloride, add
stopper, and refrigerate.
9.6 If the sample is being analyzed for N-nitrosodiphenylamine, the
analyst must immediately proceed with one of the cleanup methods in
-------
Section 10 to remove potential diphenylamine interference. Depend-
ing upon the sensitivity requirement for the analysis, the analyst
may use the entire extract for this cleanup as described, or adjust
the extract volume to 10.0 ml with methylene chloride and pipet a 2
ml aliquot onto the column in 10.2.2 or 10.3.3.
9.7 If N-nitrosodiphenylamine is of no interest, the analyst must
choose between proceeding directly to Section II, or submitting the
extract to a cleanup procedure before gas chromatography. A
solvent exchange from methylene chloride to methyl alcohol is
required for direct gas chromatography. Once the entire extract is
in methyl alcohol it cannot be treated to either of the cleanup
procedures in Section 10. Therefore, in the absence of previous
experience with the sample matrix, the analyst should remove a 2.0
ml aliquot of the extract for gas chromatography and retain the
remainder for cleanup if required later.
9.8 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the liquid to a 1000 ml graduated
cylinder. Record the sample volume to the nearest 5 ml.
10. Cleanup and Separation
10.1 If the entire extract is to be cleaned up by one of the following
procedures, it must be concentrated to 2.0 ml. To the concentrator
tube in 9.5, add a clean boiling chip and attach a two-ball
micro-Snyder column. Prewet the column by adding about 0.5 ml
methylene chloride to the top. Place the K-D apparatus on a
steaming hot (60-65°C) 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 the concentration in 5-10 minutes. At the proper rate of
distillation the balls of the column will actively chatter but the
chambers will not flood. When the apparent volume of liquid
reaches about 0.5 ml, remove the K-D and allow it to drain for at
1 *
least 10 minutes while cooling. Remove the micro- Snyder column and
rinse its lower joint into the concentrator tube with 0.2 ml of
methylene chloride. Adjust the final volume to 2.0 ml and proceed
with one of the following cleanup procedures.
10.2 Florisil Column Cleanup for Nitrosamines
10.2.1 Place 22g of activated Florisil in a 22 mm' ID chromato-
graphic column. After settling the Florisil by tapping the
column, add about a 5 mm layer of anhydrous granular sodium
sulfate to the top.
10.2.2 Preelute the column, after cooling, with 40 ml of 15% ethyl
ether/85% pentane. Discard the eluate and just prior to
exposure of the sodium sulfate layer to air, quantitatively
transfer 2.0 ml of sample extract into the column by decan-
tation using an additional 2 ml of pentane to complete the
transfer.
10.2.3 Perform the first elution with 90 ml of 15% ethyl ether/85%
pentane (V/V) and discard the eluate. This fraction will
contain any diphenylamine.
10.2.4 Perform the second elution with 100 ml of 5% acetone/95%
ethyl ether (V/V) and collect the eluate in a 500-ml K-D
flask equipped with a 10-ml concentrator tube. This
ur
-------
fraction will contain all of the nitrosamines.
10.2.5 Add 15 ml of methanol to the collected eluate and concen-
trate as in 9.5 at 70-75°C, substituting pentane for
methylene chloride.
10.2.6 Analyze by gas chromatography.
10.3 Alumina Column Cleanup for Nitrosamines
10.3.1 Place lOOg of alumina, as it comes from the manufacturer,
into a 500 ml reagent bottle and add 2 ml of distilled
water, which is free of nitrosamines and interferences. Mix
the alumina preparation thoroughly by shaking or rolling for
10 minutes and let it stand for at least 2 hours. The
preparation should be homogeneous before use. Keep the
bottle sealed tightly to ensure proper activity.
10.3.2 Place 12 grams of the alumina preparation into a 10 mm ID
chromatographic column and tap the column to settle the
alumina. Add 1-2 cm of anhydrous sodium sulfate to the top
of the alumina.
10.3.3 Preelute the column with 10 ml of 30% ethyl ether/70%
pentane (V/V). Discard the eluate (about 2 ml) and, just
prior to exposure of the sodium sulfate layer the air,
transfer 2.0 ml of sample extract onto the column by decan-
tation using an additional 2 ml of pentane to complete the
transfer.
10.3.4 Just prior to exposure of the sodium sulfate layer to the
air, add 70 ml of 2?« ethyl ether/70% pentane. Discard the
first 10 ml of elus."- - Jt collect the rest of the eluate in
S
-------
a 500-ml K-D flask equipped with a 10 ml concentrator tube.
This fraction contains N-nitrosodiphenylamine and probably a
small amount of N-nitrosodi-n-propylamine.
10.-3.5 Next elute the column with 60 ml of 50* ethyl ether/50%
pentane, collecting the eluate in a second K-D flask
equipped with a 10 ml concentrator tube. Add 15 ml methyl
alcohol to the K-D. This fraction will contain
N-nitrosodimethylamine, most of the
N-nitrosodi-n-propylamine and any diphenylamine.
10.3.6 Concentrate both fractions as in 9.5 substituting pentane
for methylene chloride.
10.3.7 Analyze by gas chromatography.
11. Gas Chromatography
11.1 N-nitrosodiphenylamine completely reacts to form diphenylamine at
normal operating temperatures of the GC injection port. There-
fore, N-nitrosodiphenylamine is actually chromatographed and
detected as diphenylamine. The determination of either of the
compounds in the original sample would be uncertain without the
use of one of the previous cleanup procedures which separate the
two compounds.
11.2 Table I summarizes some recommended gas chromatographic column
materials and operating conditions for the instrument. Included
in this table are estimated retention times and sensitivities that
should be achieved by this method. Examples of the separations
achieved by the primary column are shown in Figures 1 and 2.
Calibrate the system daily with a minimum of three injections of
-------
calibration standards.
11.3 If the extract has not been submitted to one of the cleanup proce-
dures in Section 10, it is necessary to exchange the solvent from
methylene chloride to methyl alcohol before the thermionic detec-
tor can be used. To a 1-10 ml volume of methylene chloride
extract in a concentrrator tube, add 2 ml methyl alcohol, and a
clean boiling chip. Attach a two-ball micro-Snyder column.
Prewet the column by adding about 0.5 ml methylene chloride
through the top. Place the K-D apparatus on a boiling water bath
so that the concentrator tube is partially immersed in the hot
water. Adjust the vertical position and insulate the apparatus as
necessary to complete the concentration in 5-10 minutes. At the
proper rate of distillation the balls of the column will actively
chatter but the chambers will not flood. When the apparent volume
of liquid reaches about 0.5 ml, remove the K-D and allow it to
drain for at least 10 minutes while cooling. Remove the
micro-Snyder column and rinse its lower joint into the concen-
trator tube with 0.2 ml of methyl alcohol. Adjust the final
volume to 2.0 ml.
11.4 Inject 2-5 ul of the sample extract using the solvent-flush
technique. Smaller (1.0 ul) volumes can be injected if automatic
devices are employed. Record the volume injected to the nearest
0.05 ul, and the resulting peak size, in area units.
11.5 If the peak area exceeds the linear range of the system, dilute
the extract and reanalyze.
11.6 If the peak area measurement is prevented by the presence of
-------
interferences, further cleanup is required.
12. Calculations
12.1 Determine the concentration of individual compounds according to
the formula:
Concentration, ug/1 -'(A) (B) (Vt)
(Vi) (Vs)
where A = Calibration factor for chromatographic system, in
nanograms material per area unit.
B = Peak size in injection of sample extract, in area units
V. = volume of extract injected (ul)
V. = Volume of total extract (ul)
V = Volume of water extracted (ml)
12.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
all data obtained should be reported.
13. Accuracy and Precision
The U.S. EPA Environmental Monitoring and Support Laboratory in
Cincinnati is in the process of conducting an interlaboratroy method
study to determine the accuracy and precision of this test procedure.
BIBLIOGRAPHY
"Development and Application of Test Procedures for Specific Organic Toxic
Substances in Wastewaters. Category 5 - Nitrosamines," Report for EPA .
Contract 68-03-2606 (In preparation).
iu
-------
TABLE I
GAS CHROMATOGRAPHY OF NITROSAMINES
Retention Time (min.)
Detection
Nitrosamine
N-nitrosodidimethylamine
N-nitrosodi-n-dipropylamine
N-nitrosodidiphenylamine
Column 1
4.1
12.1
12.8*
Column 2
0.88
4.2
6.4**
Limit (ug/1)
0.3
0.5
1.0
Column 1 conditions: Chromosorb WAW 80/100 mesh coated with 10% Carbowax
20M/2% KOH packed in a 180 on long x 4 mm ID glass column with helium
carrier gas at 40 ml/min flow rate. Isothermal column temperature is
110°C except where * indicates 220°C.
Column 2 conditions: Supelcoport 100/120 mesh coated with 10% SP-2250
packed in a 180 cm long x 4 mm ID glass column with helium carrier gas
at 40 ml/mi^flow rate. Isothermal column temperature is 120°C except
where ** indicates 210°C.
Detection limit is calculated from the minimum detectable GC response being
equal to five times the GC background noise, assuming a 10 ml final
volume of the 1 liter sample extract, and assuming a GC injection of 5
microliters. A nitrogen-phosphorus detector was used to collect this
data, but a Thermal Energy Analyzer exhibited equivalent sensitivity.
Ml
-------
COLUMN: 10% CARBOWAX 20M + 2% KOH OM CHROMOSORB W-AW
TEMPERATURE: 110°
DETECTOR: PHOSPHORUS/NITROGEN
Q_
o
cc
a.
O
CO
O
cc
24 6 8 10 12 14
RETENTION TIME-MINUTES
Figure 1. Gas chromatogram of nitrosamines
-------
COLUMN: 10% CARBOWAX 20M •»- 2% KOH ON CHROMOSORB YV-AW
TEMPERATURE: 220°C.
DETECTOR: PHOSPHORUS/NITROGEN
0 2 4 6 8 10 12 14 16 18
RETENTION TIME-MINUTES
Figure 2. Gas chromatogram of N-nitrosodiphenylamine
as diphenylamine
-------
ORGANOCHLORINE PESTICIDES AND PCB's
METHOD 608
1. Scope and Application
1.1 This method covers the determination of certain organochlorine
pesticides and polychlorinated biphenyls (PCBs). The following
parameters may be determined by this method:
Parameter
Aldrin
a-BHC
b-BHC
d-BHC
g-BHC
v
Chlordane
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan Sulfate
STORET No.
39330
39337
39338
39259
39340
39350
39310
39320
39300
39380
34361
34356
34351
Parameter
Endrin
Endrin Aldehyde
Heptachlor
Heptachlor Epoxide
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
STORET No
39390
34366
39410
39420
39400
34671
39488
39492
39496
39500
39504
39508
1.2 This method is applicable to the determination of these compounds
in municipal and industrial discharges. It is designed to be used
to meet the monitoring requirements of the National Pollutant
Discharge Elimination System (NPDES). As such, it presupposes a
- 113
-------
high expectation of finding the specific compounds of interest. If
the user is attempting to screen samples for any or all of the
compounds above, he must develop independent protocols for the
verification of identity.
1.3 The sensitivity of this method is usually dependent upon the level
of interferences rather than instrumental limitations. The limits
of detection listed in Table I represent sensitivities that can be
achieved in wastewaters in the absence of interferences..
1.4 This method is recommended for use only by experienced residue
analysts or under the close supervision of such qualified persons.
2. Summary of Method
2.1 A 1-liter sample of wastewater is extracted with methylene chloride
using separatory funnel techniques. The extract is dried and
concentrated to a volume of 10 ml or less. Chromatographic
conditions are described which allow for the accurate measurement
of the compounds in the extract.
2.2 If interferences are encountered, the method provides selected
general purpose cleanup procedures to aid the analyst in their
elimination.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines causing
misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the
conditions of the analysis by running method blanks. Specific
selection of reagents and purification of solvents by distillation
UT-
-------
in all-glass systems may be required.
3.2 Interferences coextracted from the samples will vary considerably
from source to source, depending upon the diversity of the
industrial complex or municipality being sampled. While general
cleanup techniques are provided as part of this method, unique
samples may require additional cleanup approaches to achieve the
sensitivities stated in Table 1.
3.3 Glassware must be scrupulously clean. Clean all glassware as soon
as possible after use by rinsing with the last solvent used. This
should be followed by detergent washing in hot water. Rinse with
tap water, distilled water, acetone and finally pesticide quality
hexane. Heavily contaminated glassware may require treatment in a
muffle furnace at 400°C for 15 to 30 minutes. Some high boiling
materials, such as PCBs, may not be eliminated by this treatment.
Volumetric ware should not be heated in a muffle furnace.
Glassware should be sealed/stored in a clean environment
immediately after drying or cooling to prevent any accumulation of
dust or other contaminants. Store inverted or capped with aluminum
foil.
3.4 Interferences by phthalate esters can pose a major problem in
.pesticide analysis. These materials elute in the 15% and 50%
fractions of the Florisil cleanup. They usually can be minimized
by avoiding contact with any plastic materials. The contamination
from phthalate esters can be completely eliminated with the use of
a microcoulometric or electrolytic conductivet detector.
77T - 11
-------
4. Apparatus and Materials
4.1 Sampling equipment, for discrete or composite sampling.
4.1.1 Grab sample bottle - amber glass, 1-liter or 1-quart
volume. French or Boston Round design is recommended.
The container must be washed and solvent rinsed before
use to minimize interferences.
4.1.2 Bottle caps - Threaded to screw on to the sample
bottles. Caps must be lined with Teflon. Foil may be
substituted if sample is not corrosive.
4.1.3 Compositing equipment - Automatic or manual compositing
system. Must incorporate glass sample containers for the
collection of a minimum of 250 ml. Sample containers
must be kept refrigerated during sampling. No tygon or
rubber tubing may be used in the system.
4.2 Separatory funnel - 2000 ml, with Teflon stopcock.
4.3 Drying column - 20 mm ID pyrex chromatographic column with coarse
frit.
4.4 Kuderna-Danish (K-D) Apparatus
4.4.1 Concentrator tube - 10 ml, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be
checked at 1.0 and 10.0 ml level. Ground glass stopper
(size 19/22 joint) is used to prevent evaporation of
extracts.
4.4.2 Evaporative flask - 500 ml (Kontes K-57001-0500 or
equivalent). Attach to concentrator tube with springs.
(Kontes K-662750-0012).
J7T
-------
4.4.3 Snyder column - three-ball macro (Kontes K503000-0121 or
equivalent).
4.4.4 Boiling chips - extracted, approximately 10/40 mesh.
4.5 Water bath - Heated, with concentric ring cover, capable of
temperature control (+ 2°C). The bath should be used in a hood.
4.6 Gas chromatograph - Analytical system complete with gas
chromatograph suitable for on-column injection and all requ-ired
accessories including electron capture or halogen-specific
detector, column supplies, recorder, gases, syringes. A data
system for measuring peak areas is recommended.
4.7 Chromatographic column - Pyrex, 400 mm x 25 mm OD, with coarse
fritted plate and Teflon stopcock (Kontes K-42054-213 or
equivalent).
5. Reagents
5.1 Preservatives:
5.1.1 Sodium hydroxide - (ACS) 10 N in distilled water.
5.1.2 Sulfuric acid (1+1) - (ACS) Mix equal volumes of cone.
H2S04 with distilled water.
5.2 Methylene chloride - Pesticide quality or equivalent.
5.3 Sodium Sulfate - (ACS) Granular, anhydrous (purified by heating at
40t)°C for 4 hrs. in a shallow tray).
5.4 Stock standards - Prepare stock standard solutions at a
concentration of 1.00 ug/ul by dissolving 0.100 grams of assayed
reference material in pesticide quality isooctane or other
appropriate solvent and diluting to volume in a 100 ml ground glass
stoppered volumetric flask. The stock solution is transferred to
"7
-------
ground glass stoppered reagent bottles, stored in a refrigerator,
and checked frequently for signs of degradation or evaporation,
especially just prior to preparing working standards from them.
5.5 Boiling chips - Hengar granules (Hengar Co.; Fisher Co.) or
equivalent.
5.6 Mercury - triple distilled.
5.7 Aluminum oxide - basic or neutral, active.
5.8 Hexane - pesticide residue analysis grade.
5.9 Isooctane (2,2,4-trimethyl pentane) - pesticide residue analysis
grade.
5.10 Acetone - pesticide residue analysis grade.
5.11 Diethyl ether - Nanograde, redistilled in glass if necessary.
5.11.1 Must be free of peroxides as indicated by EM Quant test
strips (Test strips are available from EM Laboratories,
Inc., 500 Executive Blvd., Elmsford, N.Y., 10523).
5.11.2 Procedures recommended for removal of peroxides are
provided with the test strips. After cleanup 20 ml ethyl
alcohol preservative must be added to each liter of ether.
5.12 Florisil - PR grade (60/100 mesh); purchase activated at 1250°F
and store in glass containers with glass stoppers or foil-lined
screw caps. Before use activate each batch at least 16 hours at
130°C in a foil covered glass container.
6. Calibration
6.1 Prepare calibration standards that contain the compounds of
interest, either singly or mixed together. The standards should be
prepared at concentrations covering two or more orders of magnitude
ID
-------
that will completely bracket the working range of the
chromatographic system. If the sensitivity of the detection system
can be calculated from Table I as 100 ug/1 in the final extract,
for example, prepare standards at 10 ug/1, 50 ug/1, 100 ug/1, 500
ug/1, etc., so that injections of 1-5 ul of each calibration
standard will define the linearity of the detector in the working
range.
6.2 Assemble the necessary gas chromatographic apparatus and establish
operating parameters equivalent to those indicated in Table I. By
injecting calibration standards, establish the sensitivity limit of
the detector and the linear range of the analytical system for each
compound.
6.3 The cleanup procedure in Section 10 utilizes Florisil
chromatography. Florisil from different batches rt«* sources may
vary in absorption capacity. To standardize the amount of Florisil
which is used, the use of lauric acid value (Mills, 1968) is
suggested. The referenced procedure determines the adsorption from
hexane solution of lauric acid (mg) per gram Florisil. The amount
of Florisil to be used for each column is calculated by dividing
this factor into 110 and multiplying by 20 grams.
6.4 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from the reagents.
7. Quality Control
7.1 Before processing any si."les, the analyst should demonstrate
through the analysis of i distilled water method blank, that all
HI '«?
-------
glassware and reagents are interference-free. Each time a set of
samples is extracted or there is a change in reagents, a method
blank should be processed as a safeguard against chronic laboratory
contamination.
7.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to validate the
precision of the sampling technique. Laboratory replicates should
be analyzed to validate the precision of the analysis. Fortified
samples should be analyzed to validate the accuracy of the
analysis. Where doubt exists over the identification of a peak on
the chromatogram, confirmatory techniques such as mass spectroscopy
should be used.
8. Sample Collection, Preservation, and Handling
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be prewashed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be free of tygon and other potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time of
collection until extraction. Chemical preservatives should not be
used in the field unless more than 24 hours will elapse before
delivery to the laboratory. If the samples will not be extracted
within 48 hours of collection, the sample should be adjusted to a
pH range of 6.0-8.0 with sodium hydroxide or sulfuric acid.
8.3 All samples must be extracted within 7 days and completely analyzed
-------
within 30 days of collection.
9. Sample Extraction
9.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
two-liter separatory funnel. Check the pH of the sample with
wide-range pH paper and adjust to within the range of 5-9 with
sodium hydroxide or sulfuHc acid.
9.2 Add 60 ml methylene chloride to the sample bottle, seal, and shake
30 seconds to rinse the inner walls. Transfer the solvent into the
separatory funnel, and extract the sample by shaking the funnel for
two minutes with periodic venting to release vapor pressure. Allow
the organic layer to separate from the water phase for a minimum of
ten minutes. If the emulsion interface between layers is more than
one-third the size of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool, or
centrifugation. Collect the methylene chloride extract in a 250-ml
Erlenmeyer flask.
9.3 Add a second 60-ml volume of methylene chloride to the sample
« bottle and complete the extraction procedure a second time,
combining the extracts in the Erlenmeyer flask.
9.4 Perform a third extraction in the same manner. Pour the combined
extract through a drying column containing 3-4 inches of anhydrous
sodium sulfate, and collect it in a 500-ml Kuderna-Danish (K-D)
flask equipped with a 10 ml concentrator tube. Rinse the
-------
Erlenmeyer flask and column with 20-30 ml methylene chloride to
complete the quantitative transfer.
9.5 Add 1-2 clean boiling chips to the flask and attach a three-ball
Snyder column. Prewet the Snyder column by adding about 1 ml
methylene chloride to the top. Place the K-D apparatus on a hot
water bath (60-65°C) so that the concentrator tube is partially
immersed in the hot water, and the entire lower rounded surface of
the flask is bathed in vapor. Adjust the vertical position of the
apparatus and the water temperature as required to complete the
concentration in 15-20 minutes. At the proper rate of distillation
the balls of the column will actively chatter but the chambers will
not flood. When the apparent volume of liquid reaches 1 ml, remove
the K-D apparatus and allow it to drain for at least 10 minutes
while cooling.
9.6 Increase the temperature of the hot water bath to about 80°C.
Momentarily remove the Snyder column, add 50 ml of hexane and a new
boiling chip and reattach the Snyder column. Pour about 1 ml of
hexane into the top of the Snyder column and concentrate the sol-
vent extract as before. The elapsed time of concentration should
be 5 to 10 minutes. When the apparent volume of liquid reaches 1
ml, remove the K-D apparatus and allow it to drain at least 10
minutes while cooling. Remove the Snyder column and rinse the
flask and its lower joint into the concentrator tube with 1-2 ml of
hexane, and adjust the volume to 10 ml. A 5-ml syringe is
recommended for this operation. Stopper the concentrator tube and
store refrigerated if further processing will not be performed
-------
Immediately. If the sample extract requires no further cleanup,
proceed with gas chromatographic analysis. If the sample requires
cleanup, proceed to Section 10.
9.7 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the liquid to a 1000 ml graduated
cylinder. Record the sample volume to the nearest 5 ml.
10. Cleanup and Separation
10.1 Cleanup procedures are used to extend the sensitivity of a method
by minimizing or eliminating interferences that mask or otherwise
disfigure the gas chromatographic response to the pesticides and
PCB's. The Florisil column allows for a select fractionation of
the compounds and will eliminate polar materials. Elemental sulfur
interferes with the electron capture gas chromatography of certain
pesticides but can be removed by the techniques described below.
10.2 Florisil Column Cleanup
10.2.1 Add a weight of Florisil, (nominally 21g,) predetermined by
calibration (6.3, 6.4), to a chromatographic column. Settle
the Florisil by tapping the column. Add sodium sulfate to
the top of the Florisil to form a layer 1-2 cm deep. Add 60
ml of hexane to wet and rinse the sodium sulfate and
Florisil. Just prior to exposure of the sodium sulfate to
air, stop the elution of the hexane by closing the stopcock
on the chromatography column. Discard the eluate.
10.2.2 Adjust the sample extract volume to 10 ml and transfer it
from the K-D concentrator tube to the Florisil
-------
column. Rinse the tube twice with 1-2 ml hexane, adding
each rinse to the column.
10.2.3 Place a 500 ml K-D flask and clean concentrator tube under
the chromatography column. Drain the column into the flask
until the sodium sulfate layer is nearly exposed. Elute the
column with 200 ml of 6% ethyl ether in hexane (Fraction 1)
using a drip rate of about 5 ml/min. Remove the K-D flask
and set aside for later concentration. Elute the column
again, using 200 ml of 15% ethyl ether in hexane (Fraction
2), into a second K-D flask. Perform the third elution
using 200 ml of 50% ethyl ether in hexane (Fraction 3). The
elution patterns.for the pesticides and PCB's are shown in
Table II.
10.2.4 Concentrate the eluates by standard K-D techniques (9.5),
substituting hexane for the glassware rinses and using the
water bath at about 85°C. Adjust final volume to 10 ml
with hexane. Analyze by gas chromatography.
10.3 Elemental sulfur will usually elute entirely in Fraction 1. To
remove sulfur interference from this fraction or the original
extract, pi pet 1.00 ml of the concentrated extract into a clean
concentrator tube or Teflon-sealed vial. Add 1-3 drops of mercury
and seal. Agitate the contents of the vial for 15-30 seconds.
Place the vial in an upright position on a reciprocal laboratory
shaker and shake for 2 hours. Analyze by gas chrx -;tography.
11. Gas Chromatography
11.1 Table I summarizes some recommended gas chromatoc' - ic column
-------
materials and operating conditions for the instrument. Included in
this table are estimated retention times and sensitivities that
should be achieved by this method. Examples of the separations
achieved by these columns are shown in Figures 1 through 10.
Calibrate the system daily with a minimum of three injections of
calibration standards.
11.2 Inject 2-5 ul of the sample extract using the solvent-flush
technique. Smaller (1.0 ul) volumes can be injected if automatic
devices are employed. Record the volume injected to the nearest
0.05 ul, and the resulting peak size, in area units.
11.3 If the peak area exceeds the linear range of the system, dilute the
extract and reanalyze.
11.4 If the peak area measurement is prevented by the presence of
interferences, further cleanup is required.
12. Calculations
12.1 Determine the concentration of individual compounds according to
the formula:
Concentration, ug/1 = (A) (B) (Vt)
(Vi) (Vs)
where A = Calibration factor for chromatographic system, in
nanograms material per area unit.
B = Peak size in injection of sample extract, in area units
V^ = volume of extract injected (ul)
Vt = Volume of total extract (ul)
V = Volume of water extracted (ml)
12.2 Report results in micrograms per liter without correction for
-------
recovery data. When duplicate and spiked samples are analyzed, all
data obtained should be reported.
13. Accuracy and Precision
13.1 The U.S. EPA Environmental Monitoring and Support Laboratory in
Cincinnati is in the process of conducting an interlaboratory
method study to determine the accuracy and precision of this test
procedure.
BIBLIOGRAPHY
1. "Development and Application of Test Procedures for Specific Organic
Toxic Substances in Wastewaters. Category 10-Pesticides and PCB's."
Report for EPA Contract 68-03-2606.
Mills, P. A., "Variation of Florisil Activity: Simple Method for
Measuring Absorbent Capacity and Its Use in Standardizing Florisil
Columns," Journal of the Association of Official Analytical Chemists,
51, 29 (1968).
-------
TABLE I
GAS CHROMATOGRAPHY OF PESTICIDES AND PCB's
Parameter
Retention Time (min)
Column 1 Column 2
Detection Limit
(ug/1)1
Aldrin
a-BHC
b-BHC
d-BHC
g-BHC
Chlordane
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
2.40
1.35
1.90
2.15
1.70
*
7.83
5.13
9.40
5.45
4.50
8.00
14.22
6.55
11.82
2.00
3.50
*
*
*
*
*
*
*
*
4.10
1.82
1.97
2.20
2.13
*
9.08
7.15
11.75
7.23
6.20
8.28
10.70
8.10
9.30
3.35
5.00
*
*
*
*
*
*
it
it
0.003
0.002
0.004
0.004
0.002
0.04
0.012
0.006
0.016
0.006
0.005
0.01
0.03
0.009
0.023
0.002
0.004
0.40
0.04
0.10
0.10
0.05
0.08
0.08
0.15
* Multiple peak response. See Figures 2-10.
** Detection limit is calculated from the minimum detectable GC response
being equal to five times the GC background noise, assuming a 10 ml
final volume of the 1 liter sample extract, and assuming a GC injection
of 5 micro!iters.
Column 1 conditions: Supelcoport 100/120 mesh coated with 1.5%
SP-2250/1.95% SP-2401 packed in a 180 cm long x 4 mm ID glass column
with 5% Methane/95% Argon carrier gas at 60 ml/min flow rate. Column
temperature is 200°C.
Column 2 conditions: Supelcoport 100/120 mesh coated with 3% OV-1 in a 180
cm long x 4 mm ID glass column with 5% Methane/95% Argon carrier gas at
60 ml/min flow rate. Column temperature is 200°C.
-------
TABLE II
DISTRIBUTION AND RECOVERY OF CHLORINATED PESTICIDES
AND PCBs USING FLORISIL COLUMN CHROMATOGRAPHY
Parameter
Recovery (%) by Fraction*
1(6%) 2(15%) 3(50*)
Aldrin
a-BHC
b-BHC
d-BHC
g-BHC
Chlordane
4,4'-ODD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
100
100
97
98
100
100
99
98
100
0
37
0
0
4
0
100
100
96
97
97
95
97
103
90
95
100
64
7
0
96
68
91
106
26
*From: "Development and Application of Test Procedures for Specific Organic
Toxic Substances in Wastewaters. Category 10-Pesticides and PCB's.
Report for EPA Contract 68-03-2606."
. . , , ^~<
jli - D:V
-------
COLUMN: 1.5% SP-2250*
1.95% SP-2401 DIM SUPELCOPORT
TEMPERATURE: 200°C.
DETECTOR: ELECTRON CAPTURE
4 8 12
RETENTION TIME-MINUTES
16
Figure 1. Gas chromatogram of pesticides
-------
COLUMN: 1.5% SP-2250*
1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 200°C.
DETECTOR: ELECTRON CAPTURE
4 8 12
RETENTION TIME-MINUTES
16
Figure 2. Gas chromatogram of chlordane
JL
-------
COLUMN: 1.5% SP-2250+
1.95?'« SP-2401 ON SUPELCOPORT
TEMPERATURE: 200'C.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18 22
RETENTION TIME-MINUTES
Figure 3. Gas chromatogram of toxaphene
26
-------
COLUMN: 1.5% SP-2250+ 1.95%
EMPERATURE: 160aC.
DETECTOR: ELECTRON CAPTURE
o SP-2401 ON SUPELCOPORT
2 6 10 14 18
RETENTION TIME-MINUTES
Figure 4. Gas chromatogram of PCB-1016
22
-------
COLUMN: 1.5% SP-2250+ 1.95% SP-2401 DIM SUPELCOPORT
TEMPERATURE: 160'C.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18
RETENTION TIME-MINUTES
Figure 5. Gas chromatogram of PCB-1221
22
- e
-------
COLUMN: 1.5JS SP-2250* 1.95°i SP-2401 ON SUPELCOPORT
TEMPERATURE: 160°C.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18
RETENTION TIME-MINUTES
Figure 6. Gas chromatogram of PCB-1232
22
24
tT -
-------
COLUMN: 1.5% SP-2250+1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 160°C.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18
RETENTION TIME-MINUTES
Ffgure 7. Gas chromatogram of PCB-1242
22
-------
COLUMN: 1.5% SP-2250* 1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 160°C.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18
RETENTION TIME-MINUTES
Figure 8. Gas chromatogram of PCB-1248
22
26
-------
COLUMN: 1.5% SP-2250 + 1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 200°C.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18
RETENTION TIME-MINUTES
Figure 9. Gas chromatogram of PCB-1254
22
-------
COLUMN: 1.5% SP-2250+ 1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 200aC.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18 22
RETENTION TIME-MINUTES
Figure 10. Gas chromatogram of PCB-1260
26
-------
NITROAROMATICS AND ISOPHORONE
METHOD 609
1. Scope and Application
1.1 This method covers the determination of certain nitroaromatics and
isophorone. The following parameters may be determined by this
method:
Parameter STORET No.
Isophorone 34408
Nitrobenzene 34447
2,4-Dinitrotoluene 34611
2,6-Dinitrotoluene 34626
1.2 This method is applicable to the determination of these compounds
in municipal and industrial discharges. It is designed to be used
to meet-the monitoring requirements of the National Pollutant
Discharge Elimination System (NPDES). As such, it presupposes a
high expectation of finding the specific compounds of interest. If
the user is attempting to screen samples for any or all of the
compounds above, he must develop independent protocols for the
verification of identity.
1.3 The sensitivity of this method is usually dependent upon the level
of interferences rather than instrumental limitations. The limits
of detection listed in Table I represent sensitivities that can be
achieved in wastewaters in the absence of interferences.
-------
1.4 This method is recommended for use only by experienced residue
analysts or under the close supervision of such qualified persons.
2. Summary of Method
2.1 A 1-liter sample of wastewater is extracted with methylene chloride
using separatory funnel techniques. The extract is dried and
exchanged to toluene while being concentrated to 1.0 ml.
Isophorone and nitrobenzene are measured by flame ionization gas
chromatography. The dinitrotoluenes are measured by electron
capture GC.
2.2 If interferences are encountered, the method provides a general
purpose cleanup procedure to aid the analyst in their elimination.
3. Interferences
3.1 Solvents,, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines causing
misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the
conditions of the analysis by running method blanks. Specific
selection of reagents and purification of solvents by distillation
in all-glass systems may be required.
3.2 Interferences coextracted from the samples will vary considerably
from source to source, depending upon the diversity of the
industrial complex or municipality being sampled. While general
clean-up techniques are provided as part of this method, unique
samples may require additional cleanup approaches to achieve the
sensitivities stated in Table I.
4. Apparatus and Materials
-------
4.1 Sampling equipment, for discrete or composite sampling.
4.1.1 Grab sample bottle - amber glass, 1-liter or 1-quart
volume. French or Boston Round design is recommended.
The container must be washed and solvent rinsed before
use to minimize interferences.
4.1.,2 Bottle caps - Threaded to screw on to the sample
bottles. Caps must be lined with Teflon. Foil may be
substituted if sample is not corrosive.
4.1.3 Compositing equipment - Automatic or manual compositing
system. Must incorporate glass sample containers for the
collection of a minimum of 250 ml. Sample containers
must be kept refrigerated during sampling. No tygon or
rubber tubing may be used in the system.
4.2 Separatory funnel - 2000 ml, with Teflon stopcock.
4.3 Drying column - 20 mm ID pyrex chromatographic column with coarse
frit.
4.4 Kuderna-Danish (K-D) Apparatus
4.4.1 Concentrator tube - 10 ml, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be
checked. Ground glass stopper (size 19/22 joint) is used
to prevent evaporation of extracts.
4.4.2 Evaporative flask - 500 ml (Kontes K-57001-0500 or
equivalent). Attach to concentrator tube with springs.
(Kontes K-662750-0012).
4.4.3 Snyder column - three-ball macro (Kontes K503000-0121 or
equivalent).
O
-------
4.4.4 Snyder column - two-ball micro (Kontes K-569001-0219 or
equivalent).
4.4.5 Boiling chips - solvent extracted, approximately 10/40
mesh.
4.5" Water bath - Heated, with concentric ring cover, capable of
temperature control (+ 2°C). The bath should be used 1n a hood.
4.6 Gas chromatograph - Analytical system complete with gas
chromatograph suitable for on-column Injection and all required
accessories including both electron capture and flame ionization
detectors, column supplies, recorder, gases, syringes. A data
system for measuring peak areas is recommended.
4.7 Chromatography column - 400 mm long x 10 mm ID, with coarse fritted
plate on bottom and Teflon stopcock.
5. Reagents
5.1 Preservatives:
5.1.1 Sodium hydroxide - (ACS) 10 N in distilled water.
5.1.2 Sulfuric acid (1+1) - (ACS) Mix equal volumes of cone.
H2S04 with distilled water.
5.2 Methylene chloride - Pesticide quality or equivalent.
5.3 Sodium sulfate - (ACS) Granular, anhydrous (purified by heating at
400°C for 4 hrs. in a shallow tray).
5.4 Stock standards - Prepare stock standard solutions at a
concentration of 1.00 ug/ul by dissolving 0.100 grams of assayed
reference material in pesticide quality isooctane or other
appropriate solvent and' diluting to volume in a 100 ml ground glass
stoppered volumetric flask. The stock solution is transferred to
-------
ground glass stoppered reagent bottles, stored in a refrigerator,
and checked frequently for signs of degradation or evaporation,
especially just prior to preparing working standards from them.
5.5 Acetone, Hexane, Methanol, Toluene - pesticide quality or
equivalent.
5.6 Florisil - PR grade (60/100 mesh); purchase activated at 1250°F
and store in glass containers with glass stoppers or foil-lined
screw caps. Before use, activate each batch overnight at 200°C
in glass containers loosely covered with foil.
6. Calibration
6.1 Prepare calibration standards that contain the compounds of
interest, either singly or mixed together. The standards should be
prepared at concentrations covering two or more orders of magnitude
that will completely bracket the working range of the
chromatographtc system, if the sensitivity of the detection system
can be calculated from Table I as 100 ug/1 in the final extract,
for example, prepare standards at 10 ug/1, 50 ug/1, 100 ug/1, 500
ug/1, etc. so that injections of 1-5 ul of each calibration
standard will define the linearity of the detector in the working
range.
6.2 Assemble the necessary gas chromatographic apparatus and establish
operating parameters equivalent to those indicated in Table I. By
injecting calibration standards, establish the sensitivity limit of
the detector and the linear range of the analytical system for each
compound.
6.3 Before using any cleanup procedure, the analyst must process a
-------
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from the reagents.
7. Quality Control
7.1 Before processing any samples, the analyst should demonstrate
through the analysis of a distilled water method blank, that all
glassware and reagents are interference-free. Each time a set of
samples is extracted or there is a change in reagents, a method
blank should be processed as a safeguard against chronic laboratory
contamination.
7.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to validate the
precision of the sampling technique. Laboratory replicates should
be analyzed to validate the precision of the analysis. Fortified
samples should be analyzed to'validate the accuracy of the
analysis. Where doubt exists over the identification of a peak on
the chromatogram, confirmatory techniques such as mass spectroscopy
should be used.
8. Sample Collection, Preservation, and Handling
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be prewashed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be free of tygon and other potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time of
collection until extraction. Chemical preservatives should not be
-------
used in the field unless more than 24 hours will elapse before
delivery to the laboratory. If the samples will not be extracted
within 48 hours of collection, the sample should be adjusted to a
pH range of 6.0-8.0 with sodium hydroxide or sulfuric acid.
8.3 All samples must be extracted within 7 days and completely analyzed
within 30 days of collection.
9. Sample Extraction
9.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
two- liter separatory funnel. Check the pH of the sample with
wide-range pH paper and adjust to within the range of 5-9 with
sodium hydroxide or sulfuric acid.
9.2 Add 60 ml methyl ene chloride to the sample bottle, seal, and shake
30 seconds to rinse the inner walls. Transfer the solvent into the
separatory funnel, and extract the sample by shaking the funnel for
two minutes with periodic venting to release vapor pressure. Allow
the organic layer to separate from the water phase for a minimum of
ten minutes. If the emulsion interface between layers is more than
one-third the size of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool, or
centrifugation. Collect the methylene chloride extract in a 250-ml
Erlenmeyer flask.
9.3 Add a second 60-ml volume of methylene chloride to the sample
bottle and complete the extraction procedure a second time,
IS
-------
combining the extracts in the Erlenmeyer flask.
9.4 Perform a third extraction in the same manner. Pour the combined
extract through a drying column containing 3-4 inches of anhydrous
sodium sulfate, and collect it in a 500-ml Kuderna-Danish (K-D)
flask equipped with a 10 ml concentrator tube. Rinse the
Erlenmeyer flask and column with 20-30 ml methylene chloride to
complete the quantitative transfer.
9.5 Add 1-2 clean boiling chips to the flask and attach a three-ball
Snyder column. Prewet the Snyder column by adding about 1 ml
methylene chloride to the top. Place the K-D apparatus on a hot
water bath (60-65°C) so that the concentrator tube is partially
immersed in the hot water, and the entire lower rounded surface of
the flask is bathed in vapor. Adjust the vertical position of the
apparatus and the water temperature as required to complete the
concentration in 15-20 minutes. At the proper rate of distillation
the balls of the column will actively chatter but the chambers will
not flood. When the apparent volume of liquid reaches 1 ml, remove
the K-D apparatus and allow it to drain for at least 10 minutes
while cooling. Remove the Snyder column and rinse the flask and
its lower joint into the concentrator tube with 1-2 ml of methylene
chloride. A 5-ml syringe is recommended for this operation.
9.6 Add 1.0 ml toluene to the concentrator tube, and a clean boiling
chip. Attach a two-ball micro-Snyder column. Prewet the
micro-Snyder column by adding about 0.5 ml of methylene chloride to
the top. Place this micro-K-D apparatus on a water bath
(60-65°C) so that the concentrator tube is partially immersed in
134,
-------
the hot water. Adjust the vertical position of the apparatus and
water temperature as required to complete the concentration in 5 to
10 minutes. At the proper rate of distillation the balls will
actively chatter but the chambers will not flood. When the
apparent volume of liquid reaches 0.5 ml, remove the K-D apparatus
and allow it to drain for at least 10 minutes while cooling.
