United States
Environmental Protection
Agency
Office of Water
Engineering and Analysis Division (4303)
Washington. D.C. 20460
EPA-821-B-94-001
February 1995
oEPA Analytical Methods for the
Determination of Pollutants
in Pharmaceutical Manufacturing
Industry Wastewater
. Printed on Recycled paper
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S4EPA Analytical Methods for the
Determination of Pollutants
in Pharmaceutical Manufacturing
Industry Wastewater
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Acknowledgments
This methods compendium was prepared under the direction of William A. Telliard of the Engineering and
Analysis Division within EPA's Office of Water. This document was prepared under EPA Contract No. 68-
C3-0337 by the Environmental Programs Division of DynCorp.
Disclaimer
This methods compendium has been reviewed by the Engineering and Analysis Division, U.S. Environ-
mental Protection Agency, and approved for publication. Mention of trade names or commercial products
does not constitute and endorsement or recommendation for use.
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Table of Contents
Introduction v
Method 1665
Semi-Volatile Organic Compounds Specific to the Pharmaceutical
Manufacturing Industry by Isotope Dilution QC/MS 1
Method 1666
Volatile Organic Compounds Specific to the Pharmaceutical Manufacturing
Industry by Isotope Dilution GC/MS 37
Method 1667
Formaldehyde, Isobutyraldehyde, and Furfural by Derivatization
and High-Pressure Liquid Chromatography 79
Method 1671
Volatile Organic Compounds Specific to the Pharmaceutical Manufacturing
Industry by GC/FID 97
Method 1673
Polyethylene glycol)-600 by Derivatization and High-Pressure
Liquid Chromatography 115
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Introduction
The U.S. Environmental Protection Agency (EPA or the Agency) is proposing effluent limitations guide-
lines and standards for promulgation at 40 CFR Part 439 for the Pharmaceutical Manufacturing Industrial
category to control the discharge of pollutants into surface waters of the United States. This compendium
of test procedures (methods) supports the proposal. The purpose of publishing the compendium is to pro-
vide a single source of methods that are unique to the proposed rule. These methods or the referenced
methods must be used for filing permit applications and for compliance monitoring under the National Pol-
lutant Discharge Elimination System (NPDES) program.
Two of the methods included in this compendium are based on isotope dilution methods: "Method 1624:
Volatile Organic Compounds by Isotope Dilution GC/MS" and "Method 1625: Semivolatile Organic Com-
pounds by Isotope Dilution GC/MS. Two additional methods included in this compendium are based
upon draft Method 8315, "Formaldehyde by High-Pressure Liquid Chromatography" (January 1990).
This compendium includes only those methods that are unique to the Pharmaceutical Manufacturing
Industry rulemaking. Other methods allowed under the proposed rule have been promulgated at 40 CFR
Part 136.
Questions concerning the methods in this compendium should be directed to:
W.A. Telliard
U.S. EPA
Engineering and Analysis Division
Office of Science and Technology
401 M Street, SW
Washington, DC 20460
1. United States Environmental Protection Agency, Office of Water Regulations and Standards, Industrial
Technology Division, Office of Water, June 1989.
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Method 1665
Semi-Volatile Organic Compounds
Specific to the Pharmaceutical
Manufacturing Industry by
Isotope Dilution GC/MS
January 1995
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Method 1665
Semi-Volatile Organic Compounds Specific to the
Pharmaceutical Manufacturing Industry by
Isotope Dilution GC/MS
1. Scope and Application
1.1 This method is designed to meet the survey and monitoring requirements of the U.S. EPA Engineer-
ing and Analysis Division (EAD). The method is used to determine the semivolatile organic pollut-
ants specific to the pharmaceutical manufacturing industry (PMI) that are amenable to extraction and
analysis by capillary column gas chromatography-mass spectrometry (GC/MS).
1.2 The chemical compounds listed in Table 1 may be determined in waters, soils, and municipal sludges
by this method or the method referenced.
1.3 The detection limits of the method are usually dependent on the level of interferences rather than
instrumental limitations. The limits provided in Table 2 are the minimum amounts that can be reli-
ably quantified with no interferences present.
1.4 The GC/MS portions of the method are for use only by analysts experienced with GC/MS or under
the close supervision of such qualified persons. Laboratories unfamiliar with analysis of environ-
mental samples by GC/MS should run the performance tests in Reference 1 before beginning.
1.5 This method is "performance-based." The analyst is permitted to modify the method to overcome
interferences or to lower the cost of measurements, provided that all performance criteria in this
method are met The requirements for establishing method equivalency are given in Section 9.1.2.
2. Summary of Method
2.1 The percent solids content of a sample is determined. Stable, isotopically labeled analogs of the com-
pounds of interest are added to the sample. If the solids content is less than 1%, a 1-L sample is
extracted at pH 12-13 with methylene chloride using continuous extraction techniques. If the solids
content is 30% or less, the sample is diluted to 1% solids with reagent water, homogenized ultrasoni-
cally, and extracted at pH 12-13 with methylene chloride using continuous extraction techniques. If
the solids content is greater than 30%, the sample is extracted using ultrasonic techniques. Each
extract is dried over sodium sulfate, concentrated to a volume of S mL, cleaned up using gel-perme-
ation chromatography (GPC), if necessary, and concentrated. Extracts are concentrated to 1 mL if
GPC is not performed, and to 0.5 mL if GPC is performed. An internal standard is added to the
extract, and a 1-uL aliquot of the extract is injected into the gas chromatograph (GC). The com-
pounds are separated by GC and detected by a mass spectrometer (MS). The labeled compounds
serve to correct the variability of the analytical technique.
2.2 Identification of a pollutant (qualitative analysis) is performed as follows: The GC/MS system is cal-
ibrated and the mass spectrum and retention time for each standard are stored in a user-created
library. A compound is identified when its retention time and mass spectrum agree with the library
retention time and spectrum.
2.3 Quantitative analysis is performed in one of two ways by GC/MS using extracted-ion current profile
(EICP) areas: (1) The GC/MS system is calibrated and the analyte concentration is determined using
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Method 1665
an isotope dilution technique; or (2) For analytes lacking a labeled analog, the GC/MS system is cal-
ibrated and the compound concentration is determined using an internal standard technique.
2.4 The quality of the analysis is assured through reproducible calibration and testing of the extraction
and GC/MS systems.
3. Definitions
There are no method-specific definitions to be noted for this document.
4. Interferences
4.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or ele-
vated baselines causing misinterpretation of chromatograms and spectra. All materials used in the
analysis shall be demonstrated to be free from interferences under the conditions of analysis by run-
ning method blanks initially and with each sample batch (samples started through the extraction pro-
cess on a given 8-hour shift, to a maximum of 20). Specific selection of reagents and purification of
solvents by distillation in all-glass systems may be required. Glassware and, where possible, other
equipment are cleaned by solvent rinse and baking at 450°C for a minimum of 1 hour.
4.2 Interferences co-extracted from samples will vary considerably from source to source, depending on
the diversity of the site being sampled.
5. Safety
The toxicity or carcinogenicity of each compound or reagent used in this method has not been precisely
determined; however, each chemical compound should be treated as a potential health hazard. Exposure to
these compounds should be reduced to the lowest possible level. The laboratory is responsible for main-
taining a current awareness file of OSHA regulations regarding the safe handling of the chemicals speci-
fied in this method. A reference file of material safety data sheets should also be made available to all
personnel involved in these analyses. Additional information on laboratory safety can be found in Refer-
ences 3-5.
6. Equipment and Supplies
6.1 Sampling equipment for discrete or composite sampling.
6.1.1 Sample bottles and caps.
6.1.1.1 Liquid samples (waters, sludges, and similar materials that contain less than five
percent solids)—Sample bottle, amber glass, 1.1-L minimum, with screw-cap.
6.1.1.2 Solid samples (soils, sediments, sludges, filter cake, compost, and similar materials
that contain more than 5% solids)—Sample bottle, wide mouth, amber glass, 500-
mL minimum.
6.1.1.3 If amber bottles are not available, samples shall be protected from light.
6.1.1.4 Bottle caps—Threaded to fit sample bottles. Caps shall be lined with polytetraflu-
oroethylene (FTFE).
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Method 1665
6.1.1.5 Cleaning.
6.1.1.5.1 Bottles are washed with detergent and water, rinsed with reagent water,
then solvent rinsed or baked at 450°C for a minimum of 1 hour before
use.
6.1.1.5.2 Cap liners are washed with detergent and water, rinsed with reagent
water (see Section 7.5.1) and then solvent, and then baked for at least
1 hour at approximately 200°C.
6.1.2 Compositing equipment—Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept at 0^40C
during sampling. Only glass or PTFE tubing shall be used. If the sampler uses a peristaltic
pump, a minimum length of compressible silicone rubber tubing may be used only in the
pump. Before use, the tubing shall be thoroughly rinsed with methanol, followed by
repeated rinsings with reagent water (Section 7.5.1) to minimize sample contamination. An
integrating flow meter is used to collect proportional composite samples.
6.2 Equipment for determining percent moisture.
6.2.1 Oven, capable of maintaining a temperature of 110°C (±5°C).
6.2.2 Desiccator.
6.3 Sonic disrupter—375-watt with pulsing capability and %" disrupter hom (Ultrasonics, Inc, Model
375C, or equivalent).
6.4 Extraction apparatus.
6.4.1 Continuous liquid-liquid extractor—PTFE or glass connecting joints and stopcocks without
lubrication, 1.5- to 2-L capacity (Hershberg-Wolf Extractor, Ace Glass 6841-10, or equiva-
lent).
6.4.2 Beakers.
6.4.2.1 1.5- to 2-L borosilicate glass beakers calibrated to 1 L.
6.4.2.2 400- to 500-mL borosilicate glass beakers.
6.4.2.3 Spatulas—Stainless steel.
6.4.3 Filtration apparatus.
6.4.3.1 Glass funnel—125- to 250-mL.
6.4.3.2 Filter paper for above (Whatman 41, or equivalent).
6.5 Drying column—15- to 20-mm inside diameter (i.d.) Pyrex chromatographic column equipped with
coarse-glass frit or glass-wool plug.
6.6 Concentration apparatus.
6.6.1 Concentrator tube—Kuderna-Danish (K-D) 10-mL, graduated (Kontes K-570050-1025, or
equivalent) with calibration verified. Ground-glass stopper (size 19/22 joint) is used to pre-
vent evaporation of extracts.
6.6.2 Evaporation flask—K-D 500-mL (Kontes K-570001-0500, or equivalent), attached to con-
centrator tube with springs (Kontes K-662750-0012, or equivalent).
6.6.3 Snyder column—K-D three-ball macro (Kontes K-503000-0232, or equivalent).
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Method 1665
6.6.4 Snyder column—K-D two-ball micro (Kontes K-469002-0219, or equivalent).
6.6.5 Boiling chips—Approximately 10/40 mesh, extracted with methylene chloride and baked at
450°C for a minimum of 1 hour.
6.6.6 Nitrogen evaporation device—Equipped with a water bath that can be maintained at 35-
40°C. The N-Evap by Organomation Associates, Inc., South Berlin, MA (or equivalent) is
suitable.
6.7 Water bath—Heated, with concentric ring cover, capable of temperature control (±2 *C), installed in a
fume hood.
6.8 Sample vials—Amber glass, 2- to 5-mL with PTFE-lined screw-cap.
6.9 Balances.
6.9.1 Analytical—Capable of weighing 0.1 mg.
6.9.2 Top loading—Capable of weighing 10 mg.
6.10 Automated gel-permeation chromatograph (Analytical Biochemical Labs, Inc., Columbia, MO,
Model GPC Autoprep 1002, or equivalent).
6.10.1 Column—600 to 700 mm long x 25 mm i.d., packed with 70 g of SX-3 Bio-beads (Bio-Rad
Laboratories, Richmond, CA, or equivalent).
6.10.2 UV detectors—254-nanometers (nm), preparative or semi-prep flow cell.
6.10.2.1 Schimadzu, 5-mm path length.
6.102.2 Beckman-Altex 152W, 8-uL micro-prep flow cell, 2-mm path length.
6.10.2.3 Pharmacia UV-1,3-mm flow cell.
6.10.2.4 LDC Milton-Roy UV-3, monitor #1203.
6.11 Gas chromatograph—Shall have splitless or on-column injection port for capillary column, tempera-
ture program with 30°C hold, and shall meet all of the performance specifications in Section 13.
6.11.1 Column—30 m (±5 m) long x 0.32 mm (±0.02 mm) i.d., with a 1-jjm coating of 5% phenyl,
94% methyl, 1% vinyl silicone bonded-phase on a fused-silica capillary column (Restek Rtx-
5, or equivalent). Temperature program: 5 minutes at 30°C; 30-280°C at 8°C per minute;
isothermal at 280°C until N,N-dimethylaniline elutes. Gas Velocity: 30 cm/sec (±5 cm/sec)
at 30°C.
6.12 Mass spectrometer—70 eV electron impact ionization, shall repetitively scan from 35 to 450 dalton
in 0.95-1.00 second, and shall produce a unit resolution (valleys between mass over charge [m/z]
441-442 less than 10% of the height of the 441 peak), background corrected mass spectrum from
50 ng decafluorotriphenylphosphine (DFTPP) introduced through the GC inlet. The spectrum shall
meet the mass-intensity criteria in Table 3 (Reference 6). The mass spectrometer shall be interfaced
to the GC such that the end of the capillary column terminates within 1 cm of the ion source but does
not intercept the electron or ion beams. All portions of the column that connect the GC to the ion
source shall remain at or above the column temperature during analysis to preclude condensation of
less volatile compounds.
6.13 Data system—Shall collect and record MS data, store mass-intensity data in spectral libraries, pro-
cess GC/MS data, generate reports, and shall compute and record response factors.
6.13.1 Data acquisition—Mass spectra shall be collected continuously throughout the analysis and
stored on a mass-storage device.
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Method 1665
6.13.2 Mass spectral libraries—User-created libraries containing mass spectra obtained from analy-
sis of authentic standards shall be prepared.
6.13.3 Data processing—The data system shall be used to search, locate, identify, and quantify the
compounds of interest in each GC/MS analysis. Software routines shall be employed to
compute retention times and peak areas. Displays of spectra, mass chromatograms, and
library comparisons are required to verify results.
6.13.4 Response factors and multipoint calibrations—The data system shall be used to record and
maintain lists of response factors (response ratios for isotope dilution)and multi-point cali-
bration curves (Section 10). Computations of relative standard deviation (coefficient of vari-
ation) are used for testing calibration linearity. Statistics on initial (Section 9.2) and on-going
(Section 13.7) performance shall be computed and maintained.
7. Reagents and Standards
7.1 Reagents for adjusting sample pH.
7.1.1 Sodium hydroxide—Reagent grade, 6 normal (N) in reagent water.
7.1.2 Sulfuric acid—Reagent grade, 6 N in reagent water.
7.2 Sodium sulfate—Reagent grade, powdered anhydrous, rinsed with methylene chloride (20 mL/g),
baked at 450°C for a minimum of 1 hour, cooled in a desiccator, and stored in a pre-cleaned glass
bottle with screw-cap that prevents moisture from entering.
7.3 Methylene chloride—Distilled in glass (Burdick and Jackson, or equivalent).
7.4 GPC calibration solution—Containing 300 mg/mL com oil, 15 mg/mL bis(2-ethylhexyl) phthalate,
1.4 mg/mL pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mg/mL sulfur.
7.5 Reference matrices.
7.5.1 Reagent water—Water in which the compounds of interest and interfering compounds are
not detected by this method.
7.5.2 High-solids reference matrix—Playground sand or similar material in which the compounds
of interest and interfering compounds are not detected by this method.
7.6 Standard solutions—Purchased as solutions or mixtures with certification to their purity, concentra-
tion, and authenticity, or prepared from materials of known purity and composition. If compound
purity is 96% or greater, the weight may be used without correction to compute the concentration of
the standard. When not being used, standards are stored in the dark at -20 to -10°C in screw-capped
vials with PTFE-lined lids. A mark is placed on the vial at the level of the solution so that solvent
evaporation loss can be detected. The vials are brought to room temperature prior to use. Any pre-
cipitate is redissolved and solvent is added if solvent loss has occurred.
7.7 Preparation of stock solutions—Prepare in methylene chloride, benzene, p-dioxane, or a mixture of
these solvents per the steps below. Observe the safety precautions in Section 5. The working range
for most compounds is 10-200 ug/mL. Compounds with a reduced MS response may be prepared at
higher concentrations.
7.7.1 Dissolve an appropriate amount of assayed reference material in a suitable solvent. For
example, weigh 400 mg N,N-dimethylaniline in a 10-mL ground-glass-stoppered volumetric
flask and fill to the mark with benzene. After the N,N-dimethylaniline is completely dis-
solved, transfer the solution to a 15-mL vial with PTFE-lined cap.
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Method 1665
7.7.2 Stock standard solutions should be checked for signs of degradation prior to the preparation
of calibration or performance test standards. Quality control check samples may be available
from the National Institute of Standards and Technology, Bethesda, Maryland.
7.7.3 Stock standard solutions shall be replaced after six months, or sooner if comparison with
quality control check standards indicates a change in concentration.
7.8 Labeled compound spiking solution—From stock standard solutions prepared as above, or from mix-
tures, prepare the spiking solution at a concentration of 200 ug/mL, or at a concentration appropriate
to the MS response of each compound.
7.9 Secondary standard—Using stock solutions (Section 7.7), prepare a secondary standard containing
all of the compounds hi Table 1 at a concentration of 400 ug/mL, or higher concentration appropriate
to the MS response of the compound.
7.10 Internal standard solution—Prepare 2,2'-difluorobiphenyl (DFB) at a concentration of 10 mg/mL in
benzene.
7.11 DFTPP solution—Prepare at 50 jag/mL in acetone.
7.12 Solutions for obtaining authentic mass spectra (Section 7.6)—Prepare mixtures of compounds at
concentrations that will assure authentic spectra are obtained for storage in libraries.
7.13 Calibration solutions—Combine 0.5 mL of the solution from Section 7.8 with 25,50,125,250, and
500 |jL of the solution from Section 7.9 and bring each to a total volume of 1.00 mL. This will pro-
duce calibration solutions of nominal 10,20,50,100 and 200 ug/mL of the pollutants and a constant
nominal 100 ug/mL of the labeled compounds. Spike each solution with 10 uL of the internal stan-
dard solution (Section 7.10). These solutions permit the relative response (labeled to unlabeled) to
be measured as a function of concentration (Section 10.4).
7.14 Precision and recovery standard—Used for determination of initial (Section 9.2) and ongoing (Sec-
tion 13.7) precision and recovery. This solution shall contain the pollutants and labeled compounds
at a concentration 1000 times the spike level shown in Table 4 for each native and labeled analyte.
7.15 Stability of solutions—All standard solutions (Sections 7.8-7.14) shall be analyzed within 48 hours
of preparation and on a monthly basis thereafter for signs of degradation. Standards will remain
acceptable if the peak area at the quantitation mass relative to the DFB internal standard remains
within ±15% of the area obtained in the initial analysis of the standard.
8. Sample Collection, Preservation, and Handling
8.1 Collect samples in glass containers following conventional sampling practices (Reference 8). Aque-
ous samples that flow freely are collected in refrigerated bottles using automatic sampling equip-
ment. Solid samples are collected as grab samples using wide-mouth jars.
8.2 Maintain samples at 0-4°C from the time of collection until extraction. If residual chlorine is present
in aqueous samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods 330.4 and 330.5
may be used to measure residual chlorine (Reference 9).
8.3 Begin sample extraction within seven days of collection, and analyze all extracts within 40 days of
extraction.
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Method 1665
9. Quality Assurance/Quality Control
9.1 Each laboratory that uses this method is required to operate a formal quality assurance program (Ref-
erence 7). The minimum requirements of this program consist of an initial demonstration of labora-
tory capability, analysis of samples spiked with labeled compounds to evaluate and document data
quality, and analysis of standards and blanks as tests of continued performance. Laboratory perfor-
mance is compared to established performance criteria to determine if the results of analyses meet
the performance characteristics of the method. If the method is to be applied routinely to samples
containing high-solids with very little moisture (e.g., soils, filter cake, compost), the high-solids ref-
erence matrix (Section 7.5.2) is substituted for the reagent water (Section 7.5.1) in all performance
tests, and the nigh-solids method (Section 11) is used for these tests.
9.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable accuracy
and precision with this method. This ability is established as described in Section 9.2.
9.1.2 In recognition of advances that are occurring in analytical technology, and to allow the ana-
lyst to overcome sample matrix interferences, the analyst is permitted certain options to
improve separations or lower the costs of measurements. These options include alternative
extraction, concentration, cleanup procedures, and changes in columns and detectors. Alter-
native techniques, such as the substitution of spectroscopy or immunoassay, and changes that
degrade method performance are not allowed. If an analytical technique other than the tech-
niques specified in this method is used, that technique must have a specificity equal to or bet-
ter than the specificity of the techniques in this method for the analytes of interest.
9.1.2.1 Each time a modification is made to this method, the analyst is required to repeat
the procedure in Section 9.2. If the detection limit of the method will be affected
by the change, the laboratory is required to demonstrate that the MDL (40 CFR
Part 136, Appendix B) is lower than one-third the regulatory compliance level. If
calibration will be affected by the change, the analyst must recalibrate the instru-
ment per Section 10.
9.1.2.2 The laboratory is required to maintain records of modifications made to this
method. These records include the information below, at a minimum.
9.1.2.2.1 The names, titles, addresses, and telephone numbers of the analyst(s)
who performed the analyses and modification, and of the quality con-
trol officer who witnessed and will verify the analyses and modifica-
tion.
9.1.2.2.2 A listing of pollutant(s) measured, by name and CASRN.
9.1.2.2.3 A narrative stating the reason(s) for the modification.
9.1.2.2.4 Results from all quality control (QC) tests comparing the modified
method to this method, including:
(a) Calibration (Section 10.5 or 10.6)
(b) Calibration verification (Section 13.5)
(c) Initial precision and accuracy (Section 9.2)
(d) Labeled compound recovery (Section 9.3)
(e) Analysis of blanks (Section 9.5)
(f) Accuracy assessment (Section 9.4)
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Method 1665
9.1.2.2.5 Data that will allow an independent reviewer to validate each determi-
nation by tracing the instrument output (peak height, area, or other sig-
nal) to the final result, including:
(a) Sample numbers and other identifiers
(b) Extraction dates
(c) Analysis dates and times
(d) Analysis sequence/run chronology
(e) Sample weight or volume (Section 11)
(f) Extract volume prior to each cleanup step (Section 11.9)
(g) Extract volume after each cleanup step (Section 11.9)
(h) Final extract volume prior to injection (Section 12)
(i) Injection volume (Section 12)
(j) Dilution data, differentiating between dilution of a sample or an
extract (Section 15.4)
(k) Instrument and operating conditions
(I) Column (dimensions, liquid phase, solid support, film thickness,
etc.)
(m) Operating conditions (temperature, temperature program, flow
rates, etc.)
(n) Detector (type, operating condition, etc.)
(o) Chromatograms, printer tapes, and other recording of raw data
(p) Quantitation reports, data system outputs, and other data necessary
to link raw data to the results reported
9.1.3 Analyses of blanks are required to demonstrate freedom from contamination. The proce-
dures and criteria for analysis of a blank are described in Section 9.5.
9.1.4 The laboratory shall spike all samples with labeled compounds to monitor method perfor-
mance. This test is described in Section 9.3. When results of these spikes indicate atypical
method performance for samples, the samples are diluted to bring method performance
within acceptable limits (Section 15).
9.1.5 The laboratory shall, on an ongoing basis, demonstrate through calibration verification and
the analysis of the precision and recovery standard (Section 7.14) that the analysis system is
in control. These procedures are described in Sections 13.1,13.5, and 13.7.
9.1.6 The laboratory shall maintain records to define the quality of data that is generated. Devel-
opment of accuracy statements is described in Section 9.4.
9.2 Initial precision and accuracy—To establish the ability to generate acceptable precision and accuracy,
the analyst shall perform the operations described in this subsection.
9.2.1 For low-solids (aqueous) samples, extract, concentrate, and analyze four 1-L aliquots pre-
pared by spiking 1 mL of the precision and recovery standard (Section 7.14) into 1 L of
reagent water, according to the procedure in Section 11. For high-solids samples, 30-g ali-
quots of the high-solids reference matrix, spiked with 1 mL of the precision and recovery
standard, are used.
9.2.2 Compute the average recovery (X) in ug/L and the standard deviation of the recovery (s) in
Hg/L for each compound, by isotope dilution for pollutants with a labeled analog, and by
internal standard for labeled compounds and pollutants with no labeled analog.
10
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Method 1665
9.2.3 For each compound, compare s and X with the corresponding limits for initial precision and
accuracy in Table 4. If s and X for all compounds meet the acceptance criteria, system per-
formance is acceptable and analysis of blanks and samples may begin. If, however, any of
the compounds fail, the analysis system is not performing properly. In this event, correct the
problem and repeat the test (Section 9.2.1) or follow the procedure in Section 9.2.3.1
9.2.3.1 The test may be repeated without adjustment if the analyst believes that it is war-
ranted. In this event, compute s and X for only those PMI analytes that failed the
test the first time (Sections 9.2.2 and 9.2.3). If these PMI analytes now pass, sys-
tem performance is acceptable for all compounds and analysis of samples and
blanks may begin. If, however, any of the same PMI analytes fail again, the
extraction/concentration processes are not being performed properly for these
compounds. In this event, correct the problem, and begin again.
9.3 The laboratory shall spike all samples with labeled compounds to assess method performance on the
sample matrix.
9.3.1 Analyze each sample according to the method beginning in Section 11.
9.3.2 Compute the percent recovery (P) of the labeled compounds using the internal standard
method (Section 10.5).
9.3.3 Compare the labeled compound recovery for each compound with the corresponding limits
in Table 4. If the recovery of any compound falls outside these limits, method performance
is unacceptable for that compound in that sample. To overcome such difficulties, water sam-
ples are diluted, and smaller amounts of soils, sludges, and sediments are reanalyzed per Sec-
tion 15.
9.4 As part of the QA program for the laboratory, method accuracy for samples shall be assessed and
records shall be maintained. After the analysis of five samples of a given matrix type (e.g., water,
soil, sludge, sediment) for which the labeled compounds pass the tests in Section 9.3, compute the
average percent recovery (P) and the standard deviation of the percent recovery (Sp) for the labeled
compounds only. Express the accuracy assessment as a percent recovery interval from P - 2spto
P + 2sp for each matrix.