Remove the micro-Snyder column and rinse its lower joint into the
concentrator tube with a small volume of toluene. Adjust the final
volume to 1.0 ml and stopper the concentrator tube and store
refrigerated if further processing will not be performed
immediately. Unless the sample is known to require cleanup,
proceed with gas chromatographic analysis.
9.7 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the liquid to a 1000 ml graduated
cylinder. Record the sample volume to the nearest 5 ml.
10. Cleanup and Separation
10.1 Prepare a slurry of lOg of activited Florisil in 10£ methylene
chloride in hexane (V/V). Use it to pack a 10 mm ID chromatography
column, gently tapping the column to settle the Florisil. Add 1 cm
anhydrous sodium sulfate to the top of the Florisil.
10.1.2 Just prior to exposure of the sodium sulfate layer to the
air transfer the 1 ml sample extract onto the column
using an additional 2 ml of toluene to complete the
transfer.
10.1.3 Just prior to exposure of the sodium sulfate layer to the
air, add 30 ml 10% methylene chloride in hexane and
nr- 137
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continue the elution of the column. Elution of the
column should be at a rate of about 2 ml per minute.
Discard the eluate from this fraction.
10.1.4 Next elute the column with 30 ml of 10% acetone/90%
methylene chloride (V/V) into a 500 ml K-D flask equipped
with a 10 ml concentrator tube. Concentrate the
collected fraction by the K-D technique prescribed in 9.5
and 9.6, including the solvent exchange to 1 ml toluene.
This fraction should contain the nitroaromatics and
isophorone.
10.1.5 Analyze by gas chromatography.
11. Gas Chromatography
11.1 Isophorone and nitrobenzene are analyzed by injection of a portion
of the extract into a gas chromatograph with a flame ionization
detector. The dinitrotoluenes are analyzed by a separate injection
into an electron capture gas chromatograph. Table I summarizes
some recommended gas chromatographic column materials and operating
conditions for the instruments. Included in this table are
estimated retention times and sensitivities that should be achieved
by this method. Examples of the separations achieved by the
primary column are shown in Figures 1 and 2. Calibrate the system
daily with a minimum of three injections of calibration standards.
11.2 Inject 2-5 ul of the sample extract using the solvent-flush
technique. Smaller (1.0 ul) volumes can be injected if automatic
devices are employed. Record the volume injected to the nearest
0.05 ul, and the resulting peak size, in area units.
-------
11.3 If the peak area exceeds the linear range of the system, dilute the
extract and reanalyze.
11.4 If the peak area measurement is prevented by the presence of
interferences, further cleanup is required.
12. Calculations
12.1 Determine the concentration of individual compounds according to
the formula:
Concentration, ug/1 = (A) (B) (Vt)
(Vi) (Vs)
where A = Calibration factor for chromatographic system, in
nanograms material per area unit.
B * Peak size in injection of sample extract, in area units
V. = volume of extract injected (ul)
V. = Volume of total extract (ul)
V = Volume of water extracted (ml)
12.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed, all
data obtained should be reported.
13. Accuracy and Precision
The U.S. EPA Environmental Monitoring and Support Laboratory in
Cincinnati is in the process of conducting an inter!aboratory method
study to determine the accuracy and precision of this test procedure.
BIBLIOGRAPHY
"Development and Application of Test Procedures for Specific Organic Toxic
Substances in Wastewaters. Category 4-Nitroaromatics and Isophorone,"
Report for EPA Contract No. 68-03-2624 (In preparation).
jr - 13
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TABLE I
GAS CHROMATOGRAPHY OF .NITROAROMATICS AND ISOPHORONE
Retention Time (min.) Detection Limit (ug/1)
Compound
Isophorone
Ni trobenzene
2,4-Dinitrotoluene
2,6-Dinitrotoluene
Column 1
4.49
3.31
5.35
3.52
Column 2
5.72
4.31
6.54
4.75
EC
-
- •
0.06
0.06
FID
5
5
-
-
Column 1 conditions: Gas-Chrom Q 80/100 mesh coated with 1.95% QF-1/1.5%
OV-17 packed in a 4' x 1/4" OD glass column. FID analysis for IP and NB
requires nitrogen carrier gas at 44 ml/min and 85°C column
temperature. EC analysis for the DNTs requires 10% Methane/90% Argon
carrier gas at 44 ml/min flow rate and 145°C column temperature.
Column 2 conditions: Gas-Chrom Q 80/100 mesh coated with 3% OV-101 packed
in a 10' x 1/4" OD glass column. FID analysis of IP and NB requires
nitrogen carrier gas at 44 ml/min flow rate and 100°C column
temperature. EC analysis for the DNTs requires 10% Methane/90% Argon
carrier gas at 44 ml/min flow rate and 150°C column temperature.
Detection limit is calculated from the minimum detectable GC response being
equal to five times the GC background noise, assuming a 10 ml final
volume of the 1 liter sample extract, and assuming a GC injection of 5
microliters.
JIT- I MO
-------
COLUMN: 1.5% OV-17+ 1.95% QF-1 ON GAS CHROM Q
I TEMPERATURE: 85°C.
DETECTOR: FLAME IONIZAT10N
LU
J_
_L
02468
RETENTION TIME-MINUTES
Figure 1. Gas chromatogram of nitrobenzene and
isophorone
Hi-
-------
1.95°i
COLUMN: 1.5% OV-17
TEMPERATURE: 145°C.
DETECTOR: ELECTRON CAPTURE
QF-1 ON GAS CHROM Q
2 46 8 10
RETENTION TIME-MINUTES
Figure 2. Gas chromatogram of dinitrotoluenes
WO-
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POLYNUCLEAR AROMATIC HYDROCARBONS
METHOD 610
1. Scope and Application
1.1 This method covers the determination of certain polynuclear
aromatic hydrocarbons (PAH). The following parameters may be
determined by this method:
Parameter STORET No. Parameter STORET No.
Acenaphthene 34205 Chrysene 34320
Acenaphthylene 34200 Dibenzo(ah-)anthracene 34556
Anthracene 34220 Fluoranthene 34376
Benzo(a)anthracene 34526 Fluorene 34381
Benzo(a)pyrene 34247 Indeno(l,2,3-cd)pyrene 34403
Benzo(b)fluoranthene 34230 Naphthalene 34696
Benzo(ghi)perylene 34521 Phenanthrene 34461
Benzo(k)fluoranthene 34242 Pyrene 34469
1.2 This method is applicable to the determination of these compounds
in municipal and industrial discharges. It is designed to be used
to meet the monitoring requirements of the National Pollutant
Discharge Elimination System (NPDES). As such, it presupposes a
high expectation of finding the specific compounds of interest. If
the user is attempting to screen samples for any or all of the
compounds above, he must develop independent protocols for the
verification of identity.
1.3 This method contains both liquid and gas chromatographic
approaches, depending upon the needs of the analyst. The gas
-------
chromatographic procedure cannot adequately resolve the following
four pairs of compounds: anthracene and phenanthrene; chrysene and
benzo(a)anthracene; benzo(b)fluoranthene and benzo(k)fluoranthene;
and dibenzo(ah)anthracene and indeno(l,2,3-cd)pyrene. Unless the
purposes of the analysis can be served by reporting a sum for an
unresolved pair, the liquid chromatographic approach must be used
for these compounds. The liquid chromatographic method will
resolve all of the 16 compounds listed above.
1.4 The sensitivity of this method is usually dependent upon the level
of interferences rather than instrumental limitations. The limits
of detection listed in Table I for the liquid chromatographic
approach represent sensitivities that can be achieved in
wastewaters in the absence of interferences.
1.5 This method is recommended for use only by experienced residue
analysts or under the close supervision of such qualified persons.
2. Summary of Method
2.1 A 1-liter sample of wastewater is extracted with methylene chloride
using separatory funnel techniques. The extract is dried and
concentrated to a volume of 10 ml or less. Chromatographic
conditions are described which allow for the accurate measurement
of the compounds in the extract by either High Performance Liquid
Chromatography (HPLC) or gas chromatography.
2.2 If interferences are encountered, the method provides a selected
general purpose cleanup procedure to aid the analyst in their
elimination.
3. Interferences
77T
-------
3.1 Solvents, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines causing
misinterpretation of the chromatograms. All of these materials
must be demonstrated to be free from interferences under the
conditions of the analysis by running method blanks. Specific
selection of reagents and purification of solvents by distillation
in all-glass systems may be required.
3.2 Interferences coextracted from the samples will vary considerably
from source to source, depending upon the diversity of the
industrial complex or municipality being sampled. While a general
clean-up technique is provided as part of this method, unique
samples may require additional clean-up approaches to achieve the
sensitivities stated in Table 1.
3.3 The extent of interferences that may be encountered using liquid
chromatographic techniques has not been fully assessed. Although
the chromatographic conditions described allow for a unique
resolution of the specific PAH compounds covered by this method,
other PAH compounds may interfere.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete or composite sampling.
4.1.1 Grab sample bottle - amber glass, 1-liter or 1-quart
volume. French or Boston Round design is recommended. The
container must be washed and solvent rinsed before use to
minimize interferences.
4.1.2 Bottle caps - Threaded to screw on to the sample bottles.
Caps must be lined with Teflon. Foil may be substituted if
-------
sample is not corrosive.
4.1.3 Compositing equipment - Automatic or manual compositing
system. Must incorporate glass sample containers for the
collection of a minimum of 250 ml. Sample containers must
be kept refrigerated during sampling. No tygon or rubber
tubing may be used in the system.
4.2 Separatory funnel - 2000 ml, with Teflon stopcock.
4.3 Drying column - 20 mm ID pyrex chromatographic column with coarse
frit.
4.4 Kuderna-Danish (K-D) Apparatus
4.4.1 Concentrator tube - 10 ml, graduated (Kontes K-570050-1025
or equivalent). Calibration must be checked. Ground glass
stopper (size 19/22 joint) is used to prevent evaporation of
extracts.
4.4.2 Evaporative flask - 500 ml (Kontes K-57001-0500 or
equivalent). Attach to concentrator tube with springs.
(Kontes K-662750-0012).
4.4.3 Snyder column - three-ball macro (Kontes K503000-0121 or
equivalent).
4.4.4 Snyder column - two-ball micro (Kontes K-569001-0219 or
equivalent).
4.4.5 Boiling chips - solvent extracted, approximately 10/40 mesh.
4.5 Water bath - Heated, with concentric ring cover, capable of
temperature control (_+ 2°C). The bath should be used in a hood.
4.6 HPLC Apparatus:
4.6.1 Gradient pumping system, constant flow.
-------
4.6.2 Reverse phase column, HC-ODS Sil-X, 250 mm x 2.6 mm ID
(Perkin Elmer No. 809-0716 or equivalent).
4.6.3 Fluorescence detector, for excitation at 280 ran and emission
at 389 nm.
4.6.4 UV detector, 254 nm, coupled to fluorescence detector.
4.6.5 Strip chart recorder compatible with detectors, (A data
system for measuring peak areas is recommended).
4.7 Gas chromatograph - Analytical system complete with gas
chromatograph suitable for on-column injection and all required
accessories including dual flame ionization detectors, column
supplies, recorder, gases, syringes. A data system for measuring
peak areas is recommended.
4.8 Chromatographic column - 250 mm long x 10 mm ID with coarse fritted
•disc at bottom and Teflon stopcock.
5. Reagents
5.1 Preservatives:
5.1.1 Sodium hydroxide - (ACS) 10 N in distilled water.
5.1.2 Sulfuric acid - (ACS) Mix equal volumes of cone. H2$04
with distilled water.
5.1.3 Sodium thiosulfate - (ACS) Granular.
5.2 Methylene chloride, Pentane, Cyclohexane, High Purity Water-HPLC
quality, distilled in glass.
5.3 Sodium sulfate - (ACS) Granular, anhydrous (purified by heating at
400°C for 4 hrs. in a shallow tray).
5.4 Stock standards - Prepare stock standard solutions at a
concentration of 1.00 ug/ul by dissolving 0.100 grams of assayed
-------
reference material In pesticide quality isooctane or other
appropriate solvent and diluting to volume in a 100 ml ground glass
stoppered volumetric flask. The stock solution is transferred to
ground glass stoppered reagent bottles, stored in a refrigerator,
and checked frequently for signs of degradation or evaporation,
especially just prior to preparing working standards from them.
5.5 Acetonitrile - Spectral quality.
5.6 Silica gel - 100/120 mesh desiccant (Davison Chemical grade 923 or
equivalent). 'Before use, activate for at least 16 hours at 130°C
in a foil covered glass container.
6. Calibration
6.1 Prepare calibration standards that contain the compounds of
interest, either singly or mixed together. The standards should be
prepared at concentrations covering two or more orders of magnitude
that will completely bracket the working range of the
chromatographic system. If the sensitivity of the detection system
can be calculated from Table I as 100 ug/1 in the final extract,
for example, prepare standards at 10 ug/1, 50 ug/1, 100 ug/1, 500
ug/1, etc. so that injections of 1-5 ul of each calibration
standard will define the linearity of the detector in the working
range.
6.2 Assemble the necessary HPLC or gas chromatographic apparatus and
establish operating parameters equivalent to those indicated in
Table I or II. By injecting calibration standards, establish the
sensitivity limit of the detectors and the linear range of the
analytical systems for each compound.
-------
6.3 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from the reagents.
7. Quality Control
7.1 Before processing any samples, the analyst should demonstrate
through the analysis of a distilled water method blank, that all
glassware and reagents are interference-free. Each time a set of
samples is extracted or there is a change in reagents, a method
blank should be processed as a safeguard against chronic laboratory
contamination.
7.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to validate the
precision of the sampling technique. Laboratory replicates should
be analyzed to validate the precision of the analysis. Fortified
samples should be analyzed to validate the accuracy of the
analysis. Where doubt exists over the identification of a peak on
the chromatogram, confirmatory techniques such as fraction
collection and GC-mass spectroscopy should be used.
8. Sample Collection, Preservation, and Handling
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be prewashed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be free of tygon and other potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time of
-------
collection until extraction. Chemical preservatives should not be
used in the field unless more than 24 hours will elapse before
delivery to the laboratory. If the samples will not be extracted
within 48 hours of collection, adjust the sample to a pH range of
6.0-8.0 with sodium hydroxide or sulfuric acid and add 35 mg sodium
thiosulfate per part per million of free chlorine per liter.
8.3 All samples must be extracted within 7 days and completely analyzed
within 30 days of collection.
9. Sample Extraction
9.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
two-liter separatory funnel. Check the pH of the sample with
wide-range pH paper and adjust to within the range of 5-9 with
sodium hydroxide or sulfuric acid.
9.2 Add 60 ml methylene chloride to the sample bottle, seal, and shake
30 seconds to rinse the inner walls. Transfer the solvent into the
separatory funnel, and extract the sample by shaking the funnel for
two minutes with periodic venting to release vapor pressure. Allow
the organic layer to separate from the water phase for a minimum of
ten minutes. If the emulsion interface between layers is more than
one-third the size of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool, or
centrifugation. Collect the methylene chloride extract in a 250-ml
Erlenmeyer flask.
JIT- 148
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9.3 Add a second 60-ml volume of methylene chloride to the sample
bottle and complete the extraction procedure a second time,
combining the extracts in the Erlenmeyer flask.
9.4 Perform a third extraction in the same manner. Pour the combined
extract through a drying column containing 3-4 inches of anhydrous
sodium sulfate, and collect it in a 500-ml Kuderna-Danish (K-D)
flask equipped with a 10 ml concentrator tube. Rinse the
Erlenmeyer flask and column with 20-30 ml methylene chloride to
complete the quantitative transfer.
9.5 Add 1-2 clean boiling chips to the flask and attach a three-ball
Snyder column. Prewet the Snyder column by adding about 1 ml
methylene chloride to the top. Place the K-D apparatus on a hot
water bath (60-65°C) so that the concentrator tube is partially
immersed in the hot water, and the entire lower rounded surface of
the flask is bathed in vapor. Adjust the vertical position of the
apparatus and the water temperature as required to complete the
concentration in 15-20 minutes. At the proper rate of distillation
the balls of the column will actively chatter but the chambers will
not flood. When the apparent volume of liquid reaches 1 ml, remove
the K-D apparatus and allow it to drain for at least 10 minutes
while cooling. Remove the Snyder column and rinse the flask and
its lower joint into the concentrator tube with 1-2 ml of methylene
chloride. A 5-ml syringe is recommended for this operation.
Stopper the concentrator tube and store refrigerated if further
processing will not be performed immediately.
9.6 Determine the original sample volume by refilling the sample bottle
-------
to the mark and transferring the liquid to a 1000 ml graduated
cylinder. Record the sample volume to the nearest 5 ml.
9.7 If the sample requires cleanup before chromatographlc analysis,
proceed to Section 10. If the sample does not require cleanup, or
if the need for cleanup is unknown, analyze an aliquot of the
extract according to Section 11 or Section 12.
10. Cleanup and Separation
10.1 Before the silica gel cleanup technique can be utilized, the
extract solvent must be exchanged to cyclohexane. Add a 1-10 ml
aliquot of sample extract (in methylene chloride) and a boiling
chip to a clean K-0 concentrator tube. Add 4 ml cyclohexane and
attach a micro-Snyder column. Prewet the micro-Snyder column by
adding 0.5 ml methylene chloride to the top. Place the micro-K-D
apparatus on a boiling (100°C) 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 5-10 minutes. At the proper
rate of distillation the balls of the column will actively chatter
but the chambers will not flood. When the apparent volume of the
liquid reaches 0.5 ml, remove the K-D apparatus and allow .it to
drain for at least 10 minutes while cooling. Remove the
micro-Snyder column and rinse its lower joint into the concentrator
tube with a minimum of cyclohexane. Adjust the extract volume to
about 2 ml.
10.2 Silica Gel Column Cleanup for PAHs.
10.2.1 Prepare a slurry of lOg activated silica gel in methylene
-------
chloride and place this in a 10 mm ID chromatography
column. Gently tap the column to settle the silica gel and
elute the methylene chloride. Add 1-2 cm of anhydrous
sodium sulfate to the top of the silica gel.
10.2.2 Preelute the column with 40 ml pentane. Discard the eluate
and just prior to exposure of the sodium sulfate layer to
the air, transfer the 2 ml cyclohexane sample extract onto
the column, using an additional 2 ml of cyclohexane to
complete the transfer.
10.2.3 Just prior to exposure of the sodium sulfate layer to the
air, add 25 ml pentane and continue elution of the column.
Discard the pentane eluate.
10.2.4 Elute the column with 25 ml of 40% methylene chloride/60%
pentane and collect the eluate in a 500 ml K-D flask
equipped with a 10 ml concentrator tube. Elution of the
column should be at a rate of about 2 ml/min.
10.2.5 Concentrate the collected fraction to less than 10 ml by K-D
techniques, as in 9.5, using pentane to rinse the walls of
the glassware. Proceed with HPLC or gas chromatographic
analysis.
11. High Performance Liquid Chromatography HPLC
11.1 To the extract in the concentrator tube, add 4 ml acetonitrile and
a new boiling chip, then attach a micro-Snyder column. Increase
the temperature of the hot water bath to 95-100°C. Concentrate
the solvent as above. After cooling, remove the micro-Snyder
column and rinse its lower joint into the concentrator tube with
-------
about 0.2 ml acetonitrile. Adjust the extract volume to 1.0 ml.
11.2 Table I summarizes the recommended HPLC column materials and
operating conditions for the instrument. Included in this table
are estimated retention times and sensitivities that should be
achieved by this method. An example of the separation achieved by
this column is shown in Figure 1. Calibrate the system daily with
a minimum of three injections of calibration standards.
11.3 Inject 2-5 ul of the sample extract with a high pressure syringe or
sample injection loop. Record the volume injected to the nearest
0.05 ul, and the resulting peak size, in area units.
11.4 If the peak area exceeds the linear range of the system, dilute the
extract and reanalyze.
11.5 If the peak area measurement is prevented by the presence of
interferences, further cleanup is required.
11.6 The UV detector is recommended for the determination of naphthalene
and acenaphthylene and the fluorescence detector is recommended for
the remaining PAHs.
12. Gas Chromatography
12.1 The gas chromatographic procedure will not resolve certain isomeric
pairs as indicated in Table II. The liquid chromatographic
procedure (Section 11) must be used for these materials.
12.2 To achieve maximum sensitivity with this method, the extract must
be concentrated to 1.0 ml. Add a clean boiling chip to the
methylene chloride extract in the concentrator tube. Attach a
two-ball micro-Snyder column. Prewet the micro-Snyder column by
adding about 0.5 ml of methylene chloride to the top. Place this
KT
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micro-K-D apparatus on a hot water bath (60-65°C) so that the
concentrator tube is partially immersed in the hot water. Adjust
the vertical position of the apparatus and water temperature as
required to complete the concentration in 5 to 10 minutes. At the
proper rate of distillation the balls will actively chatter but the
chambers will not flood. When the apparent volume of liquid
reaches 0.5 ml, remove the K-D apparatus and allow it to drain for
at least 10 minutes while cooling. Remove the micro-Snyder column
and rinse its lower joint into the concentrator tube with a small
volume of methylene chloride. Adjust the final volume to 1.0 ml
and stopper the concentrator tube.
12.3 Table II describes the recommended gas chromatographic column
material and operating conditions for the instrument. Included in
this table are estimated retention times that should be achieved by
this method. Calibrate the gas chromatographic system daily with a
minimum of three injections of calibration standards.
12.4 Inject 2-5 ul of the sample extract using the solvent-flush
technique. Smaller (1.0 ul) volumes can be injected if automatic
devices are employed. Record the volume injected to the nearest
0.05 ul, and the resulting peak size, in area units.
12.5 If the peak area exceeds the linear range of the system, dilute the
extract and reanalyze.
12.6 If the peak area measurement is prevented by the presence of
interferences, further cleanup is required.
13. Calculations
13.1 Determine the concentration of individual compounds according to
TV-
-------
the formula:
Concentration, ug/1 - (A) (B) (Vt)
(Vi) (Vs)
where A = Calibration factor for chromatographic system, in
nanograms material per area unit.
B = Peak size in injection of sample extract, in area units
V. = volume of extract injected (ul)
V. = Volume of total extract (ul)
V = Volume of water extracted (ml)
13.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed, all
data obtained should be reported.
14. Accuracy and Precision
14.1 The U.S. EPA Environmental Monitoring and Support Laboratory in
Cincinnati is in the process of conducting an inter!aboratory
method study to determine the accuracy and precision of this test
procedure.
BIBLIOGRAPHY
"Development and Application of Test Procedures for Specific Organic Toxic
Substances in Wastewaters, Category 9-PAHs." Report for EPA Contract
68-03-2624 (In preparation).
'13.
-------
TABLE I
High Performance Liquid Chromatography of PAH's
Detection Limit (ug/1)
Compound Retention Time (min) UV Fluorescence
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo( a) anthracene
Chrysene
Benzo (b )f 1 uorant hene
Benzo(k)fluoranthene
Benzo (a)pyrene
Dibenzo(a,h) anthracene
Benzo (ghi)perylene
Indenof 1 ,2,3-cd)pyrene
16.17
18.10
20.14
20.89
22.32
23.78
25.00
25.94
29.26
30.14
32.44
33.91
34.95
37.06
37.82
39.21
2.5
5.0
3.0
0.5
0.25
0.10
0.50
0.10
0.20
0.20
1.0
0.30
0.25
1.0
0.75
0,30
20.0
100.0
4.0
2.0
1.2
1.5
0.05
0.05
0.04
0.5
0.04
0.04
0.04
0.08
0.2
0.1
HPLC conditions: Reverse phase HC-ODS Sil-X 2.6 x 250 mm Perkin-Elmer
column; isocratic elution for 5 min. using 40% acetonitrile/60% water,
then linear gradient elution to 100% acetonitrile over 25 minutes; flow
rate is 0.5 ml/min.
Detection limit is calculated from the minimum detectable HPLC response
being equal to five times the background noise, assuming an equivalent
of a 2 ml final volume of the 1 liter sample extract, and assuming an
HPLC injection of 2 microliters.
-------
TABLE II
Gas Chromatography of PAHs
Compound Retention Time (Min)
Naphthalene 4.5
Acenaphthylene 10.4
Acenaphthene 10.8
Fluorene 12.6
Phenanthrene 15.9
Anthracene 15.9
Fluoranthene 19.8
Pyrene 20.6
Benzo(a)anthracene 20.6
Chrysene 24.7
Benzo(b)fluoranthene 28.0
Benzo(k)fluoranthene 28.0
Benzo(a)pyrene 29.4
Dibenzo(a,h)anthracene 36.2
Indeno(l,2,3-cd)pyrene 36.2
Benzo(ghi)pery!ene 38.6
6C conditions: Chromosorb W-AW-DCMS 100/120 mesh coated with 3% OV-17,
packed in a 61 x 2 mm ID glass column, with nitrogen carrier gas at 40
ml/min flow rate. Column temperature was held at 100°C for 4 minutes,
then programmed at 8°/minute to a final hold at 280°C.
-------
COLUMN: HC-ODS S1L-X
MOBILE PHASE: 40% TO 100% ACETONITRILE IN WATER
DETECTOR: FLUORESCENCE
28
36
40
RETENTION TIME-MINUTES
Figure 1. Liquid chromatogram of polynuclear aromatics
-------
HALOETHERS
METHOD 611
1. Scope and Application
1.1 This method covers the determination of certain haloethers. The
following parameters may be determined by this method:
Parameter STORET No.
Bis(2-chloroethyl) ether 34273
Bis(2-chloroethoxy) methane 34278
Bis(2-chloroisopropyl) ether 34283
4-Bromophenyl phenyl ether 34636
4-Chlorophenyl phenyl ether 34641
1.2 This method is applicable to the determination of these compounds
in municipal and industrial discharges. It is designed to be used
to meet the monitoring requirements of the National Pollutant
Discharge Elimination System (NPDES). As such, it presupposes a
high expectation of finding the specific compounds of interest. If
the user is attempting to screen samples for any or all of the
compounds above, he must develop independent protocols for the
verification of identity.
1.3 The sensitivity of this method is usually dependent upon the level
of interferences rather than instrumental limitations. The limits
of detection listed in Table I represent sensitivities that can be
achieved in wastewaters in the absence of interferences.
1.4 This method is recommended for use only by experienced residue
-------
analysts or under the close supervision of such qualified persons.
2. Summary of Method
2.1 A 1-liter sample of wastewater is extracted with methylene chloride
using separatory funnel techniques. The extract is dried and
concentrated to a volume of 10 ml or less. Chromatographic
conditions utilizing a halide specific detector are described which
allow for the accurate measurement of the compounds in the extract.
2.2 If interferences are encountered, the method provides a selected
general purpose cleanup procedure to aid the analyst in their
elimination.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines causing
misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the
conditions of the analysis by running method blanks. Specific
selection of reagents and purification of solvents by distillation
in all-glass systems may be required.
3.2 Interferences coextracted from the samples will vary considerably
from source to source, depending upon the diversity of the
industrial complex or municipality being sampled. While general
clean-up techniques are provided as part of this method, unique
samples may require additional cleanup approaches to achieve the
sensitivities stated in Table I.
3.3 Dichlorobenzenes are known to coelute with haloethers under some
-------
gas chromatographic conditions. If these materials are present
together in a sample, it may be necessary to analyze the extract
with two different column packings to completely resolve all of the
compounds.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete or composite sampling.
4.1.1 Grab sample bottle - amber glass, 1-liter or 1-quart
volume. French or Boston Round design is recommended. The
container must be washed and solvent rinsed before use to
minimize interferences.
4.1.2 Bottle caps - Threaded to screw on to the sample bottles.
Caps must be lined with Teflon. Foil may be substituted if
sample is not corrosive.
4.1.3 Compositing equipment - Automatic or manual compositing
system. Must incorporate glass sample containers for the
collection of a minimum of 250 ml. Sample containers must
be kept refrigerated during sampling. No tygon or rubber
tubing may be used in the system.
4.2 Separatory funnel - 2000 ml, with Teflon stopcock.
4.3 Drying column - 20 mm ID pyrex chromatographic column with coarse
frit.
4.4 Kuderna-Danish (K-D) Apparatus
4.4.1 Concentrator tube - 10 ml, graduated (Kontes K-570050-1025
or equivalent). Calibration must be checked. Ground glass
stopper (size 19/22 joint) is used to prevent evaporation of
extracts.
Tf-
-------
4.4.2 Evaporative flask - 500 ml (Kontes K-57001-0500 or
equivalent). Attach to concentrator tube with springs.
(Kontes K-662750-0012).
4.4.3 Snyder column - three-ball macro (Kontes K503000-0121 or
equivalent).
4.4.4 Snyder column - two-ball micro (Kontes K-569001-0219 or
equivalent).
4.4.5 Boiling chips - solvent extracted, approximately 10/40 mesh.
4.5 Water bath - Heated, with concentric ring cover, capable of
temperature control (+; 2°C). The bath should be used in a hood.
4.6 Gas chromatograph - Analytical system complete with gas chromato-
graph suitable for on-column injection and all required accessories
including halide specific detector, column supplies, recorder,
gases, syringes. A data system for measuring peak areas is
recommended.
4.7 Chromatographic Column - 400 mm long x 19 mm ID with coarse fritted
plate on bottom and Teflon stopcock (Kontes K-420540-0224 or
equivalent).
5. Reagents
5.1 Preservatives:
5.1.1 Sodium hydroxide - (ACS) 10 N in distilled water.
5.1.2 Sulfuric acid (1+1) - (ACS) Mix equal volumes of cone.
H2S04 with distilled water.
5.2 Methylene chloride - Pesticide quality or equivalent.
5.3 Sodium Sulfate - (ACS) Granular, anhydrous (purified by heating at
400°C for 4 hrs. in a shallow tray).
-------
5.4 Stock standards - Prepare stock standard solutions at a concentra-
tion of 1.00 ug/ul by dissolving 0.100 grams of assayed reference
material in pesticide quality acetone or other appropriate solvent
and diluting to volume in a 100 ml ground glass stoppered volu-
metric flask. The stock solution is transferred to ground glass
stoppered reagent bottles, stored in a refrigerator, and checked
frequently for signs of degradation or evaporation, especially just
prior to preparing working standards from them.
5.5 Florisil - PR Grade (60/100 mesh); purchase activated at 1250°F
and store in the dark in glass containers with glass stoppers or
foil-lined screw caps. Before use, activate each batch overnight
at 130°C in a foil-covered glass container.
5.6 Hexane, Petroleum ether (boiling range 30-60°C) - pesticide
quality or equivalent.
5.7 Diethyl Ether - Nanograde, redistilled in glass, if necessary.
5.7.1 Must be free of peroxides as indicated by EM Quant test
strips. (Test strips are available from EM Laboratories,
Inc., 500 Executive Blvd., Elmsford, N.Y. 10523.)
5.7.2 Procedures recommended for removal of peroxides are provided
with the test strips. After cleanup 20 ml ethyl alcohol
preservative must be added to each liter of ether.
6. Calibration
6.1 Prepare calibration standards that contain the compounds of
interest, either singly or mixed together. The standards should be
prepared at concentrations covering two or more orders of magnitude
that will completely bracket the working range of the chromato-
-------
graphic system. If the sensitivity of the detection system can be
calculated from Table I as 100 ug/1 in the final extract, for
example, prepare standards at 10 ug/1, 50 ug/1, 100 ug/1, 500 ug/1,
etc. so that injections of 1-5 ul of each calibration standard will
define the linearity of the detector in the working range.
6.2 Assemble the necessary gas chromatographic apparatus and establish
operating parameters equivalent to those indicated in Table I. By
injecting calibration standards, establish the sensitivity limit of
the detector and the linear range of the analytical system for each
compound.
6.3 The cleanup procedure in Section 10 utilizes Florisil chromatog-
raphy. Florisil from different batches or sources may vary in
absorption capacity. To standardize the amount of Florisil which
is used, the use of lauric acid value (Mills, 1968) is suggested.
The referenced procedure determines the adsorption from hexane
solution of lauric acid (mg) per gram Florisil. The amount of
Florisil to be used for each column is calculated by dividing 110
by this ratio and multiplying by 20 grams.
6.4 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from the reagents.
7. Quality Control
7.1 Before processing any samples, the analyst should demonstrate
through the analysis of a distilled water method blank, that all
glassware and reagents are interference-free. Each time a set of
samples is extracted or there is a change in reagents, a method
-------
blank should be processed as a safeguard against chronic laboratory
contamination.
7.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to validate the
precision of the sampling technique. Laboratory replicates should
be analyzed to validate the precision of the analysis. Fortified
samples should be analyzed to validate the accuracy of the
analysis. Where doubt exists over the identification of a peak on
the chromatogram, confirmatory techniques such as mass spectroscopy
should be used.
8. Sample Collection, Preservation, and Handling
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be prewashed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be free of tygon and other potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time of collection
until extraction. Chemical preservatives should not be used in the
field unless more than 24 hours will elapse before delivery to the
laboratory. If the samples will not be extracted within 48 hours
of collection, the sample should be adjusted to a pH range of
6.0-8.0 with sodium hydroxide or sulfuric acid.
8.3 All samples must be extracted within 7 days and completely analyzed
within 30 days of collection.
-------
9. Sample Extraction
9.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
two-liter separatory funnel. Check the pH of the sample with
wide-range pH paper and adjust to within the range of 5-9 with
sodium hydroxide or sulfuric acid.
9.2 Add 60 ml methylene chloride to the sample bottle, seal, and shake
30 seconds to rinse the inner walls. Transfer the solvent into the
separatory funnel, and extract the sample by shaking the funnel for
two minutes with periodic venting to release vapor pressure. Allow
the organic layer to separate from the water phase for a minimum of
ten minutes. If the emulsion interface between layers is more than
one-third the size of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include stir-
ring, filtration of the emulsion through glass wool, or centrifu-
gation. Collect the methylene chloride extract in a 250-ml
Erlenmeyer flask.
9.3 Add a second 60-ml volume of methylene chloride to the sample
bottle and complete the extraction procedure a second time,
combining the extracts in the Erlenmeyer flask.
9.4 Perform a third extraction in the same manner. Pour the combined
extract through a drying column containing 3-4 inches of anhydrous
sodium sulfate, and collect it in a 500-ml Kuderna-Danish (K-D)
flask equipped with a 10 ml concentrator tube. Rinse the
Erlenmeyer flask and column with 20-30 ml methylene chloride to
-------
complete the quantitative transfer.
9.5 Add 1-2 clean boiling chips to the flask and attach a three-ball
Snyder column. Prewet the Snyder column by adding about 1 ml
methylene chloride to the top. Place the K-D apparatus on a hot
water bath (60-65°C) so that the concentrator tube rs partially
immersed in the hot water, and the entire lower rounded surface of
the flask is bathed in vapor. Adjust the vertical position of the
apparatus and the water temperature as required to complete the
concentration in 15-20 minutes. At the proper rate of distillation
the balls of the column will actively chatter but the chambers will
not flood. When the apparent volume of liquid reaches 1-2 ml,
remove the K-D apparatus and allow it to drain for at least 10
minutes while cooling. NOTE: Haloethers have a sufficiently high
volatility that significant losses will occur in concentration
steps if care is not exercised. It is important to maintain a
constant gentle evaporation rate and not to allow the liquid volume
to fall below 1-2 ml before removing the K-0 from the hot water
bath.
9.6 Momentarily remove the Snyder column, add 50 ml hexane and a new
boiling chip and replace the column. Raise the temperature of the
water bath to 85-90°C. Concentrate the extract as in 9.5 except
use hexane to prewet the column. Remove the Snyder column and
rinse the flask and its lower joint into the concentrator tube with
1-2 ml hexane. Stopper the concentrator tube and store refrige-
rated if further processing will not be performed immediately.
9.7 Determine the original sample volume by refilling the sample bottle
-------
to the mark and transferring the liquid to a 1000 ml graduated
cylinder. Record the sample volume to the nearest 5 ml.
9.8 Unless the sample is known to require cleanup, proceed to analysis
by gas chromatography.
10. Cleanup and Separation
10.1 Florisil Column Cleanup for Haloethers
10.1.1 Adjust the sample extract volume to 10 ml.
10.1.2 Place a charge (nominally 20 g but determined in Section
6.3) of activated Florisil in a 19 mm ID chromatography
column. After settling the Florisil by tapping the column,
add about one-half inch layer of anhydrous granular sodium
sulfate to the top.
10.1.3 Pre-elute the column, after cooling, with 50-60 ml of
petroleum ether. Discard the eluate and just prior to
exposure of the sulfate layer to air, quantitatively
transfer the sample extract into the column by decantation
and subsequent petroleum ether washings. Discard the
eluate. Just prior to exposure of the sodium sulfate layer
to the air, begin eluting the column with 300 ml of 6% ethyl
ether/94% petroleum ether. Adjust the elution rate to
approximately 5 ml/min and collect the eluate in a 500 ml
K-D flask equipped with a 10 ml concentrator tube. This
fraction should contain all of the haloethers.
10.1.4 Concentrate the fraction by K-D as in 9.5 except prewet the
Snyder column with hexane. When the apparatus is cool,
remove the column and rinse the flask and its lower joint
f- Itfc
-------
into the concentrator tube with 1-2 ml hexane. Analyze by
gas chromatography.
11. Gas Chromatography
11.1 Table I summarizes some recommended gas chromatographic column
materials and operating conditions for the instrument. Included in
this table are estimated retention times and sensitivities that
should be achieved by this method. Examples of the separations
achieved by these columns are shown in Figures 1 and 2. Calibrate
the system daily with a minimum of three injections of calibration
standards.
11.2 Inject 2-5 ul of the sample extract using the solvent-flush
technique. Smaller (1.0 ul) volumes can be injected if automatic
devices are employed. Record the volume injected to the nearest
0.05 ul, and the resulting peak size, in area units.
11.3 If the peak area exceeds the linear range of the system, dilute the
extract and reanalyze.
11.4 If the peak area measurement is prevented by the presence of inter-
ferences, further cleanup is required.
12. Calculations
12.1 Determine the concentration of individual compounds according to
the formula:
Concentration, ug/1 = (A) (B) (Vt)
(Vi) (Vs)
where A = Calibration factor for chromatographic system, in nano-
grams material per area unit.
UT-
-------
B * Peak size in injection of sample extract, in area units
"V.. = volume of extract injected (ul)
V. = Volume of total extract (ul)
V » Volume of water extracted (ml)
12.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed, all
data" obtained should be reported.
13. Accuracy and Precision
The U.S. EPA Environmental Monitoring and Support Laboratory in
Cincinnati is in the process of conducting an interlaboratory method
study to determine the accuracy and precision of this test procedure.
BIBLIOGRAPHY
1. "Development and Application of Test Procedures for Specific Organic
Toxic Substances in Wastewaters. Category 2-Haloethers." Report for
EPA Contract 68-03-2633 (In preparation).
2. Mills, P.A., "Variation of Florisil Activity: Simple Method for
Measuring Absorbent Capacity and Its Use in Standardizing Florisil
Columns," Journal of the Association of Official Analytical Chemists,
51, 29 (1968).
-------
TABLE I
Gas Chromatography of Haloethers
Compound
Retention Time (min)
Column 1 Column 2
Detection
Limit (ug/L)
Bis(2-chlorpisopropyl) ether
Bis(2-chloroethyl) ether
Bis(2-chloroethoxy) methane
4-Chlorophenyl phenyl ether
4-Bromophenyl phenyl ether
8.41
9.32
13.1
19.4
21.2
9.70
9.06
9.97
15.0
16.2
0.9
0.5
0.4
2.2
1.1
Column 1 conditions: Supelcoport 100/120 mesh coated with 3% SP-1000 packed
in 1.8 m long x 2.1 mm ID glass column with ultra-high purity helium
carrier/gas at 40 ml/min flow rate. Column temperature is 60°C for 2
minutes after injection then program at 8°C/min to 230°C and hold
for 4 minutes. Under these conditions R.T. of Aldrin is 22.6 minutes.
Column 2 conditions: Tenax-GC 60/80 mesh packed in a 1.8 m long x 2.1 mm ID
glass column with helium carrier gas at 40 ml/min flow rate. Column
temperature 150°C for 4 minutes after injection then program at
16°C/min to 310°C. Under these conditions R.T. of Aldrin is 18.4
minutes.