For example, if P = 90% and sp = 10% for five analyses of compost, the accuracy interval is
expressed as 70-110%. Update the accuracy assessment for each compound in each matrix on a reg-
ular basis (e.g., after each five to ten new accuracy measurements).
9.5 Blanks—Reagent water and high-solids reference matrix blanks are analyzed to demonstrate free-
dom from contamination.
9.5.1 Extract and concentrate a 1-L reagent water blank or a high-solids reference matrix blank
with each sample lot (samples started through the extraction process on the same 8-hour
shift, to a maximum of 20 samples). Analyze the blank immediately after analysis of the
precision and recovery standard (Section 7.14) to demonstrate freedom from contamination.
9.5.2 If any of the compounds of interest (Table 1) or any potentially interfering compound is
found in an aqueous blank at greater than the ML (assuming a response factor of 1 relative to
the internal standard for compounds not listed in Table 1), analysis of samples is halted until
the source of contamination is eliminated and a blank shows no evidence of contamination at
this level.
11
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Method 1665
9.6 The specifications contained in this method can be met if the apparatus used is calibrated properly,
then maintained in a calibrated state. The standards used for calibration (Section 10), calibration ver-
ification (Section 13.5), and initial (Section 9.2) and ongoing (Section 13.7) precision and recovery
should be identical, so that the most precise results will be obtained. The GC/MS instrument in par-
ticular will provide the most reproducible results if dedicated to the settings and conditions required
for the analyses of semivolatiles by this method.
9.7 Depending on specific program requirements, field replicates may be collected to determine the pre-
cision of the sampling technique, and spiked samples may be required to determine the accuracy of
the analysis when the internal standard method is used.
10. Calibration
10.1 Assemble the GC/MS system and establish the operating conditions in Section 6.11.1. Analyze stan-
dards per the procedure in Section 12 to demonstrate that the analytical system meets the minimum
levels in Table 2, and the mass-intensity criteria in Table 3 for 50 ng DFTPP.
10.2 Mass-spectral libraries—Detection and identification of compounds of interest are dependent upon
spectra stored in user created libraries.
10.2.1 Obtain a mass spectrum of each pollutant, labeled compound, and the internal standard by
analyzing an authentic standard either singly or as part of a mixture in which there is no
interference between closely eluted components. Examine the spectrum to determine that
only a single compound is present. Fragments not attributable to the compound under study
indicate the presence of an interfering compound.
10.2.2 Adjust the analytical conditions and scan rate (for this test only) to produce an undistorted
spectrum at the GC peak maximum. An undistorted spectrum will usually be obtained if five
complete spectra are collected across the upper half of the GC peak. Software algorithms
designed to "enhance" the spectrum may eliminate distortion, but may also eliminate authen-
tic masses or introduce other distortion.
10.2.3 The authentic reference spectrum is obtained under DFTPP tuning conditions (Section 10.1
and Table 3) to normalize it to spectra from other instruments.
10.2.4 The spectrum is edited by saving the five most intense mass-spectral peaks and all other
mass spectral peaks greater than 10% of the base peak. The spectrum may be further edited
to remove the common interfering m/z's. If five mass spectral peaks cannot be obtained
under the scan conditions given in Section 6.12, the mass spectrometer may be scanned to an
m/z lower than 35 to gain additional spectral information. The spectrum obtained is stored
for reverse search and for compound confirmation.
10.3 Analytical range—Demonstrate that 20 ng anthracene or phenanthrene produces an area at m/z 178
approximately one-tenth that required to exceed the linear range of the system. The exact value must
be determined by experience for each instrument. It is used to match the calibration range of the
instrument to the analytical range and detection limits required, and to diagnose instrument sensitiv-
ity problems (Section 16.3). The 20 ug/mL calibration standard (Section 7.13) can be used to dem-
onstrate this performance.
10.3.1 Polar compound detection—Demonstrate that unlabeled pentachlorophenol and benzidine
are detectable at the 50 |ig/mL level (per all criteria in Section 14). The 50 ng/mL calibration
standard (Section 7.13) can be used to demonstrate this performance.
12
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Method 1665
10.4 Calibration with isotope dilution—Isotope dilution is used when (1) labeled compounds are avail-
able, (2) interferences do not preclude its use, and (3) the quantitation m/z (Table 5) EICP area for the
compound is in the calibration range. Alternative labeled compounds and quantitation m/z's may be
used based on availability. If any of the above conditions preclude isotope dilution, the internal stan-
dard method (Section 10.5) is used.
Note: Because the composition and purity of commercially supplied isotopically labeled standards may
vary, the primary m/z of the labeled analogs given in Table 5 should be used as guidance. The
appropriate m/z of the labeled analogs should be determined prior to use for sample analysis.
10.4.1 A calibration curve encompassing the concentration range is prepared for each compound to
be determined. The relative response (pollutant-to-labeled) versus concentration in standard
solutions is plotted or computed using a linear regression. The example in Figure 1 shows a
calibration curve for aniline using aniline-d7 as the isotopic diluent. Also shown are the
±10% error limits (dotted lines). Relative response (RR) is determined according to the pro-
cedures described below. A minimum of five data points are employed for calibration.
10.4.2 The relative response of a pollutant to its labeled analog is determined from isotope ratio val-
ues computed from acquired data. Three isotope ratios are used in this process:
area m,
R =
x
R =
x T
1
aream2/z
aream
,. '^
m
area m^/z I at RT. 1
Where:
Rx = The isotope ratio measured for the pure pollutant
Ry = The isotope ratio measured for the labeled compound
Rftr The isotope ratio of an analytical mixture of pollutant and
labeled compounds
The m/z's are selected such that R, > R^ If Rm is not between 2R, and 0.5R,, the method
does not apply and the sample is analyzed by the internal standard method.
10.4.3 Capillary columns usually separate the pollutant-labeled pair, with the labeled compound
eluted first (Figure 2). This is indeed the case for the PMI analytes treated here.
10.4.4 Special precautions are taken when the pollutant-labeled pair is not separated, or when
another labeled compound with interfering spectral masses overlaps the pollutant (a case that
can occur with isomeric compounds). In this case, it is necessary to determine the respective
13
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Method 1665
contributions of the pollutant and labeled compounds to the respective EICP areas. If the
peaks are separated well enough to permit the data system or operator to remove the contri-
butions of the compounds to each other, the equations in Section 10.4.2 apply. This usually
occurs when the height of the valley between the two GC peaks at the same m/z is less than
10% of the height of the shorter of the two peaks. If significant GC and spectral overlap
occur, RR is computed using the following equation:
RR =
(R -R }(R
\ y mj\
(R -R Y/?
^ m *A
where R, is measured as shown in Figure 3A, Ry is measured as shown in Figure 3B, and R,,,
is measured as shown in Figure 3C. For the example,
46100
V -4780
Ry= 43600= °-06078
Rm= 48300= L019
RR= 1.115
The data from these analyses are reported to three significant figures (see Section 15.6).
Therefore, in order to prevent rounding errors from affecting the values to be reported, all
calculations performed prior to the final determination of concentrations should be carried
out using at least four significant figures.
1 0.4.5 To calibrate the analytical system by isotope dilution, analyze a 1 .0-|jL aliquot of each of the
calibration standards (Section 7.13) using the procedure in Section 12. Compute the RR at
each concentration.
10.4.6 Linearity — If the ratio of relative response to concentration for any compound is constant
(less than 20% coefficient of variation) over the five-point calibration range, an averaged rel-
ative response/concentration ratio may be used for that compound; otherwise, the complete
calibration curve for that compound shall be used over the five-point calibration range.
10.5 Calibration by internal standard — Used when criteria for isotope dilution (Section 10.4) cannot be
met The internal standard to be used for both acid and base/neutral analyses is 2,2'-difluorobiphe-
nyl. The internal standard method is also applied to determination of compounds having no labeled
analog, and to measurement of labeled compounds for intra-laboratory statistics (Sections 9.4 and
13.7.4).
14
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Method 1665
10.5.1 Response factors—Calibration requires the determination of response factors (RF) which are
defined by the following equation:
A xC.
-1 if
A. xC
IS S
Where:
As = The area of the characteristic mass for the compound in the
daily standard
AX = The area of the characteristic mass for the internal standard
Qy = The concentration of the internal standard (/jg/mL)
Cs —The concentration of the compound in the daily standard
(Hg/mL)
10.5.1.1 The response factor is determined for at least five concentrations appropriate to the
response of each compound (Section 7.13); nominally, 10,20,50,100, and 200 ug/
mL. The amount of internal standard added to each extract is the same (100 ug/
mL) so that Cu remains constant. The RF is plotted versus concentration for each
compound in the standard (CJ to produce a calibration curve.
10.5.1.2 Linearity—If the response factor (RF) for any compound is constant (less than
35% coefficient of variation) over the five-point calibration range, an averaged
response factor may be used for that compound; otherwise, the complete calibra-
tion curve for that compound shall be used over the five-point range.
10.6 Combined calibration—By using calibration solutions (Section 7.13) containing the pollutants,
labeled compounds, and the internal standard, a single set of analyses can be used to produce calibra-
tion curves for the isotope dilution and internal standard methods. These curves are verified each
shift (Section 13.5) by analyzing the 100 ug/mL calibration standard (Section 7.13). Recalibration is
required only if calibration verification (Section 13.5) criteria cannot be met.
11. Sample Extraction, Concentration, and Cleanup
Samples containing less than 1% solids are extracted directly using continuous liquid/liquid extraction
techniques (Section 11.2.1 and Figure 4). Samples containing 1-30% solids are diluted to the 1% level
with reagent water (Section 11.2.2) and extracted using continuous liquid/liquid extraction techniques.
Samples containing greater than 30% solids are extracted using ultrasonic techniques (Section 11.2.5).
11.1 Determination of percent solids.
11.1.1 Weigh 5-10 g of sample into a tared beaker.
11.1.2 Dry overnight (12 hours minimum) at 110°C(±5°C), and cool in a desiccator.
15
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Metfjod 1665
11.1.3 Determine percent solids as follows:
... weight of dry sample
% sollds = weight of vet sample X 10°
1 1 .2 Preparation of samples for extraction.
11.2.1 Samples containing less than 1% solids — Extract sample directly using continuous liquid/
liquid extraction techniques.
11.2.1.1 Measure 1.00 L (±0.01 L) of sample into a clean 1.5- to 2.0-L beaker.
11.2.1 2 Dilute aliquot — For samples which are expected to be difficult to extract, concen-
trate, or clean up, measure an additional 100.0 mL (±1.0 mL) into a clean 1.5- to
2.0-L beaker and dilute to a final volume of 1.00 L (±0.1 L) with reagent water.
11 .2.1 .3 Spike 0.5 mL of the labeled compound spiking solution (Section 7.8) into the sam-
ple aliquots. Proceed to preparation of the QC aliquots for low-solids samples
(Section 11.2.3).
112.2 Samples containing 1-30% solids.
1 1 .2.2.1 Mix sample thoroughly.
Using the percent solids found in 11.1.3, determine the weight of sample required
to produce 1 L of solution containing 1% solids as follows:
, • z.
sample weight = % solids grams
11 .22.3 Discard all sticks, rocks, leaves, and other foreign material prior to weighing.
Place the weight determined in 11.2.2.2 in a clean 1.5- to 2.0-L beaker.
11 .22.4 Dilute aliquot — For samples that are expected to be difficult to extract, concen-
trate, or clean up, weigh an amount of sample equal to one-tenth the amount deter-
mined in 11.2.2.2 into a second clean 1.5- to 2.0-L beaker. When diluted to 1.0 L,
this dilute aliquot will contain 0.1% solids.
11 .2.2.5 Bring the sample aliquot(s) above to a volume of 100-200 mL with reagent water.
112.2.6 Spike 0.5 mL of the labeled compound spiking solution (Section 7.8) into each
sample aliquot.
11 .2.2.7 Using a clean metal spatula, break any solid portions of the sample into small
pieces.
11.2.2.8 Place the %" horn on the ultrasonic probe approximately Vi" below the surface of
each sample aliquot and pulse at 50% for 3 minutes at full power. If necessary,
remove the probe from the solution and break any large pieces using the metal
spatula or a stirring rod and repeat the sonication. Clean the probe with methylene
chloride:acetone (1:1) between samples to preclude cross-contamination.
11 22.9 Bring the sample volume to 1 .0 L (±0. 1 L) with reagent water.
16
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Method 1665
11.2.3 Preparation of QC aliquots for samples containing low solids (<30%).
11.2.3.1 For each sample or sample lot (to a maximum of 20) to be extracted at the same
time, place three 1.0-L (±0.01 L) aliquots of reagent water in clean 1.5- to 2.0-L
beakers.
11.2.3.2 Spike 0.5 mL of the labeled compound spiking solution (Section 7.8) into one
reagent water aliquot. This aliquot will serve as the blank.
11.2.3.3 Spike 1.0 mL of the precision and recovery standard (Section 7.14) into the two
remaining reagent water aliquots.
11.2.4 Stir and equilibrate all sample and QC solutions for 1-2 hours. Extract the samples and QC
aliquots per Section 11.3.
11.2.5 Samples containing greater than 30% solids.
11.2.5.1 Mix the sample thoroughly.
11.2.5.2 Discard all sticks, rocks, leaves and other foreign material prior to weighing.
Weigh 30 g (±0.3 g) into a clean 400- to 500-mL beaker.
11.2.5.3 Dilute aliquot—For samples which are expected to be difficult to extract, concen-
trate, or clean up, weigh 3 g (±0.03 g) into a clean 400- to 500-mL beaker.
11.2.5.4 Spike 0.5 mL of the labeled compound spiking solution (Section 7.8) into each
sample aliquot.
11.2.5.5 QC aliquots—For each sample or sample lot (to a maximum of 20) to be extracted
at the same time, place three 30 g (±0.3 g) aliquots of the high-solids reference
matrix in clean 400- to 500-mL beakers.
11.2.5.6 Spike 0.5 mL of the labeled compound spiking solution (Section 7.8) into one
nigh-solids reference matrix aliquot. This aliquot will serve as the blank.
11.2.5.7 Spike 1.0 mL of the precision and recovery standard (Section 7.14) into the two
remaining high-solids reference matrix aliquots. Extract, concentrate, and clean up
the high-solids samples and QC aliquots per Sections 11.4 through 11.8.
11.3 Continuous extraction of low-solids (aqueous) samples—Place 100-150 mL methylene chloride in
each continuous extractor and 200-300 mL in each distilling flask.
11.3.1 Pour the sample(s), blank, and QC aliquots into the extractors. Rinse the glass containers
with 50-100 mL methylene chloride and add to the respective extractors. Include all solids
in the extraction process.
11.3.2 Base/neutral extraction—Adjust the pH of the waters in the extractors to 12 -13 with 6N
NaOH while monitoring with a pH meter. Begin the extraction by heating the flask until the
methylene chloride is boiling. When properly adjusted, one to two drops of methylene chlo-
ride per second will fall from the condenser tip into the water. Test and adjust the pH of the
waters during the first to second hour and during the fifth to tenth hour of extraction. Extract
for 24-48 hours.
11.3.3 Remove the distilling flask, estimate and record the volume of extract (to the nearest 100
mL), and pour the contents through a drying column containing 7-10 cm anhydrous sodium
sulfate. Rinse the distilling flask with 30-50 mL of methylene chloride and pour through the
drying column. Collect the solution in a 500-mL K-D evaporator flask equipped with a 10-
17
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Method 1665
mL concentrator tube. Seal, label as the base/neutral fraction, and concentrate per Sections
11.5 to 11.6.
Note: All of the PMI analytes listed in Table 1 will be extracted from basic media. Acid extraction is not
necessary unless other analytes are being sought.
11.3.4 Acid extraction—Adjust the pH of the waters in the extractors to 2 or less using 6N sulfuric
acid. Charge clean distilling flasks with 300-400 mL of methylene chloride. Test and adjust
the pH of the waters during the first 1-2 hours and during the fifth to tenth hour of extraction.
Extract for 24—48 hours. Repeat Section 11.3.3, except label as the acid fraction.
11.4 Ultrasonic extraction of high-solids samples.
11.4.1 Add 60 g of powdered anhydrous sodium sulfate to the sample and QC aliquot(s) and mix
thoroughly.
11.4.2 Add 100 mL (±10 mL) of acetone:methylene chloride (1:1) to the sample and mix thor-
oughly.
11.4.3 Place the %" horn on the ultrasonic probe approximately Vi" below the surface of the solvent
but above the solids layer and pulse at SO percent for three minutes at full power. If neces-
sary, remove the probe from the solution and break any large pieces using the metal spatula
or a stirring rod and repeat the sonication.
11.4.4 Decant the extracts through Whatman 41 filter paper (or equivalent) using glass funnels and
collect in 500- to 1000-mL graduated cylinders.
11.4.5 Repeat the extraction steps (Section 11.4.2 to Section 11.4.4) twice more for each sample and
QC aliquot. On the final extraction, swirl the sample or QC aliquot, pour into its respective
glass funnel, and rinse with acetone:methylene chloride. Record the total extract volume.
11.4.6 Pour each extract through a drying column containing 7-10 cm of anhydrous sodium sulfate.
Rinse the graduated cylinder with 30-50 mL of methylene chloride and pour through the dry-
ing column. Collect each extract in a 500-mL K-D evaporator flask equipped with a 10-mL
concentrator tube. Seal and label as the high-solids semivolatile fraction. Concentrate and
clean up the samples and QC aliquots per Sections 11.5 through 11.8.
11.5 Macro concentration—Concentrate the extracts in separate 500-mL K-D flasks equipped with 10-mL
concentrator tubes.
11.5.1 Add one or two clean boiling chips to the flask and attach a three-ball macro Snyder column.
Prewet the column by adding approximately 1 mL of methylene chloride through the top.
Place the K-D apparatus in a hot water bath so that the entire lower rounded surface of the
flask is bathed with steam. Adjust the vertical position of the apparatus and the water tem-
perature as required to complete the concentration in 15 to 20 minutes. At the proper rate of
distillation, the balls of the column will actively chatter but the chambers will not flood.
When the liquid has reached an apparent volume of 1 mL, remove the K-D apparatus from
the bath and allow the solvent to drain and cool for at least 10 minutes. 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.
18
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Method 1665
11.5.2 For performance standards (Sections 9.2 and 13.7) and for blanks (Section 9.5), combine the
acid and base/neutral extracts for each at this point. Do not combine the acid and base/neu-
tral extracts for aqueous samples.
11.6 Micro-concentration.
11.6.1 Kuderna-Danish (K-D)—Add a clean boiling chip and attach a two-ball micro Snyder col-
umn to the concentrator tube. Prewet the column by adding approximately 0.5 mL methyl-
ene chloride through the top. Place the apparatus in the hot water bath. Adjust the vertical
position and the water temperature as required to complete the concentration in 5-10 min-
utes. At the proper rate of distillation, the balls of the column will actively chatter but the
chambers will not flood. When the liquid reaches an apparent volume of approximately
0.5 mL, remove the apparatus from the water bath and allow to drain and cool for at least 10
minutes. Remove the micro Snyder column and rinse its lower joint into the concentrator
tube with approximately 0.2 mL of methylene chloride. Adjust the final volume to 5.0 mL if
the extract is to be cleaned up by GPC, to 1.0 mL if it does not require clean-up, or to 0.5 mL
if it has been cleaned up.
11.6.2 Nitrogen blowdown—Place the concentrator tube in a warm water bath (35°C) and evapo-
rate the solvent volume using a gentle stream of clean, dry nitrogen (filtered through a col-
umn of activated carbon).
Caution: New plastic tubing must not be used between the carbon trap and the sample, since it may intro-
duce interferences.
The internal wall of the tube must be rinsed down several times with methylene chloride dur-
ing the operation. During evaporation, the tube solvent level must be kept below the water
level of the bath. The extract must never be allowed to become dry. Adjust the final volume
to 5.0 mL if the extract is to be cleaned up by GPC, to 1.0 mL if it does not require clean-up,
or to 0.5 mL if it has been cleaned up.
11.7 Transfer the concentrated extract to a clean screw-cap vial. Seal the vial with a PTFE-lined lid, and
mark the level on the vial. Label with the sample number and fraction, and store in the dark at -20 to
-10°C until ready for analysis.
11.8 GPC setup and calibration.
11.8.1 Column packing.
11.8.1.1 Place 75 g (±5 g) of SX-3 Bio-beads in a 400- to 500-mL beaker.
11.8.1.2 Cover the beads and allow to swell overnight (12 hours minimum).
11.8.1.3 Transfer the swelled beads to the column and pump solvent through the column,
from bottom to top, at 4.5-5.5 mL/min prior to connecting the column to the detec-
tor.
11.8.1.4 After purging the column with solvent for 1-2 hours, adjust the column head pres-
sure to 7-10 psig, and purge for 4-5 hours to remove air from the column. Main-
tain a head pressure of 7-10 psig. Connect the column to the detector.
11.8.2 Column calibration.
11.8.2.1 Load 5 mL of the calibration solution (Section 7.4) into the sample loop.
19
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Method 1665
11.8.2.2 Inject the calibration solution and record the signal from the detector. The elution
pattern will be com oil, bis(2-ethylhexyl)phthalate, pentachlorophenol, perylene,
and sulfur.
11.8.2.3 Set the "dump time" to allow >85% removal of the corn oil and >85% collection of
the phthalate.
11.8.2.4 Set the "collect time" to the peak minimum between perylene and sulfur.
11.8.2.5 Verify the calibration with the calibration solution after every 20 extracts. Calibra-
tion is verified if the recovery of the pentachlorophenol is greater than 85%. If cal-
ibration is not verified, the system shall be recalibrated using the calibration
solution, and the previous 20 samples shall be re-extracted and cleaned up using
the calibrated GPC system.
11.9 Extract cleanup.
11.9.1 Filter the extract or load through the filter holder to remove particulates. Load the 5.0-mL
extract onto the column. The maximum capacity of the column is 0.5-1.0 g. If necessary,
split the extract into multiple aliquots to prevent column overload.
11.9.2 Elute the extract using the calibration data determined in 11.8.2. Collect the eluate in a clean
400- to 500-mL beaker.
11.9.3 Concentrate the cleaned up extract to 5.0-mL per Section 11.5.
11.9.4 Rinse the sample loading tube thoroughly with methylene chloride between extracts to pre-
pare for the next sample.
11.9.5 If a particularly dirty extract is encountered, a 5.0-mL methylene chloride blank shall be run
through the system to check for carry-over.
11.9.6 Concentrate the extract to 0.5 mL and transfer to a screw-cap vial per Sections 11.6 and 11.7.
Concentrating these extracts to 0.5 mL will compensate for the loss of one-half of the extract
in the cleanup process.
12. GC/MS Analysis
12.1 Establish the operating conditions given in Section 6.11.1 for analysis of extracts.
12.2 Bring the concentrated extract (Section 11.7) or standard (Sections 7.13-7.14) to room temperature
and verify that any precipitate has redissolved. Verify the level on the extract (Sections 7.6 and 11.7)
and bring to the mark with solvent if required.
12.3 Add the internal standard solution (Section 7.10) to the extract (use 1.0 uL of solution per 0.1 mL of
extract) immediately prior to injection to minimize the possibility of loss by evaporation, adsorption,
or reaction. Mix thoroughly.
12.4 Inject a volume of the standard solution or extract such that 100 ng of the internal standard will be
injected, using on-column or splitless injection. For 1-mL extracts, this volume will be 1.0 pL. Start
the GC column initial isothermal hold upon injection. Start MS data collection after the solvent peak
elutes. Stop data collection after the internal standard (DFB) elutes. Return the column to the initial
temperature for analysis of the next sample.
20
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Method 1665
13. System and Laboratory Performance
13.1 At the beginning of each 8-hour shift during which analyses are performed, GC/MS system perfor-
mance and calibration are verified for all pollutants and labeled compounds. For these tests, analysis
of the calibration standard (Section 7.14) shall be used to verify all performance criteria. Adjustment
and/or recalibration (per Section 10) shall be performed until all performance criteria are met. Only
after all performance criteria are met may samples, blanks, and precision and recovery standards be
analyzed.
13.2 DFTPP spectrum validity—Inject 1 jjL of the DFTPP solution (Section 7.11) either separately or
within a few seconds of injection of the standard (Section 13.1) analyzed at the beginning of each
shift. The criteria in Table 3 shall be met.
13.3 Retention times—The absolute retention time of 2,2-difluorobiphenyl shall be within the range of
1092 to 1268 seconds and the relative retention times of all pollutants and labeled compounds shall
fall within the limits given in Table 2 or within ±10% of that specified.
13.4 GC resolution—All native PMI analytes shall be fully resolved and the valley height between any
two analytes shall not exceed 10% of the taller of the two peaks.
13.5 Calibration verification—Compute the concentration of each pollutant (Table 1) by isotope dilution
(Section 10.4) for those compounds that have labeled analogs. Compute the concentration of each
pollutant that has no labeled analog by the internal standard method (Section 10.5). Compute the
concentration of the labeled compounds by the internal standard method. These concentrations are
computed based on the calibration data determined in Section 10.
13.5.1 For each pollutant and labeled compound being tested, compare the concentration with the
calibration verification limit in Table 4. If all compounds meet the acceptance criteria, cali-
bration has been verified and analysis of blanks, samples, and precision and recovery stan-
dards may proceed. If, however, any compound fails, the measurement system is not
performing properly for that compound. In this event, prepare a fresh calibration standard or
correct the problem causing the failure and repeat the test (Section 13.1), or recalibrate (Sec-
tion 10).
13.6 Multiple peaks—Each compound injected shall give a single, distinct GC peak.
13.7 Ongoing precision and accuracy.
13.7.1 Analyze the extract of the precision and recovery standard (Section 9) prior to analysis of
samples from the same lot.
13.7.2 Compute the concentration of each pollutant (Table 1) by isotope dilution (Section 10.4) for
those compounds which have labeled analogs. Compute the concentration of each pollutant
that has no labeled analog by the internal standard method (Section 10.5). Compute the
concentration of the labeled compounds by the internal standard method.
13.7.3 For each pollutant and labeled compound, compare the concentration with the limits for on-
going accuracy in Table 4. If all compounds meet the acceptance criteria, system perfor-
mance is acceptable and analysis of blanks and samples may proceed. If, however, any indi-
vidual concentration falls outside of the range given, system performance is unacceptable for
that compound. In this event, correct the problem, re-extract the sample lot (Section 11) and
repeat the ongoing precision and recovery test (Section 13.7).