Detection limit is calculated from the minimum detectable GC response being
equal to five times the GC background noise, assuming a 10 ml final
volume of the 1 liter sample extract, and assuming a GC injection of 5
micro!iters. These values were collected using the Tracor 700 Hall
electrolytic conductivity detector with furnace temperature 900°C,
transfer line 250°C, 95% ethanol electrolyte at 0.3 ml/min flow rate,
and hydrogen reaction gas at 60 ml/min.
HE-It 9
-------
COLUMN: 3!S SP-1000 ON SUPELCOPORT
PROGRAM: 60°C-2 MINUTES 8"/MINUTE TO 230°C.
DETECTOR: HALL ELECTROLYTIC CONDUCTIVITY
cc
III
ce
LU
X
LU
O_
25
LU
z
X
LU
X
Q.
O
o
cc
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X
CJ
t
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m
•
1
fl
I K
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X
LU
Ij
X
LU
O
cc
o
X
CJ
1
CM
CO
CO
^
1 1 1 1 1
} 2 ^ 6 8 10
LU
X
LU
&
X
O
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LU
i
o
X
CJ
fsi
to
CO
JL_
12 14
ce
LU
X
h-
LU
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LU
Q.
-J
Z»
LU
Q.
O
ce
O
CJ
_JU
16 18
ce
X ^
LU cc
-i Q
Z <
LU
X
CL.
^
Z
OMOPHE
cc
CO
f
A
Ju
J,
t
s
1 1
20 22 2
RETENTION TIME-MINUTES
Figure 1. Gas chromatogram of haloethers
-------
COLUMN: TEN AX GC
PROGRAM: 150°C.-4 MINUTES 16VMINUTE TO 310°C.
DETECTOR: HALL ELECTROLYTIC CONDUCTIVITY
ce
LU
X
04s 12 16 20
RETENTION TIME-MINUTES
Figure 2. Gas chromatogram of haloethers
24
-------
CHLORINATED HYDROCARBONS
'METHOD 612
1. Scope and Application
1.1 This method covers the determination of certain chlorinated hydro-
carbons. The following parameters may be determined by this method:
Parameter STORET No.
Hexachlorocyclopentadiene 34386
Hexachlorobenzene 39700
Hexachlorobutadiene 34391
Hexachloroethane 34396
1,2-Dichlorobenzene 34536
1,2,4-Drichlorobenzene 34551
1,3-Dichlorobenzene 34566
1,4-Dichlorobenzene 34571
2-chloronaphthalene 34581
1.2 This method is applicable to the determination of these compounds
in municipal and industrial discharges. It is designed to be used
to meet the monitoring requirements of the National Pollutant
Discharge Elimination System (NPDES). As such, it presupposes a
high expectation of finding the specific compounds of interest. If
the user is attempting to screen samples for any or all of the
compounds above, he must develop independent protocols for the
verification of identity.
1.3 The sensitivity of this method is usually dependent upon the level
of interferences rather than instrumental limitations. The limits
-------
of detection listed in Table I represent sensitivities that can be
achieved in wastewaters in the absence of interferences.
1.4 This method is recommended for use only by experienced residue
analysts or under the close supervision of such qualified persons.
2. Summary of Method
2.1 A 1-liter sample of wastewater is extracted with methylene chloride
?
using separatory funnel techniques. The extract is dried by
passing through a sodium siilfate column and concentrated to a
volume of 10 ml or less. Chromatographic conditions are described
which allow for the accurate measurement of the compounds in the
extract.
2.2 If interferences are encountered or expected, the method provides a
selected general purpose cleanup procedure to aid the analyst in
their elimination.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines causing
misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the
conditions of the analysis by running method blanks. Specific
selection of reagents and purification of solvents by distillation
in all-glass systems may be required.
3.2 Interferences coextracted from the samples will vary considerably
from source to source, depending upon the :^.=-s-:ty of the
industrial complex or municipality being si""lea. While general
clean-up techniques are provided as part OT" :--":. method, unique
•jr-
-------
samples may require additional cleanup approaches to achieve the
sensitivities stated in Table 1.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete or composite sampling.
4.1.1 Grab sample bottle - amber glass, 1-lfter or 1-quart
volume. French or Boston Round design is recommertded. The
container must be washed and solvent rinsed before use "to
minimize interferences.
4.1.2 Bottle caps - Threaded to screw on to the sample bottles.
Caps must be lined with Teflon. Foil may be substituted if
sample is not corrosive and the foil is found to be inter-
ference free.
4.1.3 Compositing equipment - Automatic or manual compositing
system. Must incorporate glass sample containers for the
collection of a minimum of 250 ml. Sample containers must
be kept refrigerated during sampling. No tygon or rubber
tubing may be used in the system.
4.2 Separatory funnel - 2000 ml, with Teflon stopcock.
4.3 Drying column - 20 mm ID pyrex chromatographic column with coarse
frit.
4.4 Kuderna-Danish (K-D) Apparatus
4.4.1 Concentrator tube - 10 ml, graduated (Kontes K-570050-1025
or equivalent). Calibration must be checked. Ground glass
stopper (size 19/22 joint) is used to prevent evaporation of
extracts.
4.4.2 Evaporative flask - 500 ml (Kontes K-57001-0500 or
nt
-------
equivalent). Attach to concentrator tube with springs.
(Kontes K-662750-0012).
4.4.3 Snyder column - three-ball macro (Kontes K503000-0121 or
equivalent).
4.4.4 Snyder column - two-ball micro (Kontes K-569001-0219 or
equivalent).
4.4.5 Boiling chips - solvent extracted, approximately 10/40 mesh.
4.5 Water bath - Heated, with Concentric ring cover, capable of
temperature control (_+ 2°C). The bath should be used in a hood.
4.6 Gas chromatograph - Analytical system complete with gas chromato-
graph suitable for on-column injection and all required accessories
including electron capture detector, column supplies, recorder,
gases, syringes. A data system for measuring peak areas is recom-
mended.
4.7 Chromatography column - 300 mm long x 10 mm ID with coarse fritted
disc at bottom and Teflon stopcock.
5. Reagents
5.1 Preservatives:
5.1.1 Sodium hydroxide - (ACS) 10 N in distilled water.
5.1.2 Sulfuric acid - (ACS) Mix equal volumes of cone. HgSO^
with distilled water.
5.2 Methylene chloride, Hexane and Petroleum ether (boiling range
30-60°C) - Pesticide quality or equivalent.
5.3 Sodium sulfate - (ACS) Granular, anhydrous (purified by heating at
400°C for 4 hrs. in a shallow tray).
5.4 Stock standards - Prepare stock standard solutions at a concentra-
-------
tion of 1.00 ug/ul by dissolving 0.100 grams of assayed reference
material in pesticide quality isooctane or other appropriate
solvent and diluting to volume in a 100 ml ground glass stoppered
volumetric flask. The stock solution is transferred to ground
glass stoppered reagent bottles, stored in a refrigerator, and
checked frequently for signs of degradation or evaporation,
especially just prior to preparing working standards from them.
5.5 Florisil - PR grade (60/100 mesh); purchase activated at 1250°F
and store in the dark in glass containers with glass stoppers or
foil-lined screw caps. Before use, activate each batch at 130°C
in foil-covered glass containers.
6. Calibration
6.1 Prepare calibration standards that contain the compounds of
interest, either singly or mixed together. The standards should be
prepared at concentrations covering two or more orders of magnitude
that will completely bracket the working range of the chromato-
graphic system. If the sensitivity of the detection system can be
calculated from Table I as 100 ug/1 in the final extract, for
example, prepare standards at 10 ug/1, 50 ug/1, 100 ug/1, 500 ug/1,
etc. so that injections of 1-5 ul of each calibration standard will
define the linearity of the detector in the working range.
6.2 Assemble the necessary gas chromatographic apparatus and establish
operating parameters equivalent to those indicated in Table I. By
injecting calibration standards, establish the sensitivity limit of
the detector and the linear range of the analytical system for each
compound.
Tf-
-------
§.3 The cleanup procedure in Section 10 utilizes Florisil chromatog-
raphy. Florisil from different batches or sources may vary in
absorption capacity. To standardize the amount of Florisil which
is used, the use of lauric acid value (Mills, 1968) is suggested.
The referenced procedure determines the adsorption from hexane
solution of lauric acid (mg) per. gram Florisil. The amount of
Florisil to be used for each column is calculated by dividing this
ratio by 110 and multiplying by 20 grams.
6.4 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from the reagents.
7. Quality Control
7.1 Before processing any samples, the analyst should demonstrate
/
through the analysis of a distilled water method blank, that all
glassware and reagents are interference-free. Each time a set of
samples is extracted or there is a change in reagents, a method
blank should be processed as a safeguard against chronic laboratory
contamination.
7.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to validate the
precision of the sampling technique. Laboratory replicates should
be analyzed to validate the precision of the analysis. Fortified
samples should be analyzed to validate the accuracy of the analysis.
Where doubt exists over the identification of a peak on the
chromatogram, confirmatory techniques such as mass spectroscopy
should be used.
m-
-------
8. Sample Collection, Preservation, and Handling
8.1 Grab samples must be collected in glass containers, leaving a
minimum headspace. Conventional sampling practices should be
followed, except that the bottle must not be prewashed with sample
before collection. Composite samples should be collected in
refrigerated glass containers in accordance with the requirements
of the program. Automatic sampling equipment must be free of tygon
and other potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time of col-
lection until extraction. Chemical preservatives should not be
used in the field unless more than 24 hours will elapse before
delivery to the laboratory. If the samples will not be extracted
within 48 hours of collection, the sample should be adjusted to a
pH range of 6.0-8.0 with sodium hydroxide or sulfuric acid.
8.3 All samples should be extracted immediately and must be extracted
within 3 days and completely analyzed within 30 days of collection.
9. Sample Extraction
9.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
two-liter separatory funnel. Check the pH of the sample with
wide-range pH paper and adjust to within the range of 5-9 with
sodium hydroxide or sulfuric acid.
9.2 Add 60 ml methylene chloride to the sample bottle, seal, and shake
30 seconds to rinse the inner walls. Transfer the solvent into the
separatory funnel, and extract the sample by shaking the funnel for
two minutes with periodic venting to release vapor pressure. Allow
-------
the organic layer to separate from the water phase for a minimum of
ten minutes. If the emulsion interface between layers is more than
one-third the size of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The opti-
mum technique depends upon the sample, but may include stirring,
filtration of the emulsion through glass wool,-or centrifugation.
Collect the methylene chloride extract in a 250-ml Erlenmeyer flask.
9.3 Add a second 60-ml volume of methylene chloride to the sample
bottle and complete the extraction procedure a second time,
combining the extracts in the Erlenmeyer flask.
9.4 Perform a third extraction in the same manner. Pour the combined
extract through a drying column containing 3-4 inches of anhydrous
sodium sulfate, and collect it in a 500-ml Kuderna-Danish (K-D)
flask equipped with a 10 ml concentrator tube. Rinse the
Erlenmeyer flask and column with 20-30 ml methylene chloride to
complete the quantitative transfer.
9.5 Add 1-2 clean boiling chips to the flask and attach a three-ball
Snyder column. Prewet the Snyder column by adding about 1 ml
methylene chloride to the top. Place the K-D apparatus on a hot
water bath (60-65°C) so that the concentrator tube is partially
immersed in the hot water, and the entire lower rounded surface of
the flask is bathed in vapor. Adjust the vertical position of the
apparatus and the water temperature as required to complete the
concentration in 15-20 minutes. At the proper rate of distillation
the balls of the column will actively chatter but the chambers will
not flood. When the apparent volume of liquid reaches 1-2 ml,
JPI17
-------
remove the K-D apparatus and allow it to drain for at least 10,-
minutes while cooling. NOTE: The dichlorobenzenes have .a,suf-
ficiently high volatility that signficant losses may occur in
concentration steps if care is not exercised. It is important to
maintain a constant gentle evaporation rate and not to allow the
liquid volume to fall below 1-2 ml before removing the K-D from the
hot water bath.
9.6 Momentarily remove the Snyder column, add 50 ml hexane and a new
boiling chip and replace the column. Raise the temperature of the
water bath to 85-90°C. Concentrate the extract as in 9.5, except
using hexane to prewet the column. Remove the Snyder column and
rinse the flask and its lower joint into the concentrator tube with
1-2 ml of hexane. A 5-ml syringe is recommended for this operation.
Stopper the concentrator tube and store refrigerated if further
processing will not be performed immediately.
9.7 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the liquid to a 1000 ml graduated
cylinder. Record the sample volume to the nearest 5 ml.
9.8 Unless the sample is known to require cleanup, proceed to analysis
by gas chromatography.
10. Cleanup and Separation
10.1 Florisil Column Cleanup for Chlorinated Hydrocarbons.
10.1.1 Adjust the sample extract to 10 ml.
10.1.2 Place a 12 gram charge of activated Florisil (see 6.3) in a
10 mm ID chromatography column. After settling the Florisil
by tapping the column, add a 1-2 cm layer of anhydrous
-------
granular sodium sulfate to the top.
10.1.3 Pre-elute the column, after cooling, with 100 ml of
petroleum ether. Discard the eluate and just prior to
exposure of the sulfate layer to air, quantitatively
transfer the sample extract into the column by decantation
and subsequent petroleum ether washings. Discard the
eluate. Just prior to exposure of the sodium sulfate layer
to the air, begin eluting the column with 200 ml petroleum
ether and collect the eluate in a 500 ml K-D flask equipped
with a 10 ml concentrator tube. This fraction should
contain all of the chlorinated hydrocarbons.
10.1.4 Concentrate the fraction by K-D as in 9.5 except prewet the
column with hexane. When the apparatus is cool, remove the
Snyder column and rinse the flask and its lower joint into
the concentrator tube with 1-2 ml hexane. Analyze by gas
chromatography.
11. Gas Chromatography
11.1 Table I summarizes the recommended gas chromatographic column
materials and operating conditions for the instrument. Included in
this table are estimated retention times and sensitivities that
should be achieved by this method. Examples of the separations
achieved by this column are shown in Figures 1 and 2. Calibrate
the system daily with a minimum of three injections of calibration
standards.
11.2 Inject 2-5 ul of the sample extract using the solvent-flush tech-
nique. Smaller (1.0 ul) volumes can be injected if automatic
jr-
-------
devices are employed. Record the volume injected to the nearest
0.05 ul, and the resulting peak size, in area units.
11.3 If the peak area exceeds the linear range of the system, dilute the
extract and reanalyze.
11.4 If the peak area measurement is prevented by the presence of inter-
ferences, further cleanup is required.
12. Calculations
12.1 Determine the concentration of individual compounds according to
the formula:
Concentration, ug/1 = (A) (B) (Vt)
(Vi) (Vs)
where A = Calibration factor for chromatographic system, in
nanograms material per area unit.
B = Peak size in injection of sample extract, in area units
V.. = volume of extract injected (ul)
V. = Volume of total extract (ul)
V = Volume of water extracted (ml)
12.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed, all
data obtained should be reported.
13. Accuracy and Precision
The U.S. EPA Environmental Monitoring and Support Laboratory in
Cincinnati is in the process of conducting an interlaboratory method
study to determine the accuracy and precision of this test procedure.
-------
BIBLIOGRAPHY
1. "Development and Application of Test Procedures for Specific Organic
Toxic Substances in Wastewaters. Category 3 - Chlorinated Hydrocarbons
and Category 8 - Phenols." Report for EPA Contract 68-03-2625 (In
preparation).
2. Mills, P.A., "Variation of Florisil Activity: Simple Method for
Measuring Absorbent Capacity and Its Use in Standardizing Florisil
Columns," Journal of the Association of Official Analytical Chemists,
51, 29 (1968).
-------
TABLE I
Gas Chromatography of Chlorinated Hydrocarbons
Compound
Retention Time (min.)
Column 1
Detection
Limit (ug/1)
1 ,3-dichlorobenzene
1 , 4-d i ch 1 orobenzene
hexachloroethane
1,2-dichlorobenzene
hexachlorobutadiene
1,2,4-trichlorobenzene
hexachlorocyclopentadiene
2-chloronaphthalene
hexachlorobenzene
4.0
4.3
4.8
5.3
11.6
12.4
1.5*
2.5*
7.0*
0.009
0.018
0.001
0.012
0.001
0.006
0.001
0.015
0.001
Column 1 conditions: Gas Chrom Q 80/100 mesh coated with 1.5% OV-1/1.5%
OV-225 packed in a 1.8 m long x 2 mm ID glass column with 5% Methane/95%
Argon carrier gas at 30 ml/min flow rate. Column temperature is 75°C
except where * indicates 160°C. Under these conditions R.T. of Aldrin
is 18.8 minutes at 160°C.
Detection limit is calculated from the minimum detectable GC response of
the electron capture detector being equal to five times the GC back-
ground noise, assuming a 10 ml final volume of the 1 liter sample
extract, and assuming a GC injection of 5 micro!iters.
-------
COLUMN: 1.5% OV-U 1.5% OV-225 ON GAS CHROM Q
TEMPERATURE: 75°C.
DETECTOR: ELECTRON CAPTURE
(VI
LU
QQ
O
cc
O
_i
X
O
{VI
8 12 16
RETENTION TIME-MINUTES
20
Figure 1. Gas chromatogram of chlorinated hydrocarbons
T-
-------
o
<
Q.
O
o
cc
O
z
o
x
LU
z
COLUMN: 1.5°i OV-1
1.5% OV-225 ON GAS CHROM Q
TEMPERATURE: 160°C.
DETECTOR: ELECTRON CAPTURE
LU
CO
O
cc
O
I
O
8 12 16
RETENTION TIME-MINUTES
Figure 2. Gas chromatogram cf :;h'orinated hydrocarbons
-------
2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN
METHOD 613
1. Scope and Application
1.1 This method covers the determination of 2,3,7,8-tetrachlorodibenzo-
p-dioxin (TCDD). The following parameter may be determined by this
method:
Parameter STORET No.
TCDO 34675
1.2' This method is applicable to the determination of TCDD in municipal
and industrial discharges. It is designed to be used to meet the
monitoring requirements of the National Pollutant Discharge
Elimination System (NPDES). As such, it presupposes the potential
for finding trace levels of TCDD in the sample. The method incor-
porates techniques that can also be used to screen samples for TCDD
using an electron capture detector.
1.3 The sensitivity of this method is usually dependent upon the level
of interferences rather than instrumental limitations. The limit
of detection listed in Table I represents the sensitivity that can
be achieved in wastewaters in the absence of interferences.
1.4 This method is recommended for use only by analysts experienced
with residue analysis and skilled in mass spectral analytical
techniques.
1.5 Because of the extreme toxicity of this compound, the analyst must
take elaborate precautions to prevent exposure to himself, or to
3-
-------
others, of materials known or believed to contain TCDD. The
appendix to this method contains guidelines and protocols that
should serve as minimum safe-handling standards for the laboratory.
2. Summary of Method
2.1 A 1-liter sample of wastewater is extracted with methylene chloride
using separatory funnel techniques. The extract is dried and ex-
changed to hexane while being concentrated to a volume of 1.0 ml or
lower. Capillary column GC/MS conditions and internal standard
techniques are described which allow for the measurement of TCDD in
the extract. Electron capture gas chromatographic conditions are
also provided to permit the analyst to use this equipment to pre-
screen samples before GC/MS analysis.
2.2 If interferences are encountered, the method provides selected
general purpose cleanup procedures to aid the analyst in their
elimination.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines causing
misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the
conditions of the analysis by running method blanks. Specific
selection of reagents and purification of solvents by distillation
in all-glass systems may be required.
3.2 Interferences coextracted from the samples will vary considerably
from source to source, depending upon the diversity of the
industrial complex or municipality being sampled. TCDD is often
-------
associated with other interfering chlorinated compounds which are
at concentrations several magnitudes higher than that of TCDD,
While general cleanup techniques are provided as part of this
method, unique samples may require additional cleanup approaches to
achieve the sensitivity stated in Table 1.
3.3 The other isomers of tetrachlorodibenzo-p-dioxin may interfere with
the measurement of 2,3,7,8-TCDD. Capillary column gas chromatog-
raphy is required to resolve those isomers that yield virtually
identical mass fragmentation patterns.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete or composite sampling. •
4.1.1 Grab sample bottle - amber glass, 1-liter or 1-quart
volume. French or Boston Round design is recommended. The
container must be washed and solvent rinsed before use to
minimize interferences.
4.1.2 Bottle caps - Threaded to screw on to the sample bottles.
Caps must be lined with Teflon. Foil may be substituted if
sample is not corrosive.
4.1.3 Compositing equipment - Automatic or manual compositing
system. Must incorporate glass sample containers for the
collection of a minimum of 250 ml. Sample containers must
be kept refrigerated during sampling. No tygon or rubber
tubing may be used in the system.
4.2 Separatory funnels - 2000 ml and 500 ml, with Teflon stopcock.
4.3 Drying column - 20 mm ID pyrex chromatographic column with coarse
frit.
-------
4.4 Kuderna-Danish (K-D) Apparatus
4.-4.1 Concentrator tube - 10 ml, graduated (Kontes K-570050- 1025
or equivalent). Calibration must be checked. Ground glass
stopper (size 19/22 joint) is used to prevent evaporation of
extracts.
4.4.2 Evaporative flask - 500 ml (Kontes K-57001-0500 or equiva-
lent). Attach to concentrator tube with springs. (Kontes
K-662750-0012).
4.4.3 Snyder column - three-ball macro (Kontes K503000-0121 or
equivalent).
4.4.4 Snyder column - two-ball micro (Kontes K-569001-0219 or
equivalent).
4.4.5 Boiling chips - solvent extracted, approximately 10/40 mesh.
4.5 Water bath - Heated, with concentric ring cover, capable of
temperature control (+ 2°C). The bath should be used in a hood.
4.6 Gas chromatograph - Analytical system complete with gas chromato-
graph suitable for on-column injection and all required accessories
including electron capture, packed and capillary column supplies,
recorder, gases, syringes. A data system for measuring peak areas
is recommended.
4.7 GC/Mass Spectrometer system - electron ionization source, capable
of selected ion monitoring in groups of two or more ions, and
related data system.
4.8 Chromatography column - 300 mm long x 10 mm ID with coarse fritted
disc at bottom and Teflon stopcock.
4.9 Chromatography column - -CO mm long x 11 mm ID with coarse fritted
?£>
-------
disc at bottom and Teflon stopcock.
4.10 Pipets - Disposable, Pasteur, 150 mm long x 5 mm ID (Fisher
Scientific Co., No. 13-678-6A or equivalent).
5. Reagents
5.1 Sodium hydroxide - (ACS) 10 N and 1 N in distilled water. Wash
the solutions with methylene chloride and with hexane.
5.2 Sulfuric acid - (ACS) (HI) and IN. For (1+1), mix equal volumes
of cone. HgSO^ with distilled water. Wash the solutions with
methylene chloride and with hexane.
5.3 Methylene chloride, hexane, benzene, tetradecane - Pesticide
quality or equivalent.
5.4 Sodium Sulfate - (ACS) Granular, anhydrous (purified by heating at
400°C for 4 hrs. in a shallow tray).
5.5 Stock standards - In a glovebox, prepare stock standard solutions
of TCDD and Cl-TCDD. The stock solutions are stored in the
glovebox, and checked frequently for signs of degradation or evapo-
ration, especially just prior to preparing working standards from
them.
5.6 Silica gel - high purity grade, 100/120 mesh, (Fisher Scientific
Co., No. S-679 or equivalent).
5.7 Alumina - neutral, 80/200 mesh (Fisher Scientific Co., No. A-540 or
equivalent). Before use activate for 24 hours at 130°C in a foil-
covered glass container.
5.8 Activated Coconut Charcoal - 50/200 mesh (Fisher Scientific Co.,
No. 5-690A or equivalent).
6. Calibration
-------
6.1 Prepare GC/MS calibration standards for the internal standard
technique that will allow for measurement of relative response
factors of at least three TCDD/37C1 TCDD ratios. The 37C1-TCDD
concentration in the standards should be fixed and selected to
yield a reproducible response at the most sensitive setting of the
mass spectrometer.
6.2 Assemble the necessary GC or GC/MS apparatus and establish
operating parameters equivalent to those indicated in Table I.
Calibrate the GC/MS system according to Eichelberger, et al.
(1975). By injecting calibration standards, establish the response
factors for TCDD vs. 37C1-TCDD.
6.3 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from the reagents.
7. Quality Control
7.1 Before processing any samples, the analyst should demonstrate
through the analysis of a distilled water method blank, that all
glassware and reagents are interference-free. Each time a set of
samples is extracted or there is a change in reagents, a method
blank should be processed as a safeguard against chronic laboratory
contamination.
7.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to validate the
precision of the sampling technique. Laboratory replicates should
be analyzed to validate the precision of the analysis. Fortified
samples should be analyzed to validate the accuracy of the analy-
-------
sis, although surrogate spikes are recommended because of the
toxicity of TCDD. Where doubt exists over the identification of a
peak on the electron capture chromatogram, mass spectroscopy must
be used for clarification or confirmation.
8. Sample Collection, Preservation, and Handling
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be prewashed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be free of tygon and other potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time of col-
lection until extraction. Chemical preservatives should not be
used in the field unless more than 24 hours will elapse before
delivery to the laboratory. If the samples will not be extracted
within 48 hours of collection, the sample should be adjusted to a
pH range of 6.0-8.0 with sodium hydroxide or" sulfuric acid.
8.3 All samples must be extracted within 7 days and completely analyzed
within 30 days of collection.
9. Sample Extraction
CAUTION: If there is a remote possibility that the sample contains TCDD
at measurable levels, all of the following operations must be performed
in a limited access laboratory with the analyst wearing full protective
covering for all exposed skin surfaces. See Appendix.
9.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
HE-
-------
two-liter separatory funnel. Check the pH of the sample with
wide-range pH paper and adjust to within the range of 5-9 with
sodium hydroxide or sulfuric acid.
9.2 Add 60 ml methylene chloride to the sample bottle, seal, and shake
30 seconds to rinse the inner walls. Transfer the solvent into the
separatory funnel, and extract the sample by shaking the funnel for
two minutes with periodic venting to release vapor pressure. Allow
the organic layer to separate from the water phase for a minimum of
ten minutes. If the emulsion interface between layers is more than
one-third the size of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include stir-
ring, filtration of the emulsion through glass wool, or centrifu-
gation. Collect the methylene chloride extract in a 500-ml
separatory funnel.
9.3 Add a second 60-ml volume of methylene chloride to the sample
bottle and complete the extraction procedure a second time,
combining the extracts in the 500-ml separatory funnel.
9.4 Perform a third extraction in the same manner. To the combined
extracts in the separatory funnel add 1CJD ml IN NaOH. Shake the
funnel for 30-60 seconds. Allow the layers to separate and draw
the organic layer into a 250 ml Erlenmeyer flask. Discard the
aqueous layer and return the organic layer to the separatory
funnel. Perform a second wash of the organic layer with 1 N NaOH
and discard the aqueous layer.
9.5 In the same manner wash the organic layer twice with 100 ml IN
-------
h^SO^, discarding the aqueous layers.
9.6 Wash the organic layer three times with 100 ml hLO, discarding
the aqueous layers.
9.7 Pour the organic layer extract through a drying column containing
3-4 inches of anhydrous sodium sulfate, and collect it in a 500-ml
Kuderna-Danish (K-D) flask equipped with a 10 ml concentrator tube.
Rinse the Erlenmeyer flask and column with 20-30 ml methylene
chloride to complete the quantitative transfer.
9.8 Add 1-2 clean boiling chips to the flask and attach a three-ball
Snyder column. Prewet the Snyder column by adding about 1 ml
methylene chloride to the top. Place the K-D apparatus on a hot
water bath (60-65°C) so that the concentrator tube is partially
immersed in the hot water, and the entire lower rounded surface of
the flask is bathed in vapor. Adjust the vertical position of the
apparatus and the water temperature as required to complete the
concentration in 15-20 minutes. At the proper rate of distil-
lation, 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 for at
least 10 minutes while cooling.
9.9 Momentarily remove the Snyder column, add 50 ml hexane and a new
boiling chip and replace the Snyder column. Increase the tempera-
ture of the water bath to 80°C. Prewet the Snyder column by
adding about 1 ml hexane to the top. Evaporate the solvent as in
9.8. Remove the Snyder column and rinse the flask and its lower
joint into the concentrator tube with 1-2 ml of hexane. A 5-ml
at-
-------
syringe is recommended for this operation.
9.10 Add a clean boiling chip and attach a micro-Snyder column. Prewet
the column by adding about 1 ml hexane to the top. Place the K-D
apparatus on the 80°C 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
the concentration in 5-10 minutes. At the proper rate of distil-
lation the balls of the column will actively chatter but the
chambers will not flood. When the apparent volume of liquid
reaches about 0.5 ml, remove the K-D apparatus and allow it to
drain for at least 10 minutes while cooling. Remove the micro
Snyder column and rinse its lower joint into the concentrator tube
with 0.2 ml hexane. Adjust the extract volume to 1.0 ml with
hexane. Stopper the concentrator tube and store refrigerated if
further processing will not be performed immediately.
9.11 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the liquid to a 1000 ml graduated
cylinder. Record the sample volume to the nearest 5 ml.
9.12 The analyst has several options available to him depending upon the
nature of the sample and the availability of resources:
9.12.1 If the appearance of the extract or previous experience with
the matrix indicates cleanup will be required the analyst
should proceed with one or more techniques as described in
paragraph 10.
9.12.2 If the analyst wishes to screen the sample for the possible
presence of TCDD before GC/MS analysis, he can analyze the
-------
extract by packed column or capillary column electron
capture detection, as in paragraph 11.
9.12.3 The analyst may proceed directly to GC/MS analysis, para-
graph 12.
10. Cleanup and Separation
10.1 Several cleanup column choices are offered to the analyst in this
section. Although any of them may be used independently, the
silica gel column (10.2) followed immediately by the alumina column
.(10.3) has been used frequently to overcome background problems
encountered by the GC/MS.
10.2 Silica Gel Column Cleanup for TCDD.
10.2.1.Fill a 400 mm long x 11 mm ID chromatography column with
silica gel to the 300 mm level, tapping the column gently to
settle the silica gel. Add 10 mm anhydrous sodium sulfate
to the top of the silica gel.
10.2.2 Preelute the column with 50 ml 20% benzene/80% hexane (V/V).
Adjust the elution rate to 1 ml/min. Discard the eluate and
just prior to exposure of the sodium sulfate layer to the
air, transfer the entire 1.0 ml sample extract onto the
column, using two 2 ml portions of 20% benzene/80% hexane to
complete the transfer.
10.2.3 Just prior to exposure of the sodium sulfate layer to the
air, add 40 ml 20% benzene/80% hexane to the column.
Collect the eluate in a 500 ml K-D flask equipped with a 10
ml concentrator tube.
10.2.4 Evaporate the fraction to 1.0 ml by standard K-D techniques
It
-------
(9.8-9.10). Analyze by ECGC (11), GC/MS (12) or continue
cleanup as described below (10.3).
10.3 Alumina Column Cleanup for TCDD
10.3.1 If the extract is not in hexane, add 0.1-0.2 ml tetradecane
keeper and concentrate it at room temperature down to this
volume using a stream of dry nitrogen gas. Dilute'to 1.0 ml
with hexane.
10.3.2 Fill a 300 mm long x 10 mm ID chromatography column with
activated alumina to the 150 mm level, tapping the column
gently to settle the alumina. Add 10 mm anhydrous sodium
sulfate to the top of the alumina.
10.3.3 Preelute the column with 50 ml hexane. Adjust the elution
rate to 1 ml/min. Discard the eluate and just prior to
exposure of the sodium sulfate layer to the air, transfer
the entire 1 ml sample extract onto the column, using two
additional 2 ml portions of hexane to complete the transfer.
10.3.4 Just prior to exposure of the sodium sulfate layer to the
air, add 50 ml 3% methylene chloride/97% hexane (V/V) and
continue the elution of the column. Discard the eluate.
10.3.4 Next elute the column with 50 ml 20% methylene chloride/80%
hexane (V/V) into a 500 ml K-D flask equipped with a 10 ml
concentrator tube. Concentrate the collected fraction to
1.0 ml by standard K-D technique (9.8-9.10). Analyze by
ECGC (11), GC/MS (12) or continue cleanup as described below
(10.4).
10.4 Charcoal and Silica-gel Column Cleanup for TCDD.
-------
10.4.1 Prepare a homogeneous mixture of 1 part activated charcoal
to 140 parts silica-gel. Fill a 5 mm ID disposable pipet to
a length of 50 mm, tapping the column to settle the mixture.
10.4.2 Preelute the column with 5 ml hexane. Discard the eluate
and just prior to exposure of the top of the column to the
air, transfer an 0.5 ml aliquot of sample extract onto the
column, using an additional 0.5 ml hexane to complete the
transfer.
10.4.3 Just prior to exposure of the top of the column to the air,
add 10 ml hexane and continue the elution of the column.
Discard the eluate.
10.4.4 Next, elute the column with 10 ml benzene into a 10 ml K-D
concentrator tube. Concentrate the eluate to 1.0 ml with
micro-K-D concentration (9.10) on a boiling water bath.
Analyze by ECGC (11) or GC/MS (12).
11. Electron Capture Screening
11.1 The sample extracts can be screened by electron capture gas chroma-
tography at the option of the analyst in an effort to reduce the
workload on the GC/MS system. Either packed or capillary column
techniques may be used for this purpose. The only acceptable
conclusions that can be reached with this technique are: (a) TCDO
is not detectable at the detection limit of the procedure: (b)
TCDD is not present above a stated concentration or control level:
and (c) the presence or absence of TCDD is unresolved.
11.2 Table I summarizes some recommended gas chromatographic column
materials and operating conditions for the instrument. Included in
IT- IS 5"
-------
this table are estimated retention times and sensitivities that
should be achieved by this method. An example of the chromatog-
raphy achieved by the packed column is shown in Figure 1 and by the
capillary column in Figure 2. Calibrate the system daily with a
minimum of three injections of calibration standards.
11.3 For packed column GC, inject 2-5 ul of the sample extract using the
solvent-flush technique. A splitless injector is recommended for
the capillary system, but for optimum peak geometry a solvent
exchange to tetradecane is required (10.3.1). Record the volume
injected to the nearest 0.05 ul, and the resulting peak size, in
area units.
11.4 If there is no measureable baseline deflection at the retention
time of TCDD, report the result as less than the detection limit of
the electron capture system.
11.5 If a measurable peak appears within the tolerances of the TCDD
retention time of the system, the analyst should proceed to GC/MS
(12).
11.6 If the complexity of the chromatogram defies interpretation, the
analyst may want to pursue cleanup (10) followed by reanalysis by
ECGC, or proceed directly to GC/MS.
12. GC/MS Analysis
12.1 Table I summarizes the recommended capillary column gas chromato-
graphic materials and operating conditions for the instrument.
Included in this table is the estimated retention time and
sensitivity that should be achieved by this method. An example of
the chromatography achieved by this column is shown in Figure 2.
-------
Calibrate the system dally, with a minimum at three Injections of
standard mixtures.
12.2 Add a known amount of Cl-TCOO to the sample extract.
12.3 Analyze samples with selected ion monitoring of at least two Ions
characteristic of TCDD (m/e 320 and m/e 322) and of CI-TCOD
(m/e 328). Proof of the presence of TOO exists if the following
conditions are met:
12.3.1 The retention time of the peak 1n the sample must match
that in the standard, within the performance specifications
of the analytical system.
12.3.2 The ratio of ions (320:322) must agree within 1W of that
in the standard.
12.3.3 The retention time of the peak maximum for the m/e 320 peak
must exactly match that of the 322 peak.
12.4 Quantitate the TCDO peak from the response relative to the. Cl
TCDD internal standard.
12.5 If a response 1s obtained for both ions but is outside the expected
ratio, then a co-eluting impurity may be suspected. In this case,
another set of ions characteristic of the TCDD molecule, should be
analyzed. A good choice of ions 1s m/e 257 and m/e 259. These
ions are useful in characterizing the molecular structure of TCDO.
Suspected impurities such as DDE, ODD or PCS residues can be
confirmed by checking for their major fragments. These materials
can be removed by the cleanup columns. If available, an analysis
of the EC chromatogram will provide insight into the complexity of
the problem and will determine the manner in which the mass
spectrometer will be used.
-------
12.6 If broad background interference restricts the sensitivity of the
GC/MS analysis, the analyst should employ cleanup procedures (10)
and reanalyze by GC/MS.
12.7 In those circumstances where these procedures do not yield a
definitive conclusion, then the use of high resolution mass
spectrometry is suggested.
13. Calculations
13.1 Determine the concentration of individual compounds according to
the formula: •
Concentration, ug/1 = (A) (V.)
(V )1 (Vs)
where A = Nanograms TCDD injected into the GC/MS from the
calibration curve.
V. = volume of extract injected (ul)
Vt = Volume of total extract (ul)
V = Volume of water extracted (ml)
13.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed, all
data obtained should be reported.
14. Accuracy and Precision
No data available at this time.
inn-
-------
BIBLIOGRAPHY
1. "Development and Application of Test Procedures for Specific Organic
Toxic Substances in Wastewaters. Category 6-Dioxin." Report for EPA
Contract 68-03-2635 (In preparation).
2. "Reference Compound to Calibrate Ion Abundance Measurements in Gas
Chromatography--Mass Spectrometry Systems," J. W. Eichelberger, L. E.
Harris, and W. L. Budde, Anal. Chem. 47, 995-1000 (1975).
'- )
-------
Table I
Gas Chromatography of TCDD
Detection
Column Retention Time (min.) Limit (ug/L)
Packed1 7.3 0.003
Glass Capillary 2 9.5 0.003
^Packed column conditions: Supelcoport 100/120 mesh coated with 1.5%
SP-2250/1.95% SP-2401 packed in a 180 cm long x 2 mm ID glass column
with 5% Methane/95% Argon carrier gas at 25 ml/min flow rate. Column
temperature is 220°C.
capillary column conditions: SP-2250 coated on a 30 m long x 0.25
mm ID glass column (Supelco No. 2-3714 or equivalent) with helium
carrier gas at 30 cm/sec linear velocity run splitless. Column
temperature is 210°C.
Detection limit is calculated from the minimum detectable GC response being
equal to five times the GC background noise, assuming a 1 ml effective
final volume of the 1 liter sample extract, and assuming a GC injection
of 5 micro! iters. Detection levels apply to both electron capture and
GC/MS detection.
-------
APPENDIX A
Safe Handling Practices for TCDD
Dow Chemical U.S.A. has issued the following precautions for safe
handling TCDD in the laboratory. In addition to these practices, the
following points are also helpful:
1. Contamination of the laboratory will be minimized by conducting all
manipulations in the hood.
2. Effluent of the gas chromatography (from the Nickel-63 detector or as a
result of splitting when capillary columns are used) should pass through
either a column of activated charcoal or bubbled through a trap contain-
ing oil or high-boiling alcohols.
3. Liquid waste can be dissolved in methanol or ethanol and irradiated with
ultraviolet light with wavelength greater than 290 nm for several days.
* * * *
PRECAUTIONS FOR SAFE HANDLING OF
2,3,7,8-TETRACHLORODIBENZO-PARA-DIOXIN (TCDD)
IN THE LABORATORY (REVISED 11/78)
The following statements on safe handling are as complete as possible on
the basis of available toxicological information. The precautions for safe
-------
handling and use are necessarily general in nature since detailed, specific
recommendations can be made only for the particular exposure and circum-
stances of each individual use. Inquiries about specific operations or uses
may be addressed to the Dow Chemical Company. Assistance in evaluating the
health hazards of particular plant conditions may be obtained from certain
consulting laboratories and from State Departments of Health or of Labor,
many of which have an industrial health service.
TCDD is extremely toxic to laboratory animals. However, it has been
handled for years without injury in analytical and biological laboratories.
Techniques used in handling radioactive and infectious materials are
applicable to TCDD.
Protective Equipment; Throw-away plastic gloves, apron or lab coat, safety
glasses and lab hood adequate for radioactive work.
Training; Workers must be trained in the proper method of getting out of
contaminated gloves and clothing without contacting the exterior surfaces.
Personal Hygiene; Thorough washing of hands and forearms after each manipu-
lation and before breaks (coffee, lunch, and shift).
Confinement; Isolated work area, posted with signs, segregated glassware
and tools, plastic-backed absorbent paper on benchtops.