13.7.4 Add results that pass the specifications in Section 13.7.3 to initial and previous ongoing data
for each compound in each matrix. Update QC charts to form a graphic representation of
continued laboratory performance (Figure 5). Develop a statement of laboratory accuracy
21
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Method 1665
for each pollutant and labeled compound in each matrix type by calculating the average per-
cent recovery (R) and the standard deviation of percent recovery (sr). Express the accuracy
as a recovery interval from R - 2sr to R + 2sr For example, if R = 95% and sr = 5%, the accu-
racy is 85-105%.
14. Qualitative Determination
Identification is accomplished by comparison of data from analysis of a sample or blank with data stored in
the mass-spectral libraries. For compounds for which the relative retention times and mass spectra are
known, identification is confirmed per Sections 14.1 and 14.2.
14.1 Labeled compounds and pollutants having no labeled analog.
14.1.1 The signals for all characteristic m/z's stored in the spectral library (Section 10.2.4) shall be
present and shall maximize within the same two consecutive scans.
14.1.2 Either (1) the background corrected EICP areas, or (2) the corrected relative intensities of the
mass-spectral peaks at the GC peak maximum shall agree within a factor of 2 (0.5 to 2 times)
for all masses stored in the library.
14.1.3 For the compounds for which the system has been calibrated (Table 1), the retention time
shall agree with those specified in Table 2, or have a relative retention time ±10% of that
specified.
14.2 Pollutants having a labeled analog.
14.2.1 The signals for all characteristic m/z's stored in the spectral library (Section 10.2.4) shall be
present and shall maximize within the same two consecutive scans.
14.2.2 Either (1) the background corrected EICP areas, or (2) the corrected relative intensities of the
mass-spectral peaks at the GC peak maximum shall agree within a factor of two for all
masses stored in the spectral library.
14.2.3 The relative retention time between the pollutant and its labeled analog shall be within the
windows specified in Table 2 or within ±10% of that specified.
14.3 The m/z's present hi the experimental mass spectrum that are not present in the reference mass spec-
trum shall be accounted for by contaminant or background ions. If the experimental mass spectrum
is contaminated, or if identification is ambiguous, an experienced spectrometrist (Section 1.4) is to
determine the presence or absence of the compound.
15. Quantitative Determination
15.1 Isotope dilution—Because the pollutant and its labeled analog exhibit the same effects upon extrac-
tion, concentration, and gas chromatography, correction for recovery of the pollutant can be made by
adding a known amount of a labeled compound to every sample prior to extraction. Relative
response (RR) values for sample mixtures are used in conjunction with the calibration curves
described in Section 10.4 to determine concentrations directly, as long as labeled compound spiking
levels are constant. For the aniline example given in Figure 1 (Section 10.4.1), RR would be equal to
1.114. For this RR value, the aniline calibration curve given in Figure 1 indicates a concentration of
27 ug/mL in the sample extract
22
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Method 1665
15.2 Internal standard — compute the concentration in the extract using the response factor determined
from calibration data (Section 10.5) and the following equation:
A x C.
is
Where:
Cgx = The concentration of the compound in the extract, and the
other terms are as defined in Section 10.5.1.
15.3 The concentration of the pollutant in the solid phase of the sample is computed using the concentra-
tion of the pollutant in the extract and the weight of the solids (Section 11), as follows:
C xV
* ,. , , n •. eX eX
Concentration in solid ((ig/xg) = - ^ -
s
Where:
Vex = The extract volume in mL
Ws = The sample weight in kg
15.4 Dilution of samples — If the EICP area at the quantitation m/z for any compound exceeds the calibra-
tion range of the system, the extract of the dilute aliquot (Section 11) is analyzed by isotope dilution.
If further dilution is required and the sample holding time has not been exceeded, a smaller sample
aliquot is extracted per Sections 15.4.1-15.4.3. If the sample holding time has been exceeded, the
sample extract is diluted by successive factors of 10, internal standard is added to give a concentra-
tion of 100 ug/mL in the diluted extract, and the diluted extract is analyzed by the internal-standard
method.
15.4.1 For samples containing 1% solids or less for which the holding time has not been exceeded,
dilute 10 mL, 1.0 mL, 0.1 mL, etc., of sample to 1 L with reagent water and extract per Sec-
tion 11.2.1.
15.4.2 For samples containing 1-30% solids for which the holding time has not been exceeded,
extract an amount of sample equal to one-hundredth the amount determined in 1 1.2.2.2.
Extract per Section 11.2.2.
15.4.3 For samples containing 30% solids or greater for which the holding time has not been
exceeded, extract 0.30 g (±0.003 g) of sample per Section 11.2.5.
15.5 Results are reported to three significant figures for all pollutants and labeled compounds found in all
standards, blanks, and samples. For aqueous samples, the units used are ng/L. For samples containing
1-30% solids, ug/L may be used if the actual sample volume before dilution to 1% solids is used in
the calculation. In this latter case, the laboratory must report the percent solids so that conversion to
other units may be made, if required. The units of fig/kg may be used for samples containing 1-30%
solids, if based on dry weight, but must be used for samples containing greater than 30% solids,
based on dry weight.
15.5.1 Results for samples that have been diluted are reported at the least dilute level at which the
area at the quantitation m/z is within the calibration range (Section 15.4), or at which no m/z
in the spectrum is saturated (Section 15.5). For compounds having a labeled analog, results
23
-------
Method 1665
are reported at the least dilute level at which the area at the quantitation m/z is within the cal-
ibration range (Section 15.4) and the labeled compound recovery is within the normal range
for the method (Section 16.4).
16. Analysis of Complex Samples
16.1 Some samples may contain high levels (>1000 jig/L) of the compounds of interest, interfering com-
pounds, and/or polymeric materials. Some samples will not concentrate to 1 mL (Section 11.6); oth-
ers will overload the GC column and/or mass spectrometer.
16.2 Analyze the dilute aliquot (Section 11) when the sample will not concentrate to 1 mL. If a dilute ali-
quot was not extracted, and the sample holding time (Section 8.3) has not been exceeded, dilute an
aliquot of an aqueous sample with reagent water, or weigh a dilute aliquot of a high-solids sample
and re-extract (Section 11); otherwise, dilute the extract (Section 1S.4) and analyze by the internal
standard method (Section 15.2).
16.3 Recovery of internal standard—The EICP area of the internal standard should be within a factor of
two of the area in the shift standard (Section 13.1). If the absolute areas of the labeled compounds
are within a factor of 2 of the respective areas in the shift standard, and the internal standard area is
less than one-half of its respective area, then loss of the internal standard in the extract has occurred.
In this case, use one of the labeled compounds to compute the concentration of a pollutant with no
labeled analog.
16.4 Recovery of labeled compounds—In most samples, labeled compound recoveries will be similar to
those from reagent water or from the high-solids reference matrix (Section 13.7). If the labeled com-
pound recovery is outside the limits given in Table 4, the extract from the dilute aliquot (Section 11)
is analyzed as in Section 15.4. If the recoveries of all labeled compounds and the internal standard
are low (per the criteria above), then a loss in instrument sensitivity is the most likely cause. In this
case, the 100 ug/mL calibration standard (Section 13.1) shall be analyzed and calibration verified
(Section 13.5). If a loss in sensitivity has occurred, the instrument shall be repaired, the performance
specifications in Section 13 shall be met, and the extract reanalyzed. If a loss in instrument sensitivity
has not occurred, the laboratory must use additional cleanup steps to remove interferences. Sugges-
tions for additional cleanup are provided in Reference 10. If all attempts to resolve interference
problems fail, the method does not apply to the sample being analyzed, and the result may not be
reported for regulatory compliance purposes.
17. Method Performance
17.1 With the exception of o-picoline (2-methylpyridine), this method was validated in a single labora-
tory. a-Picoline was validated in the interlaboratory validation of EPA Method 1625 (Reference 6).
17.2 A chromatogram of the 100 jjg/mL calibration standard (Section 7.13) is shown in Figure 6.
18. Waste Management
18.1 It is the laboratory's responsibility to comply with all federal, State, and local regulations governing
waste management, particularly the hazardous waste identification rules and land disposal restric-
tions. In addition, it is the laboratory's responsibility to protect air, water, and land resources by min-
imizing and controlling all releases from fume hoods and bench operations. Also, compliance is
required with any sewage discharge permits and regulations.
24
-------
Method 1865
18.2 Samples containing acids at a pH of less than 2 are hazardous and must be neutralized before being
poured down a drain or must be handled as hazardous waste.
18.3 For further information on waste management, consult "The Waste Management Manual for Labora-
tory Personnel," available from the American Chemical Society's Department of Government Rela-
tions and Science Policy, 1115 Sixteenth Street, NW, Washington, DC 20036.
25
-------
Method 1665
References
1. "Performance Tests for the Evaluation of Computerized Gas Chromatography/Mass Spectrometry
Equipment and Laboratories," U.S. EPA, EMSL Cincinnati, Ohio 45268, EPA-600/4-80-025 (April
1980).
2. National Standard Reference Data System, "Mass Spectral Tape Format," U.S. National Bureau of
Standards (1979 and later attachments).
3. "Working with Carcinogens," DREW, PHS, CDC, NIOSH, Publication 77-206 (1977).
4. "OSHA Safety and Health Standards, General Industry," 29 CFR 1910, OSHA 2206 (1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication, Committee
on Chemical Safety (1979).
6. "Interlaboratory Validation of U. S. Environmental Protection Agency Method 1625A, Addendum
Report," SRI International, Prepared for Analysis and Evaluation Division (WH-557), U.S. EPA,
Washington DC 20460 (January 1985).
7. "Handbook of Analytical Quality Control in Water and Wastewater Laboratories," U.S. EPA, EMSL
Cincinnati, OH 45268, EPA-600/4-79-019 (March 1979).
8. "Standard Practice for Sampling Water," ASTM Annual Book of Standards, ASTM, Philadelphia,
PA, 76 (1982).
9. "Methods 330.4 and 330.5 for Total Residual Chlorine," U.S. EPA, EMSL, Cincinnati, OH 45268,
EPA 600/4-70-020 (March 1979).
10. "Guidance Evaluation, Resolution, and Documentation of Analytical Problems Assoicated with
Compliance Monitoring." EPA 821-B-93-001. Copies available from: Analytical Methods Staff,
Engineering and Analysis Division, Office Science and Technology, U.S. Environmental Protection
Agency, 401 M St., SW, Washington, DC 20460.
26
-------
Method 1665
Table 1. Base/Neutral Extractable PMI Analytes Determined by GC/MS Using Isotope Dilution or Internal
Standard Techniques
Pollutant
Labeled Compound
CASRN1
62-53-3
127-19-5
121-6917
68-12-2
109-06-8
110-86-1
EPA-EGD
757
976
1329
785
703
1330
Analog
dr
di,
dr
dr
d
CASRN1
14545-23-4
Not Avail.
4472-41-7
93951-93-0
7291-22-7
EPA-EGD
657
1229
685
603
1230
PMI Analyte
Aniline
N,N-Dimethylacetamide
N,N-Dimethylaniline
N,N-Dimethylforrnamide
2-Methylpyridine (a-picoline)2
Pyridine
1 Chemical Abstracts Service Registry Number.
2 This semivolatile compound may be analyzed by Method 1625B or the method described herein.
27
-------
Method 1665
Table 2. Gas Chromatographic Retention Times and Minimum Levels for Base/Neutral Extractable PMI
Analytes
Retention Time
EGD
No.1 PMI Analyte
164 2,2'-Difluorobiphenyl (int std)
1230 Pyridine-ds
1330 Pyridine
685 N,N-Dimethylformamide-d7
785 N,N-Dimethylformamide
603 2-Methylpyridine-d7 (a-picoline-d7)
703 2-Methylpyridine (a-picoline)
976 N,N-Dimethylacetamide
657 Aniline-d7
757 Aniline
1229 N.N-Dimethylaniline-du
1329 N,N-Dimethylaniline
1 Three-digit EGD numbers beginning with 0,1,5, or 9 indicate a pollutant quantified by the internal standard method; begin-
ning with 2 or 6 indicate a labeled compound quantified by the internal standard method; beginning with 3 or 7 indicate a pol-
lutant quantified by isotope dilution. The initial "1° in four-digit EGD numbers is to be ignored in applying these rules.
2 Single values in this column are based on single laboratory data.
3This is a minimum level at which the entire analytical system shall give recognizable mass spectra (background-corrected) and
acceptable calibration points, taking into account method-specific sample and injection volumes. The concentration in the
aqueous or solid phase is determined using the equations in Section 15.
Mean
(sec)
1180
343
345
399
401
449
455
536
702
705
856
863
EGD
Ref
164
164
1230
164
685
164
603
164
164
657
164
1229
Relative2
1.000-1.000
0.291
1.006
0.338
1.005
0.326-0.393
1.006-1.028
0.454
0.595
1.004
0.725
1.008
MinimumLevel3
(M9/L)
5
5
5
50
2
10
28
-------
Method 1665
Table 3. DFTPP Mass-Intensity Specifications
Mass Intensity Required
51 8-82% of m/z 198
68 less than 2% of m/z 69
69 11-91% of m/z 198
70 less than 2% of m/z 69
127 32-59% of m/z 198
197 less than 1% of m/z 198
198 base peak, 100% abundance
199 4-9% of m/z 198
275 11-30% of m/z 198
441 44-110% of m/z 443
442 30-86% of m/z 198
443 14-24% of m/z 442
29
-------
Method 1665
Table 4. Acceptance Criteria for PMI Analytes
EGD
757
657
976
1329
1229
785
685
703
603
1330
1230
PMI Analyte
Aniline
Aniline-d7
N,N-Dimethylacetamide
N,N-Dimethylaniline
N.N-Dimethylaniline-dn
N,N-Dimethylformamide
N,N-Dimethylformamide-d7
2-Methylpyridine
(a-picoline)
2-Methylpyridine-d7
Pyridine
Pyridine-ds
Spike _
Level
(ug/L)
10
100
20
10
100
10
100
10
100
10
100
Initial Precision and
Accuracy ^M
Rnmnnnnri
(H9/L)
1
55
20
5
192
2
19
4
27
3
30
X Recovery
(ug/L) P(%)
8-10
75-184 70-188
d-39
6-15
47-431 31-446
9-13
65-103 64-104
7-15
55-109 53-112
7-12
54-113 52-116
Calibration
Verification
(M9/L)
8-10
75-184
d-39
6-15
47-431
9-13
65-103
7-15
55-109
7-12
54-113
Ongoing
Accuracy
(ug/L)
8-10
70-188
d-41
6-15
31-446
9-13
64-104
7-15
53-112
7-12
52-116
d = Detected (compound must be detected)
30
-------
Method 1665
Table 5. Characteristic m/z's and Response Factors for Base/Neutral Extractable PMI Analytes
Spike
Level Labeled Response
PMIAnalyte (jig/L) Analog Primary m/z1 Factor2
Aniline 2 d7 93/98 1.22
N,N-Dimethylacetamide 50 44 0.33
N,N-Dimethylaniline 10 dM 120/130 0.99
N,N-Dmethylformamide 5 d7 73/80 0.84
2-Methylpyridine (a-picoline) 5 d7 93/100 1.15
Pyridine 5 ds 79/84 1.05
1 Native/labeled
2 Referenced to 2,2'-difluorobiphenyl
31
-------
Method 1665
I I
10 —
1.0 —
0.1 —
T
2
I 1 I
10 20 50
Concentration (pg/mL)
I I
100 200
The Dotted Lines Enclose a ±10% Error
Figure 1. Relative Response Calibration Curve for Aniline
52-025-2
32
-------
Method 1665
Area at
rrig/z
Area at
m/z
Area at
rr^/z
Figure 2. Extracted Ion-Current Profiles for Chromatographically Resolved
Labeled (m^z) and Unlabeled (m^z) Pairs
52-025-4
33
-------
Method 1665
Area = 46100
(3A)
m2/z
Area = 4780
Area = 2650
(38)
m2/z
rr^/z
Area = 43600
Area = 49200
(3C)
IT12/Z
Area = 48300
52-025-5
Figure 3. Extracted Ion-Current Profiles for (3A) Unlabeled Compound, (3B) Labeled Compound, and
(3C) Equal Mixture of Unlabeled and Labeled Compounds
34
-------
Method 1665
CO
CO
"ni OR nnn _•
" OJ,UUU ^^
1
3* nnn -
1
0
•5"
0
Q. CO 1
SfS ^^
CC c 1.00 ^
II
i
A nrt — —
6/1
I 1 I 1 I I I I 1
Anthracene-dio
•
• .
• • •
•
I I I i I i I i I
2 3456789 10
Analysis Number
I I I I I I I I I
Anthracene
• • • •
• • • • •
- +3s
_ _ Qc
— —OS
- +3s
_ «3o
OS
I I I I I I I I I
6/1 6/1 6/1 6/2 6/2 6/3 6/3 6/4 6/5
Date Analyzed
52-025-3
Figure 4. Quality Control Charts Showing Area (top graph) and Relative Response of
Anthracene to Anthracene-d10 (lower graph) Plotted as Function of Time or
Analysis Number
35
-------
Method 1665
f
ffl!t:4
c J? » » ~
|||| 3
2
2
F
r
2
F*
t
C
1
i i ' i ' i ' i
500 1,000
Seconds
1,500
2,000
52-025-1
Figure 5. Chromatogram of PMI Analytes Specific to Method 1665
36
-------
Method 1666
Volatile Organic Compounds Specific to the
Pharmaceutical Manufacturing Industry by
Isotope Dilution GC/MS
January 1995
-------
Method 1666
Volatile Organic Compounds Specific to the
Pharmaceutical Manufacturing Industry by
Isotope Dilution GC/MS
1. Scope and Application
1.1 This method is designed to meet the survey and monitoring requirements of the EPA's Engineering
and Analysis Division (EAD). The method is used to determine certain volatile organic pollutants
specific to the pharmaceutical manufacturing industry (PMI) that are amenable to purge-and-trap gas
chromatography/mass spectrometry (GC/MS) or direct aqueous injection GC/MS.
1.2 The PMI analytes listed in Tables 1 and 2 may be determined in waters, soils, and municipal sludges
by this method or the method referenced.
1.3 The detection limits of the method are usually dependent on the level of interferences rather than
instrumental limitations. The minimum levels (MLs) in Tables 3 and 4 are the level that can be
attained with no interferences present.
1.4 The GC/MS portions of this method are for use only by analysts experienced with GC/MS or under
the close supervision of such qualified persons. Laboratories unfamiliar with analysis of environmen-
tal samples by GC/MS should run the performance tests in Reference 1 before beginning.
1.5 This method is performance-based. The analyst is permitted to modify the method to overcome
interferences or to lower the cost of measurements, provided that all performance criteria in this
method are met. The requirements for establishing method equivalency are given in Section 9.1.2.
2. Summary of Method
2.1 Purge-and-trap GC/MS.
Stable, isotopically labeled analogs of the compounds of interest are added to the sample and the
sample is purged with an inert gas at 45 °C in a chamber designed for soil or water samples, as appro-
priate. In the purging process, the volatile compounds are transferred from the aqueous phase into
the vapor phase, where they are passed into a sorbent column and trapped. After purging is com-
pleted, the trap is backflushed and heated rapidly to desorb the compounds into a gas chromatograph
(GC). The compounds are separated by the GC and detected by a mass spectrometer (MS) (Refer-
ences 2 and 3).
2.2 Direct aqueous injection.
Certain volatile, water-soluble organic compounds do not purge well from water and are analyzed by
direct aqueous injection.
2.2.1 The percent solids content of the sample is determined. If the solids content is known or
determined to be less than 1%, stable, isotopically labeled analogs of the compounds of
interest are added to a 5-mL sample. If the solids content of the sample is greater than 1 %, 5
mL of reagent water and the labeled compounds are added to a S-g aliquot of sample. The
mixture is sonicated in a centrifuge vial with little or no headspace for 5 minutes. During
this period the native analytes and labeled analogs will equilibrate between the solid and
-------
Method 1666
aqueous phases. In some cases, additional sonication will be necessary to establish equilib-
rium. The resulting suspension is centrifuged and the supernatant liquid analyzed.
2.2.2 One |iL or more of the aqueous solution (or supernate) is injected into the GC/MS system.
The compounds are separated by the GC and detected by the mass spectrometer (References
2 and 3). The labeled compounds serve to correct the variability of the analytical technique.
2.3 Identification of a pollutant (qualitative analysis) is performed by calibrating the GC/MS system with
authentic standards and storing mass spectrum and retention time for each standard in a user-created
library. A compound is identified when its retention time and mass spectrum agree with the library
retention time and spectrum.
2.4 Quantitative analysis is performed in one of two ways in this method by using extracted-ion current
profile (EICP) areas. (1) For those compounds listed in Table 1 and Table 2, and for other com-
pounds for which standards and labeled analogs are available, the GC/MS system is calibrated and
the compound concentration is determined using an isotope dilution technique. (2) For those com-
pounds listed in Table 1 and Table 2, and for other compounds for which authentic standards but no
labeled compounds are available, the GC/MS system is calibrated and the compound concentration is
determined using an internal standard technique.
2.5 The quality of the analysis is assured through reproducible calibration of the GC/MS system.
3. Definitions
There are no method-specific definitions to be noted for this document.
4. Interferences
4.1 Impurities in the purge gas, organic compounds outgassing from the plumbing upstream of the trap,
and solvent vapors in the laboratory account for the majority of contamination problems encountered
with this method. The analytical system is demonstrated to be free from interferences under condi-
tions of the analysis by analyzing reagent water blanks initially and with each sample batch (samples
analyzed on the same 8-hour shift), as described in Section 9.5.
4.2 Samples can be contaminated by diffusion of volatile organic compounds (particularly methylene
chloride) through the bottle seal during shipment and storage. A field blank prepared from reagent
water and carried through the sampling and handling protocol may serve as a check on such contam-
ination.
4.3 Contamination by carryover can occur when high-level and low-level samples are analyzed sequen-
tially. To reduce carryover, the purging device (Figure 1 for samples containing less than 1% solids;
Figure 2 for samples containing 1% solids or greater) in purge-and-trap analysis or the syringe in
direct aqueous injection analysis is cleaned or replaced with a clean purging device or syringe after
each sample is analyzed. When an unusually concentrated sample is encountered, it is followed by
analysis of a reagent water blank to check for carryover. Purging devices and syringes are cleaned by
washing with soap solution, rinsing with tap and distilled water, and drying in an oven at 100-125 °C.
The trap and other parts of the system are also subject to contamination; therefore, frequent bakeout
and purging of the entire system may be required.
4.4 Interferences resulting from samples will vary considerably from source to source, depending on the
diversity of the site being sampled.
40
-------
Method 1666
5. Safety
5.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been pre-
cisely determined; however, each chemical compound should be treated as a potential health hazard.
Exposure to these compounds should be reduced to the lowest possible level. The laboratory is
responsible for maintaining a current awareness file of OSHA regulations regarding the safe han-
dling of the chemicals specified in this method. A reference file of material safety data sheets should
also be made available to all personnel involved in these analyses. Additional information on labora-
tory safety can be found in References 5 through 7.
6. Equipment and Supplies
6.1 Sample bottles and septa.
6.1.1 Bottle—25- to 40-mL with screw-cap (Pierce 13075, or equivalent). Detergent wash, rinse
with tap and distilled water, and dry at > 105 °C for a minimum of 1 hour before use.
6.1.2 Septum—Polytetrafluoroethylene (FTFE)-faced silicone (Pierce 12722, or equivalent),
cleaned as above and baked at 100-200 °C for a minimum of 1 hour.
6.2 Purge-and-trap device—Consists of purging device, trap, and desorber.
6.2.1 Purging devices for water and soil samples.
6.2.1.1 Purging device for water samples—Designed to accept 5-mL samples with water
column at least 3 cm deep. The volume of the gaseous head space between the
water and trap shall be less than 15 mL. The purge gas shall be introduced less
than 5 mm from the base of the water column and shall pass through the water as
bubbles with a diameter less than 3 mm. The purging device shown in Figure 1
meets these criteria.
6.2.1.2 Purging device for solid samples—Designed to accept 5 g of solids plus 5 mL of
water. The volume of the gaseous head space between the water and trap shall be
less than 25 mL. The purge gas shall be introduced less than 5 mm from the base of
the sample and shall pass through the water as bubbles with a diameter less than 3
mm. The purging device shall be capable of being controlled at a temperature of
45 °C (±2°C) while the sample is being purged. The purging device shown in Fig-
ure 2 meets these criteria.
6.2.2 Trap—25-30 cm long x 2.5 mm i.d. minimum, containing the following:
6.2.2.1 Methyl silicone packing—1 cm (±0.2 cm), 3% OV-1 on 60/80 mesh Chromosorb
W, or equivalent.
6.2.2.2 Porous polymer—15 cm (±1.0 cm), Tenax GC (2,6-diphenylene oxide polymer),
60/80 mesh, chromatographic grade, or equivalent.
6.2.2.3 Silica gel—8 cm (±1.0 cm), Davison Chemical, 35/60 mesh, grade 15, or equiva-
lent. The trap shown in Figure 3 meets these specifications.
6.2.3 Desorber—Shall heat the trap to 175 °C (±5 °C) in 45 seconds or less. The polymer section of
the trap shall not exceed a temperature of 180 °C and the remaining sections shall not exceed
220 °C during desorb, and no portion of the trap shall exceed 225 °C during bakeout. The des-
orber shown in Figure 3 meets these specifications.
41
-------
Method 1666
6.2.4 The purge-and-trap device may be a separate unit or coupled to a GC, as shown in Figures 4
and 5.
6.3 Gas chromatograph—Shall be linearly temperature-programmable with initial and final holds, and
shall produce results that meet the calibration (Section 10), quality assurance (Section 9), and perfor-
mance tests (Section 15) of this method.
6.3.1 Column for purge-and-trap analyses—60 m long x 0.32 mm i.d. fused-silica microbore col-
umn coated with 1.5 pm of phenylmethyl polysiloxane (Restek RTX-Volatiles, or equiva-
lent).
6.3.2 Column for direct aqueous injection analyses—30 m long x 0.32 mm i.d. fused-silica
microbore column coated with 1.5 um of 95% dimethyl- 5% diphenyl polysiloxene specially
passivated for chromatography of amines (Reslek RTX-5 Amine, or equivalent).