Waste; Good technique includes minimizing contaminated waste. Plastic bag
liners should be used in waste cans. Janitors must be trained in safe hand-
ling of waste (one accidental case of chloracne resulted from handling
laboratory waste in a routine manner).
Disposal of Wastes; TCDD decomposes above 800°C. Low-level waste such as
the absorbent paper, tissues, animal remains and plastic gloves may be
burned in a good incinerator. Gross quantities (milligrams) should be
-------
packaged securely and disposed through commerical or governmental channels
which are capable of handling high-level radioactive wastes or extremely
toxic wastes. Liquids should be allowed to evaporate in a good hood and in
a disposable container. Residues may then be handled as above.
Decontamination: Personal — any mild soap with plenty of scrubbing action:
Glassware, Tools, and Surfaces ~ Chlorothene* NU Solvent is the least toxic
solvent shown to be effective. Satisfactory cleaning may be accomplished by
rinsing with Chlorothene, then washing with any detergent and water. Dish
water may be disposed to the sewer. It is prudent to minimize solvent
wastes because they may require special disposal through commercial sources
which are expensive.
Laundry; Clothing known to be contaminated should be disposed with the
precautions described under "Disposal of Wastes." Lab coats or other
clothing worn in TCDD work may be laundered. Clothing should be collected
in plastic bags. Persons who convey the bags and launder the clothing
should be advised of the hazard and trained in proper handling. The
clothing may be put into a washer without contact if the launderer knows the
problem. The washer should be run through a cycle before being used again
for other clothing.
Wipe Tests; A useful method of determining cleanliness of work surfaces and
tools is to wipe the surface with a piece of filter paper. Extraction and
analysis by gas chromatography can achieve a limit of sensitivity of 0.1
microgram per wipe. The analytical method is available upon request. Less
than 1 microgram TCDD per sample indicates acceptable cleanliness; anything
*Trademark of the Dow Chemical Company
-------
higher warrants further cleaning. More than 10 micrograms on a wipe sample
indicates an acute hazard and requires prompt cleaning before further use of
the equipment or work space. It indicates further that unacceptably sloppy
work habits have been employed in the past.
Inhalation: Any procedure that may produce airborne contamination, must be
done with good ventilation. Gross losses to a ventilation system must .not
be allowed. Handling of the dilute solutions normally used in analytical
and animal work presents no inhalation hazards except in case of an accident,
Accidents; Remove contaminated clothing immediately, taking precautions not
to contaminate skin or other articles. Wash exposed skin vigorously and
repeatedly until medical attention is obtained.
For clinical advice, contact B. B. Holder, M.D., Medical Director, Dow
Chemical U.S.A., Midland, Michigan, 48640, (telephone 517/636-2108). For
detailed safe handling precautions for specific procedures, contact L. G.
Silverstein, Industrial Hygiene Laboratory, Dow Chemical U.S.A., Midland,
Michigan, 48640 (telephone 517/636-1688).
331-
-------
COLUMN: 1.5*6 SP-2250* 1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 220°C
DETECTOR: ELECTRON CAPTURE
02468
RETENTION TIME-MINUTES
Figure 1. Gas chromatogram of TCDD
-------
73.3
320
100.0
322
50.1
324
214.1
EXTRACTED ION CURRENT PROFILE
EXTRACTED ION CURRENT PROFILE
(M/e - 322)
EXTRACTED ION CURRENT PROFILE
(M/e =324)
TOTAL ION CURRENT PROFILE
8 9 10
RETENTION TIME-MINUTES
Figure 2. Total and extracted ion current profiles of TCDD
"2T-
-------
METHOD 624
ORGANICS BY PURGE AND TRAP
1. Scope and Application
1.1 This method is designed to determine those "unambiguous priority
pollutants," associated with the Consent Decree, that are amenable
to the purge and trap method. The parameters listed in Table 1
may be determined by this method.
1.2 .This method is applicable to the determination of these compounds
in municipal and industrial discharges. It is designed to be used
to meet the survey requirements of the Effluent Guidelines
Division (EGD) and the monitoring requirements of the National
Pollutants Discharge Elimination System (NPDES).
1.3 The detection limit of this method is usually dependent upon the
level of interferences rather than instrumental limitations. The
limits listed in Table 2 represent sensitivities that can be
achieved in wastewaters.
1.4 The GC/MS parts of this method are recommended for use only by
persons experienced in GC/MS analysis or under the close
supervision of such qualified persons.
1.5 The trapping and chromatographic procedures described do not apply
to the most volatile of the priority pollutants,
dichlorodifluoromethane. An alternative three stage trap
containing charcoal is to be used -:f th's compound is to be
analyzed. See EPA Method 601 and Reference 1. Primary ion for
-------
quantitative analysis is 101. The secondary ions are 85, 87, and
103.
1.6 Although this method can be used for qualitative screening for
acrolein and acrylonitrile, the purging efficiencies are low and
erratic. For quantitative analysis of these compounds, use either
direct aqueous injection (Ref. 4-6) or EPA Method 603, Acrolein
and 'Acrylonitrile, EMSL, Cincinnati, Ohio.
2. Summary of Method
2.1 A sample of wastewater is purged with a stream of inert gas. The
gas is bubbled through a 5 ml water sample contained in a
specially designed purging chamber. The volatile organics are
efficiently transferred from the aqueous phase into the gaseous
phase where they are passed through a sorbent bed designed to trap
out the organic volatiles. After purging is complete, the trap is
backflushed while being rapidly heated in order to thermally
desorb the components into the inlet of a gas chromatograph. The
components are separated via the gas chromatograph and detected
using a mass spectrometer which is used to provide both
qualitative and quantitative information. The chromatographic
conditions as well as typical mass spectrometer operating
parameters are supplied.
3. Interferences
3.1 Interferences coextracted from the samples will vary considerably
from source to source, depending upon the diversity of the
industrial complex or municipality being sampled. Impurities in
the purge gas and orgaric compounds out-gas ing from the plumbing
-------
ahead of the trap account for the majority of contamination
problems. The analytical system must be demonstrated to be free
from interferences under the conditions of the analysis by running
method blanks. Method blanks are run by charging the purging
device with organic-free water and analyzing it in a normal
manner. The use of non-TFE plastic tubing, non-TFE thread
sealants, or flow controllers with rubber components in the
purging device should be avoided.
3.2 Samples can be contaminated by diffusion of volatile organics
(particularly methylene chloride) through the septum seal into the
sample during shipment and storage. A field blank prepared from
organic-free water and carried through the sampling and handling
protocol can serve as a check on such contamination.
3.3 Cross contamination can occur whenever high level and low level
samples are sequentially analyzed. To reduce cross contamination,
it is recommended that the purging device and sample syringe be
rinsed out twice, between samples, with organic-free water.
Whenever an unusually concentrated sample is encountered, it
should be followed by an analysis of organic-free water to check
for cross-contam- ination. For samples containing large amounts
of water soluble materials, suspended solids, high boiling
compounds or high organohalide levels it may be necessary to wash
out the purging device with a soap solution, rinse with distilled
water, and then dry in a 105°C oven between analyses.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete sampling.
-------
4.1.1 Vial, with cap - 40 ml capacity screw cap (Pierce #13075 or
equivalent). Detergent wash and dry vial at 105°C for
one hour before use.
4.1.2 Septum - Teflon-faced silicone (Pierce #12722 or
equivalent). Detergent wash and dry at 105°C for one
hour before use.
4.2 Purge and trap device - The purge and trap equipment consists of
three separate pieces of apparatus: a purging device, a trap, and
a desorber. The complete device is available commercially from
several vendors or can be constructed in the laboratory according
to the specifications of Bellar and Lichtenberg (Ref. 2,3). The
sorbent trap consists of 1/8 in. O.D. (0.105 in. I.D.) x 25 cm
long stainless steel tubing packed with 25 cm of Tenax-GC (60-80
mesh) and 8 cm of Davison Type-15 silica gel (35-60 mesh). See
Figures 1 through 4. Ten centimeters traps may be used providing
that the recoveries are comparable to the 25 on traps.
4.3 Gas chromatograph - Analytical system complete with a temperature
programmable gas chromatograph suitable for on-column injection
and all required accessories including an analytical column.
4.3.1 Column 1 - An 8 ft. stainless steel column (1/8 in. 00 0.90
to 0.105 in. ID) packed with 1% SP-1000 coated on 60/80
mesh Carbopack B preceded by a 5 cm precolumn packed with
1% SP-1000 coated on 60/80 mesh Chromosorb W. A glass
column (1/4 in OD x 2 mm ID) may be substituted. The
precolumn is necessary only during conditioning.
IF
-------
4.3.2 Column 2 - An 8 ft. stainless steel column (1/8 in OD x
0.09 to 0.105 in. ID) packed with 0.2% Carbowax 1500 coated
on 60/80 mesh Carbopack C preceded by a 1 ft. stainless
steel column (1/8 in. OD x 0.09 to 0.105 in. ID) packed
with 3% Carbowax 1500 coated on 60/80 mesh Chromosorb W. A
glass column (1/4 in. OD x 2 mm ID) may be substituted.
The precolumn is necessary only during conditioning.
4.4 Syringes - glass 5-ml hypodermic with Luer-Lok tip (3 each).
4.5 Micro syringes - 10, 25, 100 ul.
4.6 2 -way syringe valve with Luer ends (3 each, Teflon or Kel-F).
4.7 Syringe - 5-ml gas-tight with shut-off valve.
4.8 8-inch, 20-gauge syringe needle - One per each 5-ml syringe.
4.9 Mass Spectrometer - capable of scanning from 20-260 in six seconds
or less at 70 volts (nominal), and producing a recognizable mass
spectrum (background corrected) at unit resolution from 20 ng of
4-bromofluorobenzene (BFB) when injected through the GC inlet.
The mass spectrometer must be interfaced with a gas chromatograph
equipped with an injector system designed for all -glass on-column
packed column work. All sections of the transfer lines must be
glass or glass-lined and deactivated Use Sylon-CT, Supelco, (or
equivalent) to deactivate. The GC/MS interface can utilize any
separator that gives recognizable mass spectra (background
corrected) and acceptable calibration points at the limit of
detection specified for each compound in Table II.
4.10 A computer system should be interfaced to the mass spectrometer to
allow acquisition of continuous mass scans for the duration of the
IF
-------
chromatographic program. The computer system should also be
equipped with mass storage devices for saving all data from GC-MS
runs. There should be computer software available to allow
searching any GC/MS run for specific ions and plotting the
intensity of the ions with respect to time or scan number. The
ability to integrate the area under an specific ion plot peak is
essential for quantification.
5. Reagents
5.1 Sodium thiosulfate - (ACS) Granular.
5.2 Trap Materials
5.2.1 Porous polymer packing 60/80 mesh chromatographic grade
Tenax GC (2,6-diphenylene oxide).
5.2.2 Three percent OV-1 on Chromosorb-W 60/80 mesh.
5.2.3 Silica gel-(35-60 mesh) .- Davison, grade-15 or equivalent.
5.3 Activated carbon - Filtrasorb-200 (Calgon Corp.) or equivalent.
5.4 Organic-free water
5.4.1 Organic-free water is defined as water free of interference
when employed in the purge and trap procedure described
herein. It is generated by passing tap water or well water
through a carbon filter bed containing about 1 Ib. of
activated carbon.
5.4.2 A water system (Millipore Super-Q or equivalent) may be
used to generate organic-free deionized water.
5.4.3 Organic-free water may also be prepared by boiling water
for 15 minutes. Subsequently, while maintaining the
temperature at 90°C, bubble a contaminant-free inert gas
-------
through the water for one hour. While still hot, transfer
the water to a narrow mouth screw cap bottle equipped with
a Teflon seal.
5.5 Stock standards (2 mg/ml) - Prepare stock standard solutions in
methanol using assayed liquids or gases as appropriate. Because
of the toxicity of some of the organohalides, primary dilutions of
these materials should be prepared in a hood. A NIOSH/MESA
approved toxic gas respirator should be worn when the analyst
handles high concentrations of such materials.
5.5.1 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 minutes or until all alcohol
wetted surfaces have dried. Tare the flask to the nearest
0.1 mg.
5.5.2 Add the assayed reference material:
5.5.2.1 Liquids - Using a 100 ul syringe, immediately add
2 to 3 drops of assayed reference material to the
flask, then reweigh. Be sure that the drops fall
directly into the alcohol without contacting the
neck of the flask.
5.5.2.2 Gases - To prepare standards of bromomethane,
chloroethane, chloromethane, and vinyl chloride,
fill a 5 ml valved gas-tight syringe with the
reference standard to the 5.0--1 mark. Lower the
needle to 5 mm above the re:\-r" alcohol menicus.
Slowly inject the reference r, -,-srd into the neck
-------
of flask (the heavy gas will rapidly dissolve into
the methyl alcohol).
5.5.3 Reweigh the flask, dilute to volume, stopper, then mix by
inverting the flask several times. Transfer the standard
solution to a 15 ml screw-cap bottle equipped with- a Teflon
cap liner.
5.5.4 Calculate the concentration in mg per ml (equivalent to ug
per ul) from the net gain in weight.
5.5.5 Store stock standards at 4°C. Prepare fresh standards
every second day for the four gases and 2-chloroethylvinyl
ether. All other standards must be replaced with fresh
standards each week. Remove samples from cold storage
(approximately an hour prior to analysis) and bring to room
temperature by placing in a warm water bath at 20-25°C.
5.6 Surrogate Standard Dosing Solution - From stock standard solutions
prepared as above, add a volume to give 1000 ug each of
bromochloromethane, 2-bromo-l-chloropropane, and
1,4-dichlorobutane to 40 ml of organic-free water contained in a
50 ml volumetric flask, mix and dilute to volume. Prepare a fresh
surrogate standard dosing solution weekly. Dose the surrogate
standard mixture into every 5 ml sample and reference standard
analyzed.
Calibration
6.1 Using stock standards, prepare secondary dilution standards in
methanol that contain the compounds of interest, either singly or
mixed together. The str :--ds should be at concentrations such
-------
that the aqueous standards prepared in 6.2 will completely bracket
the working range of the chroma to graphic system. If the limit of
detection listed in Table 2 is 10 ug/1, for example, prepare
secondary methanolic standards at 100 ug/1, and 500 ug/1, so that
aqueous standards prepared from these secondary calibration
standards, and the primary standards, will define the linearity of
the detector in the working range.
6.2 Using both the primary and secondary dilution standards, prepare
calibration standards by carefully adding 20.0 ul of the standard
in methanol to 100, 500, or 1000 ml of organic-free water. A 25
ul syringe (Hamilton 702N or equivalent) should be used for this
operation. These aqueous standards must be prepared fresh daily.
6.3 Assemble the necessary gas chromatographic apparatus and establish
operating parameters equivalent to those indicated in Table 2. By
injecting secondary dilution standards, establish the linear range
of the analytical system for each compound and demonstrate that
the analytical system meets the limits of detection requirements
in Table 2.
6.4 Assemble the necessary purge and trap device. Pack the trap as
shown in Figure 2 and condition overnight at a nominal 180°C by
backflushing with an inert gas flow of at least 20 ml/min. Prior
to use, daily condition the traps for 10 minutes by backflushing
at 180°C. Analyze aqueous calibration standards (6.2) according
to the purge and trap procedure in Section 9. Compare the
responses to those obtained by injection of standards (6.3), to
determine the analytical precision. The analytical precision of
3.13
-------
the analysis of aqueous standards must be comparable to data
presented by Bellar arid Lichtenberg (1978, Ref. 1) before reliable
sample analysis may begin.
6.5 The internal standard approach is acceptable for the purgeable
organics when internal standards meeting the following criteria
can be selected:
6.5.1 No interference with other components in the sample.
6.5.2 Structural similarity to the compound to be determined.
6.5.3 Elutes close to the compound to be determined.
6.5.4 The amount added approximates the concentration of the
compound to be determined.
6.6 Internal Standard Method - The utilization of the internal
standard method requires the periodic determination of response
factors (RF) which are defined in equation 1.
Eq. (1) RF = (ASC1S)/(A.SCS)
Where:
A is the integrated area or peak height of the
characteristic ion for the priority pollutant standard.
A. is the integrated area or peak height of the
characteristic ion for the internal standard.
C. is the amount of the internal standard in ug.
C is the amount of the priority pollutant standard in ug.
The relative response ratio for the priority pollutants should be
known for at least two concentration values--50 ng injected to
approximate 10 ug/1 and 500 ng to approximate the 100 ug/1 level.
Those compounds that do not respond at either of these levels may
-------
be run at concentrations appropriate to their response. The
response factor (RF) must be determined over all concentration
ranges of standard (C ) which are being determined. (Generally,
the amount of internal standard added to each extract is the same
so that C. remains constant.) This should be done by preparing
a calibration curve where the response factor (RF) is plotted
against the standard concentration (C ). Use a minimum of three
concentrations over the range of interest. Once this calibration
curve has been determined, it should be verified daily by
injecting at least one standard solution containing internal
standard. If significant drift has occurred, a new calibration
curve must be constructed.
Note: EPA, through its contractors and certain of its Regional
Laboratories, is currently evaluating selected compounds
for use as internal standards in the analysis of organics
by purge and trap.
6.7 The external standard method can be used at the discretion of the
analyst and is recommended when the criteria for use of the
internal standard cannot be met. Prepare a master calibration
curve using a minimum of three standard solutions of each of the
compounds that are to be measured. Plot concentrations versus
integrated areas or peak heights (selected characteristic ion for
GC/MS). One point on each curve should approach the method
detection limit. After the raster set of instrument calibration
curves have been establishes, they should be verified daily by
injecting at least one standard solution. If significant drift
has occurred, a new calibration curve must be
-------
constructed.
7. Quality Control
7.1 Before processing any samples, the analyst should daily
demonstrate, through the analysis of an organic-free water method
blank, that the entire analytical system is interference-free.
7.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to validate the
precision of the sampling .technique. Laboratory replicates should
be analyzed to validate the precision of the analysis. Fortified
samples should be analyzed to validate the accuracy of the
analysis.
7.3 Identification of a peak on the chromatogram is done by analysis
of the entire background corrected mass spectrum.
7.4 The analyst should maintain constant surveillance of both the
performance of the analytical system and the effectiveness of the
method in dealing with each sample matrix by spiking each 5 ml
sample, standard, and blank with surrogate halocarbons. A
combination of bromochloromethane, 2-bromo-l-chloropropane, and
1,4-dichlorobutane is recommended to encompass the boiling range
of compounds covered by this method. Prepare a fresh -surrogate
standard dosing solution on a weekly basis.
8. Sample Collection, Preservation, and Handling
8.1 Grab samples must be collected in glass containers having a total
volume greater than 20 ml. Fill the sample bottles in such a
manner that no air bubbles pass through the sample as the bottle
is being filled. Seal the bottles so that no air bubbles are
-------
entrapped in it. Maintain the hermetic seal on the sample bottle
until time of analysis.
8.2 The sample must be iced or refrigerated from the time of
collection until extraction. If the sample contains residual
chlorine, add sodium thiosulfate preservative (10 mg/40 ml) to the
empty sample bottles just prior to shipping to the sample site,
fill with sample just to overflowing, seal the bottle, and shake
vigorously for 1 minute.
8.3 All samples must be analyzed within 7 days of collection.
9. Sample Extraction and Gas Chromatography
9.1 Adjust the purge gas (nitrogen or helium) flow rate to 40 ml/min.
Attach the trap inlet to the purging device, and set the device to
the purge mode. Open the syringe valve located on the purging
device sample introduction needle.
9.2 Remove the plunger from a 5 ml syringe and attach a closed syringe
valve. Open the sample bottle (or standard) and carefully pour
the sample into the syringe barrel until it overflows. Replace
the syringe plunger and compress the sample. Open the syringe
valve and vent any residual air while adjusting the sample volume
to 5.0 ml. Since this process of taking an aliquot destroys the
validity of the sample for future analysis, the analyst should
fill a second syringe at this time to protect against possible
loss of data. Add 5.0 ul of the surrogate spiking solution (7.3)
through the valve bore, then close the valve.
9.3 Attach the syringe-valve assembly to the syringe valve on the
purging device. Open the syringe valve and inject the sample into
-------
the purging chamber.
9.4 Close both valves and purge the sample for 12.0 + .05 minutes.
9.5 After the 12-minute purge time, attach the trap to the
chromatograph, and adjust the device to 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 20 to 60 ml/min for 4 minutes. If rapid heating
cannot be achieved, the gas chromatographic column must be used as
a secondary trap by cooling it to 30°C (or subambient, if
problems persist) instead of the initial program temperature of
45°C.
9.6 While the trap is being desorbed into the gas chromatograph, empty
the purging chamber using the sample introduction syringe. Wash
the chamber with two 5 ml flushes of organic-free water. After
the purging device has been emptied, continue to allow the purge
gas to vent through the chamber until the frit is dry, and ready
for the next sample.
9.7 After desorbing the sample for four minutes recondition the trap
by returning the purge and trap device to the purge mode. Wait 15
seconds 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 seven minutes, turn off the trap
heater and open the syringe valve to stop the gas flow through the
trap. When cool, the trap is ready for the next samp I.e. (Note:
If this bake out step is omitted, the amount of water entering the
GC/MS system will progressively increase causing deterioration of
or
-------
and potential shut down of the system.)
9.8 The analysis of blanks is most important in the purge and trap
technique since the purging device and the trap can be
contaminated by residues from very concentrated samples or by
vapors in the laboratory. Prepare blanks by filling a sample
bottle with organic-free water that has been prepared by passing
distilled water through a pretested activated carbon column.
Blanks should be sealed, stored at 4°C, and analyzed with each
group of samples.
10. Gas Chromatography - Mass Spectrometry
10.1 Table 2 summarizes the recommended gas chromatographic column
materials and operating conditions for the instrument. Included
in this table are estimated retention times and sensitivities that
/
should be achieved by this method. An example of the separation
achieved by Column 1 is shown in Figure 5.
10.2 GC-MS Determination - Suggested analytical conditions for
determination of the priority pollutants .amenable to purge and
trap, using the Tekmar LCS-1 and a GC/MS are given below.
Operating conditions vary from one system to another; therefore,
each analyst must optimize the conditions for each purge and trap
and GC/MS system.
10.3 Purge Parameters
Sample size - 5.0 ml.
Purge gas - Helium, high purity grade
Purge time - 12 minutes
Purge flow - 40 ml/min.
jr
-------
Trap Dimensions - -1/8 in. O.D. (0.105 in. I.D.) x 25 cm long
Trap sorbent - Tenax-GC, 60/80 mesh (15 an], plus Type 15 silica
gel, 35/60 mesh (8 cm).
Desorption flow - 20 ml/min.
Desorption time - 4 min.
Desorption temperature - 180°C
10.4 Mass Spectrometer Parameters
Electron energy - 70 volts (nominal)
Mass range - 20-27, 33-260 amu
Scan time - 6 seconds or less.
10.5 Calibration of the gas chromatography-mass spectrometry (GC-MS
system - Evaluate the system performance each day that it is to be
used for the analysis of samples or blanks by injecting 20 ng of
BFB into the GC inlet.
10.6 Analyze the 20 ug/1 standard to demonstrate instrument performance
or these compounds.
11. Qualitative and Quantitative Determination
11.1 The complete background corrected mass spectrum is compared to a
reference mass spectrum, either from an authentic sample or a
library spectrum, to provide qualitative identification. All of
the following criteria must be met.
11.1.1 The experimental mass spectrum must contain all ions
present above 1% relative abundance in the mass spectrum of
the reference with the general agreement of _+ 20% of the
relative abundance in the reference mass spectrum.
11.1.2 Ions present in the experimental mass spectrum that are not
-------
present in the reference mass spectrum must not exceed 10X
of the total ion abundance in the experimental mass
spectrun. These contaminant ions should be readily
accounted- for by common background Ions. If the
experimental mass spectrun 1s highly contaminated,
Identificaton should not be accepted.
11.1.3 The retention time at the experimental mass spectrun should
be within £ 5 spectrum numbers or * 30 seconds (whichever
Is greater) of the retention time of the authentic compound.
11.1.4 Structural Isomers that have very similar mass spectra
(phenanthrene, anthracene; benzola)pyrene, benzo(e)pyrene;
etc.) can be explicitly Identified if the resolution
between the Isomers in a standard mix is acceptable.
Acceptable resolution 1s achieved if the valley height
ZS'L'f
between Isomers is less than be44 the sum of the two peak
heights. Otherwise, structural Isomers are identified as
1 saneric pairs.
11.2 The primary Ions to be used to quantify each compound are listed
in Table 3. If the sample produces an interference for the
primary ion, use a secondary ion to quantify.
11.3 For low concentrations, or direct aqueous injection of
acrylonltrile and acrolein, the characteristic masses (Table 3)
may be used for selected ion monitoring (SIM). SIM is the use of
a mass spectrometer as a substance selective detector by measuring
the mass spectrometric response at one or several characteristic
masses in real time.
-------
11.4 Internal Standard Method Calculations - By adding a constant known
amount of internal standard (C. in ug) to every sample extract,
the concentration of priority pollutant (C ) in ug/1 in the
sample is calculated using equation 2.
(A5) (C.s)
Eq< (2) C
o = (ASJ (RP) (Vo)
Where:
V is the volume of the original sample in liters.
To quantify, add the internal standard to the 5.0 ml sample no
more than a few minutes before purging to minimize the possibility
of losses due to evaporation, adsorption, or chemical reaction.
Calculate the concentration by using the previous equations with
the appropriate response factor taken from the calibration curve.
(Ideally, the response factor will not change with concentration.)
11.5 External Standard Method Calculations - The concentration of the
unknown can be calculated from the slope and intercept of the
curve. The unknown concentration can be determined using equation
3.
(A)(V )
Eq. (3) Micrograms/liter = ng/ml =
Where:
A - mass of compound from calibration curve (ng).
V. * volume of extract injected (ul)
Vt = volume of total extract (ul)
V = volume of water extracted (ml)
-------
11.6 An alternate external standard approach for purgeables utilizes a
single point calibration. Prepare and analyze a reference
standard that closely approximates the response for each
component in a sample. Calculate the concentration in the sample
using Equation 4.
Eq. 4 micrograms per liter =
where: A is the area of the unknown
B is the concentration of standard
C is the area of the standard
11.7 Report all results to two significant figures. Report internal
standard data to two significant figures-. When duplicate and
spiked samples are analyzed, all data obtained should be
reported. Report results in micrograms per liter without
correction for recovery data. Include identification of any
components found in the blanks.
12. Precision and Accuracy
12.1 Determine the precision of the method by dosing blank water with
the compounds selected as surrogate standards -
bromochloromethane, 2-bromo-l-chloropropane, and
1,4-dichlorobutane - and running replicate analyses. Calculate
the recovery and its standard deviation. These compounds
represent early, middle, and late eluters over the range of the
Consent Decree compounds and are not, themselves, included on the
list.
-------
12.2 The sample matrix can affect the purging efficiencies of Individual
compounds; therefore, each sample must be dosed with the surrogate
standards and analyzed 1n a manner Identical to the Internal
standards in blank water. If the recovery of the surrogate
standard shows a-deviation greater than two standard deviations
(12.1), repeat the dosed sample analyses. If the deviation 1s
again greater than two standard deviations, dose another aliquot of
the same sample with the compounds of Interest at approximately two
times the measured values and analyze. Calculate the recovery for
. the Individual compounds using these data.
13. References
1. "The Analysis of Halogenated Chemical Indicators of Industrial
Contamination 1n Water by the Purge and Trap Method," U.S. EPA,
Environmental Monitoring and Support Laboratory, Cincinnati, OH,
45268, Dec. 1978.
2. "Symposium on Measurement of Organic Pollutants in Water and
Wastewater," ASTM Special Publication, 1979 (In Press).
3. "Determining Volatile Organics at Microgram-per-Litar Levels by Gas
• Chromatography," T. A. Bellar and J. J. Lichtenberg, Jour. AWWA,
66, 739-744, Dec. 1974.
4. ASTM Annual Standards - Water, part 31, Method D2908 "Standard
Recommended Practice for Measuring Water by Aqueous-Injection Gas
Chromatography."
5. ASTM Annual Standards - Water, part 31, Method 03371 "Tentative
Method of Test for Nitriles in Aqueous Solution of Gas Liquid
Chromatograph."
-------
6. "Direct Analysis of Water Samples for Organic Pollutants with Gas
Chromatography-Mass Spectrometry," Harris, L. E., Budde, W. L., and
Eichelberger, J. W. Anal. Chem., 4§, 1912 (1974).
3T-
-------
Table 1
Parameter
Acrolein
Acrylonitrile
Benzene
Bromomethane
Bromodi chloromethane
Bromoform
Carbon Tetrachloride
Chlorobenzene
Chloroethane
2-Chloroethylvinyl ether
Chloroform
Chloromethane
Dibromoch1oromethane
1,1-Dichloroethane
1,2-Dichloroethane
1,1-Dichloroethene
trans-1,2-Dichloroethene
1,2-Dichloropropane
cis-l,3-Dichloropropene
trans-1,3-Dichloropropene
Ethylbenzene
Methylene chloride
1,1,2,2-Tetrachloroethane
Tetrachlorethene
1,1,1-Trichloroethane
1,1,2-Trichloroethane
Trichloroethene
Trichlorofluoromethane
Toluene
Vinyl chloride
STORET No.
34210
34215
34236
34413
32101
32104
32102
34301
34311
34576
32106
34418
34105
34496
34531
34501
34546
34541
34561
34561
34371
34423
34516
34475
34506
34511
39180
34488
34010
39175
-------
TABLE 2
Gas Chromatography of Organlcs by Purge and Trap
Compound
chlororaethane
bromooiethane
vinyl chloride
chloroethane
methylene chloride
tr 1 ch 1 orofl uoromethane
1,1-d1chloroethene
bromochloromethane (SS)
1 , 1 -d1 ch 1 oroethane
trans- 1 ,2-d1chloroethene
nhl T.LI +f iLiin
cn i wru i u< in
1 ,2-d1chloroethane
1,1,1 -tr1 chloroethane
carbon tetrachlorlde
bromodl ch 1 oromet hane
1 ,2-d1cn1oropropane
• ^ 4k I • I 4
trans- 1 ,j-
-------
Column 2 Eight ft. stainless steel column (1/8 1n. OD x 0.1 in. ID) packed
with 0.2% Carbowax 1500 coated on 60/80 mesh Carbopack C preceded
by a 1 ft. stainless steel column (1/8 in. OD x 0.1 in. ID) packed
with 3% Carbowax 1500 coated on 60/80 mesh Chromosorb W. A glass
column (1/4 in. OD x 2 mm ID) may be substituted. Carrier gas:
helium at 40 ml/min. Temperature program: 3 min. isothermal at
60°C then 8°/min to 160°, hold at 160 until all compounds
elute.
* This is a minimum level at which the entire system must give recognizable
mass spectra (background corrected) and acceptable calibration points.
** Sensivitity refers to either qualitative screening with this method or
direct aqueous injection GC-FID (Ref. 4,5,6).
-------
TABLE 3
Compound
Characteristic Ions of Volatile Organlcs
El Ions
chloromethane
bromomethane
vinyl chloride
chloroethane
methyl ene chloride
tr1ch1orofluoromethane
I,1-d1chloroethene
bromochlorcmethane (IS)
1,1 -d1 chloroethane
trans- 1,2-dichloroethene
chloroform
1,2-di chloroethane
1,1,1-trichloroethane
carbon tetrachlorlde
bromodl chloromethane
l,2-d1chloropropane
trans-l,3-
-------
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BASE/NEUTRALS, ACIDS, AND PESTICIDES
METHOD 625
1. Scope and Application
1.1 This method covers the determination of those "unambiguous priority
pollutants" associated with the Consent Decree that are solvent
extractable and amenable to gas chromatography. The parameters
listed in Tables 1 and 2 may be determined by this method. This
method is qualitative for the parameters listed in Table 3.
1.2 This method is applicable to the determination of these compounds
in municipal and industrial discharges. It is designed to be used
to meet the survey requirements of the Effluent Guidelines Division
(EGD) and the monitoring requirements of the National Pollutants
Discharge Elimination System (NPDES).
1.3 The detection limit of this method is usually dependent upon the
level of interferences rather than instrumental limitations. The
limits listed in Tables 4, 5, and 6 represent levels that can be
achieved in wastewaters, as such they were determined by the
minimum quantity that must be injected into the system to get
confirmation by the mass spectrometric method described below.
1.4 The GC/MS parts of this method are recommended for use only by
analysts experienced with GC/MS or under the close supervision of
such qualified persons.
2. Summary of Method
2.1 A 1-liter sample of wastewater is extracted, with methylene
JI
-------
chloride using separatory funnel or continuous extraction
techniques. If emulsions are a problem, continuous extraction
techniques should be used. The extract 1s dried over sodium
sulfate and concentrated to a volume of 1 ml or less using a
Kuderna-Oanish (K-0) evaporator. Chroraatographic conditions are
described which allow for the separation of the compounds In the
extract.
2.2 Identification of a compound (qualitative analysis) is performed by
an analysis of the full, background corrected, mass spectra.
2.3 Quantitative analysis is performed'by GC/MS using either the
internal standard or external standard technique. (Base/Neutral,
and.Acids). Quantitative analysis of the pesticides is done using
electron capture gas chroaatography (Federal Register, 38, Vol.
125, Part II, pp. 17318-17323, Friday, June 29, 1973). One liter
rather than 100 ml is to be extracted.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines causing
misinterpretation of chromatograras. All of these materials must be
demonstrated to be free from interferences under the conditions of
the analysis by running method blanks. Specific selection of
reagents and. purification of solvents by distillation in all-glass
systems may be required.
3.2 Interferences coextracted from the samples will vary considerably
from source to source, depending upon the diversity of the
industrial complex or municipality being sampled.
-------
3.3 The recommended analytical procedure may not have sufficient
resolution to differentiate between certain 1somer1c pairs. These
are anthracene and phenanthrene, chrysene and benzo(a)anthracene,
and benzo(b)fluoranthene and benzo(k)f1uoranthene. The GC
retention time and mass spectral data are not sufficiently unique
to make an unambiguous distinction between these compounds.
Alternative techniques should be used -to Identify these specific
compounds. See Reference 1.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete or composite sampling.
4.1.1 Grab sample bottle - amber glass, 1-liter or 1-quart
volume. French or Boston Round design is recommended. The
container must be washed.and solvent rinsed before use to
minimize interferences.
4.1.2 Bottle caps - Threaded to fit sample bottles. Caps must be
lined with Teflon. Aluminum foil may be substituted if
sample 1s not corrosive.
4.1.3 Compositing equipment - Automatic or manual compositing
system. Must incorporate glass sample containers for the
collection of a minimum of 1000 ml. Sample containers must
be kept refrigerated during sampling. No plastic or rubber
tubing other than. Teflon may be used in the system.
4.2 Separatory funnel - 2000 ml, with Teflon stopcock (Aca Glass
7228-T-72 or equivalent).
4.3 Drying column - A 20 mm ID pyrex cnromatograpnic column equipped
with coarse glass frit or glass wool plug.
-------
4.4 Kudema-Danlsh (K-0) Apparatus
4.4.1 Concentrator tube - 10 ml, graduated (Kontes K- 57 0050- 1025
or equivalent). Calibration must be checked. Ground glass
stopper (size 19/22 Joint) 1s used to prevent evaporation of
extracts.
4.4.2 Evaporative flask - 500 ml (Kontes K-5700T-0500 or
equivalent). Attach to concentrator tube with springs.
(Kontes K-662750-0012).
4.4.3 Snyder column - three-ball macro (Kontes KS03000-0232 or
equivalent).
4.4.4 Snyder column - two-ball micro (Kontes K-569002-0219 or
equivalent).
4.4.5 Boiling chips-extracted, approximately 10/40 mesh.
4.5 Water bath - Heated, with concentric ring cover, capable of
temperature control (+ 2°C). The bath should be used 1n a hood.
4.6 Gas chromatograph - Analytical system complete with gas
chromatograph capable of on-column Injection and all required
accessories including column supplies, gases, etc.
4.6.1 Column 1 - For Base/Neutral and Pesticides a 6-foot glass
column (1/4 in 00 x 2 mm 10) packed with 32 SP-2250 coated
on 100/120 Supelcoport (or equivalent).
4.6.2 Column 2 - For Adds, a 6-foot glass column (1/4 in 00 x 2
mm ID) packed with IS SP-1240 OA coated on 100/120 mesh
Supelcoport (or equivalent).
4.7 Mass Spectrometer - Capable of scanning from 35 to 350 a.m.u.
(Base/Neutrals-Acids) or 35 to 450 a.m.u. (Pesticides) every 6
IT-
-------
seconds or less at 70 volts (nominal) and producing a recognizable
mass spectrum (background corrected) at unit resolution from 50 ng
of OFTPP when the sample 1s Introduced through the GC Inlet
(Reference 1). The mass spectrometer must be interfaced with a gas
chromatograph equipped with an Injector system designed for
splltless Injection glass capillary column use or an Injector
system designed for all -glass on-column packed column use. All
sections of the transfer lines must be glass or glass-lined and
must be deactivated. (Use Sylon-CT, Supelco, Inc., or equivalent
to deactivate.) Note: Systems utilizing a jet separator for the
GC effluent -are recommended since membrance separators may lose
sensitivity for light molecules and glass frit separators may
'Inhibit the elutlon of polynuclear aromatics. Any of these
separators may be.used provided that it gives recognizable mass
spectra (background corrected) and acceptable calibration points at
the limit- of detection specified for each individual compound
listed in Tables 4, 5, and 6.
4.3 A computer system must be interfaced to the mass • spectrometer to
allow acquisition of continuous mass scans for the duration of the
chromatographic program. The computer system should also be
equipped with mass storage devices^or saving all data from GC-MS
runs. There must be computer software available to allow searching
any GC-MS mn for specific ions and plotting the intensity of the
ions with respect to time or scan number. The ability to integrate
the area under any specific ion plot peak is essential for
quantification.
J/T
-------
4.9 Continuous 11quid-11quid extractors - Teflon or glass connnectlng
joints and stopcocks, no lubrication. (Hershberg-Wolf Extractor -
Ace Glass Co., Vine!and, N.J. P/N 6841-10 or equivalent).
5. Reagents
5.1 Sodium hydroxide - (ACS) 6N 1n distilled water.
5.2 Sulfurtc add - (ACS) 6N 1n distilled water.
5.3 Sodium sulfate - (ACS) granular anhydrous (rinsed with methylene
chloride (20 ml/g) and conditioned at 400°C for 4 hrs.)
5.4 Methylene chloride - Pesticide quality or equivalent.
5.5 Stock standards - Obtain stock standard solutions at a
concentration of 1.00 ug/ul. For example, dissolve 0.100 grans of
assayed reference material 1n pesticide quality Isooctatte or other
appropriate solvent and dilute to volume 1n a 100 ml ground glass
stoppered volumetric flask. The stock solution 1s transferred to
15 ml Teflon lined screw cap vials, stored 1n a refrigerator, and
checked frequently for signs of degradation or evaporation,
especially just prior to preparing working standards from them.
Protect PNA standards from light.
6. Calibration Standards
S'.l Prepare calibration standards that contain the compounds of
Interest, either singly or mixed together. The standards should be
prepared at concentrations that will completely bracket the working
range of the chromatographic system (tvro or more orders of
magnitude are suggested). If the limit of detection (Tables 4, 5,
or 6) can be calculated as 20 ng injected, for example, prepare
standards at 1 ug/ml, 10 ug/ral, 100 ug/ml, etc. so that injections
-------
of 1-5 ul of the calibration standards will define the linearity of
the detector in the working range.
6.2 Assemble the necessary gas chromatographic apparatus and establish
operating parameters equivalent to those indicated in Tables 4, 5,
and 6. By injecting calibration standards, establish the linear
range of the analytical system and demonstrate that the analytical
system meets the limits of detection requirements of Tables 4, 5,
and 6. If the sample gives peak areas above the working range,
dilute and reanalyze.
6.3 The internal standard approach is acceptable for all of the
semivolatile organics when internal standards meeting the following
criteria can be selected:
6.3.1 No interference with other components in the sample.
6.3.2 Structural similarity to the compound to be determined.
6.3.3 Elutes close to the compound to be determined.
6.3.4 The amount added approximates the concentration of the
compound to be determined.
6.4 Internal Standard Method - The utilization of the internal standard
method requires the periodic determination of response factors (RF)
which are defined 1n equation 1.