6.3.3 GC operating conditions.
6.3.3.1 Purge-and-trap—4 minutes at 0
-------
Method 1666
6.9 Syringe—5-mL, gas-tight, with shut-off valve.
6.10 Bottles—15-mL, screw-cap with PTFE liner.
6.11 Balances.
6.11.1 Analytical, capable of weighing 0.1 mg.
6.11.2 Top-loading, capable of weighing 10 mg.
6.12 Equipment for determining percent moisture.
6.12.1 Oven, capable of being temperature-controlled at 110 °C (±5 °C).
6.12.2 Desiccator.
6.12.3 Beakers—50- to 100-mL.
6.13 Centrifuge apparatus.
6.13.1 Centrifuge capable of rotating 10-mL centrifuge tubes at 5000 rpm.
6.13.2 Centrifuge tubes, 10-mL, with screw-caps to fit centrifuge.
6.14 Sonication apparatus capable of sonicating 10 mL centrifuge tubes and thoroughly agitating contents.
7. Reagents and Standards
7.1 Reagent water—Water in which the compounds of interest and interfering compounds are not
detected by this method. It may be generated by any of the following methods.
7.1.1 Activated carbon—Pass tap water through a carbon bed (Calgon Filtrasorb-300, or equiva-
lent).
7.12 Water purifier—Pass tap water through a purifier (Millipore Super Q, or equivalent).
7.1.3 Boil and purge—Heat tap water to 90-100 °C and bubble contaminant-free inert gas through
it for approximately 1 hour. While still hot, transfer the water to screw-cap bottles and seal
with a PTFE-lined cap.
7.2 Sodium thiosulfate—ACS granular.
7.3 Methanol—Pesticide-quality or equivalent.
7.4 Standard solutions—Purchased as solutions or mixtures with certification to their purity, concentra-
tion, and authenticity, or prepared from materials of known purity and composition. If compound
purity is 96% or greater, the weight may be used without correction to calculate the concentration of
the standard.
7.5 Preparation of stock solutions for purge-and-trap analysis.
7.5.1 Place approximately 9.5 mL of methanol in a 10-mL ground-glass-stoppered volumetric
flask. Allow the flask to stand unstoppered for approximately 10 minutes or until all metha-
nol-wetted surfaces have dried. In each case, weigh the stoppered flask, add the compound,
restopper, then immediately reweigh to prevent evaporation losses from affecting the mea-
surement.
7.5.2 Using a 100-uL syringe, permit two drops of liquid to fall into the methanol without contact-
ing the neck of the flask. Alternatively, inject a known volume of the compound into the
methanol in the flask using a microsyringe.
43
-------
Method 1666
7.5.3 Fill the flask to volume, stopper, then mix by inverting several times. Calculate the concen-
tration in milligrams per milliliter (mg/mL; equivalent to micrograms per microliter [ug/uL])
from the weight gain.
7.5.4 Transfer the stock solution to a FTFE-sealed screw-cap bottle. Store, with minimal head-
space, in the dark at -10 to -20 °C.
7.5.5 Replace standards after one month, or sooner if comparison with check standards indicate a
change in concentration. Quality control check standards that can be used to determine the
accuracy of calibration standards may be available from the National Institute of Standards
and Technology, Gaithersburg, Maryland.
7.6 Preparation of stock solutions for direct aqueous injection analysis.
7.6.1 Place approximately 9.0 mL of reagent water in a 10-mL ground-glass-stoppered volumetric
flask. Allow the flask to stand unstoppered for approximately 10 minutes or until all wetted
surfaces have dried. In each case, weigh the stoppered flask, add the compound, restopper,
then immediately reweigh to prevent evaporation losses from affecting the measurement.
7.6.2 Using a microsyringe, add sufficient liquid (about 100 mg) so that the final solution will
have a concentration of about 10 mg/mL.
7.6.3 Fill the flask to volume, stopper, then mix by inverting several times. Calculate the concen-
tration in milligrams per milliliter (mg/mL; equivalent to micrograms per microliter [ug/uL])
from the weight gain.
7.6.4 Transfer the stock solution to a PTFE-sealed screw-cap bottle. Store, with minimal head-
space, in the dark at approximately 4*C. Do not freeze.
7.6.5 Replace standards after one month, or sooner if comparison with check standards indicate a
change in concentration. Quality control check standards that can be used to determine the
accuracy of calibration standards may be available from the National Institute of Standards
and Technology, Gaithersburg, Maryland.
7.7 Labeled compound spiking solutions.
7.7.1 For purge-and-trap analysis—From stock standard solutions (Section 7.5), or from mixtures,
prepare the spiking solution to contain a concentration of labeled compound such that a 5- to
10-uL spike into each 5-mL sample, blank, or aqueous standard analyzed will result in a con-
centration of 50 ug/L of each compound with an ML of 20 jig/L or less, a concentration of
500 ug/L for each compound with an ML of 100 or 200 ug/L, and a concentration of 1 mg/L
for each compound with an ML of 500 ug/L (see Table 3). Include the internal standards
(Section 10.4.2) in this solution, if appropriate, so that a concentration of 50 ug/L in each
sample, blank, or aqueous standard will be produced.
7.7.2 For direct aqueous injection—From stock standard solutions (Section 7.6), or from mixtures,
prepare the spiking solution to contain a concentration such that a 50- to 100-uL spike into
each sample, blank, or aqueous standard analyzed will result in a concentration of 1 mg/mL
of each labeled compound. Include the internal standard in this solution so that a concentra-
tion of 1 mg/mL will be produced.
7.8 Secondary standards—Using stock solutions, prepare a secondary standard in methanol or water, as
appropriate, to contain each pollutant at a concentration of 1 mg/mL, or 2.5 mg/mL for compounds
with higher MLs.
7.8.1 Aqueous calibration standards—Using a microsyringe, add sufficient secondary standard
(Section 7.8) to five reagent water aliquots to produce concentrations in the range of interest.
44
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Method 1666
7.8.2 Aqueous performance standard—An aqueous standard containing all pollutants, internal
standards, labeled compounds, and BFB is prepared daily, and analyzed each shift to demon-
strate performance (Section 15). This standard shall contain concentrations of pollutants,
labeled compounds, BFB, and internal standards, as appropriate, within a factor of 1-5 times
the MLs of the pollutants listed in Table 3 or 4. It may be one of the aqueous calibration
standards described in Section 7.8.1.
7.8.3 A methanolic standard containing all pollutants specific to this method (Table 1) and internal
standards is prepared to demonstrate recovery of these compounds when syringe injection
and purge-and-trap analyses are compared. This standard shall contain either 100 ng/mL or
500 ug/mL of the PMI analytes, and 100 ug/mL of the internal standards (consistent with the
amounts in the aqueous performance standard in Section 7.8.2).
7.8.4 Other standards that may be needed are those for test of BFB performance (Section 10.1) and
for collection of mass spectra for storage in spectral libraries (Section 10.1.1).
8. Sample Collection, Preservation, and Handling
8.1 Grab samples are collected in glass containers having a total volume greater than 20 mL. For aque-
ous samples that pour freely, fill sample bottles so that no air bubbles pass through the sample as the
bottle is filled and seal each bottle so that no air bubbles are entrapped. Maintain the hermetic seal on
the sample bottle until time of analysis.
8.2 Samples are maintained at 0-4
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Method 1666
9.1.2 In recognition of advances that are occurring in analytical technology, and to allow the ana-
lyst to overcome sample matrix interferences, the analyst is permitted certain options to
improve separations or lower the costs of measurements. These options include alternative
concentration and cleanup procedures, and changes in columns and detectors. Alternative
techniques, such as the substitution of spectroscopy or immunoassay, and changes that
degrade method performance are not allowed. If an analytical technique other than the tech-
niques specified in this method is used, that technique must have a specificity equal to or bet-
ter than the specificity of the techniques in this method for the analytes of interest.
9.1.2.1 Each time a modification is made to this method, the analyst is required to repeat
the procedure in Section 9.2. If the detection limit of the method will be affected
by the change, the laboratory is required to demonstrate that the MDL (40 CFR
Part 136, Appendix B) is lower than one-third the regulatory compliance level. If
calibration will be affected by the change, the analyst must recalibrate the instru-
ment per Section 10.
9.1.2.2 The laboratory is required to maintain records of modifications made to this
method. These records include the information below, at a minimum.
9.1.2.2.1 The names, titles, addresses, and telephone numbers of the analyst(s)
who performed the analyses and modification, and of the quality con-
trol officer who witnessed and will verify the analyses and modifica-
tion.
9.1.2.2.2 A listing of pollutant(s) measured, by name and CAS Registry Number.
9.1.2.2.3 A narrative stating the reason(s) for the modification.
9.1.2.2.4 Results from all quality control (QC) tests comparing the modified
method to this method, including:
(a) Calibration (Section 10)
(b) Calibration verification (Section IS)
(c) Initial precision and accuracy (Section 9.2)
(d) Labeled compound recovery (Section 9.3)
(e) Analysis of blanks (Section 9.5)
(f) Accuracy assessment (Section 9.4)
9.1.2.3 Data that will allow an independent reviewer to validate each determination by
tracing the instrument output (peak height, area, or other signal) to the final result,
including:
(a) Sample numbers and other identifiers
(b) Analysis dates and times
(c) Analysis sequence/run chronology
(d) Injection logs
(e) Sample weight or volume
(f) Sample volume prior to each cleanup step, if applicable
(g) Sample volume after each cleanup step, if applicable
(h) Final sample volume prior to injection (Sections 11 and 12)
46
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Method 1666
(i) Injection volume (Sections 11 and 12)
(j) Dilution data, differentiating between dilution of a sample or an extract (Sec-
tion 16.4)
(k) Instrument and operating conditions
(I) Column (dimensions, liquid phase, solid support, film thickness, etc.)
(m) Operating conditions (temperature, temperature program, flow rates, etc.)
(n) Detector (type, operating condition, etc.)
(o) Chromatograms, printer tapes, and other recording of raw data
(p) Quantitation reports, data system outputs, and other data necessary to link raw
data to the results reported.
9.1.3 Analyses of blanks are required to demonstrate freedom from contamination and that the
compounds of interest and interfering compounds have not been carried over from a previ-
ous analysis (Section 4.3). The procedures and criteria for analysis of a blank are described
in Section 9.5.
9.1.4 The laboratory shall spike all samples with labeled compounds to monitor method perfor-
mance. This test is described in Section 9.3. When results of these spikes indicate atypical
method performance for samples, the samples are diluted to bring method performance
within acceptable limits (Section 16).
9.1.5 The laboratory shall, on an ongoing basis, demonstrate through the analysis of the aqueous
performance standard (Section 7.8.2) that the analysis system is in control. This procedure is
described in Sections 15.1 and 15.5.
9.1.6 The laboratory shall maintain records to define the quality of data that is generated. Devel-
opment of accuracy statements is described in Sections 9.4 and 15.5.2.
9.2 Initial precision and accuracy—To establish the ability to generate acceptable precision and accuracy,
the analyst shall perform the following operations for compounds to be calibrated:
9.2.1 Analyze two sets of four 5-mL aliquots (eight aliquots total) of the aqueous performance
standard (Section 7.8.2) containing Table 1 PMI analytes by purge-and-trap. Or, for Table 2
PMI analytes, analyze two sets of four aliquots (eight aliquots total) by direct aqueous injec-
tion.
9.2.2 Using results of the first set of four analyses in Section 9.2.1, compute the average recovery
(X) in percent of spike level and the standard deviation of the recovery (s) in percent of spike
level, for each compound, by isotope dilution for pollutants with a labeled analog, and by
internal standard for labeled compounds and pollutants with no labeled analog.
9.2.3 For each compound, compare s and X with the corresponding limits for initial precision and
accuracy found in Table 6. If s and X for all compounds meet the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may begin. If, however, any
individual s exceeds the precision limit or any individual X falls outside the range for accu-
racy, system performance is unacceptable for that compound.
47
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Method 1666
Note: The large number of compounds in Table 6 presents a substantial probability that one or more will
fail one of the acceptance criteria when all compounds are analyzed. To determine if the analyti-
cal system is out of control, or if the failure can be attributed to probability, proceed as follows.
9.2.4 Using the results of the second set of four analyses, compute s and X for only those com-
pounds that failed the test of the first set of four analyses (Section 9.2.3). If these compounds
now pass, system performance is acceptable for all compounds, and analysis of blanks and
samples may begin. If, however, any of the same compounds fail again, the analysis system
is not performing properly for the compound(s) in question. In this event, correct the prob-
lem and repeat the entire test (Section 9.2.1).
9.3 The laboratory shall spike all samples with labeled compounds to assess method performance on the
sample matrix.
9.3.1 Spike and analyze each sample according to the appropriate method in Section 11 or 12.
9.3.2 Compute the percent recovery (P) of the labeled compounds using the internal standard
method (Section 10.4.2).
9.3.3 Compare the percent recovery for each compound with the corresponding labeled compound
recovery limit in Table 6. If the recovery of any compound falls outside its warning limit,
method performance is unacceptable for that compound in that sample. Therefore, the sam-
ple matrix is complex and the sample is to be diluted and reanalyzed, per Section 16.
9.4 As part of the QA program for the laboratory, method accuracy for wastewater samples shall be
assessed and records shall be maintained. After the analysis of five wastewater samples for which
the labeled compounds pass the tests in Section 9.3.3, compute the average percent recovery (P) and
the standard deviation of the percent recovery (sp) for the labeled compounds only. Express the accu-
racy assessment as a percent recovery interval from P - 2sp to P + 2sp. For example, if P = 90% and
sp = 10%, the accuracy interval is expressed as 70-110%. Update the accuracy assessment for each
compound on a regular basis (e.g., after each five to ten new accuracy measurements).
9.5 Blanks—Reagent water blanks are analyzed to demonstrate freedom from carryover and contamina-
tion (Section 4).
9.5.1 The level at which the purge-and-trap system will carry greater than the ML of a pollutant of
interest (Table 1) into a succeeding blank shall be determined by analyzing successively
larger concentrations of these compounds. When a sample contains this concentration or
more, a blank shall be analyzed immediately following this sample to demonstrate no carry-
over at the ML.
9.5.2 With each sample batch (samples analyzed on the same 8-hour shift), a blank shall be ana-
lyzed immediately after analysis of the aqueous performance standard (Section 15.1) to dem-
onstrate freedom from contamination. If any of the compounds of interest (Table 1 or 2) or
any potentially interfering compound is found in a blank at greater than the ML (assuming a
response factor of 1 relative to the nearest-eluted internal standard for compounds not listed
in Tables 1 and 2), analysis of samples is halted until the source of contamination is elimi-
nated and a blank shows no evidence of contamination at this level.
9.6 The specifications contained in this method can be met if the apparatus used is calibrated properly,
then maintained in a calibrated state. The standards used for calibration (Section 7), calibration veri-
fication (Section 15.5), and initial (Section 9.2) and ongoing (Section 15.5) precision and accuracy
should be identical, so that the most precise results will be obtained. The GC/MS instrument in par-
48
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Method 1666
ticular will provide the most reproducible results if dedicated to the settings and conditions required
for the analyses of volatiles by this method.
9.7 Depending on specific program requirements, field replicates may be collected to determine the pre-
cision of the sampling technique, and spiked samples may be required to determine the accuracy of
the analysis when the internal-standard method is used.
10. Calibration
Calibration of the GC/MS system is performed by direct aqueous injection (Section 10.3) or purging the
compounds of interest and their labeled analogs from reagent water at the temperature to be used for anal-
ysis of samples (Section 10.2).
10.1 Assemble the GC/MS apparatus and establish the operating conditions to be used for sample analysis
(Section 6.3.3.1 or Section 6.3.3.2). By injecting standards into the GC, demonstrate that the analyt-
ical system meets the minimum levels in Tables 3 or 4 for the compounds for which calibration is to
be performed, and the mass-intensity criteria in Table 5 for 50 ng BFB.
10.1.1 Mass-spectral libraries—Detection and identification of the compounds of interest are
dependent upon the spectra stored in user-created libraries.
10.1.1.1 For the compounds in Tables 1 and 2, and other compounds for which the GC/MS
is to be calibrated, obtain a mass spectrum of each pollutant and labeled compound
and each internal standard by analyzing an authentic standard either singly or as
part of a mixture in which there is no interference between closely eluted compo-
nents. Examine the spectrum to determine that only a single compound is present.
Fragments not attributable to the compound under study indicate the presence of an
interfering compound. Adjust the analytical conditionsand scan rate (for this test
only) to produce an undistorted spectrum at the GC peak maximum. An undis-
torted spectrum will usually be obtained if five complete spectra are collected
across the upper half of the GC peak. Software algorithms designed to "enhance"
the spectrum may eliminate distortion, but may also eliminate authentic m/z's or
introduce other distortion.
10.1.1.2 The authentic reference spectrum is obtained under BFB tuning conditions (Sec-
tion 10.1 and Table 5) to normalize it to spectra from other instruments.
10.1.1.3 The spectrum is edited by saving the five most intense mass-spectral peaks and all
other mass-spectral peaks greater than 10% of the base peak. The spectrum may
be further edited to remove common interfering masses. If five mass-spectral
peaks cannot be obtained under the scan conditions given in Section 6.4, the mass
spectrometer may be scanned to an m/z lower than 20 to gain additional spectral
information. The spectrum obtained is stored for reverse search and for compound
confirmation.
10.2 Assemble the GC/MS apparatus and establish operating conditions given in Section 6.3.3.1. By
injecting standards into the GC, demonstrate that the analytical system meets the minimum levels in
Table 3 for the compounds for which calibration is to be performed, and the mass-intensity criteria in
Table 5 for 50 ng BFB.
10.2.1 Assemble the purge-and-trap device. Pack the trap as shown in Figure 3 and condition over-
night at 170-180 "€ by backflushing with an inert gas at a flow rate of 20-30 mL/min. Con-
dition traps daily for a minimum of 10 minutes prior to use.
49
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Method 1666
10.2.1.1 Analyze the aqueous performance standard (Section 7.8.2) according to the purge-
and-trap procedure in Section 11. Compute the area at the primary m/z (Table 7)
for each compound. Compare these areas to those obtained by injecting 1 |jL of
the methanolic standard (Section 7.5.1) to determine compound recovery. The
recovery shall be greater than 50% for the PMI analytes. Maximum allowable
recovery for the PMI analytes found in Table 1 are shown in Table 8. This recov-
ery is demonstrated initially for each purge-and-trap GC/MS system. The test is
repeated only if the purge-and-trap or GC/MS systems are modified in any way
that might result in a change in recovery.
10.2.1.2 Demonstrate that 100 ng o-xylene (or o-xylene-d10) produces an area at m/z 106 (or
116) approximately one-tenth that required to exceed the linear range of the sys-
tem. The exact value must be determined by experience for each instrument. It is
used to match the calibration range of the instrument to the analytical range and
detection limits required.
10.3 Assemble the GC/MS system for direct aqueous injection and establish the operating conditions to be
used for sample analysis (Section 6.3.3.2). By injecting standards into the GC, demonstrate that the
analytical system meets the minimum levels in Table 4 for the compounds for which calibration is to
be performed, and the mass-intensity criteria in Table 5 for 50 ng BFB.
Demonstrate that 100 ng o-xylene (or o-xylene-d10) produces an area at m/z 106 (or 116) approxi-
mately one-tenth that required to exceed the linear range of the system. The exact value must be
determined by experience for each instrument. It is used to match the calibration range of the instru-
ment to the analytical range and detection limits required.
10.4 The following calibration steps are to be performed for both the purge-and-trap PMI analytes found
in Table 1 and the direct aqueous injection PMI analytes found in Table 2, as appropriate.
10.4.1 Calibration by isotope dilution—The isotope dilution approach is used for the PMI analytes
when appropriate labeled compounds are available and when interferences do not preclude
the analysis. If labeled compounds are not available, or interferences are present, the internal
standard method (Section 10.4.2) is used. A calibration curve encompassing the concentra-
tion range of interest is prepared for each compound determined. The relative response (RR)
vs. concentration in micrograms per liter is plotted or computed using a linear regression.
An example of a calibration curve for o-xylene using o-xylene-dlo is given in Figure 6. Also
shown are the ±10% error limits (dotted lines). Relative response is determined according to
the procedures described below. A minimum of five data points are required for calibration.
10.4.1.1 The relative response (RR) of pollutant to labeled compound is determined from
isotope ratio values calculated from acquired data. Three isotope ratios are used in
this process:
Rx = The isotope ratio measured in the pure pollutant (Figure 7A)
Ry = The isotope ratio of pure labeled compound (Figure 7B)
Rm= The isotope ratio measured in the analytical mixture of the
pollutant and labeled compounds (Figure 7C)
50
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Method 1666
The correct way to calculate RR is:
m
If Rn, is not between 2Ry and 0.5R*, the method does not apply and the sample is
analyzed by the internal standard method (Section 10.4.2).
10.4.1.2 In most cases, the retention times of the pollutant and labeled compound are simi-
lar, and isotope ratios (R's) can be calculated from the EICP areas, where:
(area at rrij/z)
(area at m2/z)
If either of the areas is zero, it is assigned a value of 1 in the calculations; that is, if
area of nij/z = 50721, and area of m2/z = 0, then R = 50721/1 = 50720.
The data from these analyses are reported to three significant figures (see Section
14.6). In order to prevent rounding errors from affecting the values to be reported,
all calculations performed prior to the final determination of concentrations should
be carried out using at least four significant figures. Therefore, the calculation of R
above is rounded to four significant figures.
The m/z's are always selected such that R* > Ry. When there is a difference in
retention times (RT) between the pollutant and labeled compounds, special precau-
tions are required to determine the isotope ratios.
R» Ry, and Rm are defined as follows:
area m,
R =
x
R =
y
area mJ^ at /?T_ J
area m,
RnT
areamJzlatRTj]
51
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Method 1666
10.4.1.3 An example of the above calculations can be taken from the data plotted in Figure
7 for o-xylene and oxylene-d,0. For these data:
. ,68900
i
V 6dm= 0.00001640
96868 , ,„
RnT 82508= L174
10.4.1 .4 The RR for the above data is then calculated using the equation given in Section
10.4.1.1. For the example, rounded to four significant figures, RR = 1.174. Not all
labeled compounds elute before their pollutant analogs.
To calibrate the analytical system by isotope dilution, analyze an aliquot of each of
the aqueous calibration standards (Section 7.8.1) spiked with an appropriate con-
stant amount of the labeled compound spiking solution (Section 7.7), using the
appropriate procedure in Section 10. Compute the RR at each concentration.
10.4.1 .5 Linearity — If the ratio of relative response to concentration for any compound is
constant (less than 20% coefficient of variation) over the five-point calibration
range, an averaged relative response/concentration ratio may be used for that com-
pound; otherwise, the complete calibration curve for that compound shall be used
over the five-point calibration range.
10.4.2 Calibration by internal standard — Used when criteria for isotope dilution (Section 10.4. 1)
cannot be met. The method is applied to pollutants having no labeled analog and to the
labeled compounds. The internal standards used for volatiles analyses are bromochlo-
romethane, 1,4-difluorobenzene, chlorobenzene-ds, and tetrahydrofuran-dg. Concentrations
of the labeled compounds and pollutants without labeled analogs are computed relative to
the nearest eluting internal standard, as shown in Tables 3 and 4.
10.4.2.1 Response factors — Calibration requires the determination of response factors (RF)
which are defined by the following equation:
RF =
Where:
As = The EICP area at the characteristic m/zfor the compound
in the daily standard
AJS = The EICP area at the characteristic m/zfor the internal
standard
C{s= The concentration ( \ng/L) of the internal standard
Cs = The concentration of the pollutant in the daily standard
52
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Method 1666
10.4.2.2 The RF is determined at 10, 20, 50,100, and 200 ug/L for the pollutants (option-
ally at 5 times or more these concentrations for highly water-soluble pollutants; see
Section 7.8), in a way analogous to that for calibration by isotope dilution (Section
10.4.1). The RF is plotted against concentration for each compound in the stan-
dard (Cs) to produce a calibration curve.
10.4.2.3 Linearity—If the RF for any compound is constant (less than 35% coefficient of
variation) over the five-point calibration range, an averaged RF may be used for
that compound; otherwise, the complete calibration curve for that compound shall
be used over the five-point range.
10.4.3 Combined calibration—By adding the isotopically labeled compounds and internal standards
(Section 7.7) to the aqueous calibration standards (Section 7.8.1), a single set of analyses can
be used to produce calibration curves for the isotope-dilution and internal-standard methods.
These curves are verified each shift (Section 15.5) by analyzing the aqueous performance
standard (Section 7.8.2). Recalibration is required only if calibration and ongoing perfor-
mance (Section 15.5) criteria cannot be met.
11. Purge, Trap, and GC/MS Analysis
Samples containing less than 1% solids are analyzed directly as aqueous samples (Section 11.4). Samples
containing 1% solids or greater are analyzed as solid samples utilizing one of two methods, depending on
the levels of pollutants in the sample. Samples containing 1% solids or greater and low to moderate levels
of pollutants are analyzed by purging a known weight of sample added to 5 mL of reagent water (Section
11.5). Samples containing 1% solids or greater and high levels of pollutants are extracted with methanol,
and an aliquot of the methanol extract is added to reagent water and purged (Section 11.6).
11.1 Determination of percent solids.
11.1.1 Weigh 5 to 10 g of sample into a tared beaker.
11.1.2 Dry overnight (12 hours minimum) at 110 °C (±5 °Q, and cool in a desiccator.
11.1.3 Determine percent solids as follows:
„ ... weight of sample dry
% solids = —rr-^ —,—- x 100
weight of sample wet
11.2 Remove standards and samples from cold storage and bring to 20-25 °C.
11.3 Adjust the purge gas flow rate to 40 mL/min (±4 mL/min).
11.4 Samples containing less than 1% solids.
11.4.1 Mix the sample by shaking vigorously. Remove the plunger from a 5-mL syringe and attach
a closed syringe valve. Open the sample bottle and carefully pour the sample into the
syringe barrel until it overflows. Replace the plunger and compress the sample. Open the
syringe valve and vent any residual air while adjusting the sample volume to 5 mL (±0.1
mL). Because this process of taking an aliquot destroys the validity of the sample for future
analysis, fill a second syringe at this time to protect against possible loss of data.
11.4.2 Add an appropriate amount of the labeled compound spiking solution (Section 7.7.1)
through the valve bore, then close the valve.
53
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Method 1666
11.4.3 Attach the syringe valve assembly to the syringe valve on the purging device. Open both
syringe valves and inject the sample into the purging chamber. Purge the sample per Sec-
tion 11.7.
11.5 Samples containing 1% solids or greater and low to moderate levels of pollutants.
11.5.1 Mix the sample thoroughly using a clean spatula and remove rocks, twigs, sticks, and other
foreign matter.
11.5.2 Weigh 5 g (±1 g) of sample into a purging vessel (Figure 2). Record the weight to three
significant figures.
11.5.3 Add 5 mL (±0.1 mL) of reagent water to the vessel.
11.5.4 Using a metal spatula, break up any lumps of sample to disperse the sample in the water.
11.5.5 Add an appropriate amount of the labeled compound spiking solution (Section 7.7.1) to the
sample in the purge vessel. Place a cap on the purging vessel and shake vigorously to fur-
ther disperse the sample. Attach the purge vessel to the purging device, and purge the sam-
ple per Section 11.7.
11.6 Samples containing 1 % solids or greater and high levels of pollutants, or samples requiring dilution
by a factor of more than 100 (see Section 16).
11.6.1 Mix the sample thoroughly using a clean spatula and remove rocks, sticks, twigs, and other
foreign matter.
11.6.2 Weigh 5 g (±1 g) of sample into a calibrated IS- to 25-mL centrifuge tube. Record the
weight of the sample to three significant figures.
11.6.3 Add 10 mL of methanol to the centrifuge tube. Cap the tube and shake it vigorously for 15
to 20 seconds to disperse the sample in the methanol. Allow the sample to settle in the
tube. If necessary, centrifuge the sample to settle suspended particles.
11.6.4 Remove approximately 0.1% of the volume of the supernatant methanol using a 15- to 25-
(jL syringe. This volume will be in the range of 10-15 uL.
11.6.5 Add this volume of the methanol extract to 5 mL reagent water in a 5-mL syringe, and ana-
lyze per Section 10.4.1.
11.6.6 For further dilutions, dilute 1 mL of the supernatant methanol (Section 10.6.4) to 10 mL,
100 mL, 1000 mL, etc., in reagent water. Remove a volume of this methanol extract/
reagent water mixture equivalent to the volume in Section 10.6.4, add it to 5 mL reagent
water in a 5- mL syringe, and analyze per Section 11.4.
11.7 Purge the sample for 11 minutes (±0.1 minute) at 45 °C (±2 °C).
11.8 After the 11-minute purge time, attach the trap to the chromatograph and set the purge-and-trap
apparatus to the desorb mode (Figure 5). Desorb the trapped compounds into the GC column by
heating the trap to 170-180 °C while backflushing with carrier gas at 20-60 mL/min for 4 minutes.
Start MS data acquisition upon start of the desorb cycle, and start the GC column temperature pro-
gram 3 minutes later. Section 6.3.3.1 provides the recommended operating conditions for the gas
chromatograph. Table 3 provides the retention times and minimum levels that can be achieved
under these conditions. An example of the separations achieved by the column listed is shown in
Figure 8. Other columns may be used provided the requirements in Section 9 are met.
54
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Method 1666
11.9 After desorbing the sample for 4 minutes, recondition the trap by purging with purge gas while main-
taining the trap temperature at 170-180 °C. After approximately 7 minutes, turn off the trap heater
and stop the gas flow through the trap. When cool, the trap is ready for the next sample.
11.10 While analysis of the desorbed compounds proceeds, remove and clean the purge device. Rinse with
tap water, clean with detergent and water, rinse with tap and distilled water, and dry for a minimum of
1 hour in an oven at a temperature greater than 15Q°C.
12. Direct Aqueous Injection and GC/MS Analysis
Samples containing less than 1% solids are analyzed directly as aqueous samples (Section 12.3). Samples
containing 1% solids or greater are analyzed after equilibration with reagent water containing labeled PMI
analytes and internal standards (Section 12.4).
12.1 Determine percent solids as in Section 11.1.
12.2 Remove standards and samples from cold storage and bring to 20-25 °C.
12.3 Samples containing less than 1% solids.
12.3.1 Allow solids to settle and remove 5 mL of sample.
12.3.2 Add an appropriate amount of the labeled compound spiking solution (Section 7.7.2).
12.3.3 Inject 1 |iL or more directly into the GC injection port. The temperature of the injection
block should be great enough to immediately vaporize the entire sample. An example of the
separations achieved by the column listed is shown in Figure 9.
12.4 Samples containing 1% solids or greater.
12.4.1 Mix the sample thoroughly using a clean spatula and remove rocks, twigs, sticks and other
foreign matter.
12.4.2 Add 5 g (±1 g) of sample to a 10-mL centrifuge tube. Using a clean metal spatula, break up
any lumps of sample. Record the sample weight to three significant figures.
12.4.3 Add an appropriate amount of the labeled compound spiking solution (Section 7.7.2) to the
sample in the centrifuge tube.
12.4.4 Add a measured quantity (Y* 0.1 mL) of reagent water to the tube so as to minimize head-
space.
12.4.5 Place a cap on the centrifuge tube leaving little or no headspace. Place the tube in the sonica-
tor for a minimum of 5 minutes, turning occasionally. For most samples this should be suffi-
cient time to distribute labeled and native analytes between the solid and aqueous phases and
to establish equilibrium. Some sample matrices may require more sonication.
12.4.6 On completion of sonication, centrifuge the sample and inject 1 pJL or more of supemate
directly into the GC injection port The temperature of the injection block should be great
enough to immediately vaporize the entire sample.
12.5 Liquid samples containing high solids concentrations, such as sludges or mud, may be weighed into a
10-mL centrifuge tube, have labeled compound spiking solution (Section 7.7.2) added, and be soni-
cated as in Section 12.4.5. Centrifugation and injection are to be performed as in Section 12.4.6.
55
-------
Method 1666
13. Qualitative Determination
Identification is accomplished by comparison of data from analysis of a sample or blank with data stored in
the mass-spectral libraries. For compounds for which the relative retention times and mass spectra are
known, identification is confirmed per Sections 13.1 and 13.2.
13.1 A labeled compound or pollutant having no labeled analog (Tables 1 and 2).
13.1.1 The signals for all characteristic m/z's stored in the spectral library (Section 10.1.1) shall be
present and shall maximize within the same two consecutive scans.
1 3.1 .2 Either ( 1 ) the background corrected EICP areas or (2) the corrected relative intensities of the
mass-spectral peaks at the GC peak maximum shall agree within a factor of 2 (0.5-2 times)
for all masses stored in the library.
13.1 .3 The relative retention time shall be within the window established in the initial precision and
accuracy study (Section 9.2).
13.2 Pollutants having a labeled analog (Tables 1 and 2).
13.2.1 The signals for all characteristic m/z's stored in the spectral library (Section 10.1.1) shall be
present and shall maximize within the same two consecutive scans.
13.2.2 Either (1) the background corrected EICP areas or (2) the corrected relative intensities of the
mass-spectral peaks at the GC peak maximum shall agree within a factor of 2 for all masses
stored in the spectral library.
13.2.3 The relative retention time between the pollutant and its labeled analog shall be within the
window established in the initial precision and accuracy study (Section 9.2).
13.3 The m/z's present in the sample mass spectrum that are not present in the reference mass spectrum
shall be accounted for by contaminant or background ions. If the sample mass spectrum is contami-
nated, or if identification is ambiguous, an experienced spectrometrist (Section 1.4) is to determine
the presence or absence of the compound.
14. Quantitative Determination
14.1 Isotope dilution — Because the pollutant and its labeled analog exhibit the same effects upon purging
and desorption, or equilibration combined with gas chromatography, correction for recovery of the
pollutant can be made by adding a known amount of a labeled compound to every sample prior to
purging or equilibration. Relative response (RR) values for sample mixtures are used in conjunction
with the calibration curves described in Section 10.4.1 to determine concentrations directly, so long
as labeled compound spiking levels are constant. For the o-xylene example given in Figure 7 (Sec-
tion 10.4.1.3), RR would be equal to 1.174. For this RR value, the o-xylene calibration curve given in
Figure 6 indicates a concentration of 31.8 ug/L.
14.2 Internal standard — For the compounds for which the system was calibrated (Table 1 and Table 2)
according to Section 10.4.2, use the response factor determined during the calibration to calculate the
concentration from the equation below, where the terms are as defined in Section 10.4.2.1.
A xC.
s is
Concentration = -? — ^Tor-
is
56
-------
Method 1666
14.3 The concentration of the pollutant in the solid phase of the sample is computed using the concentra-
tion of the pollutant detected in the aqueous solution, as follows:
/- . ^ i-j/ n > YLx. aqueous cone (lig/L)
Concentration in solid (lig/kg)= -, ^—, ,. . —=-=
^6 6/ sample wt. (kg) x percent solids x DF
where:
"percent solids" is from Section 11.1.3 or Section 12.1
Y = volume of water in liters (L)from Section 12.4.4
DF = dilution factor (as a decimal number), where necessary
Where the aqueous concentration is in mg/L, the result will be in mg/kg.
14.4 Sample dilution—If the EICP area at the quantitation m/z exceeds the calibration range of the sys-
tem, the sample is diluted by successive factors of 10 until the area is within the calibration range. If
dilution of high-solids samples by greater than a factor of 100 is required for purge-and-trap analysis,
then extract the sample with methanol, as described in Section 11.6.
14.5 Dilution of samples containing high concentrations of compounds not in Table 1 or Table 2—When
any peak in the mass spectrum is saturated, dilute the sample per Section 14.4.
14.6 Report results for all pollutants and labeled compounds found in all standards, blanks, and samples to
three significant figures. For samples containing less than 1% solids, the units are micrograms or
milligrams per liter (ug/L or mg/L); and for undiluted samples containing 1% solids or greater, units
are micrograms or milligrams per kilogram (ug/kg or mg/kg).
14.6.1 Results for samples that have been diluted are reported at the least dilute level at which the
area at the quantitation m/z is within the calibration range (Section 14.4), or at which no m/z
in the spectrum is saturated (Section 14.5). For compounds having a labeled analog, results
are reported at the least dilute level at which the area at the quantitation m/z is within the cal-
ibration range (Section 14.4) and the labeled compound recovery is within the normal range
for the method (Section 16.2).
15. System Performance
15.1 At the beginning of each 8-hour shift during which analyses are performed, system calibration and
performance shall be verified for the pollutants and labeled compounds (Table 1 or Table 2). For
these tests, analysis of the aqueous performance standard (Section 7.8.2) shall be used to verify all
performance criteria. Adjustment and/or recalibration (per Section 10) shall be performed until all
performance criteria are met. Only after all performance criteria are met may blanks and samples be
analyzed.
15.2 BFB spectrum validity—The criteria in Table 5 shall be met.
15.3 Retention times.
15.3.1 Purge-and-trap analysis—The absolute retention times of the internal standards shall fall
within ±30 seconds of the following—bromochloromethane, 954 seconds; 1,4-difluoroben-
zene, 1052 seconds; chlorobenzene-ds, 1359 seconds. The relative retention times of all pol-
lutants and labeled compounds shall fall within 5% of the value given in Table 3.
57
-------
Method 1666
15.3.2 Direct aqueous injection analysis—The absolute retention time of tetrahydrofuran-dg shall be
263 seconds (±30 seconds). The relative retention times of all pollutants and labeled com-
pounds shall fall within 10% of the value given in Table 4.
15.4 GC resolution—The valley height between o-xylene and o-xylene-d10 (at m/z 106 and 116 plotted on
the same graph) shall be less than 10% of the taller of the two peaks.
15.5 Calibration verification and ongoing precision and accuracy—Compute the concentration of each
pollutant (Table 1 or Table 2) by isotope dilution (Section 10.4.1) for those compounds that have
labeled analogs. Compute the concentration of each pollutant that has no labeled analog by the inter-
nal standard method (Section 10.4.2). Compute the concentrations of the labeled compounds by the
internal standard method. These concentrations are computed based on the calibration data deter-
mined in Section 10.
15.5.1 For each pollutant and labeled compound, compare the concentration with the corresponding
limit for ongoing accuracy in Table 6. If all compounds meet the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may continue. If any individ-
ual value falls outside the range given, system performance is unacceptable for that com-
pound.
Note: The large number of compounds in Table 6 present a substantial probability that one or more will
fail the acceptance criteria when all compounds are analyzed. To determine if the analytical sys-
tern is out of control, or if the failure may be attributed to probability, proceed as follows.
15.5.1.1 Analyze a second aliquot of the aqueous performance standard (Section 7.8.2).
15.5.1.2 Compute the concentration for only those compounds that failed the first test (Sec-
tion 15.5.1). If these compounds now pass, system performance is acceptable for
all compounds, and analyses of blanks and samples may proceed. If, however, any
of the compounds fail again, the measurement system is not performing properly
for these compounds. In this event, locate and correct the problem or recalibrate
the system (Section 10), and repeat the entire test (Section 15.1) for all compounds.
15.5.2 Add results that pass the specification in Section 15.5.1.2 to initial (Section 9.2) and previous
ongoing data. Update QC charts to form a graphic representation of laboratory performance
(Figure 8). Develop a statement of accuracy for each pollutant and labeled compound by cal-
culating the average percent recovery (R) and the standard deviation of percent recovery (sr).
Express the accuracy as a recovery intervial from R - 2sr to R + 2sr. For example, if
R = 95% and sr = 5%, the accuracy is 85-105%.
16. Analysis of Complex Samples
16.1 Some samples may contain high levels (>1000 ug/kg) of the compounds of interest and of interfering
compounds. Some samples will foam excessively when purged. Others will overload the trap or the
GC column.
16.2 When the recovery of any labeled compound is outside the range given in Table 6, dilute samples by
a factor of 10 with reagent water and analyze this diluted sample. If the recovery remains outside of
the range for this diluted sample, the aqueous performance standard shall be analyzed (Section 15.1)
and calibration verified (Section 15.5). If the recovery for the labeled compound in the aqueous per-
formance standard is outside the range given in Table 6, the analytical system is out of control. In
58
-------
Method 1666
this case, the instrument shall be repaired, the performance specifications in Section 15 shall be met,
and the analysis of the undiluted sample shall be repeated. If the recovery for the aqueous perfor-
mance standard is within the range given in Table 6, then the method does not apply to the sample
being analyzed, and the result may not be reported for regulatory compliance purposes.
16.3 When a high level of the pollutant is present, reverse-search computer programs may misinterpret the
spectrum of chromatographically unresolved pollutant and labeled compound pairs with overlapping
spectra. Examine each chromatogram for peaks greater than the height of the internal standard
peaks. These peaks can obscure the compounds of interest.
17. Method Performance
17.1 This method was developed and validated in a single laboratory.
17.2 Chromatograms of the aqueous performance standards (Sections 7.8.2 and 15.1) are shown in Fig-
ures 8 and 9.
18. Waste Management
18.1 It is the laboratory's responsibility to comply with all federal, State, and local regulations governing
waste management, particularly the hazardous waste identification rules and land-disposal restric-
tions. In addition it is the laboratory's responsibility to protect air, water, and land resources by min-
imizing and controlling all releases from fume hoods and bench operations. Also, compliance is
required with any sewage discharge permits and regulations.
18.2 Samples containing acids at a pH of less than 2 are hazardous and must be neutralized before being
poured down a drain or must be handled as hazardous waste.
18.3 For further information on waste management, consult "The Waste Management Manual for Labora-
tory Personnel," available from the American Chemical Society's Department of Government Rela-
tions and Science Policy, 1115 16th Street, NW, Washington, DC 20036.
-------
Method 1666
References
1. "Performance Tests for the Evaluation of Computerized Gas Chromatography/Mass Spectrometry
Equipment and Laboratories," U.S. EPA, EMSL Cincinnati, OH 45268, EPA-600/4-80-025 (April
1980).
2. Bellar, T. A. and Lichtenberg, J. J., "Journal American Water Works Association," 66, 739 (1974).
3. Bellar, T. A. and Lichtenberg, J. J., "Semi-Automated Headspace Analysis of Drinking Waters and
Industrial Waters for Purgeable Volatile Organic Compounds," in Measurement of Organic Pollutants
in Water and Wastewater, C. E. VanHall, ed., American Society for Testing Materials, Philadelphia,
PA, Special Technical Publication 686 (1978).
4. National Standard Reference Data System, "Mass Spectral Tape Format," U.S. National Bureau of
Standards (1979 and later attachments).
5. "Working with Carcinogens," DREW, PHS, NIOSH, Publication 77-206 (1977).
6. "OSHA Safety and Health Standards, General Industry," 29 CFR 1910, OSHA 2206 (1976).
7. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication, Committee
on Chemical Safety (1979).
8. "Methods 330.4 and 330.5 for Total Residual Chlorine," USEPA, EMSL Cincinnati, OH 45268,
EPA-4-79-020 (March 1979)"Handbook of Analytical Quality Control in Water and Wastewater Lab-
oratories," U.S. EPA, EMSL Cincinnati, OH 45268, EPA-4-79-019 (March 1979).
9. "Handbook for Analytical Quality Control in Water and Wastewater Laboratories," U.S. EPA, EMSL
Cincinnati, OH 45268, EPA-600/4-79-019 (March 1979).
60
-------
Method 1666
Table 1. Volatile PMI Analytes Amenable to Purge-and-Trap and Determined by GC/MS Using Isotope-
Dilution and Internal-Standard Techniques
CASRN1
628-63-7
7141-0
123-86-4
71-36-3
75-65-0
110-82-7
141-78-6
98-01-1
142-82-5
110-54-3
78-84-2
67-63-0
108-21-4
108-20-3
107-31-3
108-10-1
109-66-0
109-99-9
75-69-4
108-38-3
106-42-3
95-47-6
EPA-EGD
977
978
979
1036
1343
1333
1736
981
1334
1335
982
1044
983
960
991
1341
984
1345
552
1332
1331
Labeled Compound
Analog
| CASRN1
EPA-EGD
,0
13C
,6
d2
J10
53001-22-2
1735-17-7
84508-45-2
33838-52-7
21666-38-6
1693-74-9
41051-88-1
56004-61-6
1243
1233
1636
1234
1235
Pollutant
PMI Analyte
n-Amyl acetate
n-Amyl alcohol
n-Butyl acetate
n-Butyl alcohol
tert-Butyl alcohol
Cyclohexane
Ethyl acetate
Furfural2
n-Heptane
n-Hexane
Isobutyraldehyde2
Isopropanol
Isopropyl acetate
Isopropylether
Methyl formate
Methylisobutyl ketone
n-Pentane3
Tetrahydrofuran
Trichlorofluoromethane
m,p-Xylene
o-xylene
1 Chemical Abstracts Service Registry Number
2 These aldehydes from the PMI may be analyzed by this method or Method 1667
3 n-Pentane is not explicitly a PMI analyte. However, the sum of the concentrations of n-pentane, n-hexane, and n-heptane are
to be used to estimate the concentration of petroleum naphtha in PMI wastewaters.
1245
1232
1231
61
-------
Method 1666
Table 2. Volatile PMI Analytes
Internal-Standard Techniques
Determined by Direct Aqueous Injection GCMS
Using Isotope-Dilution and
Pollutant Labeled Compound
PMI Analyte
Acetonitrile
Diethylamine
Dimethylamine
Dimethyl sulfoxide
Ethanol
Ethylene glycol
Formamide
Methanol
Methylamine
Methyl cellosolve
n-Propanol
Triethylamine
CASRN1
75-05-8
109-89-7
124-40-3
67-68-5
64-17-5
107-21-1
75-12-7
67-56-1
74-89-5
109-864
71-23-8
121-44-8
EPA-EGD Analog
972 dg
986
987
1037 de
1734 de
1038
988
1735 d3
989
1040
755 1-d,
990
| CASRN1 EPA-EGD
2206-26-0 1272
2206-27-1 1237
1516-08-1 1634
1849-29-2 1635
not avail. 1255
1 Chemical Abstracts Service Registry Number
62
-------
Method 1666
Table 3. Gas Chromatographic Retention Times and Minimum Levels for Volatile PMI Analytes Determined
by Purge-and-Trap GC/MS
EGD
No.1 PMI Analyte
991 Methyl formate
552 Trichlorofluoromethane
984 n-Pentane
1344 Isopropanol
1243 tert-Butyl alcohol-d10
1343 tert-Butyl alcohol
1235 n-Hexane-d14
982 Isobutyraldehyde
1335 n-Hexane
960 Isopropylether
1636 Ethyl acetate-13c
1736 Ethyl acetate
181 Bromochloromethane (int std)
1245 Tetrhydrofuran-d8
1345 Tetrahydrofuran
1233 Cyclohexane-d12
1333 Cyclohexane
1234 n-Heptane-d,6
1336 n-Butanol
1334 n-Heptane
985 1,4-Difluorobenzene (int std)
983 Isopropyl acetate
1341 Methylisobutyl ketone
978 n-Amyl alcohol
979 n-Butyl acetate
981 Furfural
207 Chlorobenzene-ds (int std)
1232 p-Xylene-d10
1332 m,p-Xylene
1231 o-Xylene-d10
977 n-Amyl acetate
1331 o-Xylene
1 Three-digit EGD numbers beginning with 0,1,5, or 9 indicate a pollutant quantified by the internal standard method; beginning
with 2 or 6 indicate a labeled compound quantified by the internal standard method; beginning with 3 or 7 indicate a pollutant
quantified by isotope dilution.The initial '1* in four-digit EGD numbers is to be ignored in applying these rules.
2 This is the minimum level at which the entire analytical system shall give recognizable mass spectra (background-corrected)
and acceptable calibration points, taking into account method-specific sample and injection volumes. The concentration in the
aqueous or solid phase is determined using the equations in Section 14.
Mean (sec) |
526
613
622
687
730
741
820
823
839
865
925
925
954
956
964
981
996
1013
1015
1033
1052
1128
1157
1202
1268
1354
1359
1368
1379
1413
1417
1424
Retention Time
EGD Ref
181
181
181
181
181
1243
181
181
1235
181
181
1636
181
181
181
181
1233
985
985
1234
985
985
985
985
207
207
207
207
1232
207
207
1231
Relative
0.551
0.642
0.652
0.720
0.765
1.016
0.860
0.863
1.023
0.907
0.970
1.000
1.000
1.002
1.012
1.028
1.015
0.963
0.964
1.020
1.000
1.072
1.100
1.143
0.933
0.996
1.000
1.007
1.008
1.040
1.043
1.008
ML2
(M9/L)
100
10
10
200
100
10
10
5
10
20
5
500
10
10
10
500
5
500
10
5
5
-------
Method 1666
Table 4. Gas Chromatographic Retention Times and Minimum Levels for Volatile PMI Analytes by Direct
Aqueous Injection GC/MS
Retention Time
No.1 PMIAnalyte Mean (sec) EGDRef Relative (mg/L)
989 Methylamine 81 1245 0.308 200
1635 Methlyateohol-dg 85 1245 0.323
1735 Methyl alcohol 85.5 1635 1.006 50
987 Dimethylamine 93 1245 0.354 200
1634 Ethyl alcohol-^ 103 1245 0.394
1734 Ethyl alcohol 104 1634 1.010 20
1272 Acetonitrile-dg 119 1245 0.452
972 Acetonitrite 121 1272 1.017 5
1255 n-PropanoM-d, 170 1245 0.464
755 n-Propanol 170.5 1255 1.003 20
986 Diethylamine 188 1245 0.717 200
1245 Tetrahydrofuran-dg(intstd) 263 1245 1.000
1040 Methyl ce)losofve(2-MethoxyethanoO 290 1245 1.103 50
990 Triethylamine 372 1245 1.414 200
1038 Elhyleneglycol 398 1245 1.513 200
988 Formamide 400 1245 1.521 1000
1237 Dimethyl sulfoxide-de 639 1245 2.431
1037 Dimethyl sulfoxkJe 643 1237 1.006 100
1 Three-digit EQD numbers beginning with 0, 1, 5, or 9 indicate a pollutant quantified by the internal standard method; beginning
with 2 or 6 indicated a labteted compound quantified by the internal standard method; beginning with 3 or 7 indicate a poUutant
quantified by isotope dilution. The initial '1 ' in four-digit EGD numbers is to be ignored in applying these rules.
2 This is the minimum level at which the entire analytical system shall give recognizable mass spectra (background-corrected)
and acceptable calforation points, taking into account method-specific sample and injection volumes. The concentration in the
aqueous or solid phase is determined using the equations in Section 14.