Eq. I RF - (AsC1s)/(A1sCs)
Where:
A is the integrated area or peak height of the
characteristic ion for the priority pollutant standard.
A. is the integrated area or peak height of the
characteristic ion for the internal standard.
ML. -
-------
C1s 1s the amount (ug) of the Internal standard.
C_ 1s the amount (ug) of the priority pollutant standard.
6.4 The relative response ratio for the priority pollutants should be
known for at least two concentration values—20 ng Injected to
approximate 10 ug/1 and 200 ng Injected to approximate the 100 ug/1
level. (Assuming 1 ml final volume and a 2 ul injection). Those
compounds that do not respond at either of these levels may be run
at concentrations appropriate to their response.
The response factor (RF) must be determined over all concentration
ranges of standard (C$) which are being determined. (Generally,
the amount of Internal standard added to each extract is the same
(20 ug) so that C1$ remains constant.)* This should be done by
preparing a calibration curve where the response factor (RF) is
plotted against the standard concentration (Cs). Use a minimum
of three concentrations over the range of interest. Once this
calibration curve has been determined, it should be verified daily
by injecting at least one standard solution containing internal
standard. If significant drift has occurred, a new calibration
curve must be constructed. To quantify, add the internal standard
to the concentrated sample extract no more than a few minutes
before injecting into the SC/MS to minimize the possibility of
losses due to evaporation, adsorption, or chemical reaction.
Calculate the concentration by using the previous equations with
the appropriate response factor taken from the calibration curve.
(Ideally, the response factor will not ch'ange with concentration.)
Suggested internal standrds and surrogate standards fall into two
23'7
-------
groups. A - Oeuterated compounds. For Aromatics; benzene-dg,
toluene-dg, and anthracene-dg. For amines; pyridine-d-, and
aniline-de. For phenols; phenol-dg. For nitroaromatics;
nitrobenzene-dg, B - Fluorinated compounds. For aromatics;
pentafluorobenzene, 1-fluoronaphthalene, 2-fluoronaphthalene,
2-fluorobiphenyl, 2,2'-difluorobiphenyl, and 1,2,3,4,5-
pentafluoroblphenyl. For phenols; pentafluorophenol,
2-perfluororaethyl phenol and 2-fluorophenol. Compounds used as
internal standards are not to be used as surrogate standards. The
internal standard must be different from the surrogate standards.
6.5 The external standard method can be used at the discretion of the
analyst and is recommended when the criteria for use of the
internal standard cannot be met. Prepare a master calibration
curve using a minimum of three standard solutions of each of the
compounds that are to be measured. Plot concentrations versus
integrated areas or peak heights (selected characteristic ion for
SC/MS). One point on each curve should approach the limit of
detection (Tables 4, 5, and 6). After the master set of instrument
calibration curves have been established, they should be verified
daily by injecting at least one standard solution. If significant
drift has occurred, a new calibration curve must be constructed.
Quality Control
7.1 Before processing any samples, demonstrate through the analysis of
a method blank, that all glassware and reagents are interfersnce-
free. Each time a set of samples is extracted or there is a change
in reagents, a method blank should be processed as a safeguard
-------
against chronic laboratory contamination.
7.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to determine the
precision of the sampling technique. Laboratory replicates should
be analyzed to determine the precision of the analysis. Fortified
samples should be analyzed to determine the accuracy of the
analysis. Field blanks should be analyzed to check for
contamination Introduced during sampling and transportation.
8. Sample Collection, Preservation, and Handling
8.1 Srab samples must be collected 1n glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected' In refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be free of tygon and other potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time of
col-lection until extraction. Chemical preservatives should not be
used in the field unless more than 24 hours will elapse before
delivery to the laboratory. If the samples will not be extracted
with 48 hours of collection, they must be preserved as follows:
8.2.1 Add 35 mg of sodium thiosulfate per 1 ppm of free chlorine
per liter of sample.
3.2.2 Adjust the pH of the water sample to a pH of 7 to 10 using
sodium hydroxide or sulfuric acid. Record the volume of
acid or base used.
8.3 All samples must be extracted within 3 days and completely analyzad
-------
within 7 days of collection.
9. Sample Extraction (Base/Neutrals. Acids, and Pesticides)
9.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
two-liter separatory funnel. Adjust the pH of the sample with 6N
NaOH to 11 or greater. Use multirange pH paper for the
measurements. Proceed to Section 10 if continuous extraction is
used.
9.2_ Add 60 ml methylene chloride to the sample bottle, cap, and shake
30 seconds to rinse the walls. Transfer the solvent into the
separatory funnel, and extract the sample by shaking the funnel for
two minutes with periodic venting to release excess vapor
pressure. Allow the organic layer to separate front the water phase
for a minimum of ten minutes. If the emulsion interface between
layers is more than one-third the size of the solvent layer, the
analyst must employ mechanical techniques to complete the phase
separation. The optimum technique depends upon the sample, but may
include stirring, filtration of the emulsion through glass wool, or
centrifugation. If the emulsion cannot be broken, that is,
recovery of less than 802 of the added solvent corrected for the
water solubility of methylene chloride, use continuous extraction.
Collect the methylene chloride extract in a 250-ml Erlenmeyer flask.
9.3 Add a second 60-ml volume of methylene chloride to the sample
bottle and complete the extraction procedure a second time,
combining the extracts in the Erlenmeyer flask.
9.4 Perform a third extraction in the same manner. Pour the combined
-------
extract through a drying column containing 3-4 Inches of anhydrous
sodlun sulfate, and collect 1t in a 500 ml K-0 flask equipped with
10 nl concentrator tube. Rinse the Erlenmeyer with 20 to 40 ml of
methylene chloride. Pour this through the drying column. Seal,
label as base/neutral fraction, and proceed with the add
extraction.
9.5 Add (Phenols) Extraction - Adjust the pH of the water, previously
extracted for base-neutrals, with 6N H^SO^ to 2 or below.
Serially extract with 60, 60, and 60 ml portions of
d1stilled-1n-glass methylene chloride. Collect and combine the
extracts in a 250-
-------
an apparent volume 1 ml, remove the K-0 apparatus and allow the
solvent to drain for at least 10 minutes while cooling. Remove the
Snyder column and rinse the flask and Its lower joint Into the
concentrator tube with 1 to 2 oil of methylene chloride. A 5-ml .
syringe 1s recommended for this operation.
9.3 Add a clean boiling chip and attach a two-ball micro-Snyder column
to the concentrator tube 1n 9.7. Prewet the column by adding about
0.5 ml methylene chloride through the top. Place the K-0 apparatus
on a warm water bath (60 to 65°C) so that the concentrator tube
1s partially Immersed 1n the water. Adjust the vertical position
of the apparatus and the water temperature as required to complete
the concentration in 5-10 minutes. At the proper rate of
•
distillation the balls of the column actively chatter but the
chambers do not flood. When the liquid reaches an apparent volume
of about 0.5 ml, remove the K-0 from the water bath and allow the
solvent to drain and cool for at least 10 minutes. Remove the
raicro-Snyder column and rinse its lower joint into the concentrator
tube witft approximately 0.2 ml of methylene chloride. . Adjust the
final volume to 1.0 ml.
9.9 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the liquid to a 1000 ml graduated
cylinder. Record the sample volume to the nearest 5 ml.
9.10 Pesticides. Perform only if pesticides are found by GC-ECD.
Carefully concentrate the 10 ml final hexane extract (no dilutions)
by "blowing down" to 0.5 ml using a clean airstream. Seal, label
as pesticide fraction. Proceed to section 11.
-------
10. Emulsions/Continuous Extraction
10.1 Place 100 to 150 ml of methylene chloride 1n the extractor and ZOO-
SCO ml methylene chloride 1n the distilling flask.
10.2 Add the aqueous sample (pH 11 or greater) to the extractor.
Extract for 24 hours. Remove the distilling flask and pour the
contents through a drying column containing 7 to 10 on of
armhydrous sodium sulfate. Collect the methylene chloride in a 500
ml K-0 evaporator flask equipped with a 10 ml concentrator tube.
Seal, label as the base/neutral fraction, and concentrate as per
section 9.7 to 9.10.
10.3 Adjust the pH of the sample 1n the continuous extractor to 2 or
below using 6H sulfurlc add. Charge a clean distilling flask with
500 ml of methyl ene chloride.. Extract for 24 hours. Remove the
distilling flask and pour the contents through a'drying column
containing 7 to 10 on of anhydrous sodium sulfate. Collect the
methylene chloride layer on a K-fl evaporator flask equipped with a
10 ml concentrator tube. Label as the add fraction. Concentrate
as per section 9.7 to 9.10.
11. Gas Chromatoqrapny-Mass Spectrometry of Base/Neutrals and Pesticide
Fraction
11.1 Tables 4, 5, and 6 summarize the recommended gas chromatographic
column materials and operating conditions for the instrument.
Included 1n this table are estimated retention times and
sensitivities that can be achieved by this method. Examples of the
separations achieved by these columns are shown 1n Figures 1
through 7. Calibrate the system daily with a minimum of one
-------
injection of calibration standards. Insure that the GC-MS system
meets the criteria in reference 1 by injecting 50 ng of DFTPP
through the GC inlet system. If the extract must be stored
overnight before analysis by GC/MS, transfer it with a disposable
pipet, from the K-0 ampul to a solvent tight container. Do not add
the internal standard at this time. The recommended container is a
standard 2 ml serin vial equipped with a Teflon lined rubber septum
and crimp cap. These cap liners are inert and methylene chloride
can be held without evaporation loss for months if caps liners are
unpierced. When the extracts are not being used for analysis,
store them with unpierced septa in the dark at less than 4°C.
11.2 Internal Standard - Add 20 ug (10 ul of 2 ug/ul) of internal
standard just prior to analysis. Mix thoroughly.
11.3 Inject 2 to 5 ul of the sample extract. The solvent-flush
technique is preferred. Smaller (1.0 ul) volumes can be injected
if automatic devices are employed. Record the volume injected to
the nearest 0.05 ul, and the resulting peak size, in area units.
11.4 If the peak area exceeds the linear range of the system, dilute the
extract and reanalyze.
11.5 At the beginning of each GC-MS run of a base-neutral fraction, the
operator should demonstrate the ability to chromatograph 100 ng of
benzldine. Only after this is accomplished should the run be
started.
11.5 At the beginning of each GC-MS run of the pesticide fraction, the
operator should demonstrate the ability to chrcmatograph TOO ng of
aldrin. Only after this should the run be started.
-------
12. 6as Chromatograohy/Hass Soectrometry of Add Fraction
12.1 Before at acid extract Is analyzed by 6C-MS, the operator should
demonstrate the ability to detect 50 ng of pentachlorophenol.
12.2 After acceptable Instrumental response 1s demonstrated, proceed
with the may sis as described for the base/neutral fraction^
(11.1 to 11.4).
12.3 Table 5 lists the retention times of the 11 aoid extractables.
Chroraatography of nltrophenols 1s. poor. The limits of detection
given refer to the amounts required to obtain MS confirmation by
the methods described below.
13. Mass Spectrometry
13.1 The complete background corrected mass spectrum 1s compared to a
reference mass spectrum, either from an authentic sample or a
library spectrum, to provide qualitative Identification. All of
the following criteria must be met.
13.1.1 The experimental mass spectrum must contain all ions
present above 1% relative abundance in the mass spectrum
of the reference with the general agreement of * 2QX of
the relative abundance 1n the reference mass spectrum.
13.1.2 Ions present in the experimental mass spectrum that are
not present in the reference mass spectrum must not
exceed 10% of the total ion abundance in the experimental
mass spectrum. These contaminant ions should be readily
accounted for by common background Ions. If the
experimental mass spectrum is highly contaminated,
identification should not be accepted.
-------
13.1.3 The retention time at the experimental mass spectrum
should be within ^ 5 spectrum numbers or * 30 seconds
(whichever 1s greater) of the retention time of the
authentic compound.
13.1.4 Structural isomers that have very similar mass spectra
(phenanthrene, anthracene; faenzo(a}pyrene,
benzo(e)pyrene, etc.) can be explicitly identified if the
resolution between the isomers in a standard mix is
acceptable. Acceptable resolution is achieved if the
2.5% f
valley height between isomers is less than -freW- the sum
of the two peak heights. Otherwise, structural isomers
are identified as isomeric pairs.
13.2 In samples that contain an inordinate number of interferences the
chemical ionization (CI) mass spectrum may make identification
easier. In Tables 7 and 8 characteristic CI ions for most of the
compounds are given. The use of chemical ionization MS to
support El is encouraged but not required.
14. Calculations (Base/Neutrals and'Acids)
14.1 When a compound has been identified, the quantification of that
compound will be based on the integrated area from the specific
ion plot of the first listed characteristic ion in Tables 7, 8
and 9. If the sample produces an interference for the first
listed ion, use a secondary ion to quantify. Quantification will
be done by the external or internal standard method.
14.2 Internal Standard - By adding a constant known amount of internal
standard (C- in ug) to every sample extract, the concentration
•ML- 2H ±
-------
of priority pollutant (CQ) 1n ug/1 in the sample is calculated
using equation 2.
r- , , .
^ 0 (A ) (RFj (Voj
Where: s
V 1s the volume of the original sample in liters.
14.3 External Standard - The concentration of the unknown can be
calculated from the slope and intercept of the calibration
curve. The unknown concentration can be determined using
equation 3.
(A)(VJ
Bj. 3 Mlcrograrcs/liter * ng/ml » ,\> Wy i
Where:
A » mass of compound from calibration curve (ng).
V, » volume of extract injected (ul)
Vt » volume of total extract (ul)
V « volume of water extracted (ml)
14.4 Report all results to two significant figures. Report results in
raicrograms per liter (Base/Neutrals and Adds) without correction
for recovery data. Report qualitative identification only for
pesticides (quantitative data is available from GC-SC). When
duplicate and spiked samples are analyzed, all data obtained
should be reported. Relative response ratios and response
factors from MS analysis should be included when reporting data.
Include identification of any components found in the blanks.
14.5 In order to minimize unnecessary GC-MS analysis of method blanks
and field blanks, the field blank may be run on a FID-GC equipped
JIT- o?^
-------
with the appropriate SP-2250 or SP-1240 OA columns. If no peaks
are seen of Intensities equal to or greater than the internal
standard, then it is not necessary to do a GC-MS analysis of the
blank. If such peaks are seen, then the field blank must be sent
for full priority pollutant analysis.
15. Accuracy and Precision
15.1 Data not available at this time.
16. Quality Assurance
16.1 GC-MS system performance evaluation is required each day the
system is used for samples or reagent blanks. A sample of 50 ng
of decafluorotriphenylphosphine (PCR, Gainesville, Florida) is
injected into the system, and the mass spectrum is acquired and
the background corrected spectra is plotted. Criteria
established in Reference 1 must be met. The analyst should also
•demonstrate that the analytical conditions employed result in
sharp total ion current peaks for 100 ng of benzidine on the
SP-2250 column when this column is used for Base/Neutrals, 100 ng
of aldrin on the SP-2250 column when used for pesticides, and 50
ng of pentachlorophenol on the SP 1240-DA column when it is
used. All plots from the performance evaluation must be retained
as proof of valid performance. See Reference 3.
-------
17. References
1. Method 610, Polynuclear Aromatic Hydrocarbons, U.S. EPA, EMSL,
Cincinnati, Ohio, 45268, 1979.
2. "Reference Compound to Calibrate Ion Abundance Measurement 1n Gas
Chromatography—Mass Spectrometry Systems," J. W. Elchelberger, L. E.
Harris and W. L. Budde, Anal. Chen. 47, 995-1000 (1975).
3. "Quality Assurance and Quality Control Procedures for Screening and
Verification of Industrial Effluents for Priority Pollutants," U.S.
EPA, EMSL, Cincinnati, Ohio, 45268, 1979.
-------
TABLE 1
Base-Neutral Extractables
Compound
Acenaphthene
Acenaphthylene
Anthracene
Benzo(a)anthracene
Benzo(b)f!uoranthene
Benzo(k)f1uoranthene
8enzo(a)pyrene
8enzo(gni)perylene
Benzldine
B1s(2-chloroethyl)ether
B1s(2-ch1oroethoxy)methane
B1s(2-«thy1hexyl)phthalate
81s(2-chloroisopropyl)ether
4-8romcpheny1 phenyl ether
Butyl benzyl phthalate
2-£h1oronaphthalene
4-Chlorophenyl phenyl ether
Chrysene
D1benzo(a,h)anthracene
D1-n-butylpnthalate
1,3-Qichlorobenzene
1,4-Oichlorobenzene
1,2-01ch1orob enzene
3,3'-01chlorobenzidine
Dlethylphthalate
Dimethylphthalate
2,4-01n1trotoluene
2,6-01nitrotoluene
Dloctylphthalate
1,2-01phenylhydrazi ne
Fluoranthene
Fluorene
Hexachlorobenzene
Hexach1orobutadiene
Hexach1oroeth ane
Hexachlorocyclopentadiene
Indeno(l,2,3-cd)pyrene
Isophorone
Naphthalene
Nitrobenzene
N-ni trosodimethy1 amine
N-nitrosodi-n-propylamine
N-n1trosod1phenylamine
Phenanthrene
Pyrene
2,3,7,8-Tetrachlorodibenzo-p-dioxin
1,2,4-Trichlorobenzene
STDRET No.
34205
34200
34220
34526
34230
34242
34247
34521
39120
34273
34278
39100
34283
34636
34292
34581
34641
34320
34556
39110
34566
34571
34536
34631
34336
34341
34611
34625
34596
34346
34376
34381
39700
34391
34396
34336
34403
34408
34696
34447
34438
34428
34433
34461
34469
34675
34551
IT-
-------
TABLE 2
Add Extractables
Compound Storet No.
4-Chloro-3Hnethylph«nol 34452
2-Chlorophenol 34536
2,4-01ch1orophenol 34601
2,4-Olmethylphenol 34606
2,4-01n1trophenol 34616
2-Methy1-4,6-/
-------
Table 3
Pesticide Extractables
Aldrln
a-BHC
b-BHC
d-BHC
g-BHC
Chlordane
4, 4 '-ODD
4, 4 '-ODE
4,4' -DOT
01eldr1n
Endosulfan I
Endosulfan II
Endosulfan Sulfate
Endrln
EndHn Aldehyde
Heptachlor
Heptachlor Epoxide
Toxaphene
PC3-1016
PCS- 1221
PCS- 1232
PCB-1242
PCS- 1248
PCB-1254
PCS- 1260
39330
39337
39338
39259
39340
39350
39310
39320
39300
39380
34361
34356
34351
39390
34366
39410
39420
39400
34671
39488
39492
39496
39500
39504
39508
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-------
TABLE 5
Gas Chromatography of Add Extractables
Retention Time*
(M1n)
Limit of Detection*
Compound (M1n) nq Injected uq/1
25
25
25
25
25
25
25
250
250
25
25
*6 foot glass column (1/4 1n. 00 x 2 mm 10} packed with 1% SP-1240 DA coated
on 100/120 mesh Supelcoport. Carrier gas: helium at 30 ml per m1n.
Temperature program: 2 m1n. Isothermal at 70°, then 8° per m1n to
200QC.
*Th1s Is a minimum level at which the entire analytical system must give
recognizable mass spectra (background corrected) and acceptable'calibration
points. NanVans Injected 1s based on a 2 ul Injection of a one liter
sample that has been extracted and concentrated to 1.0 ml.
2-Chlorophenol
2-N1tropheno1
Phenol
2, 4-Q1methy 1 phenol
2,4-01chlorophenol
2, 4, 6-Trf chlorophenol
4-Chl oro-3-methylphenol
2,4-01 nltrophenol
2-Methyl -4,6-d1n1trophenol
Pent ach 1 orophenol
4-N1trophenol
5.9
6.4
3.0
9.4
9.3
11.3
13.2
15.9
16.2
17.5
20.3
50
50
50
50
50
50
50
500
500
50
50
-------
Table 6
Gas Chromatography of Pesticide Extractables
Retention Limit of Detection*
Compound Time (min)* ng injected ug/1
a-bhc 21.1 40 10
g-bhc 22.4 40 10
b-bhc 23.4 40 10
Heptachlor 23.4 40 10
d-bhc 23.7 40 10
Aldrin 24.0 40 10
Heptachlor epoxide 25.6 40 10
Endosulfan I 26.4 40 10
Oieldrin 27.2 40 10
4,4'-ODE 27.2 40 10
Endrin 27.9 40 10
Endosulfan II 28.5 40 10
4,4'-000 28.6 40 10
4,4'-ODT 29.3 40 10
Endosulfan sulfate 29.3 40 TO
Cnlordane 19 to 30
Toxaphene 25 to 34
PCB-1242 20 to 32
PC3-1254 23 to 32
*6 foot glass column (% in OF X 2 mm ID) packed with 3% SP-2250 coated on
100/120 mesh. Supelcoport. Carrier gas: helium at 30 ml per min.
Temperature Program: Isothermal for 4 minutes at 50°C, then 8° per
minute to 270°. Hold at 270°C for 5 minutes.
#Tn1s is a minimum level at which the entire analytical system must given
recognizable background corrected mass spectra. Manograms injected is based
on a 2 ul injection of a one-liter sample that has been extracted and concen-
trated to a volume of 0.5 ml.
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-------
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e . r*» r»»
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in in in r>. r«» r*.
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•— -o c c >»
lQ<»~k 41 i— 4) 4) O,
£.<— C M 4J.C J= —-.
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J= — C I I •£ I OJ -ta> — .C
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-------
Table 9
Pesticides Characteristic Ions
Characteristic Ions
Electron Impact
Compound fritli Inti]
a-bhc 183 131 109
g-bhc 183 181 109
b-bhc 181 183 109
heptadilor 100 272 274
d-bhc 133 109 181
aldrln 66 263 220
heptachlor ep oxide 353 . 355 351
endosulfan I 201 283 273
dleldrln 79 263 279
4,4'-QOE 246 248 176
4,4'-000 235 165
endrln 81 263 82
endosulfan -II 201 283 278
4,4'-QOT 235 237 165
endosulfan sulfate 272 387 422
chlordane* 373 375 377
toxaphene** 231 233 235
PCB-1242** 224 260 294
PCB-1254** 294 330 362
* Characteristic of alpha and gamma forms of chlordane.
** . ,> theKcompounds are mixtures of
various Isomers.
-------
o
CM
3N3>IH1NVN3H
o
o
3
a
-------
10N3HdOHilN-fr
10N3HdOH01HOViN3d
10N3HdOailNia-tr'2
10N3HdOa01HO IHi-9' fr 'Z
10N3HdlAH13Wld-fr'Z
= K 10N3HdOa01HO-2
e
o
u
-------
aaa-.fr> « n
aaa-.fr>
iaa-.fr>
=s
Ninons
•^••M
MM^BM
I NVdinsoaivs
u
-------
COLUMN: 3% SP-2250 ON SUPaCOPORT
PROGRAM: 50*C. 4 MIN. 8*PER WIN TO 270"C
DETECTOR: MASS SPECTROMETER
•PEAKS GIVING THE THREE
CHARACTERISTIC IONS
20
25
30
RETENTION TIME-MINUTES
Figure 4. Gas chromatogram of chiordane
-------
COLUMN: 3% SP-2250 ON SUP&COPORT
PROGRAM: 50*C, 4 WIN. 8*PER MIN TO 27Q*C
DETECTOR: MASS SPECTROMETER
25 30 35
RETENTION TIME-MINUTES
Rgure 5. Gas chromatogram of toxaphene
-------
COLUMN: 3% SP-2250 ON SUPELCOPORT
PROGRAM: 50«C. 4 WIN. 8°PER WIN TO 270"C
DETECTOR: MASS SPECTROMETER
A m/ft 224 PRESENT
8 m/e 260 PRESENT
C m/e 294 PRESENT
20
25 30
RETENTION TIME-MINUTES
35
Figure 6. Gas chromatogram of Arochlor 1248
-------
COLUMN: 3% SP-2250 ON SUPELCOPERT
PROGRAM: 50*C, 4 WIN, 8* PER MIN TO 270aC
DETECTOR: MASS SPECTROMETER
A n/e 294 PRESENT
B m/e 330 PRESENT
C m/e 362 PRESENT
20 25 30
RETENTION TIME-MINUTES
Rgure 7. Gas chromatogram of Arochlor 1254
-------
QUALITY ASSURANCE AND QUALITY CONTROL PROCEDURES FOR
SCREENING AND VERIFICATION OF INDUSTRIAL EFFLUENTS FOR
PRIORITY POLLUTANTS
U. S. ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
CINCINNATI, OHIO 45268
June 1979
-------
INTRODUCTION
"The Sampling and Analysis Procedures for Screening of Industrial
Effluents for Priority Pollutants," (revised April, 1977) was designed to
provide qualitative/seraiquantitative determination of the priority
pollutants. In order to apply these procedures to quantitative analysis,
adequate quality assurance practices must be incorporated into the
procedures. This document provides modifications to enable implementation
of a quality assurance program that will define the accuracy and precision
of the procedures for specific parameters.
In considering modifications to be made, the following assumptions were
made:
1. The program must be well documented. It must be integrated into
field and laboratory procedures. It must include field and
laboratory blanks, equipment calibration checks, replicate and
spiked sample analyses, the analysis of check standards and
performance evaluation standards.
2. The differences in industrial subcategories require method
validation and the establishment of control parameters for each
subcategory.
3. Verification Phase analyses will seldom, if ever, require analysis
of all 114 organic priority pollutants. Thus, the quality control
program-will focus on a Smaller selected number of compounds based
on the results of screening analyses and other data.
4. For purposes of the Effluent Guidelines Verfication Phase studies,
the quality control programs provided, herein, must be applied to
the sampling sources most frequently involved in rule making, i.e.,
supply water, influent to treatment, and effluent from treatment.
Primary emphasis should be placed on the effluent from treatment
since this is where the control limits will be set,
5. Two distinct levels of quality control are required: initial method
validation and continuing performance evaluation. The initial
quality control is to demonstrate the method precision and accuracy
and thus validate the method on a common wasta stream (industrial
subcategory) where waste characterization was the basis"for
establishing the subcatagory.
5. The initial control limits will be established for each subcategory
on the effluent from treatment prior to initiation of the routine
sampling and analysis program for that subcatagory. The control
limits for supply water and influent to treatment watar will be
established over a period of time using data derived from the
ongoing continuing quality control program.
7. As more quality control data is obtained for each wasta type within
each subcatagory, the precision and accuracy of the method will be
updated to provide better defined control limits.
inr- j
-------
FOREWORD
These methods for sampling and analysis of Industrial wastes have been
prepared by the staff of the Environmental Monitoring and Support
Laboratory, at the request of the Effluent Guidelines Division, Office of
Water and Hazardous Wastes, with the cooperation of the Environmental
Research Laboratory, Athens, Georgia. The Quality Assurance/Quality Control
(QA/QC) procedures were prepared by the staff of the Environmental
Monitoring and Support Laboratory with the cooperation of the Effluent
Guidelines Division, Office of Water and Hazardous Wastes, and the
Environmental Research Laboratory, Athens, Georgia, And several EPA Regional
Laboratories.
The procedures represent the current state-of-the-art and are applicable
to the Effluent Guidelines'Survey of prirority pollutants. This survey is
divided into two phases; the Screening Phase and the Verification Phase.
The objectives of the Screening Phase are:
1. To provide a deternrination of the presence or absence of a particular
priority pollutant.
2. To provide a quantitative estimate of the concentration of priority
pollutants present.
3. To focus the verification survey to specific priority pollutants.
4. To provide direction to other best available technology (BAT)
regulatory strategies.
The objectives of the Verification Phase are:
1. To provide the confirmation of the presence or absence of the
priority pollutants Identified during the screening phase, necessary
for consideration in rule making.
2. To provide qualitative pollutant data with a known precision and
accuracy.
3. To provide pollutant data for use in rule making.
$. To serve as a basis for technology selection.
-------
DISCLAIMER
The mention of trade names or commercial products 1n this document 1s
for Illustration purposes, and does not constitute endorsement or
recomnendatlon for use by the U. S. Environmental Protection Agency.
-------
Quality Assurance and Quality Control Procedures
for Screening Analysis for Organic Priority Pollutants
110 Scope
This procedure 1s provided for use by laboratories
performing screening analyses using the GC/MS methods 624
and 625. Screening analysis provides a determination of the
presence or absence of a specific compound and a
searfquantitative estimate of the concentration of a specific
compound. Quality Assurance (QA) 1s the total program for
assuring the reliability of the monitoring data. Quality
control (QC) 1s the routtne application of procedures for
controlling the measurement process.
120 Routine Quality Assurance and Quality Control
121 Method Blank - The method blank 1s defined as an
appropriate volume of "organic-free" water which has
been processed exactly as the sample (Including
glassware, reagents, solvents, etc.). Reagents or
solvents having background levels that Interfere with
the compounds to be determined must be purified and
shown to be acceptable or replaced with some that are
acceptable prior to proceeding with analyses. Problems
encountered and corrective actions taken shall be
documented and reported for the record.
121.1 For the extractafale fractions (Base/Neutral,
Add, and Pesticide) the method blank requires
-------
extraction of 1-liter "organic-free" water. A
method blank must be extracted for each set of
field samples extracted at a given time (at
least one method blank per 20 field samples
analyzed) and whenever a new source of reagent
or solvent 1s Introduced Into the analytical
scheme. The method blank can be screened by
GC-FID. Analysis by GC-MS is required only 1f
GC-FID analysis of the field blank gives peaks
larger than the Internal standard peak.
121.2 For the volatile fraction, 5 ml of "organic-
free" water should be analyzed by the purge and
trap methodology only 1f positive Interferences
are noted during the analysis of a field blank.
If positive Interference stm occurs, repeat
the method blank analysis. If interference
persists, dismantle the system, thoroughly clean
all parts that contact the sample, purge gas,
and carrier gas. Replace or repack the sorbent
trap and change purge and carrier gas.
122 Field Blank
The field blank is defined as an appropriate volume of
sent
"organic-free" water which has been^to the sampling
site and back to the analytical laboratory in a
container and bottle identical to the type used to
collect the samples. Field blanks and samples must be
-------
shipped 1n separate containers. When received In the
lab, the field blank 1s dosed, extracted and
concentrated as If It were an actual sample.
122.1 For the extractable fractions (Base/Neutral,
Add, and Pesticide), the field blank may be
screened by GC-FID or SC-EC (pesticides only).
Full SC-MS analysis 1s to be performed 1f the
screening analysis gives any peaks larger than
.the Internal standard peak.
122.2 For the volatile fraction, analyze a 5-ml field
blank after each sample analysis. Follow the
guidelines 1n 121.2 1f positive Interferences
are noted during the analysis of a field blank.
123 GC-MS Calibration Chedc
123.1 For the Base/Neutral-Acid or Pesticide fraction,
dally analyze decaf1uorotr1pheny1phosph1ne
(OFTPP) by Injection Into the GC Inlet. See
Reference 1. (DFTPP must also be run after any
mass spectrometer tuning 1s done). This
calibration check may be done fn conjunction
with Section 124. The requirement Is that 50
nanograms of DFTPP Injected must meet the
specifications prescribed. Table 1 lists the
OFTPP key Ions and 1on abundance criteria.
122.2 For the volatile (purgeable) fraction, analyze
4-bromo-fluorcbenzene (BFB) daily fay direct
-------
injection Into the GC/MS. The requirement is
that 20 nanograms of PFB8 injected must meet the
prescribed specifications. Table 2 lists the
BFB key ions and ion abundance criteria.
124 6C Performance Check
124.1 SP-2250 column - At the beginning of each day
that base/neutral or pesticide analyses are to
be performed, inject 100 nanograms of benzidine
(base neutral analyses) or aldrin (pesticide
-analyses) either separately or as part of a
standard mixture that may also contain 50 ng of
DFTPP. Performance for base/neutrals is
acceptable if the tailing factor for benzidine
is less than 3. Performance for pesticides is
acceptable if the tailing factor for aldrin is
less than 2. Calculation of the tailing factor
is given in Reference 2 and described in Figure
1. Retain all p-lots of performance evaluation
as evidence of valid performance.
124.2 SP-1240 OA Column - At the beginning of each day
that acid fraction analyses are to be performed,
inject 100 nanograms of pentachlorophenol either
separately or as part of a standard mixture that
may also contain DFTPP. Performance is
acceptable if the tailing factor is less than
5, Calculation of the tailing factor is given
in Reference 2 and described in Figure 1.
Retain all plots of performance evaluation as
evidence of valid performance.
-------
124.3 Carbopak C with Carbowax T500 or Carbopak B with
SP-1000 column. At the beginning of each day
that purgeables analyses are to be performed,
Inject 20 nanograms of BFB either separately or
as part of a standard mixture. Performance 1s
acceptable If the tailing factor 1s less- than
2. Calculation of the tailing factor 1s given
1n Reference 2 and described in Figure 1.
Retain all plots of performance evaluation as
evidence of valid performance.
125 Performance Evaluation
Once each 20 working days; dose, extract, and analyze a
performance evaluation sample (available from
EMSL-C1nc1nnat1). Retain the results as evidence of
valid performance.
126 If the final extract 1s to be retained, or shipped to
another location, follow the appropriate guidelines
given in Appendix A.
130 Reporting of Data
13T All concentrations should be reported in ranges, i.e.
less than 10 ug/1, 10 to 100 ug/1, or greater than 100
ug/1.
132 Report the mean and standard deviation of the relative
response ratios of the standards (10 and 100 ug/1) from
the MS analysis with the range values in 131.
133 Report all quality control data with the analytical
results. This includes, but is not limited to, the
recovery of surrogate standard spikes (Method 624), the
precision for the analysis of surrogate standard spikes
or-
-------
(Method 624), the results from the analysis of
performance evaluation samples, and Identification of
components found 1n the blanks.
134 If the GC/MS data 1s to be archived on magnetic tape,
follow the guidelines given 1n Appendix 3.
200 Sampling Procedures for Screening Phase
•The Initial characterization (screening) of the varied
Industrial discharges covered by this program will be made
on an analysis of a. composite effluent sample. Any scheme
for collecting a composite sample is, in effect, a method
for mechanically integrating to obtain average
characteristics of a discharge. During the screening phase
the sample composite can be used to determine the average
characteristics which would be representative of that
discharge. Simple composite samples are those that are made
up of a series of aliquots of constant volume collected at
regular time intervals in a single container. Some
situations may require flow or time proportional sampling;
this determination will be made by the individual project
officer after considering his specific industrial category.
The determination of compositing period 24, 48, or 72 hours
will be made on a case-by-case basis. The duration of
compositing will depend on the type of sample being
collected, the type of facility being sampled, and the time
varying characteristics of the discharge. The rate of
change of flow and other characteristics of the discharge
-------
and the accuracy required will also Influence the
determination of the compositing period. For example,
longer compositing periods would be warranted when less
stable unit process operations are being sampled.
All samples and blanks must be carefully Identified using
water proof labels and water proof Ink. Include the
following Information on the label: sample number, date and
hour of sampling, complete Information as to source and
sampling point, preservative added, if any, and name of
person collecting the sample (Include address and/or phone
number).
201 Extractable Qrqanlcs (Base/Neutrals -' Acids and
Pesticides)
201.1 Collect a representative composite sample. The
maximum time Interval between aliquot samples
shall be no longer than 30 minutes. The minimum
aliquot size shall be 100 ml. The sample must
be collected with an automatic sampler using the
equipment and methods outlined below. Minimum
composite volume must be 2% gallons.
201.2 Automatic Sample Collection - A peristaltic pump
automatic sampler with timer and a single glass
compositing jug is required. The 2h - 3 gallon
compositing battle must be glass and cleaned as
outlined below. New unused tubing must be used
for the sampling line and
-------
for the pump for each Individual outfall or
sample location. Vacuum type automatic samplers
may be used provided that the sample chambers
are glass and that they are cleaned after every
use as outlined for glass composite containers.
Place the sampler or composite container in an
Insulated chest and ice. Maintain the sample at
4°C during the compositing procedure. At the
completion of the compositing period, seal the
container with a Teflon lined cap. Place the
container in an insulated shipping container,
ice, and seal, then ship to the analytical
laboratory. Maintain at 4°C during transport
and storage prior to analysis.
201.3 When sampling raw untreated industrial
discharges which are generally high in suspended
solids, it is imperative that adequate sample
flow rate be maintained throughout the sample
train in order to effectively transport the
solids. In horizontal runs, the velocity must
exceed the scour velocity, while in vertical
runs the settling or the fall velocity must be
exceeded several times to assure adequate
transport of solids in the flow. The equipment
used in sampling raw discharges then must have a
-------
minimum Intake velocity of 60 on per second (2
fps). In the sampling of treated effluents,
Just about any commercially available automatic
liquid sampler could be used.
201.4 When more than one laboratory 1s Involved 1n the
analysis of the various parameters, the sample
should, 1f at all possible, not be divided in
the field but rather at the contractors'
laboratory. For purpose of this program the
composite will be divided Into four parts, one
part for metals analysis, one for pesticides and
PCB's, one for SC/MS compounds and one for the
classic parameters.
201.5 Blend the composite sample to provide a
homogeneous mix-Sure Including a representative
suspension of any solids 1n the container. No
specific method 1s required; hand stirring with
clean glass or Teflon rods, mechanical paddles
or magnetic mixing with Teflon coated stirring
bars may be used. Metal mixing devices may not
be used.
202 Preservation of Extractable Orqanics
202.1 Do not add any preservative to the sample; just
seal and prepare for shipment. All samples mist
be carefully identified using labels supplied by
ESD. Indicate on the label whether the sample
-------
1s a raw discharge or treated effluent as
shown. If sample 1s to be run on the plasma
unit, only Indicate so at base of tag.
203 Field Blank Procedure for Automatic Samplers
203.1 Blank Water - Blank water must be as free from
organic Interferences as possible. The
analytical laboratory should supply this water
1n bulk glass containers (minimum of five
liters) for field use. The supplying laboratory
shall analyze the blank water to determine the
organic background that may be present.
203.2 Procedure - All parts of the sampling system
must be scrubbed with hot detergent water and
thoroughly rinsed with tap water and blank water
prior to use. Further rinsing with methylene
chloride is required when parts permit, i.e.,
are not susceptible to dissolution by the
solvent. (Note: Tygon plastic tubing is a
source of phthalate ester contamination. Where
Its use 1s required, i.e., 1n the peristaltic
pump, the length must be kept as short as
possible.) Teflon is acceptable and may be used
in other parts of the sampling system as in
intake lines. In the field, pump two liters of
blank water through the sampling line and pump
tubing and discard. Then pump three liters of
JUT
-------
blank water through the system and collect as a
blank 1n a 1 -gall on sample bottle that has been
prepared as described below. Seal the bottle
with a Teflon lined cap. Immediately 1ce the
blank (4°C) and maintain at 4°C during the
transport and storage prior to analysis.
203.3 Composite Container - Prepare narrow-mouth glass
sample bottles for use by washing with hot
detergent water and thoroughly rinsing with tap
water and blank water. Heat the bottles at
400°C 1n a muffle-furnace or dry heat
sterilizer for 30 minutes or alternatively;
rinse with methyl ene chloride and air dry at
room temperature protected from atmospheric or
other sources of contamination. Caps for the
bottles must be lined with Teflon which has been
solvent rinsed as above.
204 Volatile Orqanlcs (Purqeables)
204.1 Containers - Use 45 to 125 ml screw cap glass
vials with Teflon faced sHlcone septa:
204.1.1 Vials - Pierce #13074 or equivalent.
204.1.2 Septa - Pierce #12722 or equivalent.
204.2 Wash the bottles, septa, and caps with hot water
and thoroughly rinse with tap water and blank
water. Heat the bottles and septa at 105°C
for one hour, cool to room temperature 1n an
J7T-
-------
enclosed contaminant-free area. When cool, seal
bottles with septa (Teflon side down) and screw
cap. Maintain the bottles 1n this condition
until Just prior to filling with blank water or
sample.
204.3 Collect duplicates 45-125 ml samples each time
samples are collected. Two blank water samples,
sealed 1n 45 ml vials, are to accompany the
sample bottles during shipment to and from the
sampling site. If preservation for residual
chlorine is to be used, collect four samples
during each sampling period. Two should be
preserved and two not preserved. Two preserved
and two non-prssflarved blanks are to be provided-.
204.4 Filling and Sealing Bottles - Slowly fill each
container to overflowing. Carefully set the
container on a level surface. Place the septum
(Teflon side down) on the convex sample
meniscus. Seal the sample with the screw cap.
To ensure that the sample has been properly
sealed, invert the sample and lightly tap the
Hd on a solid surface. The absence of
entrapped air bubbles indicates a proper seal.
If air bubbles are present, open the bottle, add
-------
additional sample, and reseal. The sample must
remain hermetically sealed until 1t Is analyzed.
205 Preservation of Volatile Orqanlcs
205*1 Preservative (sodium thlosulfate or sodium
bisulfite) 1s used to stabilize samples
containing residual chlorine. The production of*
chloroform and other haloforms continues 1n such
samples 1f they are not stabilized. Waste
streams that have been treated with chlorine
should be tested on-s1te to determine whether or
not preservative 1s needed. If preservation 1s
required, collect both preserved and non-<
preserved samples, wrap the samples with water
proof packing material, place In an Insulated
chest and 1ce at 4°C. Maintain at 4°C
during transport and storage prior to analysis.
206 Grab Samples for Volatile Organ1cs (Purgeables)
206.1 Collect grab samples (minimum of one per day)
for the analysis of volatile organlcs. Collect
samples from the raw process discharge, the
treated effluent, and the treated effluent after
chlorlnatlon, when chlorfnation 1s practiced.
It 1s recommended that the samples be collected
from mid-channel at mid-depth. Samples should
be collected at a turbulent, well mixed section
of the channel.
ML- 37
-------
References
1. Eichelberger, J.W., L.E. Harris, and W.L. Budde, "Reference
Compound to Calibrate Ion Abundance Measurements 1n Gas
Chromatography—Mass Spectrometry Systems," Anal. Chem. 47.
995-1000 (1975).
2. McNalr, H.M., and E.J. Bonelli, "Basic Gas Chromatography,"
Consolidated Printing, p. 52, Berkeley, CA, 1969.
-------
TABLE 1
DFTPP Key Ions and Ion Abundance Criteria
Mass Ion Abundance Criteria
51 30-60% of mass 198
68 less than 25! of mass 69
70 less than 22 of mass 69
127 40-60* of mass 198
«
197 less than 1% of mass 198
198 base peak, 1002 relative abundance
199 5-9* of mass 198
275 10-30X of mass 198
365 greater than 12 of mass 198
441 less than mass 443
442 greater than 402 of mass 198
443 17-23S of mass 442
:&:
-------
TABLE 2
BFB Key Ions and Abundance Criteria
Mass Ion Abundance Criteria
50 20-40* of mass 95
75 50-70* of mass 95
95 base peak, 100* relative abundance
96 5-9* of mass 95
173 less than 1* of mass 95
174 70-90* of mass 95
175 5-9* of mass 95
176 70-90* of mass 95
177 5-9* of mass 95
OF- 2a
-------
TAIUNG FACTORa ~
A8
Example calculation: Peak HeightsDE = 100 mm
10% Peak Height a BO *10 mm
Peak Width at 10% Peak Height sACs 23 mm
A8 = 11 mm
SC =* T2 mm
Therefore: Tailing Factors— s1.1
11
Rgure 1. Tailing factor calculation
3ZT-
-------
Appendix A
Storage of Final Extracts
Each extract 1s to be washed out of Its container into a 10 ml
glass ampul and brought to 5 ml * 1 ml. Methylene chloride is
the solvent for the base-neutrals and acids, hexane for
pesticides. The cooled ampuls (-20°C) are to be sealed in a
rounded-off, fire polished manner, i.e., no thin sharp peaks of
glass that are easily, broken on handling and shipping. After
sealing the ampuls, put an indelible mark at the solvent level.
Securely attach a label or tag that gives:
Type of fraction (base-neutral, etc.)
Industrial category
Nane (of plant, city and state)
Specific source of stage of treatonent
Date sampled
Date sealed
Name of contractor and analytical laboratory
Final volume of extract.
Wrap the ampuls in packing material to prevent breakage and mail
or ship them postpaid at ambient temperature. When the samples
are safely in ampuls, the remainder of the composite sample may
be discarded.
-------
Appendix 8
Archiving SC/MS Data on Magnetic Tape
As the analyses are completed, transfer GC/MS raw data to
magnetic tape as described below. All raw GC/MS data is to be
retained on 9-track magnetic tape. When a tape is filled,
deliver the tape to the Environmental Protection Agency for
storage and later evaluation. The tape format is:
Type - 9-track, 800 BPI, 2400 foot reels
Record length - 80
Block size - multiple of 80 (specify)
Code - ASCII or EBCDIC
The data format must be approved by EPA. At the present time
there are formats used by commercially available equipment that
have been approved by EPA. Before submitting tapes to EPA, a
sample data tape with appropriate documentation should be
submitted for approval of the format and content.
Each GC/MS run on tape should contain in the following
header information at minimum.
1. Identifying run number
2. Data and time of sample run
3. Run description. Include GC column used, temp, program
(e.g. 70-270- 8), concentration factor of extract, run
type (e.g. Base/Neutral, Acid, VQA, Direct Aqueous
injectton).
4. Scan time in seconds.
32'
-------
5. Mass range scanned.
Each scan within each run should contain at minimum:
1. Identifying run number
2. Scan number
3. Date
4. Time of scan (time from start of run).
5. Total Intensity of scan (not normalized).
6. If Intensities are normalized, the mass and raw
Intensity of the base peak for the scan. Intensities
may be normalized or unnormallzed. Normalized values
must have.a dynamic range of at least. 1000;
unnormallzed values must reflect the actual absolute
Intensity value. Masses may be nominal Integer values.
Each magnetic tape reel shall be accompanied by Table of MS
response ratios and a descriptor sheet that Includes the
following information for each run. See GC/MS Data Transmittal
Form, this Appendix.
1. EPA sample number
2. Laboratory sample number
3. Fraction type (Base/Neutral, Acid, VGA, Blank, Standard
Runs, etc.)
4. Industrial category
5. Specific source or stage of treatment
6. Date sampled
7. Sampler (Contractor or EPA Region)
8. Data run
-------
9. File number on-tape
10. File name on tape
11. Comments • to Include any deviation from methods 624 or
625.
-------
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-------
Quality Assurance and Quality Control Procedures for
Verification Analysis For Organic Priority Pollutants
110 Scope
111 These procedures are provided for use by laboratories performing
verification analyses using EPA Methods 624 and 625. Verification
analyses require quantitative determinations with known precision
and accuracy, so that these data will be acceptable for use 1n
developing effluent limitation guidelines. To provide data with
this degree of reliability, a strong quality assurance and quality
control program 1s presented. These procedures are designed to
produce data with known precision and accuracy so that a
determination of confidence can be placed in the data. Quality
Assurance (QA) 1s the total program for assuring the reliability of
the monitoring data. Quality control (QC) 1s the routine
application of procedures for controlling the measurement process.
112 Initially, the methodology must be validated for each Industrial
subcategory. The requirement for validation of each subcategory is
based on the unique nature of the wastewater associated with most
of the subcategories. Since the effluent from treatment is to be
used for setting control limits, 1t will be used to develop Initial
validation data for the method prior to routine sampling and
analyses. Based on screening data, a particular subcategory may
not require verification analyses of all four fractions (Volatile*,
Base/Neutrals, Adds, and Pesticides) or for all of the individual
Consent Decree compounds, in which case, the method requires
validation only for the fraction or the selected compounds of
interest.
-------
113 The results of the validation analyses will be used to provide
Information with which to judge a laboratory's ability to Interpret
and i rip lament the method for each Industrial subcategory. Intlal
QC limits for precision and accuracy will be established using
these results, and then used in subsequent analyses as control
limits. A numerical example 1s given in Appendix C.
114 After the method 1s validated for each subcategory and routine
analysis begins, continuing QA/QC will be required to ensure that
the subsequent analyses are within the established control limits.
115 Prior to developing initial method validation and a continuing
quality control program, the analyst (individual or group if team
approach 1s used) must demonstrate the ability to perform the
required analyses. If a laboratory has not established precision
and accuracy criteria for clean water, the laboratory must perform
replicate analyses of clean water as prescribed in section 121.
Such data must be presented.to the responsible EGO project officer
prior to the beginning of the verification phase.
120 Routine Quality Assurance and Quality Control
121 Preliminary Clean Water Precision and Accuracy
121.1 Before any work is begun on actual field samples, a labora-
tory must demonstrate its ability to properly perform the
11quid-11quid extractions, the gas purge extractions, and
the required chromatography. Clean water spikes are ana-
lyzed to demonstrate the laboratory's ability to implement
-------
Methods 624 and 625, and to establish the baseline precision
and accuracy criteria for the method 1n that laboratory-
121.2 Procedure:
121.2.1 Prepare "organic-free* water for use 1n determining
preliminary precision and accuracy according to the
procedures given 1n Methods 624 and 62S.
121.2.2 Spike four replicates of clean water with each
compound of Interest at a concentration
approximately equal to 10 times the limit of
detection. In addition, spike all purgeable
allquots with a minimum of three surrogate
standards at a level of 100 ug/1. For extractable
organlcs, each replicate must be one liter; each
purgeable sample requires at least 100 ml. Oo not
dose purgeables with more than 20 ul of an
alcoholic standard per each 100 ml of water.
Analyze spiked solutions according to Method 624 or
625.
121.3 Precision - For each parameter, use the resulting observed
values of the spikes (0-j, 02, Oj, and 0^) to
calculate the standard deviation (S) of the replicates
according to Equation 1.
n(n-l)
Where:
n * number of replicatas
-------
121.4 Accuracy - For each parameter, use the resulting observed
values of the spikes (0-j, 02, 03, and 04) to
calculate the mean percent recovery (P) of the method
according to equation 2.
n
Eq. 2 P
nr
Where:
n * number of replicates
T » true value of the spike
121.5 The precision and accuracy data shall be documented for the
record as evidence that the laboratory can properly perform
the extractions and chroraatography essential for methods 624
and 625.
122 Method Blank - The method blank is defined as an appropriate volume
of "organic-free" water which has been processed exactly as the
sample (including glassware, reagents, solvents, etc.). Reagents
or solvents having background levels that interfere with the
compounds to be determined must be purified and shown to be
acceptable or replaced with some that are acceptable prior to
proceeding with analyses. Problems encountered and corrective
actions taken shall be documented and reported for the record.
122.1 For the extractable fractions (Base/Neutral, Acid, and
Pesticide) the method blank requires extraction of 1-11 tar
"organic-free11 water. A method blank must be extracted for
each set of field samples extracted at a given time (at
least one method blank per 20 field samples analyzed) and
-------
whenever a new source of reagent or solvent 1s introduced
Into the analytical scheme. The method blank can be
screened by SC-FIC. Analysis by GC-MS 1s required only if
6C-FID analysis of the field blank gives any peaks larger
than the internal standard peak.
122.2 For the volatile fraction, 5 ml of "organic-free* water
should be analyzed by the purge and trap methodology only if
positive interferences are noted during the analysis of a
field blank. If positive interference still occurs, repeat
the method blank analysis. If interference persists,
dismantle the system, thoroughly clean all parts that
contact the sample, purge gas and carrier gas. Replace or
repack the sorbent trap and change purge and carrier gas.
123 Field Blank - The field blank is defined as an appropriate
341*"
volume of "organic-free" water which has been/\to the
sampling site and back to the analytical laboratory in a
container and bottle identical to the type used to collect
the samples. Field blanks and samples must be shipped' in
separate containers. When received in the lab, the field
blank is dosed, extracted and concentrated as if it were an
actual sample.
123.1 For the extractable factors (Base/Neutral, Acid, and
Pesticide), the field blank may be screened by GC-FTD or
GC-eC (Pesticides) only). Full GC-MS analysis is to be
performed if the screening analysis gives any peaks larger
than the internal standard peak.
-------
123.2 For the volatile fraction, analyze a 5-ml field blank after
each sample analysis. Follow the guidelines in 121.2 if
positive interferences are noted during the analysis of a
field blank.
124 GC-MS Calibration Check
124.1 For the Base/Neutral-Acid or Pesticide fraction, daily
analyze decafluorotriphenylphosphine (DFTPP) by injection
into the GC inlet. See Reference 1. (DFTPP must also be
run after any mass spectrometer tuning is done). This
calibration check may be done in conjunction with Section
125. The requirement is that 50 nanograms of DFTPP injected
must meet the specifications prescribed. Table 1 lists the
DFTPP key ions and ion abundance criteria.
124.2 For the volatile (purgeable) fraction, analyze
4-brcrao-fluorobenzene (BFB) daily by direct injection into
the GC/MS. The requirement is that 20 nanograms of BFB
injected must meet the prescribed specifications. Table 2
lists the BFB key ions and ion abundance criteria. This
calibration check may be done in conjunction with Section
125.
125 GC Performance Check
125.1 SP-2250 column - At the beginning of each day that
base/neutral or pesticide fraction analyses are to be
performed, inject 100 nanograms of benzidine (for
base/neutral analyses) or aldrin (pesticide analyses) either
separately or as part of a standard mixture that may also
contain 50 ng of DFTPP. Performance for base neutrals
-------
1s acceptable 1f the tailing factor for benzidine 1s less
than 3. Performance for the pesticides 1s acceptable 1f the
tailing factor for aldrln 1s less than 2. Calculation of
the tailing factor 1s given 1n Reference 2 and described In
Figure 1. Retain all plots of performance evaluation as
evidence of valid performance.
125.2 SP-1240 DA Column - At the beginning of each day that acid
fraction analyses are to be performed, Inject 100 nanograms
of pentachlorophenol either separately or as part of a
standard mixture that may also contain DFTPP. Performance
is acceptable if the tailing factor is less than 5.
Calculation of the tailing factor is given 1n Reference 2
and described in Figure 1. Retain all plots of performance
evaluation as evidence of valid performance.
125.3 Carbopak C with Carbowax 1500 or Carbopak B with SP-1000
column. At the beginning of each day that purgeables
analysis are to be performed, inject 20 nanograms of BF3
either separately or as part of a standard mixture.
Performance is acceptable if the tailing factor is less than
2. Calculation of the tailing factor is given in Reference
2 and described in Figure 1. Retain all plots of
performance evaluation as evidence of valid performance.
126 Performance Evaluation - Ones each 20 working days; dose, extract,
and analyze a performance evaluation sample (available from
EMSL-C1ncinnati). Retain the results as evidence of valid
performance.
-------
127 Saving Extracts -If the final extract 1s to be retained, or shipped
to another location, follow the appropriate guidelines given 1n
Appendix A.
128 Archiving GC/MS Data - If the SC/MS data are to be archived on
magnetic tape, follow the guidelines In Appendix 8.
130 Method Validation
131 Extractable Orqanlcs (Base/Neutrals-Acids and Pesticides) - The
following procedures are to be applied, separately, to samples
being analyzed for the Base/Neutral-Acid group of compounds and to
samples being analyzed for the Pesticides group. The analyses
shall be performed according to the procedures given in Method
625. The validation studies must be performed under the same
conditions ordinarily applied to the samples of a given
subcategory. That is, if separatory funnels are routinely used for
extraction of the samples, the study must be conducted using
separatory funnels. If continuous extractors are used for routine
analysis of the subcategory, the validation study must be performed
using the same type of continuous extractors.
131.1 Sample pretreatment - The laboratory will receive a 24-hour
composite sample of adequate volume to carry out the
validation study and one field blank taken as described in
the sampling protocol on the same day from the same source.
Mix composite sample and withdraw a 1-liter aliquot for
analysis. Vigorously mix the sample by some type of
stirring device. Withdraw aliquots, while stirring, into a
1-liter graduated cylinder, using a siphon made of glass or
-------
Teflon. Measure and record the volume. Transfer the
aliquot to a 2-11ter separatory funnel or continuous
extractor for spiking. Initially analyze a 1-11ter aliquot
of the sample to determine the sample background so that
proper spiking levels can be selected for 131.2.1. The
remainder of the composite sample should be stored at 4°C
until the validation study 1s begun. At the same time,
analyze a 1-11ter aliquot of the field blank. Choose three
levels of priority pollutant spikes to cover the expected
concentration range of the samples 1n the subcategory.
131.2 Preparation of Allquots for Validation -Study - Withdraw
twelve 1-11ter allquots from the stirred composite sample as
described 1n 131.1, Separate Into three groups of four.
131.2.1 Spiking of AHquots - Spike two allquots of each
»
group with surrogate standards only. The other two
allquots are spiked with surrogate standards plus
the priority pollutant standards of Interest at one
of the concentration levels (See Figure 2}. Repeat
this process for each group of allquots. Select
the three spiking concentrations for the priority
pollutants based on the results of the background
analysis obtained 1n 131.1. If the Initial
background level for a particular priority
pollutant 1s X, select the three spiking levels to
give final concentrations of 2X, 10X, and 100X. If
X equals 15 ug/1, dose with 15, 135, and 1435 ug
-------
per liter. This gives final concentrations of
priority pollutants of 30, 150, and 1500 ug/1.
Spike each 1-11ter replicate with each surrogate at
a level of 100 ug/1. (Note: Consideration should
be given to the water solubility of the compounds
being spiked when selecting the spiking
concentration levels.)
131.2.2 Prepare spiking standards 1n concentrations such
that no more than 5 ml of spiking solution 1s added
for each liter of sample. This will ensure that
•the solubility of the standard In water will not be
significantly affected by the added organic
solvent. Add the spiking solution to the sample
allquots 1n the separatory funnel using a transfer
plpet. After adding the spikes, thoroughly mix the
samples and after one hour at room temperature
proceed with the extraction. ' If continuous
extractors are used, It may be necessary to spike
the 1 liter allquots before they are placed Into
the extractor. Place the aliquot in a separatory
funnel or a clean bottle, spike, and transfer to
the extractor. The bottle must be rinsed with
solvent. Malt an hour and begin the extraction.
(Note: Extractions of all samples must begin
within 48 hours of receipt in the laboratory.)
-------
131.3 Use of the Data from Spiked Samples 1n Analyses - The data
obtained from the determination of priority pollutants are
used to calculate the precision and accuracy of the method
and to establish control limits for the Individual compounds
of Interest. Surrogate spikes are added to every sample to
provide quality control on every sample .by monitoring for
matrix effects and gross sample processing errors. The
surrogate 1s not used as an Internal standard for
quantification purposes. Suggested surrogate spikes are
given 1n Section 6.4 of Method 625. If verification 1s
needed for only one fraction, only the surrogates for that
fraction shall be added.
131.4 Extract and analyse all allquots as directed 1n Method 625
or other appropriate EPA methods.
131.5 Calculation of Precision and Accuracy - The precision of the
method may be calculated from the data obtained during the
validation study. There are three spiked concentration
levels of priority pollutants as outlined 1n Figure 2. The
method precision for the background level of priority
pollutants occurring in the sample may be calculated from
the three pairs of replicate allquots which are spiked only
with surrogate standards (Al, A2; 81, 82; Cl, and C2. See
Figure 2). The precision and accuracy for the surrogates
may be calculated from all twelve replicatas sines the
spiking level is constant for both sets of six samples. The
precision and accuracy for the surrogates may be calculated
-------
for each set of six samples if there 1s an effect due to the
added priority pollutant spike. Similarly, the precision
and accuracy at each spike level of the priority pollutants
may be determined from the two replicate aliquots that
received that spike (01, D2; El, E2; Fl. and F2 See Figure
2).
a. Precision
Calculate the range (R) for each pair of replicate aliquots
1e.t duplicate analyses, according to equation 3:
Eq. 3 R
Where:
X-i and X- are each an analytical result from two
replicate aliquots.
The concentration level related to R can be represented as
in equation 4.
Eq. 4 I - (Xt + X2)
2
Where:
X is the mean of the duplicate analyses, X-j and
v
For any group of n duplicate analyses that are considered
similar to each other, their ranges (R^) and means (X.),
where 1 * 1 to n, can be used to estimate the critical
difference (Rc) between similar future duplicate analyses
or any specific concentration leva! (C). Calculate R as
shown in aquation 5.
izr r
-------
Eq. 5
1-1
From these data develop a table of RC values for various
concentration (C) values that span the concentration range of
interest. Use these Initial critical difference values to
judge the acceptability of succeeding duplicate results
generated under the same conditions. Revise and update as
additional duplicate data becomes available. When more than
15 pairs of duplicates are available within any specific
concentration level C, RC should be calculated directly
from the average range of these duplicates alone. Equation 5
reduces to
n
2Z RI
b. Accuracy for Priority Pollutants
Calculate the background concentration of
priority pollutants occurring 1n each of the
field composite samples. The calculation is
similar to equation 4, but there are six
pieces of data (AT, A2, Bl, 82, Cl, C2)
available for this calculation as shown in
Figure 2. Therefore, the calculation is as
shown in equation 6.
6
Eq. 6 I » ( £ XJ
1-1
,
'
-------
Where:
X* 1s the meanjXj, 1=1 to 6 are the analytical
results for the six 1-Hter allquots of a single
composite sample spiked with surrogates only.
Calculate the recovery of each priority pollutant
1ir each of the 1-11 ter allquots spiked with
priority pollutants (01, D2. El, E2, FT and F2)
according to equation 7:
Eq. 7 P - 100 (2 - 7)
Where:
P 1s the percent recovery of the spike
2 1s the analytically determined
concentration of the priority pollutant in
the spiked aliquot
X is the mean background concentration of
the priority pollutant and
T is the true value of the spike.
Determine the percent recoveries for each priority
pollutant at all of its concentration levels. If
there is no significant difference between the
percent recoveries for the various concentration
-------
levels, all n of the percent recovery values may be
treated together as 1n equations 8 and 9. If some
of the percent recovery values are significantly
different, each group of similar percent recoveries
must be treated Independently to develop Its own
characteristic mean percent recovery (P) and Its
associated standard devlalon (S }.
n
Eq. 3 7 «
Where:
P 1s the mean percent recovery
•
P 1s an Individual percent recovery value
n 1s the number of observations at this
concentrator! level
-------
».»
n(n-l)
c. Accuracy for Surrogates
Proceed exactly as with priority pollutants in 131.5b above,
keeping the following two differences in mind: there is no
background concentration and there are six sets of duplicate
analyses for the surrogate spikes; three sets spiked with
priority pollutant (D1, 02; £1, E2; FT, F2) and three sets
without (Al, A2; Bl, 82; C1, C2), see Figure 2. Calculate
the percent recovery as shown in equation 10.
Eq. 10 P - 100 Z
Where:
P is the percent recovery of the surrogate spike.
Z is the measured value of the surrogate spikes
in the aliquot.
T is the calculated or true value of the
surrogate spikes added to the sample.
Calculate the mean percent recovery (P) and the standard
deviation (S ) of the percent recovery of the surrogate
spikes in all of the sample aliquots according to equations
3 and 9.
132 Volatile Organics (Purgeables)
132.1 All individual grab samples collected during the specified
time period shall be composited in the laboratory, spiked
and analyzed as soon as possible. -Significant losses of
-------
some of the purgeable priority pollutants will occur in 1 to
2 days. However, in a nonreactlve matrix, most are stable
up to 14 days. The method validation study requires twelve
5-ffll allquots from each composited sample. Carefully
transfer the remaining volume of composited sample back to a
clean vial or vials and seal with no headspace as done when
collecting a sample. These should be held at 4°C until it
1s determined that there 1s no further need for the sample.
Figure 3 summarizes the validation study for volatile
organlcs.
Caution: Prepare only as many sample aliquots as can be
analyzed in the working day. .This may mean that each of the
three concentration levels will be analyzed on different
days.
132.2 Pretreatnent of Grab Samples - Individual grab samples
should be composited according to the following procedure:
a. Composite only grab sanples of equal volume.
b. Carefully pour the contents of all individual grab
sanples collected from a given source during the
specified time period into a 1000-flil round-bottom flask
which Is chilled 1n a wet ice bath.
c. Stir the mixture gently with a glass rod for
approximately one minute while in the ice bath.
d. Carefully fill 13 clean 40-ml vials or three 120-*nl
vials and four 40-ml vials with ccmpositad sample.
e. Take one 40-
-------
f. Store the remaining vials at 4°C until the validation
study 1s begun.
132.3 Spiking levels for priority pollutants and surrogate
standards - The spiking levels of the priority pollutants
are determined by the background (X) 1n the samples. The
low level spike will give a final concentration that 1s 2
times the background level. The Intermediate and high level
spikes will give final concentrations that are 10 and 100
times the background level. Concentrations 1n excess of
1000 ug/1 are likely to flood the gas chromatographic
column. Therefore, the total concentration (background plus
spike) of each Individual priority pollutant should not
exceed 900 ug/1. Even at this level the solubility of the
compounds in the sample must be considered. The spiking
level for all surrogate standards should be 100 ug/1.
132.4 Spiking Procedures
132.4.1 Preparation of Spiking Standards - Prepare
methanolic stock standard solutions of the priority
pollutants and the surrogate standards according to
the directions given in Method 624.
From the methanolic primary dilutions prepare
secondary aqueous spiking mixtures of the surrogate
standards so that 20.0 ul of the primary standard
solution, diluted to 50.0 ml 1n organic free water
will permit adding 5 ul of the resulting solution
to the 5 ml sample giving the desired surrogate
-------
concentration level of 100 ug/1.
Prepare spiking mixtures of the priority pollutant
standards 1n methanol so that 20.0 ul of the
solution added to 100.0 ml of sample will give the
desired concentration levels.
132.4.2 To minimize the solubility effect of methanol on
the constituents to be measured, do not Inject more
than 20 ul of spiking solution per 100 ml of
sample. Never use a pipet to transfer samples or
aqueous standards that are to be analyzed for
volatile purgeable compounds. Transfer samples by
pouring Into the receiving vessel.
*
132.4.3 Spiking the Sample AHquots - Take one of the
120-ml or 3 of the 40 ml sample allquots from cold
storage, equilibrate to room temperature, and fill
a 100 ml volumetric flask to mark with the sample.
Rapidly Inject 20 ul of the methanoUc solution of
priority pollutant spiking standard (concentration
2X) Into the expanded area of the flask below the
neck. Stopper and nrix by gently Inverting the
flask three times. Fill two 5-ml syringes with
spiked sample from the flask as directed 1n the
analytical protocol. Open the valve of the syringe
and Inject 5 ul of the surrogate standard spiking
solution. Inject the sample aliquot Into the
purging device and analyze according to Method 624.
-------
Take one of the 40-ral sample aliquots from cold
storage, equilibrate to room temperature and fill
two 5-ffll syringes with the sample as directed 1n
Method 624. Spike 5 ul of the surrogate standard
water solution (concentration 100 ug/1) Into the
syringe through the valve giving a final
concentration of 100 ug/1. Inject the sample
•aliquot Into the purge device and analyze according
to Method 624. See Figure 3. Repeat this
procedure twice, giving three sets of analyses of
two samples spiked with surrogate standards only
and two samples spiked with surrogate standards and
priority pollutants.
132*5 Calculation of Precision and Accuracy - The precision and
accuracy for the purgeable priority pollutants and the
surrogate standards are calculated as directed for the
semlvolatHe solvent extractable compounds In paragraphs
131.5a, b, and c.
140 Continuing Quality Assurance and quality Control
141 Extractable Organics (First Day Samples) - The following
procedures should be applied to the first day samples of the
pesticides group Base/Neutral -Add group. They should be carried
out on each first day composite sample for each new subcatagory.
This Includes samples of the Influent to treatment, the effluent
from treatment and the supply water. An outline diagram for first
-------
day ongoing quailtyassurance samples 1s given in Figure 4.
141.1 Withdraw three 1-1 Her allquots of the composite sample
according to the procedure 1n 131.1.
141.2 Spiking the Sample AHquots - Spike one of the allquots with
priority pollutant standards plus the surrogate standards
and two of the allquots with surrogate standards only.
141.3 Add a spike sufficient to approximately double the
background concentration of the priority pollutants as
determined In 131.5b-. If the original concentration Is
higher than the midpoint of the calibration curve, then the
concentration- of the spike should be approximately one-half
the original concentration. Surrogate spikes as specified
1n 131.3 should be added to all three allquots from each
sample at a concentration level of 100 ug/1.
141.4 Analyze according to Method 625.
141.5 Calculations of Precision and Accuracy
a. For the first day samples, calculate the precision of
the duplicate analyses (X^ and X^) from the two
l-11ter allquots for the priority pollutants background
and the surrogate standards. Calculate the range (R)
of the results according to equation 11.
Eq. 11 R «|X., - X2|
The concentration of each compound Is represented by
the mean of the duplicate values. Calculate the mean
(X) according to equation 12.
Eq. 12 7 » (X7 * X?)
-------
Refer to the table of critical range values developed in
131.5a, to find the concentration (C) nearest to X.
Use this RC to evaluate the acceptability of R from
Eq. 11. If R is greater than RC, the system precision
is out of control and the source of this unusual
variability should be identified and resoJved before
continuing with routine analyses. After correcting the
source of this unusual variability, reanalyze the sample
if possible. Record the results of all duplicate
analyses and periodically (after 5-10 additional
duplicate results are obtained), revise, update, and
improve the table of critical range values.
»
b. Accuracy for Surrogate Spikes
Calculate the recovery of the surrogate spikes in the
duplicates according to equation 13.
Eq. 13 P • 100 I
Where:
P is the percent recovery.
Z is the analytically determined concentration of the
surrogate standard spikes.
T is the true value of the surrogate standard spikes
added in 132.4.3.
If the percent recoveries are not within the interval ?
+ 3S as determined in 131.5c, the system should be
checked for problems. If problems exist, they must be
-------
resolved before continuing with routine analyses.
Record the recovery of all surrogate spikes and
periodically (every 5 to 10 additional data points),
revise, update and Improve the recovery criteria.
c. Accuracy for Priority Pollutant Spikes
Using the results obtained from the 1-Htar aliquot of
composite sample spiked with surrogate standards and
priority pollutant standards, calculate the recoveries
of the priority pollutants according to equation 14.
Eq. 14 P - 100 (Z-X)
Where:
P 1s the percent recovery
Z is the analytically determined
concentration, of the priority pollutant
spikes
T is 'the true value of the priority
pollutant spikes added in 132.4.3, and
X is the mean concentration of the priority
pollutant background determined by equation
9.
If the percent recovery is not within the interval
of P *, 3S , as determined in 131.5b the system
should be checked far problems. If problems
exist, they must be resolved before continuing
HUT-
-------
with routine analyses. Record the recovery of all
spikes and periodically revise, update, and
Improve the accuracy criteria.
142 Extractable Organics - (Subsequent Day Samples) - The
following procedures should be applied to each subsequent day
sample of the Base/Neutrals-Acids group and the pesticide
group. A flow diagram for each subsequent day ongoing
quality assurance samples 1s given in Figure 5.
142.1 Withdraw a. one-liter aliquot as directed in 131.1
142.2 Spike the aliquot with surrogate standards at a
concentration of 100 ug/1.
142.3 Analyze according to Method 625.
142.4 Determine the percent recovery of the surrogate
standards using Equation 10. If the percent recovery
is outside the interval 7 + 3S as determined in
131.5c, the analytical system should be checked for
problems. If problems exist, they must be resolved
before continuing further sample analyses.
142.5 A field blank for the day-must be analyzed according
to Method 625. If priority pollutants are found and
quantified, the values for the field blank should be
noted and reported along with sample results. If
significant interference problems occur, the method
blank must be analyzed to determine if interference
was introduced in the
-------
field or the laboratory. Appropriate action must
be taken to eliminate the problem before continuing
with the analysis of routine samples.
143 Volatile Organ1cs (First Day Samples) - Continuing quality
assurance for the purgeable volatile organics should be
applied to samples from Influent to treatment, effluent from
treatment, and supply water. The following procedures should
be carried out on each first day composite samples from the
three sample sources from each subcategory. An outline is
given in Figure 4.
143.1 Composite all relevant grab samples as described in
Section 132.2. Prepare six 5-ml allquots for analysis.
143.2 Spike two allquots with the priority pollutant
standards at a level twice that determined in Section
132.5 and the surrogate standards using the procedures
in Section 132.3 and 132.4. Spike four 5-flil aliquots
with surrogate standards only as 1n 132.3 and 132.4.
143.3 Analyze one of the duplicates spiked with priority
pollutants and surrogate standards and two of the four
replicates spiked with surrogate standards only. The
remaining spiked aliquots are analyzed only 1f a
problem is encountered with the analysis of the first
set of aliquots.
-------
143.4 Analyze the spiked allquots according to Method 624.
143.5 Calculate the precision and accuracy as directed for
the semivolatne solvent extractables as directed in
141.5.
144 Volatile Orqanics (Subsequent Day Samples) - The following
procedures should be applied to each subsequent day sample of
the volatile organics group. An outline is given in Figure 5.
144.1 Composite the relevant grab samples as described in
Section 132.2. Prepare two 5-ml aliquots for analysis.
144.2 Spike both aliquots with surrogate standards only to
give a concentration of 100 ug/1.
144.3 Analyze one of the aliquots according to Method 624.
The other aliquot is analyzed only if a problem is
encountered..
144.4 Determine the percent recovery of the surrogate
standards using Equation 10. If the percent recovery
is outside the interval P £ 3 S as determined in
131.5c, then the analytical system should be checked
for problems. If problems exist, they must be
resolved before continuing further sample analysis.
144.5 Analyze a field blank representing the same day that
the samples were collected. Follow the guidelines
given in 142.5.
'- 3/2.
-------
200 Sampling Procedures for Verification Phase
201 These procedures are given as an example for the collection
of sample*.
202 Extractafale Organlcs (Base/Neutrals-Adds and Pesticides)
202.1 Initial QA/QC - Three 2Vgallon samples must be taken
concurrently over the Initial period of sampling (24
hrs.) effluent from treatment If both the
Base/Neutrals-Acid group and the Pesticide group are
to be determined. If only one of the two groups 1s to
be determined, collect two 2%-gallon samples or one
5-gallon sample. The samples can be collected using
two or more'automatic Isco samplers that have the
sample lines linked together to ensure close replicate
samples.
a. Cluster the Inlet tubes to ensure that samples
are taken geographically close.
b. Link the compositors in a master/slave
arrangement, wherein one "master" sampler
supplies the sample Interval timing for the
"slave" sampler(s). The Mastar sampler
operates 1n a TIME mode and the interval
between samples is set on the SAMPLE RATE
switch (Model 1580) or the SAMPLE INTERVAL
switch (Model 1680). The "slave" sampler(s)
operate in a FLOW mode, with the FLOW
MULTIPLIER switch (Model 1580) set on 1, or
-------
the SAMPLE INTERVAL switch (Model 1689) set to 1 FLOW
PULSE. When the master sampler times out and collects
a sample, a flow pulse 1s sent to the slave
sampler(s), causing them to collect samples
simultaneously with the master sampler. (Note that
depressing the MANUAL ADVANCE pushbutton on the master
sampler will cause the slave sampler(s) to collect a
sample; therefore; the slave sampler(s) should be left
1n STANDBY until the master sampler set-up has been
completed). No modification to the equipment 1s
necessary. Use of two "slave" samplers require a "Y"
connect cable assembly for the master samples and two
• sampler-flow meter cables to connect to the slave
samplers^ . Samplers should be checked before use
1n the field to ensure that each collects the required
volume of sample + 5X,
202.2 Continuing QA/QC - One 2Jj-gallon sample Is required
for the continuing QA/QC work.
Available from Isco, Environmental Division, Building
1020, Lincoln Air Park West, Lincoln, Nebraska, 68524.
-------
202.3 Blanks - One 2-gallon aliquot 1s needed for the preliminary
QA/QC. This should be obtained by flushing the organic-free
water through all Isco samplers used. One 1-gallon blank 1s
needed for continuing QA/QC work.
203 Volatile Orqanlcs (Purgeables)
203.1 Sampling Precautions
a. Collect 2 replicates from each sample source (I.e.,
duplicate samples).
b. Fill the sample bottles 1n such a manner that no air
bubbles pass through the sample as the bottle 1s being
filled.
c. Seal the bottles so that no air bubbles are entrapped 1n
them.
d. Maintain the hermetic seal on the sample bottle and keep
at 4°C until time of analysis.
203.2 Initial QA/QC - A minimum of 1000 ml (twenty-five 40-flil
vials) must be collected over the relevant time period at
each sample point.
203.3 Continuing QA/QC - A minimum of 200 ml (five 40-ml vials)
must be collected over the relevant time period at each
sample point.
203.4 Blanks - Three 40-flil "trip" blanks (field blanks) are
required for both the Initial and continuing QA/QC work.
-------
REFERENCES
1. Elchelberger, J.W., L.E. Harris, and W.L. Budde, "Reference Compound to
Calibrate Ion Abundance Measurements 1n Gas Chromatography-Mass
Spectrometry Systems," Anal. Chem. 47. 995-1000 (1975).
2. McNalr, H.M., and E.J. BonelH, "Basic Gas Chromatography," p.52,
Consolidated Printing, Berkeley, CA 1969.
-------
TABLE 1
OFTPP Key Ions and Ion Abundance Criteria
Mass Ion Abundance Criteria
51 30-60* of mass 198
68 less than 25 of mass 69
70 less than 22 of mass 69
127 40-60* of mass 198
197 less than IX of mass 198
198 base peak, 1002 relative abundance
199 5-92 of mass 198
275 10-302 of mass 198
365 greater than IX of mass 198
441 less than mass 443
442* greater than 402 of mass 198
443 17-23* of mass 442
-------
TABLE 2
BFB Key Ions and Ion Abundance Criteria
Mass Ion Abundance Criteria
50 20-40*-of mass 95
75 50-70* of mass 95
95 base peak, 100* relative abundance
96 5-9* of mass 95
173 less than 1* of mass 95
174 70-90* of mass 95
175 5-9* of mass 95
176 70-90* of mass 95
177 5-9* of mass 95
-------
TAIUNG FACTORS —
AS
Example calculation: Peak Height a DE a 100 mm
10% Peak Height a BO a 10 mm
Peak Width at 10% Peak Height = AC s 23 mm
A3 = 11 mm
BC»12 mm
Therefore: Tailing Factors— =1.1
11
Rgure 1. Tailing factor calculation
-------
Figure 2
Summary of Initial Validation
Analyses for Extractable Samples
1. Composite the Replicate Samples.
2. Withdraw a 1-Hter aliquot. Store composite at 40C.
Separate Into three groups of 4 aliquots each.
3» Determine X the background concentration of each
Priority Pollutant.
4. Withdraw twelve 1-liter allquots from the composite.
5. a) Dose 2 of allquots with surrogate standards only at
100 ug/1. Label as Al and A2. 2
b) Dose 2 allquots with surrogate standard at 100 ug/1
and priority pollutants to give a concentration
level of 2X. Label as 01 and 02. 2
c) Analyze Al, A2, 01-, and 02 using Method 625.
6. Repeat 5a, b, and c. Label surrogate standards only
as 81 and 82. Use 10X level for priority pollutants.
Label as El and E2. 4
7. Repeat 5a, b, and c. Label surrogate standards only
as Cl and C2. Use 100X level for priority pollutants.
Label as Fl and F2. 4
Liters GS/MS
Used Runs
TOTAL 13 13
'ML
-------
Figure 3
Summary of Initial Validation
Analyses for Purgeable Samples
5-ffll
Syringes SC/MS
Used Runs
1. Composite the grab sample.
2. F111 13 clean 40-fltI vials or 3 clean 120-ml vials +
4 clean 40-nl vials with conposlte, cap, and store at 4QC.
3. a) F111 a 5-ffll syringe from one 40-nil vial. 1
b) Analyze and determine X the background concentration 1
of each priority pollutant.
4. a) F111 two 5-ffll syringes from one 40-flil vial 2
b) Dose with surrogate standards at 1QO ug/1.