Table 5. BFB Mass-Intensity Specifications
m/z Intensity Required
50 15-40% of m/z 95
75 30-60% of m/z 95
95 base peak, 100%
96 5-9% of m/Z 95
173 less than 2% of m/z 174
1 74 greater than 50% of m/z 95
175 5-9% of m/z 174
176 95-101% of m/z 174
177 5-9% of m/z 176
64
-------
Method 1666
Table 6. Acceptance Criteria for PMI Analytes
Acceptance Criteria for Performance Tests (% of Spike Level)
EGO
No. PMI Analyte
972 Acetonrtrile
977 Amyl acetate
978 Amyl alcohol
979 n-Butyl acetate
1036 n-6utyl alcohol
1343 tert-Butyl alcohol
1333 Cydohexane
986 DiethylamJne
987 Dimethyiamine
1037 Dimethyl sulfoxide
1734 Ethanol
1736 Ethyl acetate
1038 Ethylene glycol
988 Formamide
981 Furfural
1334 n-Heptane
1335 n-Hexane
982 IsobutyraJdehyde
1044 Isopropanol
983 Isopropyl acetate
960 Isopropyl ether
1735 Methanol
989 Methylamine
1040 Methyl cellosotve
991 Methyl formate
1 341 Methylisobutyl ketone
984 n-Pentane
755 n-Propand
975 Tetrahydrofuran
552 Trichlorofluoromethane
990 Triethylamine
1332 m.p-Xytene
1331 o-Xytene
Spike Level
50mg/L
10ng/L
200jig/L
10ng/L
200|xg/L
50ng/L
10jig/L
250mg/L
250mg/L
250mg/L
50mg/L
10jig/L
250mg/L
500mg/L
100»ig/L
Labeled and Native
PMI Analyte
Initial Precision and Labek
Accuracy Am
s X Recov
Labeled and
KlPMI Native PMI
lyte Analyte Ongoing
ery (P) Accuracy (R)
5 94-103 99-110 93-103
14 80-107
75 16-166
20 88-129
108 d-190
121 d-202
78-109
10-172
86-130
d-199
d-212
26 82-134 8-156 80-136
31 71-132
38 61-136
68-134
58-139
16 84-116 59-122 83-118
15 66-96 103-121 65-97
48 60-157 58-159 57-160
195 d-310
113 60-286
186 d-282
d-326
51-296
d-297
10fig/L 37 87-161 14-128 84-164
10U0/L
10ng/L
100ng/L
10ng/L
10jig/L
50mg/L
250mg/L
250mg/L
50jig/L
10»ig/L
10ufl/L
50mg/L
34 86-154 5-157 84-157
54 67-176
284 d-418
32 82-147
21 86-127
63-180
d-441
80-150
84-129
26 57-109 100-120 55-111
36 61-133
13 99-125
73 20-165
42 78-162
52 51-155
59-136
98-126
14-171
75-165
47-159
25 42-93 54-149 40-95
10ufl/L 89 35-214 42-178 28-221
20jig/L
250mg/L
20WJ/L
10|ig/L
9 90-108
31 71-133
89-109
69-135
15 90-121 76-109 89-122
16 84-116 84-100 82-118
-------
Method 1666
Table 7. Characteristic m/z's for Volatile PMI Analytes
Spike Level Primary m/z
PMI Analyte ftig/L)
Acetonitrile
n-Amyl acetate 10
n-Amyl alcohol 100
n-Butyl acetate 10
n-Butyl alcohol 200
tert-Butyl alcohol 50
Cyclohexane 10
Diethylamine
Dimethylamine
Dimethyl sulfoxide
Ethanol
Ethyl acetate 10
Ethylene glycol
Formamide
Furfural 100
n-Heptane 10
n-Hexane 10
Isobutyraldehyde 10
Isopropanol 100
Isopropyl acetate 10
Isopropyl ether 10
Methanol
Methylamine
Methyl cellosolve (2-methoxyethanol)
Methyl formate 50
Methylisobutyl ketone 10
n-Pentane 10
n-Propanol
Tetrahydrofuran 10
Trichlorofluoromethane 20
Tn'ethylamine
m,p-Xylene 20
o-Xylene 10
Labeled (Native/
(mg/L) Analog Labeled)
50 dg 41/44
43
70
43
56
d,0 59/66
d12 56/96
250 58
250 44
250 de 47/50
50 d5 31/33
13Q 43/44
250 31
500 45
96
d16 71/82
d,4 57/66
72
45
43
45
50 dg 31/33
250 30
250 45
60
43
43
50 1-d, 31/32
d8 72/80
101
250 86
d,0 106/116
d10 106/116
Reference
Compound
1272
207
985
207
985
1234
1233
1245
1245
1237
1634
1636
1245
1245
207
1234
1235
181
181
985
181
1635
1245
1245
181
181
1255
1245
181
1245
1232
1231
66
-------
Method 1666
Table 8. Maximum Recoveries for PMI Analytes by Purge-and-Trap GC/MS
PMI Analyte Maximum Recovery (%)
n-Amyl acetate 110
n-Amyl alcohol 300
n-Butyl acetate 120
n-Butyl alcohol 440
tert-Butyl alcohol 130
Cyclohexane 120
Ethyl acetate 130
Furfural 170
n-Heptane 140
n-Hexane 140
Isobutyraldehyde 150
Isopropanol 250
Isopropl acetate 130
Isopropyl ether 120
Methyl formate 120
Methylisobutyl ketone 130
n-Pentante 130
Tetrahydrofuran 150
Trichlorofluoromethane 110
m,p-Xylene 120
o-Xylene 120
67
-------
Method 1666
Optional
Foam Trap
Exit 1/4 in. O.D.
—14mmO.D.
Inlet 1/4 in. O.D.
Exit 1/4 In. O.D.
10 mm Glass Frit,
Medium Porosity
Sample Inlet
2-Way Syringe Valve
17-cm, 20-Gauge Syringe Needle
6 mm O.D. Rubber Septum
Inlet, 1/4 in. O.D.
Stainless Steel, 1/16 in. O.D.
13x
Molecular Sieve
Purge-Gas Filter
Purge-Gas
Flow Control
Figure 1. Purging Device for Waters
52-025-6
68
-------
Method 1666
Purge Inlet Fitting
Sample Outlet Fitting
3 in. Long x 6 mm O.D. Glass Tubing
40-mLVial
Septum
Cap
52-025-7
Figure 2. Purging Device for Soils or Waters
-------
Method 1666
Packing Detail
Construction Detail
5 mm Glass Wool
7.7 cm Silica Gel
15cmTenaxGC
•- 1cm3%OV-1
£- 5 mm Glass Wool
Trap Inlet
Compression
Fitting Nut
and Ferrules
14ft,7y/ft
Resistance Wire
Wrapped Solid
Thermocouple/
Controller
Sensor
Electronic
Temperature
Control and
Pyrometer
Tubing, 25 cm
0.105in.l.D.
0.125in.O.D.
Stainless Steel
Figure 3. Trap Construction and Packings
52-025-8
70
-------
Method 1666
Carrier-Gas
Flow Control
Pressure
Regulator
Purge-Gas
Flow Control
13x Molecular
Sieve Filter
Liquid Injection Ports
,— Column Oven
Optional 4-Port Column
A Selection Valve
Trap Inlet
6-Port
Valve
Confirmatory Column
To Detector
Analytical Column
Vent
Purging
Device
Note:
All lines between trap
and GC should be heated
to 80°C.
52-025-9
Figure 4. Schematic of Purge-and-Trap Device—Purge Mode
71
-------
Method 1666
Carrier-Gas
Flow Control
Pressure
Regulator
Purge-Gas
Flow Control
13x Molecular
Sieve Filter
Optional 4-Port Column
Selection Valve
Liquid Injection Ports
i— Column Oven
"JULJU"*-
Confirmatory Column
To Detector
Analytical Column
Inlet
Trap,
200°C
Purging
Device
Note:
All lines between trap
and GC should be heated
to 80°C.
Figure 5. Schematic of Purge-and-Trap Device—Desorb Mode
52-025-10
72
-------
I I
Method 1666
10 —
1.0 —
0.1 —
I
2
I
5
I I I
10 20 50
Concentration (pg/L)
I I
100 200
The Dotted Lines Enclose a ±10% Error Window.
Figure 6. Relative Response Calibration Curve for o-Xylene
52-025-11
73
-------
Method 1666
(A)
Area=168920
M/Z116
M/Z106
(B)
Area=60960
M/Z116
M/Z106
(C)
M/Z106
M/Z116
96868
82508
M/Z116
M/Z106
Figure 7. Extracted Ion Current Profiles for (A) o-Xylene, (B) o-Xylene-d10,and
(C) a Mixture of o-Xylene and o-Xylene-d10
52-025-12
74
-------
Method 1666
170000 -
160000 -
150000 -
140000 -
130000 -
120000 -
110000 -
100000 -
90000 -
80000 -
70000 -
60000 -
50000 -
40000 -
30000 -
20000 -
10000 -
0
File
Operator
Acquired
Instrument
Sample Name
Misc Info
Vial Number
D:\DATA\HOTPURGBAUG1994\082694\HPSTD1.D
rln
26 Aug 94 2:27 pm using AcqMethod HOTPURGE
HP-1
Hotpurge Method Std. 08/26/94
hotpurge analytes (50 ppb IS/SS)
2
"UUJ
LMj
10
15
20
Figure 8. Chromatogram of Aqueous Performance
Standard of Analytes from Table 1
52-025-13
75
-------
Method 1666
100 -
%FS
Sample
Lab File ID
Analyzed
instrument ID
STD. 400/2000
4K15A02
11/15/94
VG04
10.0
12.0
Figure 9. Chromatogram of Aqueous Performance
Standard of Analytes from Table 2
52425-14
76
-------
Method 1666
&
120,000
100,000
I I I I
<•
80,000-
1 2
o-Xylene-d10
-- +3s
1 1
34
^
6
Analysis
Number
1
7
1
8
1
10
-3s
,.<
1.00'
-3s
6/1 6/1 6/1 6/1 6/2 6/2 6/3 6/3 6/4 6/5
Date
Analyzed
52-025-15
Figure 10. Quality Control Charts Showing Area (top graph) and Relative Response of
o-Xylene to o-Xylene-d10 (lower graph) Plotted as Function of Time or Analysis
Number
77
-------
Method 1667
Formaldehyde, Isobutyraldehyde, and Furfural
by Derivatization and
High-Pressure Liquid Chromatography
January 1995
-------
Method 1667
Formaldehyde, Isobutyraldehyde, and Furfural by Derivatization and
High-Pressure Liquid Chromatography
1. Scope and Application
1.1 This method is designed to meet the survey and monitoring requirements of the EPA's Engineering
and Analysis Division (EAD). The method is used to determine certain organic pollutants specific to
the pharmaceutical manufacturing industry (PMI) that can be derivatized and analyzed by high-pres-
sure liquid chromatography (HPLC).
1.2 The chemical compounds listed in Table 1 may be determined in waters, soils, and municipal sludges
by the method.
1.3 The detection limits of the method are usually dependent on the level of interferences rather than
instrumental limitations. The limits in Table 2 are the minimum levels that can be reliably quantified
by this method with no interferences present.
Furfural (2-furaldehyde) forms two relatively stable geometric isomers upon derivatization with 2,4-
dinitrophenylhydrazine (DNPH). The first isomer (probably anti-) elutes after the formaldehyde
derivative and before the isobutryaldehyde derivative. The second isomer (probably syn-) elutes
after the isobutyraldehyde derivative. Experience with this system has shown that the best quantita-
tive results (lowest method detection limits) are obtained using the area from the first eluted peak
rather than that from the second peak or the sum of the two areas. This method is for use only by ana-
lysts experienced with HPLC or under the close supervision of such qualified persons.
1.4 This method is performance-based. The analyst is permitted to modify the method to overcome
interferences or to lower the cost of measurements, provided that all performance criteria in this
method are met. The requirements for establishing method equivalency are given in Section 9.1.2.
2. Summary of the Method
2.1 For solid wastes or for aqueous wastes containing significant amounts of solid material, the aqueous
phase, if any, is separated from the solid phase and stored for later analysis. If necessary, the particle
size of the solids in the waste is reduced. The solid phase is extracted with an amount of extraction
fluid equal to 20 times the weight of the solid phase. The extraction fluid employed is a function of
the alkalinity of the solid phase of the waste. Following extraction, the aqueous extract is separated
from the solid phase by filtration employing 0.6- to 0.9-um glass fiber-filter.
2.2 If compatible (i.e., multiple phases will not form on combination), the initial aqueous phase of the
waste is added to the aqueous extract, and these liquids are analyzed together. If incompatible, the
liquids are analyzed separately and the results are mathematically combined to yield a volume-
weighted average concentration.
2.3 A measured volume of aqueous sample or an appropriate amount of solids leachate is buffered to
pH = 5 and derivatized with 2,4-dinitrophenylhydrazine (DNPH), using either the solid-sorbent or
methylene chloride derivatization/extraction option. If the solid-sorbent option is used, the deriva-
tive is extracted using solid-sorbent cartridges, followed by elution with ethanol. If the methylene
81
-------
Method 1667
chloride option is used, the derivative is extracted with methylene chloride. The methylene chloride
extracts are concentrated using the Kudema-Danish (K-D) procedure and solvent exchanged into
methanol prior to HPLC analysis. Liquid chroraatographic conditions are described that permit the
separation and measurement of formaldehyde, isobutyraldehyde, and furfural derivatives in the
extract by absorbance detection at 365 nm.
2.4 The quality of the analysis is assured through reproducible calibration and testing of the derivatiza-
tion/extraction procedure and the HPLC system.
3. Definitions
There are no method-specific definitions to be noted for this document.
4. Interferences
4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample processing hardware that lead to discrete artifacts and/or elevated baselines in chromato-
grams. All of these materials must be routinely demonstrated to be free from interferences under the
conditions of the analysis by analyzing laboratory reagent blanks as described in Section 9.3.
4.1.1 Glassware must be scrupulously cleaned. Clean all glassware as soon as possible after use
by rinsing with the last solvent used. This should be followed by detergent washing with hot
water, and rinses with tap water and reagent water. It should then be drained, dried, and
heated in a laboratory oven at 130 ^C) for several hours before use. Solvent rinses with meth-
anol may be substituted for the oven heating. After drying and cooling, glassware should be
stored in a clean environment to prevent any accumulation of dust or other contaminants.
4.1.2 The use of high-purity reagents and solvents helps to minimize interference problems. Purifi-
cation of solvents by distillation in all-glass systems may be required.
4.2 Analysis of formaldehyde is complicated by its ubiquitous occurrence in the environment. Acetic
acid, even high-purity acetic acid, is often contaminated with formaldehyde. For this reason, a phtha-
late buffer is used in this method instead of an acetate buffer. Wherever acetic acid is used, it must be
demonstrated to be formaldehyde free.
4.3 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the matrix being sampled. If matrix interferences occur, some additional
cleanup may be necessary.
4.4 The extent of interferences that may be encountered using liquid chromatographic techniques has not
been fully assessed. Although the HPLC conditions described allow for resolution of the specific
compounds covered by this method, other matrix components may interfere.
5. Safety
5.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been pre-
cisely determined; however, each chemical compound should be treated as a potential health hazard.
Exposure to these compounds should be reduced to the lowest possible level. The laboratory is
responsible for maintaining a current awareness file of OSHA regulations regarding the safe han-
dling of the chemicals specified in this method. A reference file of material safety data sheets should
also be made available to all personnel involved in these analyses. Additional information on labora-
tory safety can be found in References 1 through 3.
82
-------
Method 1667
5.2 Formaldehyde has been classified as a potential carcinogen. Primary standards of formaldehyde
should be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator should be worn
when high concentrations are handled.
6. Equipment and Supplies
6.1 Reaction vessel—250-mL Florence flask.
6.2 Separatory funnel—250-mL, with polytetrafluoroethylene (PTFE) stopcock.
6.3 Kuderna-Danish (K-D) apparatus.
6.3.1 Concentrator tube—10-mL, graduated (Kontes K-570050 or equivalent). A ground-glass
stopper is used to prevent evaporation of extracts.
6.3.2 Evaporation flask—500-mL (Kontes K-570001-500 or equivalent). Attach to concentrator
tube with springs, clamps, or equivalent.
6.3.3 Snyder column—Three-ball macro (Kontes K-503000-0121 or equivalent).
6.3.4 Snyder column—-Two-ball micro (Kontes K569001-0219 or equivalent).
6.3.5 Springs—'/a" (Kontes K-662750 or equivalent).
6.4 Vials—10-mL, 25-mL, glass with PTFE-lined screw-caps or crimp-tops.
6.5 Boiling chips—Solvent-extracted with methylene chloride, approximately 10/40 mesh (silicon car-
bide or equivalent).
6.6 Balance—Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.7 pH meter—Capable of measuring to the nearest 0.01.
6.8 High-performance liquid chromatograph (modular).
6.8.1 Pumping system—Isocratic, with constant flow control capable of 1.00 mL/min.
6.8.2 High-pressure injection valve with 20-uL loop.
6.8.3 Column—250 mm long x 4.6 mm inside diameter (i.d.), 5-um particle size, C18 (or equiva-
lent).
6.8.4 Absorbance detector—365 nm.
6.8.5 Strip-chart recorder compatible with the detector. Use of a data system is recommended.
6.9 Glass-fiber filter paper, 0.6- to 0.9-um.
6.10 Solid-sorbent cartridges—Packed with 500 mg C18 (Baker or equivalent).
6.11 Vacuum manifold—Capable of simultaneous extraction of up to 12 samples (Supelco or equivalent).
6.12 Sample reservoirs—50-mL capacity (Supelco or equivalent).
6.13 Pipet—Capable of accurately delivering 0.10 mL of solution (Pipetman or equivalent).
6.14 Water bath—Heated, with concentric ring cover, capable of temperature control of ± 2 °C at 80-90 °C
The bath should be used under a hood.
-------
Method 1667
7. Reagents and Standards
7.1 Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the Committee on Analytical Reagents of the Ameri-
can Chemical Society, where such specifications are available. Other grades may be used, provided
it is first ascertained that the reagent is of sufficiently high purity to permit its use without lessening
the accuracy of the determinations.
7.2 Reagent water—Water in which the compounds of interest and interfering compounds are not
detected by this method. It may be generated by any of the methods in this subsection.
7.2.1 Activated carbon—Pass tap water through a carbon bed (Calgon Filtrasorb-300, or equiva-
lent).
7.2.2 Water purifier—Pass tap water through a purifier (Millipore Super Q, or equivalent).
7.2.3 Boil and purge—Heat tap water to 90-100 °C and bubble contaminant-free inert gas through
it for approximately 1 hour. While still hot, transfer the water to screw-cap bottles and seal
with a PTFE-lined cap.
7.3 Methylene chloride—HPLC grade or equivalent.
7.4 Methanol—HPLC grade or equivalent.
7.5 Ethanol (absolute)—HPLC grade or equivalent.
7.6 2,4-Dinitrophenylhydrazine (DNPH, 70% w/w) in reagent water.
7.7 Formalin (37.6% w/w) in reagent water.
7.8 Acetic acid (glacial), demonstrated to be formaldehyde-free.
7.9 Potassium acid phthalate.
7.10 Sodium hydroxide solutions, 1 N, and 5 N.
7.11 Sodium chloride.
7.12 Sodium sulfate solution, 0.1 M.
7.13 Hydrochloric acid, 0.1 N.
7.14 Extraction fluid—Dilute 64.3 mL of 1.0 N sodium hydroxide and 5.1 mL of glacial acetic acid to 900
mL with reagent water. Further dilute to 1 L with reagent water. The pH should be 4.93 ± 0.02. If
not, adjust with acid or base.
7.15 Stock standard solutions.
7.15.1 Stock formaldehyde (approximately 1.00 mg/mL)—Prepare by diluting 265 uL formalin to
100 mL with reagent water.
Standardization of formaldehyde stock solution—Transfer a 25-mL aliquot of a 0.1 M
sodium sulfate solution to a beaker and record the pH. Add a 25-mL aliquot of the formalde-
hyde stock solution (Section 7.15.1) and record the pH. Titrate this mixture back to the orig-
inal pH using 0.1 N hydrochloric acid. The formaldehyde concentration is calculated using
the following equation:
84
-------
Method 1667
Concentration (mg/mL) = 30.03 x (NHCl) x (mLHCl) x25
where:
NHCL = Normality of the HCl solution
mL HCl= mL of standardized HCl solution
30.03 = Molecular weight of formaldehyde
7.15.2 Stock formaldehyde, isobutyraldehyde, and furfural—Prepare by adding 265 uL of formalin,
0.100 g of isobutyraldehyde, and 0.100 g of furfural to 90 mL of reagent water and dilute to
100 mL. The concentrations of isobutyraldehyde and furfural in this solution are 1.00 mg/
mL. Calculate the concentration of formaldehyde in this solution using the results of the
assay performed in Section 7.15.1.
7.15.3 Stock standard solutions must be replaced after six months, or sooner if comparison with
check standards indicates a problem.
7.15.4 Aqueous performance standard—An aqueous performance standard containing formalde-
hyde (nominally 100 ug/L), isobutyraldehyde at 100 ug/L, and furfural at 100 ug/L shall be
prepared daily and analyzed each shift to demonstrate performance (Section 9).
7.15.5 Preparation of calibration standards.
7.15.5.1 Prepare calibration standard solutions of formaldehyde, isobutyraldehyde, and fur-
fural in reagent water from stock standard solution (Section 7.15.2). Prepare these
solutions at the following concentrations (in fig/mL) by serial dilution of the stock
standard solution: 50,20,10. Prepare additional calibration standard solutions at
the following concentrations, by dilution of the appropriate 50,20, or 10 ug/mL
standard: 5,0.5, 2,0.2,1,0.1. Make further dilutions if appropriate.
7.16 Reaction solutions.
7.16.1 DNPH (1.00 mg/mL)—Dissolve 142.9 mg 70% (w/w) reagent in 100 mL of absolute etha-
nol. Slight heating or sonication may be necessary to effect dissolution.
7.16.2 Phthalate buffer (0.1 N)—Prepare by dissolving 20.42 g of potassium acid phthalate in 1 L of
reagent water. Adjust pH to 5 by addition of sodium hydroxide or hydrochloric acid, as nec-
essary.
7.16.3 Sodium chloride solution (saturated)—Prepare by mixing an excess of the reagent-grade
solid with reagent water.
8. Sample Collection, Preservation, and Storage
8.1 Grab samples are collected in glass containers having a total volume greater than 20 mL. For aque-
ous samples that pour freely, fill sample bottles so that no air bubbles pass through the sample as the
bottle is filled and seal each bottle so that no air bubbles are entrapped. Maintain the hermetic seal on
the sample bottle until time of analysis.
8.2 Samples are maintained at 0-4 tl from the time of collection until analysis. Samples must be deriva-
tized within five days of collection and analyzed within three days of derivatization.
-------
Method 1667
9. Quality Control
9.1 Each laboratory that uses this method is required to operate a formal quality assurance program (Ref-
erence 4). The minimum requirements of this program consist of an initial demonstration of labora-
tory capability and analysis of standards and blanks as tests of continued performance. Laboratory
performance is compared to established performance criteria to determine if the results of analyses
meet the performance characteristics of the method.
9.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable accuracy
and precision with this method. This ability is established as described in Section 9.2.
9.1.2 In recognition of advances that are occurring in analytical technology, and to allow the ana-
lyst to overcome sample matrix interferences, the analyst is permitted certain options to
improve separations or lower the costs of measurements. These options include alternative
extraction, concentration, cleanup procedures, and changes in columns and detectors. Alter-
native techniques, such as substitution of immunoassay, and changes that degrade method
performance are not allowed. If an analytical technique other than the techniques specified
in this method is used, that technique must have a specificity equal to or better than the spec-
ificity of the techniques in this method for the analytes of interest.
9.1.2.1 Each time a modification is made to this method, the analyst is required to repeat
the procedure in Section 9.2. If the detection limit of the method will be affected
by the change, the laboratory is required to demonstrate that the method detection
limit (MDL) (40 CFR Part 136, Appendix B) is lower than one-third the regulatory
compliance level. If calibration will be affected by the change, the analyst must
recalibrate the instrument per Section 10.
9.122 The laboratory is required to maintain records of modifications made to this
method. These records include the information below, at a minimum.
9.1.2.2.1 The names, titles, addresses, and telephone numbers of the analyst(s)
who performed the analyses and modification, and of the quality con-
trol officer who witnessed and will verify the analyses and modifica-
tion.
9.1.2.2.2 A list of pollutant(s) measured, including name and CAS Registry
Number.
9.1.2.2.3 A narrative stating the reason(s) for the modification.
9.1.2.2.4 Results from all quality control (QC) tests comparing the modified
method to this method, including:
(a) Calibration (Section 10.1.2)
(b) Calibration verification (Section 10.1.2.2)
(c) Initial precision and accuracy (Section 9.2)
(d) Analysis of blanks (Section 9.3)
(e) Accuracy assessment (Section 9.5)
9.1.2.2.5 Data that will allow an independent reviewer to validate each determi-
nation by tracing the instrument output (peak height, area, or other sig-
nal) to the final result. These data are to include:
(a) Sample numbers and other identifiers
86
-------
Method 1667
(b) Extraction dates
(c) Analysis dates and times
(d) Analysis sequence/run chronology
(e) Sample weight or volume (Section 11)
(f) Extract volume prior to each cleaning step (Section 11.1.2)
(g) Final extract volume prior to injection (Section 11.3.4.5 or Section
11.3.5.5)
(h) Injection volume (Section 12)
(i) Dilution data, differentiating between dilution of a sample or an
extract
(j) Instrument and operating conditions
(k) Column and operating conditions (nature of column, dimensions,
flow rates, solvents, etc.)
(I) Detector operating conditions (wavelength, etc.)
(m) Chromatograms, printer tapes, and other recording of raw data
(n) Quantitation reports, data system outputs, and other data necessary
to link raw data to the results reported
9.1.3 Analyses of blanks are required to demonstrate freedom from contamination and that the
compounds of interest and interfering compounds have not been carried over from a previ-
ous analysis (Section 4). The procedures and criteria for analysis of a blank are described in
Section 9.3.
9.1.4 The laboratory shall, on an ongoing basis, demonstrate through the analysis of the aqueous
performance standard (Section 7.15.4) that the analysis system is in control. This procedure
is described in Section 10.
9.1.5 The laboratory shall maintain records to define the quality of data that is generated.
9.2 Initial precision and accuracy—To establish the ability to generate acceptable precision and accuracy,
the analyst shall perform the following operations for compounds to be calibrated.
9.2.1 Analyze four aliquots of the aqueous performance standard (Section 7.15.4) according to the
method beginning in Section 11. Use the solid-sorbent option or the methylene chloride
option, whichever will be used routinely.
9.2.2 Using results from Section 9.2.1, compute the average percent recovery (X) and the standard
deviation of the recovery (s) for each compound.
9.2.3 For each compound, compare s and X with the corresponding limits for initial precision and
accuracy found in Table 3. If s and X for all compounds meet the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may begin. If, however, any
individual s exceeds the precision limit or any individual X falls outside the range for accu-
racy, system performance is unacceptable for that compound. This is an indication that the
analytical system is not performing properly for the compound(s) in question. In this event,
correct the problem and repeat the entire test (Section 9.2.1).
87
-------
Method 1667
9.3 Blanks—Reagent water blanks are analyzed to demonstrate freedom from contamination.
"With each sample lot (samples analyzed on the same 8-hour shift), a blank shall be analyzed immedi-
ately after analysis of the aqueous performance standard (Section 9.1.4) to demonstrate freedom from
contamination. If any of the compounds of interest or any potentially interfering compound is found
in a blank at greater than 10 ug/L, analysis of samples is halted until the source of contamination is
eliminated and a blank shows no evidence of contamination at this level.
9.4 The specifications contained in this method can be met if the apparatus used is calibrated properly,
then maintained in a calibrated state. The standards used for calibration (Section 7.15.5), calibration
verification (Section 10.1.2.2) and for initial (Section 9.2) and ongoing (Section 9.1.4) precision and
accuracy should be identical, so that the most precise results will be obtained.
9.5 Depending on specific program requirements, field replicates may be collected to determine the pre-
cision of the sampling technique, and spiked samples may be required to determine the accuracy of
the analysis.