Label as Al and A2.
c) Analyze Al and A2 using Method 624. 2
d) Fill a 100 ml volumetric to mark using one 120-ffll
or three 40nnl vials.
e) Dose with 20 ul of priority pollutants to give a
concentration of 2X.
f) Fill two Snnl syringes from the 100-ml volumetric. 2 .
g) Dose each syringe with surrogate standards at a
concentration of 100 ug/1. Label as 01 and 02.
h) Analyze 01 and 02 using Method 624. 2
5. Repeat 4. Label surrogates only as B1 and 82. Use 10X
level for priority pollutant. Label priority pollutants
plus surrogate standards as £1 and E2. 4 4
6. Repeat 4. Label surrogates only as C1 and C2. Use 100X
level for priority pollutants. Label priority pollutants
as F! and F2. 4 4
TOTAL 13 13
-------
Figure 4
Summary of Ongoing Quality Assurance
for First Day Samples
Liters GC/MS
Extractables Used Runs
1. Composite the Sample.
2. a) Withdraw three 1-liter aliquots. 3
b) Dose two aliquots with surrogate standards
only at 100 ug/1.
c) Dose one aliquotvith surrogate standards and
the priority pollutants of interest to give a
concentration of 2X, Section 131.5.
d) Analyze using Method 625. 3
TOTAL "I "T
5 ml
syringes GC/MS
Purgeables Used Runs
1. Composite the Sample.
2. a) Fill 4 clean 4-ml vials or 1 clean 120-ml vial
•*• 1 clean 40nnl vial with composite. Store at 4°C.
3. a) Fill four 5-ml syringes from one 40-ml vial. 4
b) Dose each with surrogate standards at 100 ug/1.
c) Fill a 100 ml volumetric to mark using the 120-ml vial
or three 40-flil vials.
d) Dose with 20 ul of priority pollutants to give a
concentration of 2X, Section 132.5.
e) Fill two 5-ml syringes from the 100 ml volumetric. 2
f) Analyze two 5-ml syringes containing surrogate
standards only and one 5-ml syringes containing surrogate 3
standards and priority pollutants using method 624.
TOTAL ~T~ "T"
-------
Figure 5
Suanary of Ongoing Quality
Assurance for Subsequent Day Samples
Liters GC/MS
Extractables Used Runs
1. Composite the sample.
2. a) Withdraw a 1-liter aliquot.
b) Oose with surrogate stand
c) Analyze using Method 525.
b) Oose with surrogate standards only at TOO ug/1.
Subtotal r ^H
TOTAL (x29 days) 29 29*
5-ml
Syringes 6C/MS
Purqeables Used Runs
1. Composite the sample.
2. a) F111 two 5-fl»l syringes with composite. 2
b) Dose with surrogate standards at 100 ug/1.
c) Analyze one 5-ral sample. 1 •
Subtotal 2 ]
TOTAL (x29 days) SB 29*
*Assunring that field blank shows no priority pollutants.
If field blank were to be analyzed by GC/MS each subsequent
day, the total would be 53.
^3
-------
Appendix A
Storage of Final Extracts
Each extract 1s to be washed out of Its container Into a 10-ml glass ampul
and brought to 5 ml £ 1 ml. Methylene chloride 1s the solvent for the
base-neutrals and acids,, hexane for pesticides. The cooled ampuls (-20°C)
are to be sealed 1n a rounded-off, fire polished manner, I.e., no thin sharp
peaks of glass that are easily broken on handling and shipping. After
sealing the ampuls, put an Indelible mark at the solvent level. Securely
attach a label or tag that gives:
Type of fraction (base-neutral, etc.)
Industrial category
Name (of plant, city and state)
Specific source or stage of treatment
Date sampled
Date sealed
Name of contractor and analytical laboratory
Wrap the ampuls 1n .packing material to prevent breakage and mall or ship
than postpaid at snblent temperature. When the samples are safely 1n
ampuls, the remainder of the composite sample may be discarded.
IT-
-------
Appendix B
Archiving SC/MS data on Magnetic Tape
When the analysts are completed, transfer GC/MS raw data to magnetic tape as
described below. All raw SC/MS data 1s to be retained on 9-trade magnetic
tape. When a tape 1s filled, deliver the tape to the Environmental
Protection Agency for storage and later evaluation. The tape format Is:
Type • 9-tradc, 800 BPI, 2400 foot reels
Record Length - 80
Block size - multiple of 80 (specify)
Code - ASCII or EBCDIC
The data format must be approved by EPA. At the present time there are
formats used by commercially available equipment that have been approved by
EPA. Before submitting tapes to EPA, a sample data tape with appropriate
documentation should be submitted for approval of the format and content.
Each SC/MS run on tape .should contain the following header Information at
minimum.
1. Identifying run number
2. Data and time of sample run
3. Run description. Include GC column used, temp, program
(e.g. 70-270 £8), concentration factor of extract, run type
. (e.g. Base/Neutral, Add, VOA, Direct Aqueous Injection).
4. Scan time in seconds.
5. Mass range scanned.
-------
Each scan within each run should contain at minimum:
1. Identifying run number.
2. Scan number
3. Date
4. Time of scan (time from start of run).
5. Total Intensity of scan (not normalized).
6. If Intensities'are normalized, the mass and raw Intensity of
the base peak for the scan.
7. Mass/Intensity pairs for the scan. Intensities may be
normalized or urmormallzed* Normalized values must have a
dynamic range of at least 1000; unnormallzed values must
reflect the actual absolute Intensity value. Masses may be
nominal irrterfer values.
t
Each magnetic tape reel shall be accompanied by Table of MS response ratios
and a descriptor sheet that Includes the following Information for each
run. See Figure GC-MS Transnrfttal Form, this Appendix.
1. EPA sample number
2. 'Laboratory sample number
3. Fraction type (base/Neutral, Add, VOA, Blank, Standard Runs, etc.)
4. Industrial Category
5. Specific source or stage of treatment
6. Date sanpled
7. Sanpler (Contractor or EPA Region)
3. Data run
9. File number on tape
10. File name on tape
11. Comments - to Include any deviation from method 624 or 625.
-------
GC/US DATA TRANSMITTAL FORM
1
2
3
4
5
6
7
3
9
10
11
12
^P» ^h^^BI
U.«_.
fotfcMMI
l««M>lil
IwalKtaM»«
ln»#t in
OMt
u
I
I
Imitm-
CvHXCMi W
t»» liMiimi
DM
•OT
GC/US DATA TRANSMITTAL FORM
MOT!
1
2
3
4
5
6
7
3
9
10
11
12
-------
APPENDIX C
Numerical Example of Verification Phase Results
The following 1s an example of the calculations and results of a
.verification study.
Surrogate Standard
(ug/1)
Range Mean 2 Recovery
Satnole Added Found (Eft. 3) (Eg. 4) (Eg. 10)
Al 95 93 98
A2 95 97 4 95 102
81 95 96 101
82 95 98 2 97 103
Cl 95 90 95
C2 95 94 4 92 99
01 95 99 104
02 95 95 4 97 100
El 95 89 94
EZ 95 91 2 90 96
Fl 95 94 99
FZ 95 96 2 95 101
Critical Difference (Eq. 5) Rc - 9.8 at 95 ug/1.
Mean % Recovery (Eq. 8) ? •* 99*
Standard Oev. of P (Eq. 9) Sp « 3.1 3Sp - 9.4
Acceptable Range of Recovery 90 to 109*
During the same verification study, the following data were
obtained for one of the priority pollutants studied.
Priority Pollutant Percent Mean
Back- ' Range Mean Recovery Recovery' Std.Dev.
Sample ground Added Found (Eg. 3) (Eo. 4) (Eo. 7) (Eg. 8) (Eo. 9)
2 10 -
3 12.5
1 13.5
42
1 17.5 50 46 5.9
82
5 106 86 84 3.3
96
20 1150 94 95 1.2
X
Al
A2
81
82
Cl
C2
01
D2
El
E2
Fl
F2
X
X
X
X
X
X
X
12
12
12
12
12
12
0
0
0
0
0
0
0
12
12
no
no
1200
1200
12
9
n
n
14
13
14
17
18
102
107
1160
114Q
-------
Critical Difference (Eq. 5) Rc - 6.7 at 12 ug/1
for priority pollutants Rc » 3.3 at 17.5 ug/1
Rc » 16.4 at 106 ug/1
Rc • 65 at 1150 ug/1
Mean Value of X (Eq. 6) X « 12.0 ug/1 (background concentration)
Is there a significant difference 1n the recoveries between the 100X and 10X
levels? Apply a two tailed student's t-test with a confidence level of 95*.
v
(3.3)2 . 3.51
2 degrees of freedom
d • XIOQ - XJQ - 95-84 « 11
• d/Sd • 11/3.51 « 3.70
Since 3.7 1s less than 4.3 (t-value, 0.95, 2 degrees of freedom) there 1s no
significant difference between the 100X and 10X levels. Apply equations 8
and 9 to the four recoveries for these two levels. The mean recovery (eq.8)
1s 89S with a standard devlalon (eq. 9) of 6.5 (3 degrees of freedom). Test
the 2X level against this mean recovery and standard deviation.
S
-------
For the Surrogate Standards, on day one, the range between a and b (Eq. 11) 1s
3, and the mean concentration (Eq. 12) 1s 91.5 ug/1. Since the critical
difference 1s 9.8 at 95 ug/1, this range 1s acceptable. The recoveries of the
Surrogate Standards 1n a, b, and c (Eq. 3) are all 1n the acceptable range of
^90 to 109S. Therefore, the accuracy 1s acceptable.
For the Priority Pollutant, on day one, the range between a and b 1s
acceptable, (less than 6.7) and the recovery 1s acceptable (acceptable range
of 28 to 64*).
The following 1s an example of the results for day 2 to 30 of the 30-day study:
Priority
Pollutant (ug/1)
Day Found
2 14'
3 15
4 20
5 11
6 17
7 18
8 10
9 14
10 13
11 12
12 20
30 15
Surrogate Standard (ug/1)
Added Found % Recovery
100
100
00
100
100
100
100
100
100
100
100
100
90
99
100
107
100
100
75
92
93
94
95
95
90
99
100
107
100
100
75
92
93
94
95
-Update Recovery for SS*
-Not Acceptable
01sregard
Results
-Update Recovery for
SS**
95 -Give final statistic on
Recovery of SS.
* Includes 20 results, 12 validation, 3 first day and days 2 through 6
Mean - 99 Std. Dev. » 4.6 Acceptable range 85-1132
** Includes 25 results; day 8 result not included.
• Mean » 98 Std. Dev. » 4.6 Acceptable range 84 -112S
-------
Appendix D
Possible Sources of Standards
_Aldr1ch Chemical Co., Milwaukee, Wise.;
-Ana4abs, Inc., North Haven, Conn.;
j. T. Baker Chemical Co., PhUHpsburgh, N.J.;
_Chenrfcal Procurement Laboratories, College Point, N.Y.;
•-Columbia Organlcs Catalog A-7, Columbia, S.C.
Eastman Kodak Co., Rochester, N.Y.;
JCac.Rare & Fine Chemicals, Plainvlew, N.Y.;
• Nanogens International, P.O. Box 487, Freedom, CA 95019
Pfaltz & Bauer'Chemical Co., Stanford, Conn.;
RFR Corp., Hope, R.I.; "Chemical Standards for Air-Water-Industry-Foods"
(1975).
"Analytical Reference Standards and Supplemental Data for Pesticides and
-Other Selected Organic Compounds," EPA-660/9-76-012 (May 1975), Health
Effects Research Laboratory, Environmental Toxicology Division, Research
"Triangle Park, 1C. /I nmfTi ffirnlan VinV rnn ?-u-nr'nMlg -\*A~*»m&**+—
'ITTChemical Co., 2135 Howard St., Hartville, OH 44632
Tridom Chemical Inc., Hauttauge, N.Y.
-NOTE: These sources are not to be interpreted as being endorsed by the EPA.
-------
GLOSSARY
EICP - Extracted Ion Current Profile. EICP 1s a reduction of majss
spectrotnetrlc data acquired by continuous, repetitive measurement
of spectra that plots the change 1n relative abundance of one or
more Ions as a function of time.
Equivalent - A piece of apparatus, gc liquid phase, etc. 1s
equivalent 1f Its performance gives results Identical to or better
than that specified.
FID - Flame Ion1zat1on Detector
Field Blank - Organic-free water that has been transported to the
sampling site and bade to the analytical laboratory in a container
and bottle Identical to the type used to collect the samples.
Fie^d blanks and samples must be shipped 1n separate containers.
When received 1n the lab, the Field Blank 1s dosed, extracted and
concentrated as 1f .1t were an actual sample.
Limit of Detection - 10 ug/1 unless otherwise specified. For the
8C/MS Methods, 624 and 625, this 1s an operational Hm1t that must
be met. The entire analytical system must give recognizable mass
spectra and calibration points from standards representing this
concentration. For Method 624, the condition (at 10 ug/1) must be
met.when sparging 50 'ng of a component from 5.0 ml of water. For
Method 625, the conditions (at 10 ug/1) must be met when Injecting
standards at a concentration of 10 ug per ml.
Method Blank - For Method 625, a 1-Hter volume of organic-free water
that is extracted and concentrated to the final volume of 1 ml.
For Method 624, a 5.0 ml aliquot of organic-free water.
Organic-Free Water - Water that gives no interferences when analyzed
as a Method Blank. See Method 624, Section 5.4, for the
preparation of organic-free water.
Sample Blank - Identical to Field Blank.
Significant Difference - Check for difference in magnitude by using a
two-tailed Student's t-test at a 95S Confidence Level.
Significant Drift - A 255 or larger change from an averaged
response. Must be based on a constantly updated average.
-------
SIM - Selected Ion Monitoring - Measuring the mass spectrometrlc
response to one or several Ions 1n real time.
Surrogate Standard - Compounds not expected to occur in the
environmental sample that are added to the sample to monitor for
unusual matrix effects, gross sample processing errors, etc. The
compounds chosen should be chemically similar to the pollutants
being measured. Surrogate Standards are not used as Internal
standards.
Transfer P1pet - Class A, to deliver, plpet.
-------
Chapter IV
QUALITY ASSURANCE AND QUALITY CONTROL PROCEDURES FOR
SCREENING AND VERIFICATION OF INDUSTRIAL EFFLUENTS FOR
PRIORITY POLLUTANTS
U. S. ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
CINCINNATI, OHIO 45268
June 1979
-------
INTRODUCTION
"The Sampling and Analysis Procedures for Screening of Industrial
Effluents for Priority Pollutants," (revised April, 1977) was designed to
provide qualitative/semiquantitative determination of the priority
pollutants. In order to apply these procedures to quantitative analysis,
adequate quality assurance practices must be incorporated into the
procedures. This document provides modifications to enable implementation
of a quality assurance program that will define the accuracy and precision
of the procedures for specific parameters.
In considering modifications to be made, the following assumptions were
made:
1. The program must be well documented. It must be integrated into
field and laboratory procedures. It must include field and
laboratory blanks, equipment calibration checks, replicate and
spiked sample analyses, the analysis of check standards and
performance evaluation standards.
2. The differences in industrial subcategories require method
validation and the establishment of control parameters for each
subcategory.
3. Verification Phase analyses will seldom, if ever, require analysis
of all 114 organic priority pollutants. Thus, the quality control
program will focus on a smaller selected number of compounds based
on the results of screening analyses and other data.
4. For purposes of the Effluent Guidelines Verfication Phase studies,
the quality control programs provided, herein, must be applied to
the sampling sources most frequently involved in rule making, i.e.,
supply water, influent to treatment, and effluent from treatment.
Primary emphasis should be placed on the effluent from treatment
since this is where the control limits will be set.
5. Two distinct levels of quality control are required: initial method
validation and continuing performance evaluation. The initial
quality control is to demonstrate the method precision and accuracy
and thus validate the method on a common waste stream (industrial
subcategory) where waste characterization was the basis for
establishing the subcategory.
6. The initial control limits will be established for each subcategory
on the effluent from treatment prior to initiation of the routine
sampling and analysis program for that subcategory. The control
limits for supply water and influent to treatment water will be
established over a period of time using data derived from the
ongoing continuing quality control program.
7. As more quality control data is obtained for each waste type within
each subcategory, the precision and accuracy of the method will be
updated to provide better defined control limits.
-------
FOREWORD
These methods for sampling and analysis of industrial wastes have been
prepared by the staff of the Environmental Monitoring and Support
Laboratory, at the request of the Effluent Guidelines Division, Office of
Water and Hazardous Wastes, with the cooperation of the Environmental
Research Laboratory, Athens, Georgia. The Quality Assurance/Quality Control
(QA/QC) procedures were prepared by the staff of the Environmental
Monitoring and Support Laboratory with the cooperation of the Effluent
Guidelines Division, Office of Water and Hazardous Wastes, the Environmental
Research Laboratory, Athens, Georgia, and several EPA Regional Laboratories.
The procedures represent the current state-of-the-art and are applicable
to the Effluent Guidelines Survey of prirority pollutants. This survey is
divided into two phases; the Screening Phase and the Verification Phase.
The objectives of the Screening Phase are:
1. To provide a determination of the presence or absence of a particular
priority pollutant.
2. To provide a quantitative estimate of the concentration of priority
pollutants present.
3. To focus the verification survey to specific priority pollutants.
4. To provide direction to other best available technology (BAT)
regulatory strategies.
The objectives of the Verification Phase are:
1. To provide the confirmation of the presence or absence of the
priority pollutants identified during the screening phase, necessary
for consideration in rule making.
2. To provide qualitative pollutant data with a known precision and
accuracy.
3. To provide pollutant data for use in rule making.
4. To serve as a basis for technology selection.
-------
DISCLAIMER
The mention of trade names or commercial products in this document is
for illustration purposes, and does not constitute endorsement or
recommendation for use by the U. S. Environmental Protection Agency.
-------
CONTENTS
Quality Assurance and Quality Control Procedures for
Screening Analysis for Organic Priority Pollutants
110 Scope 1
120 Routine Quality Assurance and Quality Control 1
121 Method Blank , 1
122 Field Blank 2
123 GC-MS Calibration Check * , 3
124 GC Performance Check.. 4
125 Performance Evaluation 5
130 Reporting of Data 5
200 Sampling Procedures for Screening Phase 6
201 Extractable Organics (Base Neutrals - Acids and Pesticides).. 7
202 Preservation of Extractable Organics 9
203 Field Blank Procedure for Automatic Samplers 10
204 Volatile Organics (Purgeables) 11
205 Preservation of Volatile Organics 13
206 Grab Samples for Volatile Organics 13
Quality Assurance and Quality Control Procedures
for Verification Analysis of Organic Priority Pollutants
110 Scope 14
120 Routine Quality Assurance and Quality Control 15
121 Preliminary Clean Water Precision and Accuracy 15
122 Method Blank 17
123 Field Blank 18
124 GC-MS Calibration Check 19
125 GC Performance Check 19
126 Performance Evaluation 20
127 Saving Extracts 21
128 Archiving GC/MS Data ..21
130 MethodXEvaluation 21
131 Extractable Organics (Base Neutrals-Acids and Pesticides)....21
132 Volatile Organics (Purgeables) 29
140 Continuing Quality Assurance and Quality Control 33
141 Extractable Organics (First Day Samples) 33
-------
CONTENTS (continued)
142 Extractable Organics (Subsequent Day Samples) 37
143 Volatile Organics (First Day Samples) 38
144 Volatile Organics (Subsequent Day Samples) 39
200 Sampling Procedures for Verification Phase 40
202 Extractable Organics Base/Neutrals-Acids and Pesticides) 40
203 Volatile Organics (Purgeables) 42
References 43
Appendices
A Storage of Final Extracts ....51
B Archiving GC/MS Data on Magnetic Tape 52
C Numerical Example of Verification Phase Results 55
D Possible Sources of Standards 58
Gl ossary 59
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TABLES
Number
1 DFTPP Key Ions and Ion Abundance Criteria 44
2 BFB Key Ions and Ion Abundance Criteria 45
FIGURES
Number
1 Tailing Factor Calculation 46
2 Summary of Initial Validation Analyses for Extractable Samples 47
3 Summary of Initial Validation Analyses for Purgeable Samples 48
4 Summary of Ongoing Quality Assurance for First Day Samples 49
5 Summary of Ongoing Quality Assurance for Subsequent Day Samples 50
VII
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Quality Assurance and Quality Control Procedures
for Screening Analysis for Organic Priority Pollutants
110 Scope
This procedure is provided for use by laboratories
performing screening analyses using the 6C/MS methods 624
and 625. Screening analysis provides a determination of the
presence or absence of a specific compound and a
semiquantitative estimate of the concentration of a specific
compound. Quality Assurance (QA) is the total program for
assuring the reliability of the monitoring data. Quality
control (QC) is the routine application of procedures for
controlling the measurement process.
120 Routine Quality Assurance and Quality Control
121 Method Blank - The method blank is defined as an
appropriate volume of "organic-free" water which has
been processed exactly as the sample (including
glassware, reagents, solvents, etc.). Reagents or
solvents having background levels that interfere with
the compounds to be determined must be purified and
shown to be acceptable or replaced with some that are
acceptable prior to proceeding with analyses. Problems
encountered and corrective actions taken shall be
documented and reported for the record.
121.1 For the extractable fractions (Base/Neutral,
Acid, and Pesticide) the method blank requires
-------
extraction of 1-liter "organic-free" water. A
method blank must be extracted for each set of
field samples extracted at a given time (at
least one method blank per 20 field samples
analyzed) and whenever a new source of reagent
or solvent is introduced into the analytical
scheme. The method blank can be screened by
GC-FID. Analysis by GC-MS is required only if
GC-FID analysis of the field blank gives peaks
larger than the internal standard peak.
121.2 For the volatile fraction, 5 ml of "organic-
free" water should be analyzed by the purge and
trap methodology only if positive interferences
are noted during the analysis of a field blank.
If positive interference still occurs, repeat
the method blank analysis. If interference
persists, dismantle the system, thoroughly clean
all parts that contact the sample, purge gas,
and carrier gas. Replace or repack the sorbent
trap and change purge and carrier gas.
122 Field Blank
The field blank is defined as an appropriate volume of
"organic-free" water which has been to the sampling
site and back to the analytical laboratory in a
container and bottle identical to the type used to
collect the samples. Field blanks and samples must be
-------
shipped in separate containers. When received in the
lab, the field blank is dosed, extracted and
concentrated as if it were an actual sample.
122.1 For the extractable fractions (Base/Neutral,
Acid, and Pesticide), the field blank may be
screened by 6C-FID or 6C-EC (pesticides only).
Full GC-MS analysis is to be performed if the
screening analysis gives any peaks larger than
the internal standard peak.
122.2 For the volatile fraction, analyze a 5-ml field
blank after each sample analysis. Follow the
guidelines in 121.2 if positive interferences
are noted during the analysis of a field blank.
123 GC-MS Calibration Check
123.1 For the Base/Neutral-Acid or Pesticide fraction,
daily analyze decafluorotriphenylphosphine
(DFTPP) by injection into the GC inlet. See
Reference 1. (DFTPP must also be run after any
mass spectrometer tuning is done). This
calibration check may be done in conjunction
with Section 124. The requirement is that 50
nanograms of DFTPP injected must meet the
specifications prescribed. Table 1 lists the
DFTPP key ions and ion abundance criteria.
122.2 For the volatile (purgeable) fraction, analyze
4-bromo-fluorobenzene (BFB) daily by direct
osr-3
-------
injection into the GC/MS. The requirement is
that 20 nanograms of PFBB injected must meet the
prescribed specifications. Table 2 Itsts the
BFB key ions and ion abundance criteria.
124 GC Performance Check
124.1 SP-2250 column - At the beginning of each day
that base/neutral or pesticide analyses are to
be performed, inject 100 nanograms of benzidine
(base neutral analyses) or aldrin (pesticide
analyses) either separately or as part of a
standard mixture that may also contain 50 ng of
DFTPP. Performance for base/neutrals is
acceptable if the tailing factor for benzidine
is less than 3. Performance for pesticides is
acceptable if the tailing factor for aldrin is
less than 2. Calculation of the tailing factor
is given in Reference 2 and described in Figure
1. Retain all plots of performance evaluation
as evidence of valid performance.
124.2 SP-1240 DA Column - At the beginning of each day
that acid fraction analyses are to be performed,
inject TOO nanograms of pentachlorophenol either
separately or as part of a standard mixture that
may also contain DFTPP. Performance is
acceptable if the tailing factor is less than
5. Calculation of the tailing factor is given
JSt- 4
-------
in Reference 2 and described in Figure 1.
Retain all plots of performance evaluation as
evidence of valid performance.
124.3 Carbopak C with Carbowax 1500 or Carbopak B with
SP-1000 column. At the beginning of each day
that purgeables analyses are to be performed,
inject 20 nanograms of BFB either separately or
as part of a standard mixture. Performance is
acceptable if the tailing factor is less than
2. Calculation of the tailing factor is given
in Reference 2 and described in Figure 1.
Retain all plots of performance evaluation as
evidence of valid performance.
125 Performance Evaluation
Once each 20 working days; dose, extract, and analyze a
performance evaluation sample (available from
EMSL-Cincinnati). Retain the results as evidence of
valid performance.
126 If the final extract is to be retained, or shipped to
another location, follow the appropriate guidelines
given in Appendix A.
130 Reporting of Data
131 All concentrations should.be reported in ranges, i.e.
less than 10 ug/1, 10 to 100 ug/1, or greater than 100
ug/1.
132 Report the mean and standard deviation of the relative
-------
response ratios of the standards (10 and 100 ug/1) from
the MS analysis with the range values in 131.
133 Report all quality control data with the analytical
results. This includes, but is not limited to, the
recovery of surrogate standard spikes (Method 624), the
precision for the analysis of surrogate standard spikes
(Method 624), the results from the analysis of
performance evaluation samples, and identification of
components found in the blanks.
134 If the 6C/MS data is to be archived on magnetic tape,
follow the guidelines given in Appendix B.
200 Sampling Procedures for Screening Phase
The initial characterization (screening) of the varied
industrial discharges covered by this program will be made
on an analysis of a composite effluent sample. Any scheme
for collecting a composite sample is, in effect, a method
for mechanically integrating to obtain average
characteristics of a discharge. During the screening phase
the sample composite can be used to determine the average
characteristics which would be representative of that
discharge. Simple composite samples are those that are made
up of a series of aliquots of constant volume collected at
regular time intervals in a single container. Some
situations may require flow or time proportional sampling;
this determination will be made by the individual project
officer after considering his specific industrial category.
The determination of compositing period 24, 48, or 72 hours
-------
will be made on a case-by-case basis. The duration of
compositing will depend on the type of sample being
collected, the type of facility being sampled, and the time
varying characteristics of the discharge. The rate of
change of flow and other characteristics of the discharge
and the accuracy required will also influence the
determination of the compositing period. For example,
longer compositing periods would be warranted when less
stable unit process operations are being sampled.
All samples and blanks must be carefully identified using
water proof labels and water proof ink. Include the
following information on the label: sample number, date and
hour of sampling, complete information as to source and
sampling point, preservative added, if any, and name of
person collecting the sample (include address and/or phone
number).
201 Extractable Organics (Base/Neutrals - Acids and
Pesticides)
201.1 Collect a representative composite sample. The
maximum time interval between aliquot samples
shall be no longer than 30 minutes. The minimum
aliquot size shall be TOO ml. The sample must
be collected with an automatic sampler using the
equipment and methods outlined below. Minimum
composite volume must be 2h gallons.
201.2 Automatic Sample Collection - A peristaltic pump
automatic sampler with timer and a single glass
- 7
-------
compositing jug is required. The 2*5-3 gallon
compositing bottle must be glass and cleaned as
outlined below. New unused tubing must be used
for the sampling line and for the pump for each
individual outfall or sample location. Vacuum
type automatic samplers may be used provided
that the sample chambers are glass and that they
are cleaned after every use as outlined for
glass composite containers. Place the sampler
or composite container in an insulated chest and
ice. Maintain the sample at 4°C during the
compositing procedure. At the completion of the
compositing period, seal the container with a
Teflon lined cap. Place the container in an
insulated shipping container, ice, and seal,
then ship to the analytical laboratory.
Maintain at 4°C during transport and storage
prior to analysis.
201.3 When sampling raw untreated industrial
discharges which are generally high in suspended
solids, it is imperative that adequate sample
flow rate be maintained throughout the sample
train in order to effectively transport the
solids. In horizontal runs, the velocity must
exceed the scour velocity, while in vertical
runs the settling or the fall velocity must be
exceeded several times to assure adequate
-------
transport of solids in the flow. The equipment
used in sampling raw discharges then must have a
minimum intake velocity of 60 cm per second (2
fps). In the sampling of treated effluents,
just about any commercially available automatic
liquid sampler could be used.
201.4 When more than one laboratory is involved in the
analysis of the various parameters, the sample
should, if at all possible, not be divided in
the field but rather at the contractors'
laboratory. For purpose of this program the
composite will be divided into four parts, one
part for metals analysis, one for pesticides and
PCB's, one for GC/MS compounds and one for the
classic parameters.
201.5 Blend the composite sample to provide a
homogeneous mixture including a representative
suspension of any solids in the container. No
specific method is required; hand stirring with
clean glass or Teflon rods, mechanical paddles
or magnetic mixing with Teflon coated stirring
bars may be used. Metal mixing devices may not
be used.
202 Preservation of Extractable Organics
202.1 Do not add any preservative to the sample; just
seal and prepare for shipment. All samples must
be carefully identified using labels supplied by
EGD. Indicate on the label whether the sample
3ST-9
-------
is a raw discharge or treated effluent as
shown. If sample is to be run on the plasma
unit, only indicate so at base of tag.
203 Field Blank Procedure for Automatic Samplers
203.1 Blank Water - Blank water must be as free from
organic interferences as possible. The
analytical laboratory should supply this water
in bulk glass containers (minimum of five
liters) for field use. The supplying laboratory
shall analyze the blank water to determine the
organic background that may be present.
203.2 Procedure - All parts of the sampling system
must be scrubbed with hot detergent water and
thoroughly rinsed with tap water and blank water
prior to use. Further rinsing with methylene
chloride is required when parts permit, i.e.,
are not susceptible to dissolution by the
solvent. (Note: Tygon plastic tubing is a
source of phthalate ester contamination. Where
its use is required, i.e., in the peristaltic
pump, the length must be kept as short as
possible.) Teflon is acceptable and may be used
in other parts of the sampling system as in
intake lines. In the field, pump two liters of
blank water through the sampling line and pump
tubing and discard. Then pump three liters of
"- 10
-------
blank water through the system and collect as a
blank in a 1-gallon sample bottle that has been
prepared as described below. Seal the bottle
with a Teflon lined cap. Immediately ice the
blank (4°C) and maintain at 4°C during the
transport and storage prior to analysis.
203.3 Composite Container - Prepare narrow-mouth glass
sample bottles for use by washing with hot
detergent water and thoroughly rinsing with tap
water and blank water. Heat the bottles at
400°C in a muffle-furnace or dry heat
sterilizer for 30 minutes or alternatively;
rinse with methylene chloride and air dry at
room temperature protected from atmospheric or
other sources of contamination. Caps for the
bottles must be lined with Teflon which has been
solvent rinsed as above.
204 Volatile Orgam'cs (Purgeables)
204.1 Containers - Use 45 to 125 ml screw cap glass
vials with Teflon faced silicone septa:
204.1.1 Vials - Pierce #13074 or equivalent.
204.1.2 Septa - Pierce #12722 or equivalent.
204.2 Wash the bottles, septa, and caps with hot water
and thoroughly rinse with tap water and blank
water. Heat the bottles and septa at 105°C
for one hour, cool to room temperature in an
-------
enclosed contaminant-free area. When cool, seal
bottles with septa (Teflon side down) and screw
cap. Maintain the bottles in this condition
until just prior to filling with blank water or
sample.
204.3 Collect duplicates 45-125 ml samples each time
samples are collected. Two blank water samples,
sealed in 45 ml vials, are to accompany the
sample bottles during shipment to and from the
sampling site. If preservation for residual
chlorine is to be used, collect four samples
during each sampling period. Two should be
preserved and two not preserved. Two preserved
and two non-preserved blanks are to be provided.
204.4 Filling and Sealing Bottles - Slowly fill each
container to overflowing. Carefully set the
container on a level surface. Place the septum
(Teflon side down) on the convex sample
meniscus. Seal the sample with the screw cap.
To ensure that the sample has been properly
sealed, invert the sample and lightly tap the
lid on a solid surface. The absence of
entrapped air bubbles indicates a proper seal.
If air bubbles are present, open the bottle, add
-------
additional sample, and reseal. The sample must
remain hermetically sealed until it is analyzed.
205 Preservation of Volatile Organics
205.1 Preservative (sodium thiosulfate or sodium
bisulfite) is used to stabilize samples
containing residual chlorine. The production of
chloroform and other haloforms continues in such
samples if they are not stabilized. Waste
streams that have been treated with chlorine
should be tested on-site to determine whether or
not preservative is needed. If preservation is
required, collect both preserved and non-
preserved samples. Wrap the samples with water
proof packing material, place in an insulated
chest and ice at 4°C. Maintain at 4°C
during transport and storage prior to analysis.
206 Grab Samples for Volatile Organics Q^rgeables)
206.1 Collect grab samples (minimum of one per day)
for the analysis of volatile organics. Collect
samples from the raw process discharge, the
treated effluent, and the treated effluent after
chlorination, when chlorination is practiced.
It is recommended that the samples be collected
from mid-channel at mid-depth. Samples should
be collected at a turbulent, well mixed section
of the channel.
-------
Quality Assurance and Quality Control Procedures for
Verification Analysis For Organic Priority Pollutants
110 Scope
111 These procedures are provided for use by laboratories performing
verification analyses using EPA Methods 624 and 625. Verification
analyses require quantitative determinations with known precision
and accuracy, so that these data will be acceptable for use in
developing effluent limitation guidelines. To provide data with
this degree of reliability, a strong quality assurance and quality
control program is presented. These procedures are designed to
produce data with known precision and accuracy so that a
determination of confidence can be placed in the data. Quality
Assurance (QA) is the total program for assuring the reliability of
the monitoring data. Quality control (QC) is the routine
application of procedures for controlling the measurement process.
112 Initially, the methodology must be validated for each industrial
subcategory. The requirement for validation of each subcategory is
based on the unique nature of the wastewater associated with most
of the subcategories. Since the effluent from treatment is to be
used for setting control limits, it will be used to develop initial
validation data for the method prior to routine sampling and
analyses. Based on screening data, a particular subcategory may
not require verification analyses of all four fractions (Volatiles,
Base/Neutrals, Acids, and Pesticides) or for all of the individual
Consent Decree compounds, in which case, the method requires
validation only for the fraction or the selected compounds of
interest.
'-14
-------
113 The results of the validation analyses will be used to provide
information with which to judge a laboratory's ability to interpret
and implement the method for each industrial subcategory. Intial
QC limits for precision and accuracy will be established using
these results, and then used in subsequent analyses as control
limits. A numerical example is given in Appendix C.
114 After the method is validated for each subcategory and routine
analysis begins, continuing QA/QC will be required to ensure that
the subsequent analyses are within the established control limits.
115 Prior to developing initial method validation and a continuing
quality control program, the analyst (individual or group if team
approach is used) must demonstrate the ability to perform the
required analyses. If a laboratory has not established precision
and accuracy criteria for clean water, the laboratory must perform
replicate analyses of clean water as prescribed in section 121.
Such data must be presented to the responsible EGD project officer
prior to the beginning of the verification phase.
120 Routine Quality Assurance and Quality Control
121 Preliminary Clean Water Precision and Accuracy
121.1 Before any work is begun on actual field samples, a labora-
tory must demonstrate its ability to properly perform the
liquid-liquid extractions, the gas purge extractions, and
the required chromatography. Clean water spikes are ana-
lyzed to demonstrate the laboratory's ability to implement
-15
-------
Methods 624 and 625, and to establish the baseline precision
and accuracy criteria for the method in that laboratory'.
121.2 Procedure:
121.2.1 Prepare "organic-free" water for use in determining
preliminary precision and accuracy according to the
procedures given in Methods 624 and 625.
121.2.2 Spike four replicates of clean water with each
compound of interest at a concentration
approximately equal to 10 times the limit of
detection. In addition, spike all purgeable
aliquots with a minimum of three surrogate
standards at a level of 100 ug/1. For extractable
organics, each replicate must be one liter; each
purgeable sample requires at least 100 ml. Do not
dose purgeables with more than 20 ul of an
alcoholic standard per each 100 ml of water.
Analyze spiked solutions according to Method 624 or
625.
121.3 Precision - For each parameter, use the resulting observed
values of the spikes (0^ 02, 03, and 04) to
calculate the standard deviation (S) of the replicates
according to Equation 1.
n(n-l)
n = number of replicates
-16
-------
121.4 Accuracy - For each parameter, use the resulting observed
values of the spikes (0-p C^, Og, and 04) to
calculate the mean percent recovery (P) of the method
according to equation 2.
Eq. 2
Where:
P = 100 (£ 0-j )
nT
n a number of replicates
T = true value of the spike
121.5 The precision and accuracy data shall be documented for the
record as evidence that the laboratory can properly perform
the extractions and chromatography essential for methods 624
and 625.
122 Method Blank - The method blank is defined as an appropriate volume
of "organic-free" water which has been processed exactly as the
sample (including glassware, reagents, solvents, etc.). Reagents
or solvents having background levels that interfere with the
compounds to be determined must be purified and shown to be
acceptable or replaced with some that are acceptable prior to
proceeding with analyses. Problems encountered and corrective
actions taken shall be documented and reported for the record.
122.1 For the extractable fractions (Base/Neutral, Acid, and
Pesticide) the method blank requires extraction of 1-liter
"organic-free" water. A method blank must be extracted for
each set of field samples extracted at a given time (at
least one method blank per 20 field samples analyzed) and
lt-17
-------
whenever a new source of reagent or solvent is introduced
into the analytical scheme. The method blank can be
screened by GC-FIC. Analysis by 6C-MS is required only if
GC-FID analysis of the field blank gives any peaks larger
than the internal standard peak.
122.2 For the volatile fraction, 5 ml of "organic-free" water
should be analyzed by the purge and trap methodology only if
positive interferences are noted during the analysis of a
field blank. If positive interference still occurs, repeat
the method blank analysis. If interference persists,
dismantle the system, thoroughly clean all parts that
contact the sample, purge gas and carrier gas. Replace or
repack the sorbent trap and change purge and carrier gas.
123 Field Blank - The field blank is defined as an appropriate
volume of "organic-free" water which has been to the sampling
site and back to the analytical laboratory in a container and
bottle identical to the type used to collect the samples.
Field blanks and samples must be shipped in separate
containers. When received in the lab, the field blank is
dosed, extracted and concentrated as if it were an actual
sample.
123.1 For the extractable factors (Base/Neutral, Acid, and
Pesticide), the field blank may be screened by GC-FID or
GC-EC (Pesticides) only). Full GC-MS analysis is to be
performed if the screening analysis gives any peaks larger
than the internal standard peak.
ISf-18
-------
123.2 For the volatile fraction, analyze a 5-ml field blank after
each sample analysis. Follow the guidelines in 121.2 if
positive interferences are noted during the analysis of a
field blank.
124 GC-MS Calibration Check
124.1 For the Base/Neutral-Acid or Pesticide fraction, daily
analyze decafluorotriphenylphosphine (DFTPP) by injection
into the GC inlet. See Reference 1. (DFTPP must a-lso be
run after any mass spectrometer tuning is done). This
calibration check may be done in conjunction with Section
125. The requirement is that 50 nanograms of DFTPP injected
must meet the specifications prescribed. Table 1 lists the
DFTPP key ions and ion abundance criteria.
124.2 For the volatile (purgeable) fraction, analyze
4-bromo-fluorobenzene (BFB) daily by direct injection into
the GC/MS. The requirement is that 20 nanograms of BFB
injected must meet the prescribed specifications. Table 2
lists the BFB key ions and ion abundance criteria. This
calibration check may be done in conjunction with Section
125.
125 GC Performance Check
125.1 SP-2250 column - At the beginning of each day that
base/neutral or pesticide fraction analyses are to be
performed, inject 100 nanograms of benzidine (for
base/neutral analyses) or aldrin (pesticide analyses) either
separately or as part of a standard mixture that may also
contain 50 ng of DFTPP. Performance for base neutrals
-------
is acceptable if the tailing factor for benzidine is less
than 3. Performance for the pesticides is acceptable if the
tailing factor for aldrin is less than 2. Calculation of
the tailing factor is given in Reference 2 and described in
Figure 1. Retain all plots of performance evaluation as
evidence of valid performance.
125.2 SP-1240 DA Column - At the beginning of each day that acid
fraction analyses are to be performed, inject 100 nanograms
of pentachlorophenol either separately or as part of a
standard mixture that may also contain DFTPP. Performance
is acceptable if the tailing factor is less than 5.
Calculation of the tailing factor is given in Reference 2
and described in Figure 1. Retain all plots of performance
evaluation as evidence of valid performance.