10. Calibration
10.1 Establish liquid chromatographic operating parameters to produce a retention time equivalent to that
indicated in Table 2 for formaldehyde derivative. Suggested chromatographic conditions are pro-
vided in Section 12.1. Prepare derivatized calibration standards according to the procedure in Section
10.1.1. Calibrate the chromatographic system using the external standard technique (Section 10.1.2).
10.1.1 Process each calibration standard solution through the derivatization option used for sample
processing (Section 11.3.4 or 11.3.5).
10.1.2 External standard calibration procedure.
10.1.2.1 Analyze each derivatized calibration standard using the chromatographic condi-
tions specified in Section 12.1, and tabulate peak area against concentration
injected. The results may be used to prepare calibration curves for formaldehyde,
isobutyraldehyde, and furfural.
10.1.2.2 The working calibration curve must be verified at the beginning of each 8-hour
shift by the measurement of one or more calibration standards. If the response for
any analyte varies from the previously established responses by more than 10%,
the test must be repeated using a fresh calibration standard after it is verified that
the analytical system is in control. Alternatively, a new calibration curve may be
prepared for that compound. If an autosampler is available, it is convenient to pre-
pare a calibration curve daily by analyzing standards along with test samples.
11. Sample Extraction, Cleanup, and Derivatization
11.1 Extraction of solid samples.
11.1.1 All solids must be homogeneous. When the sample is not dry, determine the dry weight of
the sample using a representative aliquot.
11.1.1.1 Determination of dry weight—In certain cases, sample results are desired based on
a dry weight basis. When such data is desired, a portion of the sample is weighed
out at the same time as the portion used for the analytical determination.
88
-------
Method 1667
Warning: The drying oven should be contained in a hood or vented. Significant laboratory contamination
_ may result from drying a heavily contaminated hazardous waste sample. _
11 .1 .1 .2 Immediately after weighing the sample for extraction, weigh 5-1 0 g of the sample
into a tared crucible. Determine the percent dry weight of the sample by drying
overnight at 105 °C. Allow to cool in a desiccator before weighing.
g of dry sample
of sample
*100
11 .1 .2 Measure 25 g of solid into a 500-mL bottle with a PTFE-lined screw-cap or crimp-top, and
add 500 mL of extraction fluid (Section 6.13). Extract the solid by rotating the bottle at
approximately 30 rpm for 18 hours. Filter the extract through glass-fiber filter paper and
store in a sealed bottle at 4 °C. Each mL of extract represents 0.050 g of solid.
11.2 Cleanup and separation.
11.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedures recommended in this method have been used for the analysis of various sample
types. If particular circumstances demand the use of an alternative cleanup procedure, the
analyst must meet the specifications in Section 9.1.2.
11 .2.2 If the sample is not clean, or the complexity is unknown, the entire sample should be centri-
fuged at 2500 rpm for 10 minutes. Decant the supernatant liquid from the centrifuge bottle
and filter through glass-fiber filter paper into a container that can be tightly sealed.
11.3 Derivatization.
11.3.1 For aqueous samples, measure a 50- to 100-mL aliquot of sample. Quantitatively transfer
the sample aliquot to the reaction vessel (Section 6.1).
11.3.2 For solid samples, 1-10 mL of leachate (Section 11.1.2 or Section 11.2.2) will usually be
required. The amount used for a particular sample must be determined through preliminary
experiments.
11 .3.3 Derivatization and extraction of the derivative can be accomplished using the solid-sorbent
(Section 11.3.4) or methylene chloride option (Section 11.3.5).
11 .3.4 Solid Sorbent Option
11.3.4.1 Add 4 mL of phthalate buffer (Section 7.16.2) and adjust the pH to 5.0 ±0.1 with
sodium hydroxide or hydrochloric acid. Add 10 mL of DNPH reagent, adjust the
total volume to approximately 100 mL with reagent water, seal the container and
place on a wrist-action shaker at room temperature for 1 hour. Samples or stan-
dards containing high analyte concentrations may require more DNPH reagent for
complete reaction.
11 .3.4.2 Assemble the vacuum manifold and connect to a water aspirator or vacuum pump.
Assemble solid sorbent cartridges containing a minimum of 1.5 g of C18 sorbent,
using connectors supplied by the manufacturer, and attach the sorbent train to the
vacuum manifold. Condition each cartridge by passing 10 mL dilute phthalate
buffer (10 mL 5 N phthalate buffer dissolved in 250 mL of reagent water) through
the sorbent cartridge train.
89
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Method 1667
11.3.4.3 Remove the reaction vessel from the shaker and add 10 mL of saturated sodium
chloride solution to the vessel.
11.3.4.4 Add the reaction solution to the sorbent train and apply a vacuum so that the solu-
tion is drawn through the cartridges at a rate of 3 to 5 mL/min. Release the vacuum
after the solution has passed through the sorbent.
11.3.4.5 Elute each cartridge train with approximately 9 mL of absolute ethanol, directly
into a 10-mL volumetric flask. Dilute the solution to volume with absolute etha-
nol, mix thoroughly, and place in a tightly sealed vial until analyzed.
11.3.5 Methylene chloride option.
11.3.5.1 Add 5 mL of phthalate buffer (Section 7.16.2) and adjust the pH to 5.0 ±0.1 with
sodium hydroxide or hydrochloric acid. Add 10 mL of DNPH reagent, adjust the
volume to approximately 100 mL with reagent water, seal the container, and place
on a wrist-action shaker at room temperature for 1 hour. Samples or standards with
high analyte concentrations may require more DNPH reagent for complete reac-
tion.
11.3.5.2 Extract the solution with three 20-mL portions of methylene chloride, using a 250-
mL separatory funnel, and combine the methylene chloride layers. If an emulsion
forms upon extraction, remove the entire emulsion and centrifuge at 2000 rpm for
10 minutes. Separate the layers and proceed with the next extraction.
11.3.5.3 Assemble a K-D concentrator by attaching a 10-mL concentrator tube to a 500-mL
evaporator flask. Wash the K-D apparatus with 25 mL of extraction solvent to
complete the quantitative transfer.
11.3.5.4 Add one or two clean boiling chips to the evaporation flask and attach a three-ball
Snyder column. Prewet the Snyder column by adding about 1 mL of methylene
chloride to the top. Place the K-D apparatus on a hot water bath (80-90 °C) so that
the concentrator tube is partially immersed in the hot water and the entire lower
rounded surface of the flask is bathed with hot vapor. Adjust the vertical position
of the apparatus and the water temperature, as required, to complete the concentra-
tion in 10-15 minutes. At the proper rate of distillation, the balls of the column
will actively chatter, but the chambers will not flood with condensed solvent.
When the apparent volume of the liquid reaches 10 mL, remove the K-D apparatus
and allow it to drain and cool for at least 10 minutes.
11.3.5.5 Prior to liquid chromatographic analysis, the solvent must be exchanged to metha-
nol. The analyst must ensure quantitative transfer of the extract concentrate. The
exchange is performed as described below.
11.4 After cooling and draining as described in Section 11.3.5.4, momentarily remove the Snyder column
and add 5 mL of methanol and a new boiling chip. Attach the micro Snyder column. Concentrate
the extract using 1 mL of methanol to prewet the Snyder column. Place the K-D apparatus on the
water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical
position of the apparatus and the water temperature, as required, to complete the concentration.
When the apparent volume of the liquid reaches less than 5 mL, remove the K-D apparatus and allow
it to drain and cool for at least 10 minutes.
Remove the Snyder column and rinse the flask and its lower joint with 1-2 mL of methanol and add
to the concentrator tube. A 5-mL syringe is recommended for this operation. Adjust the extract vol-
ume to 10-mL with methanol. Stopper the concentrator tube and store refrigerated at 4
-------
Method1667
processing will not be performed immediately. If the extract will be stored longer than two days, it
should be transferred to a vial with a PTFE-lined screw-cap or crimp-top. Proceed with the liquid
chromatographic analysis if further cleanup is not required.
12. High-Pressure Liquid Chromatography
12.1 Chromatographic conditions.
Column: C18,250 mm long x 4.6 mm i.d., 5-um particle size (or equivalent).
Mobile Phase: Methanol/water, 75:25 (v/v), isocratic at 30 °C.
How Rate: l.OmL/min.
UV Detector: 365 nm.
Injection Vol.: 20 uL.
12.2 Analysis.
12.2.1 Analyze samples by HPLC using conditions described in Section 12.1. Table 2 lists the
retention times and MDLs that were obtained under these conditions. Other HPLC columns,
chromatographic conditions, or detectors may be used if the requirements of Section 9 are
met.
12.2.2 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-time variations of standards over the course of a day.
Three times the standard deviation of the retention time for a compound can be used to cal-
culate a suggested window size; however, the experience of the analyst should weigh heavily
in the interpretation of chromatograms.
12.2.3 If the peak area exceeds the linear range of the calibration curve, a smaller sample volume
should be used. Alternatively, the final solution may be diluted with ethanol or methanol, as
appropriate, and reanalyzed.
12.2.4 If the peak area measurement is prevented by the presence of observed interferences, further
cleanup may be required.
12.3 Calculations.
12.3.1 Calculate each response factor (RF) as follows (mean value based on 5 points):
concentration of standard
RF =
area of the signal
5
RF.
mean RF = RF =
91
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Method 1667
12.3.2 Calculate the concentration of formaldehyde, isobutyraldehyde, and furfural as follows:
\ig\mL = RFx. area of signal x concentration factor
where:
final volume of extract
concentration factor = /m-rifl/ sampie (or leachate) volume
12.3.2.1 For solid samples, a factor must be included in the equation to account for the
weight of the sample used and, if desired, to correct for dry weight.
13. Method Performance
13.1 The MDLs listed in Table 2 were obtained using reagent water and methylene chloride extraction.
Similar results can be obtained using the solid-sorbent method.
13.2 This method has been tested for linearity of recovery from spiked reagent water and has been demon-
strated to be applicable over the range from the ML to 50 times the ML.
13.3 A representative chromatogram is presented as Figure 1.
14. Waste Management
14.1 It is the laboratory's responsibility to comply with all federal, State, and local regulations governing
waste management, particularly the hazardous waste identification rules and land-disposal restric-
tions. In addition, it is the laboratory's responsibility to protect air, water, and land resources by min-
imizing and controlling all releases from fume hoods and bench operations. Also, compliance is
required with any sewage discharge permits and regulations.
14.2 Samples containing acids at a pH of less than 2 are hazardous and must be neutralized before being
poured down a drain or must be handled as hazardous waste.
14.3 For further information on waste management, consult "The Waste Management Manual for Labora-
tory Personnel," available from the American Chemical Society's Department of Government Rela-
tions and Science Policy, 1115 Sixteenth Street, NW, Washington, DC 20036.
92
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Method 1667
References
1. "Working with Carcinogens," DHEW, PHS, NIOSH, Publication 77-206 (1977).
2. "OSHA Safety and Health Standards, General Industry," 29 CFR 1910, OSHA 2206, (1976).
3. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication, Committee
on Chemical Safety (1979).
4. "Handbook of Analytical Quality Control in Water and Wastewater Laboratories," U.S. EPA, EMSL
Cincinnati, OH 45268, EPA-600/4-79-019 (March 1979).
93
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Method 1667
Table 1. PMI Analytes to Which This Method Applies
PMI Analyte CASRN1
Formaldehyde 50-00-0
Furfural 98-01-1
Isobutyraldehyde 78-84-2
1 Chemical Abstracts Service Registry Number.
Table 2. Retention Times and Minimum Levels (MLs) for PMI Analytes
Retention Time1 ML2
PMI Analyte (seconds) (ug/L)
Formaldehyde 326 50
Furfural 495 50
Isobutyraldehyde 714 50
1 Retention times are for the DNPH derivative.
o
This is the minimum level at which the entire analytical system shall give a recognizable signal and an acceptable cal-
ibration point, taking into account method-specific sample and injection volumes.
Table 3. Requirements for Initial Precision and Accuracy
Spike Average Percent Standard Deviation
PMI Analyte Level (ug/L) Recovery (X) (s)
Formaldehyde 20 25-187 81
Furfural 100 70-102 16
Isobutyraldehyde 10 45-121 38
94
-------
Method 1667
250 -
200 -
150 -
100 -
50 -
10 PPM STANDARD. Amount: 1.000.
Acquired on 17-08-94 at 00:43:45 Reported on 08-17-94 at 10:40:44
10
I
15
20
Figure 1. Chromatogram of the 2,4-DNPH Derivative of Formaldehyde,
Furfural, and Isobutyraldehyde
52-025
95
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Method 1671
Volatile Organic Compounds Specific to the
Pharmaceutical Manufacturing
Industry by GC/FID
January 1995
-------
Method 1671
Volatile Organic Compounds Specific to the
Pharmaceutical Manufacturing Industry by GC/FID
1. Scope and Application
1.1 This method is for survey and monitoring under the Clean Water Act. The method is used to deter-
mine certain non-purgeable volatile organic pollutants specific to the pharmaceutical manufacturing
industry (PMI) that are amenable to direct aqueous injection gas chromatography (GC) and detection
by a flame ionization detector (FID).
1.2 The PMI analytes listed in Table 1 may be determined in waters, soils, and municipal sludges by this
method.
1.3 The detection limits of Method 1671 are usually dependent on the level of interferences rather than
instrumental limitations. The minimum levels (MLs) in Table 2 are the level that can be attained
with no interferences present.
1.4 This method is recommended for use by, or under the supervision of, analysts experienced in the
operation of gas chromatographs and in the interpretation of chromatograms.
1.5 This method is performance-based. The analyst is permitted to modify the method to overcome
interferences or to lower the cost of measurements, provided that all performance criteria in this
method are met. The requirements for establishing method equivalency are given in Section 9.2.
2. Summary of the Method
2.1 The percent solids content of the sample is determined. If the solids content is less than 1%, internal
standard(s) is added to a 5-mL sample. If the solids content of the sample is greater than 1 %, 5 mL of
reagent water and internal standard(s) are added to a 5-g aliquot of sample.
The mixture is sonicated in a centrifuge tube with little or no headspace for 5 minutes. During this
period the analytes and the internal standard will equilibrate between the solid and aqueous phases.
In some cases, additional sonication will be necessary to establish equilibrium. The resulting suspen-
sion is centrifuged and the supernatant liquid analyzed.
22 An appropriate amount of the aqueous solution (or supernate) is injected into the GC. The com-
pounds are separated by the GC and detected by the FID.
3. Definitions
There are no definitions specific to this method.
4. Interferences
4.1 Impurities in the carrier gas, organic compounds outgassing from the GC plumbing, and solvent
vapors in the laboratory account for the majority of contamination problems encountered with this
99
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Method 1671
method. The analytical system is demonstrated to be free from interferences under conditions of the
analysis by analyzing reagent water blanks initially and with each sample batch (samples analyzed
on the same 8-hour shift), as described in Section 9.4.
4.2 Samples can be contaminated by diffusion of volatile organic compounds through the bottle seal dur-
ing shipment and storage. A field blank prepared from reagent water and carried through the sam-
pling and handling protocol may serve as a check on such contamination.
4.3 Contamination by carryover can occur when high-level and low-level samples are analyzed sequen-
tially. To reduce carryover, the syringe is cleaned or replaced with a new syringe after each sample is
analyzed. When an unusually concentrated sample is encountered, it is followed by analysis of a
reagent water blank to check for carryover. Syringes are cleaned by washing with soap solution,
rinsing with tap and distilled water, and drying in an oven at 100-125°C. Other parts of the system
are also subject to contamination; therefore, frequent bakeout and purging of the entire system may
be required.
4.4 Interferences resulting from samples will vary considerably from source to source, depending on the
diversity of the site being sampled.
5. Safety
The toxicity or carcinogenicity of each analyte, compound, or reagent used in this method has not
been precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The laboratory
is responsible for maintaining a current awareness file of OSHA regulations regarding the safe han-
dling of the chemicals specified in this method. A reference file of material safety data sheets should
also be made available to all personnel involved in these analyses. Additional information on labora-
tory safety can be found in References 2-4.
6. Equipment and Supplies
6.1 Sample bottles and septa
6.1.1 Bottles—25- to 40-mL with polytetrafluoroethylene (PTFE)-lined screw-cap (Pierce 13075,
or equivalent). Detergent wash, rinse with tap and distilled water, and dry at >105°C for a
minimum of 1 hour before use.
6.1.2 Septa—PTFE-faced silicone (Pierce 12722, or equivalent), cleaned as above and baked at
100-200°C for a minimum of 1 hour.
6.2 Gas chromatograph—Shall be Linearly temperature programmable with initial and final holds, and
shall produce results which meet the calibration (Section 10), quality assurance (Section 9), and per-
formance tests (Section 13) of this method.
6.2.1 Column—30 m long x 0.32 mm i.d. fused-silica microbore column coated with 4-um of
bonded poly(dimethylpolysiloxane) (Supelco SPB-1 Sulfur, or equivalent).
6.2.2 GC operating conditions.
Temperatures:
Column—2 minutes at 40°C, 10°C per minute to 180°C.
Injection port—200°C
100
-------
Method 1671
FED—300°C
Carrier gas—Hydrogen at a head pressure of 10 psig.
An injector split may be used in order to optimize peak shape and repeatability.
6.3 Syringes—5-mL, gas-tight glass hypodermic, with Luer-lok tips.
6.4 Micro syringes—10-, 25-, and 100-|jL.
6.5 Syringe valves—2-way with Luer ends, PTFE.
6.6 Bottles—15-mL, screw-cap with PTFE liner.
6.7 Balances.
6.7.1 Analytical, capable of weighing 0.1 mg.
6.7.2 Top-loading, capable of weighing 10 mg.
6.8 Equipment for determining percent moisture.
6.8.1 Oven, capable of being temperature-controlled at 110°C (±5°C).
6,8.2 Desiccator.
6.8.3 Beakers—50-, 100-mL.
6.9 Centrifuge apparatus.
6.9.1 Centrifuge capable of rotating 10-mL centrifuge tubes at 5000 rpm.
6.9.2 Centrifuge tubes, 10-mL, with screw-caps (PTFE-lined) to fit centrifuge.
6.10 Sonication apparatus capable of sonicating 10-mL centrifuge tubes and thoroughly agitating con-
tents.
7. Reagents and Standards
7.1 Reagent water: Water in which the compounds of interest and interfering compounds are not
detected by this method. It may be generated by any of the following methods:
7.1.1 Activated carbon—pass tap water through a carbon bed (Calgon Filtrasorb-300, or equiva-
lent).
7.1.2 Water purifier—Pass tap water through a purifier (Millipore Super Q, or equivalent).
7.1.3 Boil and purge—Heat tap water to between 90 and 100°C and bubble contaminant-free inert
gas through it for approximately 1 hour. While still hot, transfer the water to screw-cap bot-
tles and seal with a PTFE-lined cap.
7.2 Sodium thiosulfate—ACS granular.
7.3 Standard solutions—Purchased as solutions or mixtures with certification to their purity, concentra-
tion, and authenticity, or prepared from materials of known purity and composition. If compound
purity is 96% or greater, the weight may be used without correction to calculate the concentration of
the standard.
7.3.1 Place approximately 8 mL of reagent water in a 10-mL ground-glass-stoppered volumetric
flask. Allow the flask to stand unstoppered for approximately 10 minutes or until all wetted
101
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Method 1671
surfaces have dried. For each analyte, weigh the stoppered flask, add the compound, restop-
per, then immediately reweigh to prevent evaporation losses from affecting the measure-
ment.
7.3.2 Liquids—Using a microsyringe, add sufficient liquid (about 100 mg) so that the final solu-
tion will have a concentration of about 10 mg/mL.
7.3.3 Gases—Fill a valved 5-mL gas-tight syringe with the compound. Lower the needle to
approximately 5 mm above the meniscus. Slowly introduce the compound above the surface
of the meniscus. The gas will dissolve in the solvent. Repeat if necessary to reach desired
concentration.
7.3.4 Fill the flask to volume, stopper, then mix by inverting several times. Calculate the concen-
tration in milligrams per milliliter (mg/mL, equivalent to micrograms per microliter [|ig/uL])
from the weight gain.
7.3.5 Transfer the stock solution to a PTFE-sealed screw-cap bottle. Store, with minimal head-
space, in the dark at approximately 4°C. Do not freeze.
7.3.6 Replace standards after one month, or sooner if comparison with check standards indicate a
change in concentration. Quality control check standards that can be used to determine the
accuracy of calibration standards may be available from the National Institute of Standards
and Technology, Gaithersburg, MD.
7.4 Secondary standards—Using standard solutions (Section 7.3), prepare a secondary standard to con-
tain each pollutant at a concentration of 100 mg/L or 500 mg/L for compounds with higher MLs.
Where necessary, a concentration of 1000 mg/L may be used.
7.4.1 Aqueous calibration standards—Using a syringe or a microsyringe, add sufficient secondary
standard (Section 7.4) to five reagent water aliquots to produce concentrations in the range of
interest
7.4.2 Aqueous performance standard—An aqueous standard containing all pollutants and internal
standard(s) is prepared daily, and analyzed each shift to demonstrate performance (Section
13). This standard shall contain concentrations of pollutants and internal standard(s), as
appropriate, within a factor of 1 to 5 times the MLs of the pollutants listed in Table 1. It may
be one of the aqueous calibration standards described in Section 7.4.1.
8. Sample Collection, Preservation, and Handling
8.1 Grab samples are collected in glass containers having a total volume greater than 20 mL. For aque-
ous samples that pour freely, fill sample bottles so that no air bubbles pass through the sample as the
bottle is filled and seal each bottle so that no air bubbles are entrapped. Maintain the hermetic seal on
the sample bottle until time of analysis.
8.2 Maintain samples at 4°C from the time of collection until analysis. Do not freeze. If an aqueous
sample contains residual chlorine, add sodium thiosulfate preservative (10 rag/40 mL) to the empty
sample bottles just prior to shipment to the sample site. EPA Methods 330.4 and 330.5 may be used
for measurement of residual chlorine (Reference 5). If preservative has been added, shake the bottle
vigorously for 1 minute immediately after filling.
8.3 For aqueous samples, experimental evidence indicates that some PMI analytes are susceptible to
rapid biological degradation under certain environmental conditions. Refrigeration alone may not be
102
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Method 1671
adequate to preserve these compounds in wastewaters for more than seven days. For this reason, a
separate sample should be collected, acidified, and analyzed when compounds susceptible to rapid
biological degradation are to be determined. Collect about 500 mL of sample in a clean container.
Adjust the pH of the sampleto about 2 by adding hydrochloric acid (1+1) while stirring. Check pH
with narrow range (1.4 to 2.8) pH paper. Fill a sample bottle as described in Section 8.1. If residual
chlorine is present, add sodium thiosulfate to a separate sample bottle and fill as in Section 8.1.
8.4 All samples shall be analyzed within 14 days of collection.
9. Quality Assurance/Quality Control
9.1 Each laboratory that uses this method is required to operate a formal quality assurance program (Ref-
erence 6). The minimum requirements of this program consist of an initial demonstration of labora-
tory capability (Section 9.5) and analysis of standards (Sections 9.6 and 13) and blanks (Section 9.4)
as tests of continued performance. Each time a batch of samples is analyzed or there is a change in
reagents or procedures, a method blank must be analyzed as a safeguard against contamination.
9.2 In recognition of advances that are occurring in analytical technology, and to allow the analyst to
overcome sample matrix interferences, the analyst is permitted certain options to improve separa-
tions or lower the costs of measurements. These options include alternative concentration and
cleanup procedures, and changes in columns and detectors. Alternative techniques, such as the sub-
stitution of spectroscopy or immunoassay, and changes that degrade method performance are not
allowed. If an analytical technique other than the techniques specified in this method is used, that
technique must have a specificity equal to or better than the specificity of the techniques in this
method for the analytes of interest.
9.2.1 If the detection limit of the method will be affected by the change, the laboratory is required
to demonstrate that the MDL (40 CFR Part 136, Appendix B) is lower than one-third the reg-
ulatory compliance level. If calibration will be affected by the change, the analyst must reca-
librate the instrument per Section 10.
9.2.2 The laboratory is required to maintain records of modifications made to this method. These
records include the information in this subsection, at a minimum.
9.2.2.1 The names, titles, addresses, and telephone numbers of the analyst(s) who per-
formed the analyses and modification, and of the quality control officer who wit-
nessed and will verify the analyses and modification.
9.2.2.2 A listing of pollutant(s) measured, by name and CAS Registry Number.
9.2.2.3 A narrative stating the reason(s) for the modification.
9.2.2.4 Results from all quality control (QC) tests comparing the modified method to this
method including:
(a) calibration (Section 10);
(b) calibration verification (Section 13);
(c) initial precision and accuracy (Section 9.5);
(d) analysis of blanks (Section 9.4); and
(e) accuracy assessment (Section 9.6 and 13).
703
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Method 1671
9.2.2.5 Data that will allow an independent reviewer to validate each determination by
tracing the instrumentoutput (peak height, area, or other signal) to the final result.
These data are to include:
(a) sample numbers and other identifiers;
(b) analysis dates and times;
(c) injection logs;
(d) analysis sequence/run chronology;
(e) sample weight or volume;
(f) sample volume prior to each cleanup step, if applicable;
(g) sample volume after each cleanup step, if applicable;
(h) final sample volume prior to injection;
(i) injection volume;
(j) dilution data, differentiating between dilution of a sample or an extract;
(k) instrument and operating conditions;
(I) column (dimensions, liquid phase, solid support, film thickness, etc,);
(m) operating conditions (temperature, temperature program, flow rates, etc.);
(n) detector (type, operating condition, etc,);
(o) chromatograms, printer tapes, and other recording of raw data; and
(p) quantitation reports, data system outputs, and other data necessary to link raw
data to the results reported.
9.3 With each sample batch, a matrix spike (MS) and matrix spike duplicate (MSD) are analyzed to
assess precision and accuracy of the analysis. The relative percent difference (RPD) between the MS
and MSD shall be less than 30% and compound recoveries shall fall within the limits specified in
Table 3. Where RPDs are found to be 30% or greater, or where compound recoveries fall outside of
the limits specified in Table 3, the analytical process is considered to be out of control and sample
analysis shall cease until the analytical process is returned to control as evidenced by RPDs of less
than 30% and compound recoveries within the limits specified.
94 Analyses of blanks are required to demonstrate freedom from contamination and that the compounds
of interest and interfering compounds have not been carried over from a previous analysis (Section
4.3).
94.1 With each sample batch (samples analyzed on the same 8-hour shift), a blank shall be ana-
lyzed immediately after analysis of the aqueous performance standard (Sections 9.6 and 13)
to demonstrate freedom from contamination. If any of the compounds of interest or any
potentially interfering compound is found in a blank at greater man the ML (assuming a
response factor of 1 relative to the nearest-eluted internal standard for compounds not listed
in Table 1), analysis of samples is halted until the source of contamination is eliminated and
a blank shows no evidence of contamination at this level.