125.3 Carbopak C with Carbowax 1500 or Carbopak B with SP-1000
column. At the beginning of each day that purgeables
analysis are to be performed, inject 20 nanograms of BFB
either separately or as part of a standard mixture.
Performance is acceptable if the tailing factor is less than
2. Calculation of the tailing factor is given in Reference
2 and described in Figure 1. Retain all plots of
performance evaluation as evidence of valid performance.
126 Performance Evaluation - Once each 20 working days; dose, extract,
and analyze a performance evaluation sample (available from
EMSL-Cincinnati). Retain the results as evidence of valid
performance.
20
-------
127 Saving Extracts -If the final extract is to be retained, or shipped
to another location, follow the appropriate guidelines given in
Appendix A.
128 Archiving GC/MS Data - If the 6C/MS data are to be archived on
magnetic tape, follow the guidelines in Appendix B.
130 Method Validation
131 Extractable Organics fBase/Neutrals-Acids and Pesticides) - The
following procedures are to be applied, separately, to samples
being analyzed for the Base/Neutral-Acid group of compounds and to
samples being analyzed for the Pesticides group. The analyses
shall be performed according to the procedures given in Method
625. The validation studies must be performed under the same
conditions ordinarily applied to the samples of a given
subcategory. That is, if separatory funnels are routinely used for
extraction of the samples, the study must be conducted using
separatory funnels. If continuous extractors are used for routine
analysis of the subcategory, the validation study must be performed
using the same type of continuous extractors.
131.1 Sample pretreatment - The laboratory will receive a 24-hour
composite sample of adequate volume to carry out the
validation study and one field blank taken as described in
the sampling protocol on the same day from the same source.
Mix composite sample and withdraw a 1-liter aliquot for
analysis. Vigorously mix the sample by some type of
stirring device. Withdraw aliquots, while stirring, into a
1-liter graduated cylinder, using a siphon made of glass or
- 21
-------
Teflon. Measure and record the volume. Transfer the
aliquot to a 2-liter separatory funnel or continuous
extractor for spiking. Initially analyze a 1-liter aliquot
of the sample to determine the sample background so that
proper spiking levels can be selected for 131.2.1. The
remainder of the composite sample should be stored at 4°C
until the validation study is begun. At the same time,
analyze a 1-liter aliquot of the field blank. Choose three
levels of priority pollutant spikes to cover the expected
concentration range of the samples in the subcategory.
131.2 Preparation of Aliquots for Validation Study - Withdraw
twelve 1-liter aliquots from the stirred composite sample as
described in 131.1. Separate into three groups of four.
131.2.1 Spiking of Aliquots - Spike two aliquots of each
group with surrogate standards only. The other two
aliquots are spiked with surrogate standards plus
the priority pollutant standards of interest at one
of the concentration levels (See Figure 2). Repeat
this process for each group of aliquots. Select
the three spiking concentrations for the priority
pollutants based on the results of the background
analysis obtained in 131.1. If the initial
background level for a particular priority
pollutant is X, select the three spiking levels to
give final concentrations of 2X, 10X, and 100X. If
X equals 15 ug/1, dose with 15, 135, and 1485 ug
-DT-22
-------
per liter. This gives final concentrations of
priority pollutants of 30, 150, and 1500 ug/1.
Spike each 1-liter replicate with each surrogate at
a level of 100 ug/1. (Note: Consideration should
be given to the water solubility of the compounds
being spiked when selecting the spiking
concentration levels.)
131.2.2 Prepare spiking standards in concentrations such
that no more than 5 ml of spiking solution is added
for each liter of sample. This will ensure that
the solubility of the standard in water will not be
significantly affected by the added organic
solvent. Add the spiking solution to the sample
aliquots in the separatory funnel using a transfer
pipet. After adding the spikes, thoroughly mix the
samples and after one hour at room temperature
proceed with the extraction. If continuous
extractors are used, it may be necessary to spike
the 1 liter aliquots before they are placed into
the extractor. Place the aliquot in a separatory
funnel or a clean bottle, spike, and transfer to
the extractor. . The bottle must be rinsed with
solvent. Wait an hour and begin the extraction.
(Note: Extractions of all samples must begin
within 48 hours of receipt in the laboratory.)
-------
131.3 Use of the Data from Spiked Samples in Analyses - The data
obtained from the determination of priority pollutants are
used to calculate the precision and accuracy of the method
and to establish control limits for the individual compounds
of interest. Surrogate spikes are added to every sample to
provide quality control on every sample by monitoring for
matrix effects and gross sample processing errors. The
surrogate is not used as an internal standard for
quantification purposes. Suggested surrogate spikes are
given in Section 6.4 of Method 625. If verification is
needed for only one fraction, only the surrogates for that
fraction shall be added.
131.4 Extract and analyse all aliquots as directed in Method 625
or other appropriate EPA methods„
131.5 Calculation of Precision and Accuracy - The precision of the
method may be calculated from the data obtained during the
validation study. There are three spiked concentration
levels of priority pollutants as outlined in Figure 2. The
method precision for the background level of priority
pollutants occurring in the sample may be calculated from
the three pairs of replicate aliquots which are spiked only
with surrogate standards (Al, A2; Bl, B2; Cl, and C2. See
Figure 2). The precision and accuracy for the surrogates
may be calculated from all twelve replicates since the
spiking level is constant for both sets of six samples. The
precision and accuracy for the surrogates may be calculated
-------
for each set of six samples if there is an effect due to the
added priority pollutant spike. Similarly, the precision
and accuracy at each spike level of the priority pollutants
may be determined from the two replicate aliquots that
received that spike (Dl, D2; El, E2; Fl, and F2 See Figure
2).
a. Precision
Calculate the range (R) for each pair of replicate aliquots
ie., duplicate analyses, according to equation 3:
Eq. 3 R =|x] - X2|
Where:
X, and %2 are eacn an analytical result from two
replicate aliquots.
The concentration level related to R can be represented as
in equation 4.
Eq. 4 I = (X1 + X2)
2
Where:
X is the mean of the duplicate analyses, X, and
V
For any group of n duplicate analyses that are considered
similar to each other, their ranges (Rj and means (X.).
where i = 1 to n, can be used to estimate the critical
difference (Rc) between similar future duplicate analyses
or any specific concentration level (C). Calculate R as
shown in equation 5.
37- 25
-------
Eq. 5 R - 3.27(C)
n
From these data develop a table of R values for various
C
concentration (C) values that span the concentration range of
interest. Use these Initial critical difference values to
judge the acceptability of succeeding duplicate results
generated under the same conditions. Revise and update as
additional duplicate data becomes available. When more than.
15 pairs of duplicates are available within any specific
concentration level C, R should be calculated directly
from the average range of these duplicates alone. Equation 5
reduces to
R = 3.27(C) £ R-J
n (.1=1
b. Accuracy for Priority Pollutants
Calculate the background concentration of
priority pollutants occurring in each of the
field composite samples. The calculation is
similar to equation 4, but there are six
pieces of data (Al, A2, Bl, 82, Cl, C2)
available for this calculation as shown in
Figure 2. Therefore, the calculation is as
shown in equation 6.
6
Eq. 6 *= ( £ X.)
"- 26
-------
Where:
«•
X is the mean X., i-1 to 6 are the analytical
results for the six 1-liter aliquots of a single
composite sample spiked with surrogates only.
Calculate the recovery of each priority pollutant
in each of the 1-liter aliquots spiked with
priority pollutants (01, D2. El, E2, Fl and F2)
according to equation 7:
Eq. 7 P » 100 (Z - I)
T
Where:
P is the percent recovery of the spike
Z is the analytically determined
concentration of the priority pollutant in
the spiked aliquot
X is the mean background concentration of
the priority pollutant and
T is the true value of the spike.
Determine the percent recoveries for each priority
pollutant at all of its concentration levels. If
there is no significant difference between the
percent recoveries for the various concentration
J7-27
-------
levels, all n of the percent recovery values may be
treated together as in equations 8 and 9. If some
of the percent recovery values are significantly
different, each group of similar percent recover ie.s
must be treated independently to develop its own
characteristic mean percent recovery (P) and its
associated standard deviaion (S ).
n
Eq. 8 "P * £ Pi
Where:
P is the mean percent recovery
P is an individual percent recovery value
n is the number of observations at this
concentraton level
'28
-------
n(n-l)
c. Accuracy for Surrogates
Proceed exactly as with priority pollutants in 131.5b above,
keeping the following two differences in mind: there is no
background concentration and there are six sets of duplicate
analyses for the surrogate spikes; three sets spiked with
priority pollutant (Dl, D2; El, E2; Fl, F2) and three sets
without (Al, A2; Bl, B2; Cl, C2), see Figure 2. Calculate
the percent recovery as shown in equation 10.
Eq. 10 P = 100 Z
Where:
P is the percent recovery of the surrogate spike.
Z is the measured value of the surrogate spikes
in the aliquot.
T is the calculated or true value of the
surrogate spikes added to the sample.
Calculate the mean percent recovery (P) and the standard
deviation (S ) of the percent recovery of the surrogate
spikes in all of the sample aliquots according to equations
8 and 9.
132 Volatile Organics (Purgeables)
132.1 All individual grab samples collected during the specified
time period shall be composited in the laboratory, spiked
and analyzed as soon as possible. Significant losses of
17 29
-------
some of the purgeable priority pollutants will occur in 1 to
2 days. However, in a nonreactive matrix, most are stable
up to 14 days. The method validation study requires twelve
5-ml aliquots from each composited sample. Carefully
transfer the remaining volume of composited sample back to a
clean vial or vials and seal with no headspace as done when
collecting a sample. These should be held at 4°C until it
is determined that there is no further need for the sample.
Figure 3 summarizes the validation study for volatile
organ ics.
Caution: Prepare only as many sample aliquots as can be
analyzed in the working day. This may mean that each of the
three concentration levels will be analyzed on different
days.
132.2 Pretreatment of Grab Samples - Individual grab samples
should be composited according to the following procedure:
a. Composite only grab samples of equal volume.
b. Carefully pour the contents of all individual grab
samples collected from a given source during the
specified time period into a 1000-ml round-bottom flask
which is chilled in a wet ice bath.
c. Stir the mixture gently with a glass rod for
approximately one minute while in the ice bath.
d. Carefully fill 13 clean 40-ml vials or three 120-ml
vials and four 40-ml vials with composited sample.
e. Take one 40-ml vial for immediate analysis to determine
the background of the purgeable priority pollutants.
30
-------
f. Store the remaining vials at 4°C until the validation
study is begun.
132.3 Spiking levels for priority pollutants and surrogate
standards - The spiking levels of the priority pollutants
are determined by the background (X) in the samples. The
low level spike will give a final concentration that is 2
times the background level. The intermediate and high level
spikes will give final concentrations that are 10 and 100
times the background level. Concentrations in excess of
1000 ug/1 are likely to flood the gas chromatographic
column. Therefore, the total concentration (background plus
spike) of each individual priority pollutant should not
exceed 900 ug/1. Even at this level the solubility of the
compounds in the sample must be considered. The spiking
level for all surrogate standards should be 100 ug/1.
132.4 Spiking Procedures
132.4.1 Preparation of Spiking Standards - Prepare
methanolic stock standard solutions of the priority
pollutants and the surrogate standards according to
the directions given in Method 624.
From the methanolic primary dilutions prepare
secondary aqueous spiking mixtures of the surrogate
standards so that 20.0 ul of the primary standard
solution, diluted to 50.0 ml in organic free water
will permit adding 5 ul of the resulting solution
to the 5 ml sample giving the desired surrogate
31
-------
concentration level of 100 ug/1.
Prepare spiking mixtures of the priority pollutant
standards in methanol so that 20.0 ul of the
solution added to 100.0 ml of sample will give the
desired concentration levels.
132.4.2 To minimize the solubility effect of methanol on
the constituents to be measured, do not inject more
than 20 u.l of spiking solution per 100 ml of
sample. Never use a pipet to transfer samples or
aqueous standards that are to be analyzed for
volatile purgeable compounds. Transfer samples by
pouring into the receiving vessel.
132.4.3 Spiking the Sample Aliquots - Take one of the
120-ml or 3 of the 40 ml sample aliquots from cold
storage, equilibrate to room temperature, and fill
a 100 ml volumetric flask to mark with the sample.
Rapidly inject 20 ul of the methanolic solution of
priority pollutant spiking standard (concentration
2X) into the expanded area of the flask below the
neck. Stopper and mix by gently inverting the
flask three times. Fill two 5-ml syringes with
spiked sample from the flask as directed in the
analytical protocol. Open the valve of the syringe
and inject 5 ul of the surrogate standard spiking
solution. Inject the sample aliquot into the
purging device and analyze according to Method 624.
'4? 32
-------
Take one of the 40-ml sample aliquots from cold
storage, equilibrate to room temperature and fill
two 5-ml syringes with the sample as directed in
Method 624. Spike 5 ul of the surrogate standard
water solution (concentration 100 ug/1) into the
syringe through the valve giving a final
concentration of 100 ug/1. Inject the sample
aliquot into the purge device and analyze according
to Method 624. See Figure 3. Repeat this
procedure twice, giving three sets of analyses of
two samples spiked with surrogate standards only
and two samples spiked with surrogate standards and
priority pollutants.
132.5 Calculation of Precision and Accuracy - The precision and
accuracy for the purgeable priority pollutants and the
surrogate standards are calculated as directed for the
semivolatile solvent extractable compounds in paragraphs
131.5a, b, and c.
140 Continuing Quality Assurance and Quality Control
141 Extractable Organics (First Day Samples) - The following
procedures should be applied to the first day samples of the
pesticides group Base/Neutral-Acid group. They should be carried
out on each first day composite sample for each new subcategory.
This includes samples of the influent to treatment, the effluent
from treatment and the supply water. An outline diagram for first
'IT-33
-------
day ongoing qualityassurance samples is given in Figure 4.
141.1 Withdraw three 1-liter aliquots of the composite sample-
according to the procedure in 131.1.
141.2 Spiking the Sample Aliquots - Spike one of the aliquots with
priority pollutant standards plus the surrogate standards
and two of the aliquots with surrogate standards only.
141.3 Add a spike sufficient to approximately double the
background concentration of the priority pollutants as
determined in 131.5b. If the original concentration is
higher than the midpoint of the calibration curve, then the
concentration of the spike should be approximately one-half
the original concentration. Surrogate spikes as specified
in 131.3 should be added to all three aliquots from each
sample at a concentration level of 100 ug/1.
141.4 Analyze according to Method 625.
141.5 Calculations of Precision and Accuracy
a. For the first day samples, calculate the precision of
the duplicate analyses (X, and X-) from the two
1-liter aliquots for the priority pollutants background
and the surrogate standards. Calculate the range (R)
of the results according to equation 11.
Eq. 11 R =
Xl -
The concentration of each compound is represented by
the mean of the duplicate values. Calculate the mean
(X) according to equation 12.
Eq. 12 X = (Xi + X?)
- 34
-------
Refer to the table of critical range values developed in
131. 5a, to find the concentration (C) nearest to X.
Use this R to evaluate the acceptability of R from
Eq. 11. If R is greater than R , the system precision
is out of control and the source of this unusual
variability should be identified and resolved before
continuing with routine analyses. After correcting the
source of this unusual variability, reanalyze the sample
if possible. Record the results of all duplicate
analyses and periodically (after 5-10 additional
duplicate results are obtained), revise, update, and
improve the table of critical range values.
b. Accuracy for Surrogate Spikes
Calculate the recovery of the surrogate spikes in the
duplicates according to equation 13.
Eq. 13 P - 100 Z
Where :
P is the percent recovery.
Z is the analytically determined concentration of the
surrogate standard spikes.
T is the true value of the surrogate standard spikes
added in 132.4.3.
If the percent recoveries are not within the interval P
+_ 3S as determined in 131. 5c, the system should be •
checked for problems. If problems exist, they must be
' 35
-------
resolved before continuing with routine analyses.
Record the recovery of all surrogate spikes and
periodically (every 5 to 10 additional data points),
revise, update and improve the recovery criteria.
c. Accuracy for Priority Pollutant Spikes
Using the results obtained from the 1-liter aliquot of
composite sample spiked with surrogate standards and
priority pollutant standards, calculate the recoveries
of the priority pollutants according to equation 14.
Eq. 14 P = 100 (Z-X)
T
v Where:
P is the percent recovery
Z is the analytically determined
concentration of the priority pollutant
spikes
T is the true value of the priority
pollutant spikes added in 132.4.3, and
X is the mean concentration of the priority
pollutant background determined by equation
9.
If the percent recovery is not within the interval
of P _+ 3S , as determined in 131.5b the system
should be checked for problems. If problems
exist, they must be resolved before continuing
-OT 36
-------
with routine analyses. Record the recovery of all
spikes and periodically revise, update, and
improve the accuracy criteria.
142 Extractable Organics - (Subsequent Day Samples) - The
following procedures should be applied to each subsequent day
sample of the Base/Neutrals-Acids group and the pesticide
group. A flow diagram for each subsequent day ongoing
quality assurance samples is given in Figure 5.
142.1 Withdraw a one-liter aliquot as directed in 131.1
142.2 Spike the aliquot with surrogate standards at a
concentration of 100 ug/1.
142.3 Analyze according to Method 625.
142.4 Determine the percent recovery of the surrogate
standards using Equation 10. If the percent recovery
is outside the interval P + 3S as determined in
131.5c, the analytical system should be checked for
problems. If problems exist, they must be resolved
before continuing further sample analyses.
142.5 A field blank for the day must be analyzed according
to Method 625. If priority pollutants are found and
quantified, the values for the field blank should be
noted and reported along with sample results. If
significant interference problems occur, the method
blank must be analyzed to determine if interference
was introduced in the
"5SL 37
-------
field or the laboratory. Appropriate action must
be taken to eliminate the problem before continuing
with the analysis of routine samples.
143 Volatile Organics (First Day Samples) - Continuing quality
assurance for the purgeable volatile organics should be
applied to samples from influent to treatment, effluent from
treatment, and supply water. The following procedures should
be carried out on each first day composite samples from the
three sample sources from each subcategory. An outline is
given in Figure 4.
143.1 Composite all relevant grab samples as described in
Section 132.2. Prepare six 5-ml aliquots for analysis.
143.2 Spike two aliquots with the priority pollutant
standards at a level twice that determined in Section
132.5 and the surrogate standards using the procedures
in Section 132.3 and 132.4. Spike four 5-ml aliquots
with surrogate standards only as in 132.3 and 132.4.
143.3 Analyze one of the duplicates spiked with priority
pollutants and surrogate standards and two of the four
replicates spiked with surrogate standards only. The
remaining spiked aliquots are analyzed only if a
problem is encountered with the analysis of the first
set of aliquots.
f-38
-------
143.4 Analyze the spiked aliquots according to Method 624.
143.5 Calculate the precision and accuracy as directed for
the semivolatile solvent extractables as directed in
141.5.
144 Volatile Organics (Subsequent Days Samples) - The following
procedures should be applied to each subsequent day sample of
the volatile organics group. An outline is given in Figure 5.
144.1 Composite the relevant grab samples as described in
Section 132.2. Prepare two 5-ml aliquots for analysis.
144.2 Spike both aliquots with surrogate standards only to
give a concentration of 100 ug/1.
144.3 Analyze one of the aliquots according to Method 624.
The other aliquot is analyzed only if a problem is
encountered.
144.4 Determine the percent recovery of the surrogate
standards using Equation 10. If the percent recovery
fs outside the interval P + 3 S_ as determined in
~ P
131.5c, then the analytical system should be checked
for problems. If problems exist, they must be
resolved before continuing further sample analysis.
144.5 Analyze a field blank representing the same day that
the samples were collected. Follow the guidelines
given in 142.5.
39
-------
200 Sampling Procedures for Verification Phase
201 These procedures are given as an example for the collection
of samples.
202 Extractable Organics (Base/Neutrals-Acids and Pesticides)
202.1 Initial QA/QC - Three 2*s-galien samples must be taken
concurrently over the initial period of sampling (24
hrs.) effluent from treatment if both the
Base/Neutrals-Acid group and the Pesticide group are
to be determined. If only one of the two groups is to
be determined, collect two 2H-gal1on samples or one
5-gallon sample. The samples can be collected using
two or more automatic Isco samplers that have the
sample lines linked together to ensure close replicate
samples.
a. Cluster the inlet tubes to ensure that samples
are taken geographically close.
b. Link the compositors in a master/slave
arrangement, wherein one "master" sampler
supplies the sample interval timing for the
"slave" sampler(s). The Master sampler
operates in a TIME mode and the interval
between samples is set on the SAMPLE RATE
switch (Model 1580) or the SAMPLE INTERVAL
switch (Model 1680). The "slave" sampler(s)
operate in a FLOW mode, with the FLOW
MULTIPLIER switch (Model 1580) set on 1, or
i.r-40
-------
the SAMPLE INTERVAL switch (Model 1689) set to 1 FLOW
PULSE. When the master sampler times out and collects
a sample, a flow pulse is sent to the slave
sampler(s), causing them to collect samples
simultaneously with the master sampler. (Note that
depressing the MANUAL ADVANCE pushbutton on the master
sampler will cause the slave sampler(s) to collect a
sample; therefore, the slave sampler(s) should be left
in STANDBY until the master sampler set-up has been
completed). No modification to the equipment is
necessary. Use of two "slave" samplers require a "Y"
connect cable assembly for the master samples and two
sampler-flow meter cables to connect to the slave
samplers' . Samplers should be checked before use
in the field to ensure that each collects the required
volume of sample +_ 5%.
202.2 Continuing QA/QC - One 2%-gallon sample is required
for the continuing QA/QC work.
Available from Isco, Environmental Division, Building
1020, Lincoln Air Park West, Lincoln, Nebraska, 68524.
41
-------
202.3 Blanks - One 2-gallon aliquot is needed for the preliminary
QA/QC. This should be obtained by flushing the organic-free
water through all Isco samplers used. One 1-gallon blank is
needed for continuing QA/QC work.
203 Volatile Organics (Purqeables)
203.1 Sampling Precautions
a. Collect 2 replicates from each sample source (i.e.,
duplicate samples).
b. Fill the sample bottles in such a manner that no air
bubbles pass through the sample as the bottle is being
filled.
c. Seal the bottles so that no air bubbles are entrapped in
them.
d. Maintain the hermetic seal on the sample bottle and keep
at 4°C until time of analysis.
203.2 Initial QA/QC - A minimum of 1000 ml (twenty-five 40-ml
vials) must be collected over the relevant time period at
each sample point.
203.3 Continuing QA/QC - A minimum of 200 ml (five 40-ml vials)
must be collected over the relevant time period at each
sample point.
203.4 Blanks - Three 40-ml "trip" blanks (field blanks) are
required for both the initial and continuing QA/QC work.
JST-42
-------
REFERENCES
1. Eichelberger, O.W., I.E. Harris, and W.L. Budde, "Reference Compound to
Calibrate Ion Abundance Measurements in Gas Chromatography-Mass
Spectrometry Systems," Anal. Chem. 47. 995-1000 (1975).
2. McNair, H.M., and E.J. Bonelli, "Basic Gas Chromatography," p.52,
Consolidated Printing, Berkeley, CA 1969.
IT'43
-------
TABLE 1
OFTPP Key Ions and Ion Abundance Criteria
Mass Ion Abundance Criteria
51 30-60% of mass 198
68 less than 2% of mass 69
70 less than 2% of mass 69
127 40-60* of mass 198
197 less than 1% of mass 198
198 base peak, 100% relative abundance
199 5-9% of mass 198
275 10-30% of mass 198
365 greater than 1% of mass 198
441 less than mass 443
442 greater than 40% of mass 198
443 17-23% of mass 442
IF-44
-------
TABLE 2
BFB Key Ions and Ion Abundance Criteria
Mass Ion Abundance Criteria
50 20-40% of mass 95
75 50-70% of mass 95
95 base peak, 100% relative abundance
96 5-9% of mass 95
173 less than 1% of mass 95
174 70-90% of mass 95
175 5-9% of mass 95
176 70-90% of mass 95
177 5-9% of mass 95
- 45
-------
TAILING FACTOR= —
AB
Example calculation: Peak Height = DE = 100 mm
10% Peak Heights BD =10 mm
Peak Width at 10% Peak Height s AC = 23 mm
AB = 11 mm
12
Therefore: Tailing Factor = — =1.1
11
Figure 1 . Tailing factor calculation
46
-------
Figure 2
Summary of Initial Validation
Analyses for Extractable Samples
1. Composite the Replicate Samples.
2. Withdraw a 1-liter aliquot. Store composite at 4°C.
Separate into three groups of 4 aliquots each.
3. Determine X the background concentration of each
Priority Pollutant.
4. Withdraw twelve 1-liter aliquots from the composite.
5. a) Dose 2 of aliquots with surrogate standards only at
100 ug/1. Label as Al and A2. 2
b) Dose 2 aliquots with surrogate standard at 100 ug/1
and priority pollutants to give a concentration
level of 2X. Label as Dl and 02. 2
c) Analyze Al, A2, Dl, and D2 using Method 625.
6. Repeat 5a, b, and c. Label surrogate standards only
as Bl and 82. Use 10X level for priority pollutants.
Label as El and E2. 4
7. Repeat 5a, b, and c. Label surrogate standards only
as Cl and C2. Use 100X level for priority pollutants.
Label as Fl and F2. 4
Liters GS/MS
Used Runs
TOTAL 13 13
JT-47
-------
Figure 3
Summary of Initial Validation
Analyses for Purgeable Samples
5-ml
Syringes GC/MS
Used Runs
1. Composite the grab sample.
2. Fill 13 clean 40-ml vials or 3 clean 120-ml vials +
4 clean 40-ml vials with composite, cap, and store at 4°C.
3. a) Fill a 5-ml syringe from one 40-ml vial. 1
b) Analyze and determine X the background concentration
of each priority pollutant.
4. a) Fill two 5-ml syringes from one 40-ml vial 2
b) Dose with surrogate standards at 100 ug/1.
Label as Al and A2.
c) Analyze Al and A2 using Method 624.
d) Fill a 100 ml volumetric to mark using one 120-ml
or three 40-ml vials.
e) Dose with 20 ul of priority pollutants to give a
concentration of 2X.
f) Fill two 5-ml syringes from the 100-ml volumetric. 2
g) Dose each syringe with surrogate standards at a
concentration of 100 ug/1. Label as Dl and D2.
h) Analyze Dl and D2 using Method 624.
5. Repeat 4. Label surrogates only as 81 and 82. Use 10X
level for priority pollutant. Label priority pollutants
plus surrogate standards as El and E2. 4
6. Repeat 4. Label surrogates only as Cl and C2. Use 100X
level for priority pollutants. Label priority pollutants
as Fl and F2. 4
TOTAL 13 13
35T-48
-------
Figure 4
Summary of Ongoing Quality Assurance
for First Day Samples
Liters GC/MS
Extractables Used Runs
1. Composite the Sample.
2. a) Withdraw three 1-liter aliquots. 3
b) Dose two aliquots with surrogate standards
only at 100 ug/1.
c) Dose one aliquot ith surrogate standards and
the priority pollutants, of interest to give a
concentration of 2X, Section 131.5.
d) Analyze using Method 625.
TOTAL ~3~
5 ml
syringes GC/MS
Purgeables Used Runs
1. Composite the Sample.
2. a) Fill 4 clean 4-ml vials or 1 clean 120-ml vial
+ 1 clean 40-ml vial with composite. Store at 4°C.
3. a) Fill four 5-ml syringes from one 40-ml vial. 4
b) Dose each with surrogate standards at 100 ug/1.
c) Fill a 100 ml volumetric to mark using the 120-ml vial
or three 40-ml vials.
d) Dose with 20 ul of priority pollutants to give a
concentration of 2X, Section 132.5.
e) Fill two 5-ml syringes from the 100 ml volumetric. 2
f) Analyze two 5-ml syringes containing surrogate
standards only and one 5-ml syringes containing surrogate 3
standards and priority pollutants using method 624.
TOTAL ~5~ ~T~
49
-------
Figure 5
Summary of Ongoing Quality
Assurance for Subsequent Day Samples
Extractables
1.
2.
Composite the sample.
Liters
Used
1
GC/MS
Runs
a) Withdraw a 1-liter aliquot.
b) Dose with surrogate standards only at 100 ug/1.
c) Analyze using Method 625.
Subtotal ~~T
TOTAL (x29 days) 29
5-ml
Syringes
Used
Purgeables
1. Composite the sample.
2. a) Fill two 5-ml syringes with composite.
b) Dose with surrogate standards at 100 ug/1.
c) Analyze one 5-ml sample.
Subtotal
TOTAL (x29 days) 58
GC/MS
Runs
*Assuming that field blank shows no priority pollutants.
If field blank were to be analyzed by GC/MS each subsequent
day, the total would be 58.
50
-------
Appendix A
Storage of Final Extracts
Each extract is to be washed out of its container into a 10-ml glass ampul
and brought to 5 ml ^ 1 ml. Methylene chloride is the solvent for the
base-neutrals and acids, hexane for pesticides. The cooled ampuls (-20°C)
are to be sealed in a rounded-off, fire polished manner, i.e., no thin sharp
peaks of glass that are easily broken on handling and shipping. After
sealing the ampuls, put an indelible mark at the solvent level. Securely
attach a label or tag that gives:
Type of fraction (base-neutral, etc.)
Industrial category
Name (of plant, city and state)
Specific source or stage of treatment
Date sampled
Date sealed
Name of contractor and analytical laboratory
Wrap the ampuls in packing material to prevent breakage and mail or ship
them postpaid at ambient temperature. When the samples are safely in
ampuls, the remainder of the composite sample may be discarded.
51
-------
Appendix B
Archiving GC/MS data on Magnetic Tape
When the analyses are completed, transfer GC/MS raw data to magnetic tape as
described below. All raw GC/MS data is to be retained on 9-track magnetic
tape. When a tape is filled, deliver the tape to the Environmental
Protection Agency for storage and later evaluation. The tape format is:
Type - 9-track, 800 BPI, 2400 foot reels
Record Length - 80
Block size - multiple of 80 (specify)
Code - ASCII or EBCDIC
The data format must be approved by EPA. At the present time there are
formats used by commercially available equipment that have been approved by
EPA. Before submitting tapes to EPA, a sample data tape with appropriate
documentation should be submitted for approval of the format and content.
Each GC/MS run on tape should contain the following header information at
minimum.
1. Identifying run number
2. Data and time of sample run
3. Run description. Include GC column used, temp, program
(e.g. 70-270 @8), concentration factor of extract, run type
(e.g. Base/Neutral, Acid, VOA, Direct Aqueous injection).
4. Scan time in seconds.
5. Mass range scanned.
"TV"- 52
-------
Each scan within each run should contain at minimum:
1. Identifying run number.
2. Scan number
3. Date
4. Time of scan (time from start of run).
5. Total intensity of scan (not normalized).
6. If intensities are normalized, the mass and raw intensity of
the base peak for the scan.
7. Mass/intensity pairs for the scan. Intensities may be
normalized or unnormalized. Normalized values must have a
dynamic range of at least 1000; unnormalized values must
reflect the actual absolute intensity value. Masses may be
nominal interfer values.
Each magnetic tape reel shall be accompanied by Table of MS response ratios
and a descriptor sheet that includes the following information for each
run. See Figure GC-MS Transmittal Form, this Appendix.
1. EPA sample number
2. Laboratory sample number
3. Fraction type (base/Neutral, Acid, VOA, Blank, Standard Runs, etc.)
4. Industrial Category
5. Specific source or stage of treatment
6. Date sampled
7. Sampler (Contractor or EPA Region)
8. Data run
9. File number on tape
10. File name on tape
11. Comments - to include any deviation from method 624 or 625.
- 53
-------
GC/MS DATA TRANSMITTAL FORM
(continues on pan,* 2)
1
2
3
4
5
6
7
8
9
10
n
12
EPA Numb*
Lab Numbef
Type Fraction
Inriustnat
Caieqoiy
Specific Sou ice of
Siaqe Ot Tieatment
Date
Sampled
SMnptet-
Contracior 01
EPA Laboratoiv
Date
Run
GC/MS DATA TRANSMITTAL FORM
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APPENDIX C
Numerical Example of Verification Phase Results
The following is an example of the calculations and results of a
verification study.
Surrogate Standard
(ug/1)
Sample Added
Al
A2
81
B2
Cl
C2
Dl
D2
El
E2
Fl
F2
95
95
95
95
95
95
95
95
95
95
95
95
Found
93
97
96
98
90
94
99
95
89
91
94
96
Range
(Eg. 3)
4
2
4
4
2
2
Mean
(Eg. 4)
95
97
92
97
90
95
% Recovery
(Eg. 10)
98
102
101
103
95
99
104
100
94
96
99
101
Critical Difference (Eq. 5} Rc =
Mean % Recovery (Eq. 8) P =
Standard Dev. of P (Eq. 9) Sp =
Acceptable Range of Recovery
9.8 at 95 ug/1.
99%
3.1 3SD »
90 to 109X
9.4
During the same verification study, the following data were
obtained for one of the priority pollutants studied.
Priority Pollutant Percent Mean
Back- Range Mean Recovery Recovery Std.Dev.
Sample ground Added Found (Eq. 3) (Eq. 4) (Eq. 7) (Eq. 8) (Eq. 9)
X
Al
A2
Bl
82
Cl
C2
Dl
D2
El
E2
Fl
F2
X
X
X
X
X
X
X
12
12
12
12
12
12
0
0
0
0
0
0
0
12
12
no
no
1200
1200
12
9
n
n
14
13
14
17
18
102
107
1160
1140
2
3
1
1
5
20
10
12.5
13.5
17.5
106
1150
42
50
82
86
96
94
46
84
95
5.9
3.3
1.2
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Critical Difference (Eq. 5) Rc = 6.7 at 12 ug/1
for priority pollutants Rc s 3.3 at 17.5 ug/1
Rc =» 16.4 at 106 ug/1
Rc = 65 at 1150 ug/1
Mean Value of X (Eq. 6) X = 12.0 ug/1 (background concentration)
Is there a significant difference in the recoveries between the 100X and 10X
levels? Apply a two tailed student's t-test with a confidence level of 95%.
Sd - MoO- SlO2 - 7(1.2)2+ (3.3)2 = 3.51
tf v 2 degrees of
degrees of freedom
- *10 - 95-84 = 11
texp = d/Sd = 11/3.51 = 3'. 70
Since 3.7 is less than 4.3 (t-value, 0.95, 2 degrees of freedom) there is no
significant difference between the 100X and 10X levels. Apply equations 8
and 9 to the four recoveries for these two levels. The mean recovery (eq.8)
is 89% with a standard deviaion (eq. 9) of 6.5 (3 degrees of freedom). Test
the 2X level against this mean recovery and standard deviation.
Sd = ,/(6.5)2 + (5.9)2 = 8.78 (4 degrees of freedom)
d = 89-46 * 43
« 43/8.78 = 4.90
Since 4.90 is greater than 2.78 (t value, 0.95, 4 degrees of freedom) there
is a significant difference between the 2X and the 100X, 10X levels.
2X 100X, IPX
Mean % Recovery (Eq. 8) "41T §9
Standard deviation (Eq. 9) 5.9 6.5
3 Sn 18 20
Acceptable Range (%) 28 to 64 69 to 109
During day one of the 30-day study the following data were obtained.
Surrogate Std. Priority Pollutant
(ug/1) (ug/1)
Recovery % Recovery
Sample Added Found (Eq. 13) Added Found (Eq. 14)
a 100 93 93 0 13
b 100 90 90 0 15
c 100 105 105 12 20 50%
T3T 56
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For the Surrogate Standards, on day one, the range between a and b (Eq. 11) is
3, and the mean concentration (Eq. 12) is 91.5 ug/1. Since the critical
difference is 9.8 at 95 ug/1, this range is acceptable. The recoveries-of the
Surrogate Standards in a, b, and c (Eq. 3) are all in the acceptable range of
90 to 10955. Therefore, the accuracy is acceptable.
For the Priority Pollutant, on day one, the range between a and b is
acceptable, (less than 6.7) and the recovery is acceptable (acceptable range
of 28 to 64*).
The following is an example of the results for day 2 to 30 of the 30-day study:
Priority
Pollutant (ug/1)
Day Found
2 14
3 15
4 20
5 11
6 17
7 18
8 10
9 14
10 13
11 12
12 20
30 15
Surrogate Standard (ug/1)
Added Found % Recovery
100
100
00
100
100
100
100
100
100
100
100
100
90
99
100
107
100
100
75
92
93
94
95
95
90
99
100
107
100
100
75
92
93
94
95
-Update Recovery for SS*
-Not Acceptable
Disregard
Results
•Update Recovery for
SS**
95 -Give final statistic on
Recovery of SS.
* Includes 20 results, 12 validation, 3 first day and days 2 through 6
Mean =99 Std. Dev. = 4.6 Acceptable range 85-113%
** Includes 25 results; day 8 result not included.
Mean = 98 Std. Dev. = 4.6 Acceptable range 84 -112%
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Appendix 0
Possible Sources of Standards
Aldrich Chemical Co., Milwaukee, Wise.;
Analabs, Inc., North Haven, Conn.;
J. T. Baker Chemical Co., Phillipsburgh, N.J.;
Chemical Procurement Laboratories, College Point, N.Y.;
Columbia Organics Catalog A-7," Columbia, i.C.
Eastman Kodak Co., Rochester, N.Y.;
K&K Rare & Fine Chemicals, Plainview, N.Y.;
Nanogens International, P.O. Box 487, Freedom, CA 95019
Pfaltz & Bauer Chemical Co., Stamford, Conn.;
RFR Corp., Hope, R.I.; "Chemical Standards for Air-Water-Industry-Foods"
(1975).
"Analytical Reference Standards and Supplemental Data for Pesticides and
Other Selected Organic Compounds," EPA-660/9-76-012 (May 1976), Health
Effects Research Laboratory, Environmental Toxicology Division, Research
Triangle Park, NC.
R K Chemical Co., 2135 Howard St., Hartville, OH 44632
Tridom Chemical Inc., Hauttauge, N.Y.
NOTE: These sources are not to be interpreted as being endorsed by the EPA.
J7-58
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GLOSSARY
EICP - Extracted Ion Current Profile. EICP is a reduction of mass
spectr^/aetrfc data acquired by continuous, repetitive measurement of
spectra that plots the change in relative abundance of one or more ions
as a function of time.
Equivalent - A piece of apparatus, gc liquid phase, etc. is
equivalent if its performance gives results identical to or better than
that specified. Contact Equivalency Staff, EMSL, Cincinnati for guide-
lines in equivalency for Alternate Test Procedures.
FID - Flame lonization Detector
Field Blank - Organic-free water that has been transported to the
sampling site and back to the analytical laboratory in a container and
bottle identical to the type used to collect the samples. Field blanks
and samples must be shipped in separate containers. When received in
the lab, the Field Blank is dosed, extracted and concentrated as if it
were an actual sample.
Limit of Detection - 10 ug/1 unless otherwise specified. For the
GC/MS Methods, 624 and 625, this is an operational limit that must be
met. The entire analytical system must give recognizable mass spectra
and calibration points from standards representing this concentration.
For Method 624, the condition (at 10 ug/1) must be met when sparging 50
ng of a component from 5.0 ml of water. For Method 625, the conditions
(at 10 ug/1) must be met when injecting standards at a concentration of
10 ug per ml.
Method Blank - For Method 625, a 1-liter volume of organic-free water
that is extracted and concentrated to the final volume of 1 ml. For
Method 624, a 5.0 ml aliquot of organic-free water.
Organic-Free Water - Water that gives no interferences when analyzed
as a Method Blank. See Method 624, Section 5.4, for the preparation of
organic-free water.
Sample Blank - Identical to Field Blank.
Significant Difference - Check for difference in magnitude by using a
two-tailed Student's t-test at a 95% Confidence Level.
Significant Drift - A 25% or larger change from an averaged
response. Must be based on a constantly updated average.
IT- 59
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SIM - Selected Ion Monitoring - Measuring the mass spectrometr*) c
response to one or several iojis in real time.
Surrogate Standard - Compounds not expected to occur in the
environmental sample that are added to the sample to monitor for
unusual matrix effects, gross sample processing errors, etc. The
compounds chosen should be chemically similar to the pollutants
being measured. Surrogate Standards are not used as internal
standards.
Transfer Pipet - Class A, to deliver, pipet.
RIMmMQfTICt-Bn
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