9J5 Initial precision and accuracy—To establish the ability to generate acceptable precision and accuracy,
the analyst shall perform the following operations for compounds to be calibrated.
704
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Method 1671
9.5.1 Analyze two sets of four 5-mL aliquot* (eight aliquot* total) of the aqueous performance
standard (Section 7,4.2) containing the PMI analytes listed in Table 1.
9.5.2 Using the first set of four analyses, compute the average recovery (X) in percent of spike
level and standard deviation of the recovery (s) in percent of spike level, for each compound.
9.5.3 For each compound, compare s and X with the corresponding limits for initial precision and
accuracy found in Table 3. If s and X for all compounds meet the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may begin. If, however, any
individual s exceeds the precision limit or any individual X falls outside the range for accu-
racy, system performance is unacceptable for that compound.
9J5A Using the results of the second set of analyses, compute s and X for only those compounds
that failed the test of the first set of four analyses (Section 9.5.3). If these compounds now
pass, the system performance is acceptable for all compounds, and analysis of blanks and
samples may begin. If, however, any of the same compounds fail again, the analysis system
is not performing properly for the compound(s) in question. In this event, correct the prob-
lem and repeat the entire test (Section 9.5).
9.6 The laboratory shall, on an ongoing basis, demonstrate through the analysis of the aqueous perfor-
mance standard (Section 7.4.2) that the analysis system is in control. This procedure is described in
Section 13.
9.7 Where available, field replicates may be used to validate the precision of the sampling technique.
9.8 The laboratory shall maintain records to define the quality of data that is generated.
10. Calibration
10.1 Inject standards into the GC and adjust the sensitivity to detect an amount of each compound less
than or equal to one-third of the ML listed in Table 2 for the analyte.
10.2 Internal standard calibration procedure. The analyst must select one or more internal standards that
are similar in analytical behavior to the compounds of interest. The analyst must further demonstrate
that the measurement of the internal standards) is not affected by method or matrix interferences.
Because of these limitations, no internal standard that would be applicable to all samples can be
required in the method. The method was developed using tetrahydrofuran (THF) as an internal stan-
dard. Where THF is not present in the sample matrix and no interference precludes its use, THF is to
be used as an internal standard for application of this method.
10.2.1 Prepare aqueous calibration standards at a minimum of five concentration levels for each
analyte by carefully adding an appropriate amount of secondary standard to reagent water or
to the matrix under study. One of me concentrations should be at or below the ML. The con-
centration range should bracket the concentrations expected in the samples and should not
exceed the dynamic range of the GC/FID instrument. These aqueous standards must be pre-
pared daily.
10.2.2 Prepare a spiking solution containing the internal standard(s) using the procedures described
in Sections 7.3 and 1A and add an appropriate amount of internal standard to each aqueous
calibration standard.
10.2.3 Using injections appropriate to optimize system sensitivity and separation of the analytes,
analyze each calibration standard and tabulate peak height or area responses against concen-
105
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Method 1671
tration for each compound and internal standard. Calculate response factors (RF) for each
compound as follows:
where:
As = Response for the analyte to be measured
A £5 = Response for the nearest eluting internal standard
Qy = Concentration of the nearest eluting internal standard
Cs = Concentration of the analyte to be measured
If the RF value over the working range is a constant (less than 10% relative standard deviation), the RF can
be assumed to be invariant and the average RF can be used for calculations. Alternatively, the results can
be used to plot a calibration curve of response ratios, Ag/Ajg, against concentration.
11. Sample Preparation
Samples containing less than 1% solids are analyzed directly as aqueous samples. Samples containing 1%
solids or greater are analyzed after equilibration with reagent water containing internal standard(s).
11 .1 Determination of percent solids.
11 .1 .1 Weigh 5-10 g of sample into a tared beaker.
11.1.2 Dry overnight (12 hours minimum) at 110°C (±5°C), and cool in a desiccator.
1 1 .1 .3 Determine the percent solids as follows :
weight of sample dry
%sollds = weigh, of sample ve,*100
11 .2 Remove standards and samples from cold storage and bring to 20-25°C.
11.3 Samples containing less than 1% solids.
1 1 .3.1 Allow sou' ds to settle and remove 5 mL of sample.
11 .3.2 Add an appropriate amount of internal standard spiking solution.
11.3.3 Inject a sample directly into the GC. The temperature of the injection block should be great
enough to immediately vaporize the entire sample. An example of the separations achieved
by the column listed is shown in Figure 1 .
Note: Use of a 0.2-yL injection has been found to improve method sensitivity over a larger injection
combined with a split sample. Where possible, splitless injection should be used. All require-
ments of this Method must be met regardless of type of injection used. _
106
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Method 1671
11.4 Samples containing 1% solids or greater.
11.4.1 Mix the sample thoroughly using a clean spatula and remove rocks, twigs, sticks, and other
foreign matter.
11.4.2 Add 5 g (±1 g) of sample to a tared 10-mL centrifuge tube. Using a clean metal spatula,
break up any lumps of sample. Record the sample weight to three significant figures.
11.4.3 Add an appropriate amount of internal standard spiking solution to the sample in the centri-
fuge tube.
11.4.4 Add a measured quantity (Y ± 0.1 mL) of reagent water to the tube so as to minimize head
space.
11.4.5 Place a cap on the centrifuge tube leaving little or no headspace. Place the tube in the sonica-
tor for a minimum of 5 minutes, turning occasionally. For most samples this should be suffi-
cient time to distribute the analytes and standard(s) between the solid and aqueous phases
and to establish equilibrium. Some sample matrices may require more sonication.
11.4.6 On completion of sonication, centrifuge the sample and inject the same amount of supemate
into the GC that was injected for the calibration standards.
11.5 For liquid samples containing high-solids concentrations, such as sludges or muds, weigh approxi-
mately 5 g (to three significant figures) into a 10-mL centrifuge tube, add an appropriate amount of
internal standard solution, sonicate, centrifuge, and inject as in Section 11.4.6.
12. Quantitative Determination
12.1 The calibration curve or averaged response factor determined during calibration is used to calculate
the concentration. For calculation using the averaged RF, the equation below is used, and the terms
are as defined in Section 10.2.3.
As X Cis
Concentration^ -j =-=,
Ais x RF
12.2 The concentration of the pollutant in the solid phase of the sample is computed using the concentra-
tion of the pollutant detected in the aqueous solution, as follows:
YL x aqueous cone (mg/L)
Concentration in solid ([ig/kg) = sample wt (kg) x percent solids x DF
where:
percent solids is from Section 11.1
7 = Volume of water in liters (L)from 11.4.4
DF = Dilution factor (as a decimal number), if necessary
12.3 Sample dilution—If the calibration range of the system is exceeded, the sample is diluted by succes-
sive factors of 10 until the sample concentration is within the calibration range.
12.4 Report results for all pollutants found in standards, blanks, and samples to three significant figures.
For samples containing less than 1% solids, the units are milligrams per liter (mg/L); and for samples
containing 1% solids or greater, units are milligrams per kilogram (mg/kg).
107
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Method 1671
13. System Performance
13.1 At the beginning of each 8-hour shift during which analyses are performed, system calibration and
performance shall be verified. Acceptance criteria for each compound (R) are found in Table 3.
Adjustment and/or recalibration shall be performed until all performance criteria are met. Only after
all performance criteria are met may blanks and samples be analyzed.
13.2 Where THF is used as the internal standard, the absolute retention time of THF shall be 416 seconds
(± 30 seconds). The relative retention times of all pollutants shall fall within 10% of the value given
in Table 2.
14. Method Performance
14.1 This method was developed and validated in a single laboratory.
14.2 A chromatogram of the aqueous performance standard is shown in Figure 1.
15. Waste Management
15.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations governing
waste management, particularly die hazardous waste identification rules and land-disposal restric-
tions. In addition, it is the laboratory's responsibility to protect air, water, and land resources by min-
imizing and controlling all releases from fume hoods and bench operations. Also, compliance is
required with any sewage discharge permits and regulations.
15.2 Samples containing acids at a pH of less than 2 are hazardous and must be neutralized before being
poured down the drain or must be handled as hazardous waste.
15.3 For further information on waste management consult "The Waste Management Manual for Labora-
tory Personnel" and "Less is Better—Laboratory Chemical Management for Waste Reduction,"
available from the American Chemical Society's Department of Government Relations and Science
Policy, 1155 16th Street, N.W., Washington, DC 20036.
108
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Method 1671
References
1. "Standard Test Method for Volatile Alcohols in Water by Direct Aqueous-Injection Gas Chromatog-
raphy." 1994 Annual Book of ASTM Standards, Volume 11.02 (Water (II)). ASTM, 1916 Race
Street, Philadelphia, PA 19103-1187.
2. "Working with Carcinogens," DREW, PHS, NIOSH, Publication 77-206 (1977).
3. "OSHA Safety and Health Standards, General Industry," 29 CFR 1910, OSHA 2206 (1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication, Committee
on Chemical Safety (1979).
5. "Methods 330.4 and 330.5 for Total Residual Chlorine," USEPA, EMSL Cincinnati, OH 45268.
6. "Method of Analytical Quality Control in Water and Wastewater Laboratories," USEPA, EMSL Cin-
cinnati, OH 45268, EPA-4-79-019 (March, 1979).
109
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Method 1671
Table 1. Non-Purgeable Water Soluble PMI Analytes to be Analyzed by Direct Aqueous Injection GC/FID
and Internal Standard Techniques
1
PMI Analyte
Acetonitrile
Diethylamine
Dimethylamine
Dimethyl sulfoxide
Ethanol
Ethylene glycol
Formamide
Methanol
Methylamine
Methyl cellosolve (2-methoxyethanol)
n-Propanol
Tn'ethylamine
Chemical Abstracts Service Registry Number
CASRN
75-05-8
109-89-7
124-40-3
67-68-5
64-17-5
107-21-1
75-12-7
67-56-1
74-89-5
109-86-4
71-23-8
121-44-8
i1
EPA-EGD
972
986
987
1037
134
1038
988
135
989
1040
955
990
110
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Method 1671
Table 2. Gas Chromatographic Retention Times and Minimum Levels for Non-purgeable Water Soluble PMI
Analytes by Direct Aqueous Injection GC/FID
EGD
No. PMI Analyte
989 Methylamine
135 Methanol
987 Dimethylamine
134 Ethanol
972 Acetonitrile
955 n-Propanol
986 Diethylamine
975 Tetrahydrofuran(intstd)
1040 Methyl cellosolve (2-Methoxyethanol)
988 Fomnamide
1038 Ethylene glycol
990 Triethylamine
1037 Dimethyl sulfoxide
Retention Time
ML1
Mean (sec)
128
139
165
188
203
307
341
416
429
473
495
518
676
EGD Ref
975
975
975
975
975
975
975
975
975
975
975
975
975
Relative
0507
0334
0396
0.452
0.488
0.737
OB19
1.000
1.030
1.136
1.189
1244
1.624
(mg/L)
50
50
50
50
50
50
50
20
100
100
50
20
1
This is a minimum level at which the entire analytical system shall give an acceptable calibration point, taking into account
method-specific sample and injection volumes. The concentration in the aqueous or solid phase is determined using the equa-
tions in Section 12.
111
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Method 1671
Table 3. Acceptance Criteria for Performance Tests
Acceptance Criteria (% of Spike Level)
Initial Precision and Accuracy
EGDNo. PMIAnalyte Spike Level
972 Acetonitrile 50
986 Diethylamine 50
987 Dimethylamine 50
1037 Dimethyl sulfoxide 50
134 Ethanol 50
1038 Ethylene glycol 100
988 Formamkte 200
135 Methanol 50
989 Methylamine 50
1040 Methyl celtosolve (2-Methoxyethanol) 50
955 n-Propanol 50
990 Triethylamine 50
s
30.2
14.8
27.2
6.1
18.6
21.6
12.3
20.4
18.0
13.6
24.9
46.1
X
85-146
65-100
98-153
31-100
93-131
100-149
96-121
79-121
79-116
64-92
87-137
72-165
Ongoing
Accuracy
R
83-148
63-100
96-155
30-100
92-132
100-150
95-122
78-122
78-117
64-93
85-139
68-168
112
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Method 1671
100% -i
%FS -
I/
Analysis: 4 MDL3,8,1
Created at 10:36 on 01/Dec/94
Sample*: 8
Injections: 1
Sample Name :MDL #3
2 345678 9 10 11 12
Rgure 1. Chromatogram of Aqueous Performance
Standard of Analytes from Table 1
52-025-17
113
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Method 1673
Polyethylene glycol)-600 by Derivatization and
High-Pressure Liquid Chromatography
January 1995
-------
Method 1673
Polyethylene glycol)-600 by Derivatization and
High-Pressure Liquid Chromatography
1. Scope and Application
1.1 This method is designed to meet the survey and monitoring requirements of the EPA's Engineering
and Analysis Division (BAD). The method is used to determine poly(ethylene glycol)-600 (PEG-
600, Chemical Abstracts Service Registry Number 25322-68-3), a substance used in the pharmaceu-
tical manufacturing industry (PMI), that can be derivatized and analyzed by high pressure liquid
chromatography (HPLC).
1.2 PEG-600 is a mixture of oligomers with a molecular weight centered around 600 Da. The exact
composition may vary from manufacturer to manufacturer and even between batches from the same
manufacturer. This method has been developed for aqueous samples and is not, in its present form
applicable to solids or sludges.
1.3 The detection limits of the method are usually dependent on the level of interferences rather than
instrumental limitations. The detection limit provided in Table 1 is the minimum level that can be
reliably quantified by this method with no interferences present.
1.4 This method is for use only by analysts experienced with HPLC or under the close supervision of
such qualified persons.
2. Summary of the Method
2.1 One liter of aqueous sample is placed into a liquid-liquid extractor and a known quantity of surrogate
is added. Extraction with dichloromethane is carried out over an 18-hour period. The dichlo-
romethane extracts are dried over anhydrous sodium sulfate, evaporated to a small volume and dried
again. Remaining dichloromethane is removed and the water-free extract is derivatized using 3,5-
dinitrobenzoyl chloride and pyridine in tetrahydrofuran. The tetrahydrofuran solution is diluted with
diethyl ether, extracted to remove side products, evaporated, and solvent exchanged with acetonitrile/
water and chromatographed on a reverse-phase C18 column with a solvent gradient of 40 percent ace-
tonitrile/water to 100 percent acetonitrile. Detection is performed at 254 nanometers. The PEG-600
derivative is identified by its retention time relative to that of the derivatized surrogate and quantified
by external standard techniques. Derivatized samples must be stored at 4°C in an amber container
and analyzed within 96 hours of preparation.
2.2 The quality of the analysis is assured through reproducible calibration and testing of the derivatiza-
tion/extraction procedure and the HPLC system.
1. This Chemical Abstracts Service Registry Number (CASRN) is common to all poly(ethylene glycol) oligomers
and mixtures and does not specifically identify PEG-600.
117
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Method 1673
3. Definitions
There are no method specific definitions to be noted for this document.
4. Interferences
4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other
sample processing hardware that lead to discrete artifacts and/or elevated baselines in chromato-
grams. All of these materials must be routinely demonstrated to be free from interferences under the
conditions of the analysis by analyzing laboratory reagent blanks as described in Section 9.3.
4.1.1 Glassware must be scrupulously cleaned. Clean all glassware as soon as possible after use
by rinsing with the last solvent used. This should be followed by detergent washing with hot
water, and rinses with tap water and reagent water. It should then be drained, dried, and
heated in a laboratory oven at 130 degrees celsius (°C) for several hours before use. Solvent
rinses with acetone may be substituted for the oven heating. After drying and cooling, glass-
ware should be stored in a clean environment to prevent any accumulation of dust or other
contaminants.
4.1.2 The use of high purity reagents and solvents helps to minimize interference problems. Purifi-
cation of solvents by distillation in all glass systems may be required.
4.2 Matrix interferences may be caused by contaminants that are coextracted from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature and diversity of the matrix being sampled. If matrix interferences occur, some additional
cleanup may be necessary.
4.3 The extent of interferences that may be encountered using liquid chromatographic techniques has not
been fully assessed. Although the HPLC conditions described allow for resolution of PEG-600,
other matrix components may interfere.
5. Safety
5.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been pre-
cisely determined; however, each chemical compound should be treated as a potential health hazard.
Exposure to these compounds should be reduced to the lowest possible level. The laboratory is
responsible for maintaining a current awareness file of OSHA regulations regarding the safe han-
dling of the chemicals specified in this method. A reference file of Material Safety Data Sheets
should also be made available to all personnel involved in these analyses. Additional information on
laboratory safety can be found in References 1 through 3.
6. Equipment and Supplies
6.1 Reaction vessel—5-mL screw-cap vial with V-shaped chamber (Aldrich Zl 8,302-4, or equivalent)
with polytetrafluoroethylene (PTFE) lined cap (Aldrich Zll,511-8, or equivalent).
6.2 Liquid-liquid extractor—Capable of extracting 1 L of sample (Aldrich Zl 0,156-7, or equivalent)
6.3 Kudema-Danish (K-D) apparatus.
6.3.1 Concentrator tube—10-mL graduated (Kontes K-570050 or equivalent). A ground-glass
stopper is used to prevent evaporation of extracts.
118
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Method 1673
6.3.2 Evaporation flask — 500-mL (Kontes K-57000 1-500 or equivalent). Attach to concentrator
tube with springs, clamps, or equivalent.
6.3.3 Snyder column— Three-ball macro (Kontes K-503000-0121 or equivalent).
6.3.4 Snyder column — Two-ball micro (Kontes K569001-0219 or equivalent).
6.3.5 Springs — One-half inch (Kontes K-662750 or equivalent).
6.4 Vials and bottles — 10-mL, 25-mL, 100-mL, and 1-L amber glass with PTFE-lined screw-caps.
6.5 Boiling chips — Solvent-extracted with methylene chloride, approximately 10/40 mesh (silicon car-
bide or equivalent).
6.6 Balance — Analytical, capable of accurately weighing to the nearest 0. 1 mg.
6.7 High-performance liquid chromatograph (modular).
6.7.1 Pumping system — Solvent programmable, with flow control capable of 2.00 milliliter per
minute (mL/min).
6.7.2 High-pressure injection valve with 50-microliter (uL) loop or autosampler.
6.7.3 Column — 250 millimeter (mm) long x 4.6 mm inside diameter (ID), 5 jjm particle size, Beta-
sil C18 (or equivalent).
6.7.4 Absorbance detector — 254 nm.
6.7.5 Strip chart recorder compatible with the detector. Use of a data system is recommended.
6.8 Glass-fiber filter paper, 0.6-0.9 urn.
6.9 Pipette — Capable of accurately delivering 0.1-1.0 mL of solution.
6.1 0 Water bath — Heated, with concentric ring cover, capable of temperature control of ± 2°C. The bath
should be used under a hood.
6.11 Sand bath — Heated, with temperature control of ± 5°C.
6.12 Volumetric flasks — 100-mL, 1-L.
6.13 Microsyringes — lOO-pL,
7. Reagents and Standards
7.1 Reagent grade or pesticide grade chemicals shall be used in all tests. Other grades may be used, pro-
vided it is first ascertained that the reagent is of sufficiently high purity to permit its use without less-
ening the accuracy of the determinations.
7.2 Reagent water: Water in which the compounds of interest and interfering compounds are not
detected by this method. It may be generated by any of the methods in this subsection.
7.2.1 Activated carbon: pass tap water through a carbon bed (Calgon Filtrasorb-300, or equiva-
lent).
7.2.2 Water purifier: Pass tap water through a purifier (Millipore Super Q, or equivalent).
7.2.3 Boil and purge: Heat tap water to between 90 and 100°C and bubble contaminant free inert
gas through it for approximately 1 hour. While still hot, transfer the water to screw-cap bot-
tles and seal with a PTFE-lined cap.
7.3 Dichloromethane — Pesticide grade or equivalent.
119
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Method 1673
7.4 Acetonitrile—Pesticide grade or equivalent.
7.5 Diethyl ether—Pesticide grade or equivalent.
7.6 Tetrahydrofuran, anhydrous—Can be prepared by distillation from potassium benzophenone ketyl,
or other, similar methods.
7.7 Surrogate—Di(ethylene glycol) monohexyl ether (CASRN 112-59-4)
7.8 Sodium sulfate, anhydrous.
7.9 Hydrochloric acid solution—Dilute 100 mL of concentrated hydrochloric acid to approximately one
liter with reagent water.
7.10 Sodium bicarbonate solution—Dissolve 10 g of sodium bicarbonate in approximately 1 L of reagent
water.
7.11 Saturated sodium chlroide solution—Prepare a saturated solution in reagent water by adding reagent
sodium chloride until no more will dissolve at room temperature.
7.12 Pyridine, anhydrous (CASRN 110-86-l)(Aldrich 27,097-0, or equivalent).
7.13 3,5-Dinitrobenzoyl chloride (CASRN 99-33-2) in anhydrous tetrahydrofuran (10 mg/mL)—Weigh
1 g of 3,5-dinitrobenzoyl chloride into a 100-mL volumetric flask and fill to the mark with anhydrous
tetrahydrofuran. Store the resulting solution in an amber bottle protected from moisture.
7.14 Stock standard solutions.
7.14.1 Stock PEG-600 (approximately 10.0 mg/mL)—Prepare by weighing 1.0 g of PEG-600 into a
100-mL volumetric flask and diluting to volume with tetrahydrofuran (if aliquots of this
solution are to be derivatized, then anhydrous tetrahydrofuran must be used).
7.14.2 Secondary standard—1.0,2.5,5.0,7.5 and 10.0 mg/L. Measure 0.1,0.25,0.5,0.75, and 1.0
mL of stock solution (Section 7.14.1) into separate 1-L volumetric flasks and dilute to vol-
ume with reagent water. Store in 1-L amber bottles.
7.14.3 Surrogate standard—Prepare by weighing 1.0 g of di(ethylene glycol) monohexyl ether into
a 100-mL volumetric flask and diluting to volume with tetrahydrofuran (if aliquots of this
solution are to be derivatized, then anhydrous tetrahydrofuran must be used). Store in a 100-
mL amber bottle protected from moisture.
7.14.4 Stock standard solution and surrogate solution must be replaced after six months, or sooner,
if comparison with check standards indicates a problem.
7.14.5 Aqueous performance standard—An aqueous performance standard containing PEG-600 at
2.5 mg/L and surrogate at Img/L, shall be prepared daily, and analyzed each shift to demon-
strate performance (Section 9).
8. Sample Collection, Preservation, and Storage
8.1 Grab samples are collected in glass containers having a total volume greater than one liter. Fill sam-
ple bottles so that no air bubbles pass through the sample as the bottle is filled and seal each bottle so
that no air bubbles are entrapped. Maintain the hermetic seal on the sample bottle until time of anal-
ysis.
8.2 Samples are maintained at 0-4°C from the time of collection until analysis. Samples must be
extracted within five days of collection, derivatized within seven days of extraction, and analyzed
within four days of derivatization.
120
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Method 1673
9. Quality Control
9.1 Each laboratory that uses this method is required to operate a formal quality assurance program (Ref-
erence 4). The minimum requirements of this program consist of an initial demonstration of labora-
tory capability and analysis of standards and blanks as tests of continued performance. Laboratory
performance is compared to established performance criteria to determine if the results of analyses
meet the performance characteristics of the method.
9.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable accuracy
and precision with this method. This ability is established as described in Section 9.2.
9.1.2 In recognition of advances that are occurring in analytical technology, and to allow the ana-
lyst to overcome sample matrix interferences, the analyst is permitted certain options to
improve separations or lower the costs of measurements. These options include alternate
extraction, concentration, cleanup procedures, and changes in columns and detectors. Alter-
native techniques, such as substitution of immunoassay, and changes that degrade method
performance are not allowed. If an analytical technique other than the techniques specified
in this method is used, that technique must have a specificity equal to or better than the spec-
ificity of the techniques in this method for the analytes of interest.
9.1.2.1 Each time a modification is made to this method, the analyst is required to repeat
the procedure in Section 9.2. If the detection limit of the method will be affected
by the change, the laboratory is required to demonstrate that the method detection
limit (MDL) (40 CFR Part 136, Appendix B) is lower than one-third the regulatory
compliance level. If calibration will be affected by the change, the analyst must
recalibrate the instrument per Section 10.
9.1.2.2 The laboratory is required to maintain records of modifications made to this
method. These records include the information below, at a minimum.
9.1.2.2.1 The names, titles, addresses, and telephone numbers of the analyst(s)
who performed the analyses and modification, and of the quality con-
trol officer who witnessed and will verify the analyses and modifica-
tion.
9.1.2.2.2 A Listing of pollutant(s) measured, by name and CAS Registry Number.
9.1.2.2.3 A narrative stating the reason(s) for the modification.
9.1.2.2.4 Results from all quality control (QC) tests comparing the modified
method to this method, including:
(a) calibration (Section 10);
(b) calibration verification (Section 10.1.3.1);
(c) initial precision and accuracy (Section 9.2);
(d) analysis of blanks (Section 9.3);
(e) accuracy assessment (Section 9.5).
9.1.2.2.5 Data that will allow an independent reviewer to validate each determi-
nation by tracing the instrument output (peak height, area, or other sig-
nal) to the final result. These data are to include:
(a) sample numbers and other identifiers;
(b) extraction dates;
121
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Method 1673
(c) analysis dates and times;
(d) analysis sequence/run chronology;
(e) sample weight or volume;
(f) extract volume prior to each cleaning step (Section 11.1.2);
(g) final extract volume prior to injection;
(h) injection volume;
(I) dilution data, differentiating between dilution of a sample or an
extract;
(j) instrument and operating conditions;
(k) column and operating conditions (nature of column, dimensions,
flow rates, solvents, etc.)
(I) detector operating conditions (wavelength, etc.)
(m) chromatograms, printer tapes, and other recording of raw data; and
(n) quantitation reports, data system outputs, and other data necessary
to fink raw data to the results reported.
9.1.3 Analyses of blanks are required tn demonstrate fregdnm from rnntaminarirm and that PFfi-
600 and interfering compounds have not been carried over from a previous analyses (Section
4). The procedures and criteria for analysis of a blank are described in Section 93.
9.1 A The laboratory shall, on an ongoing basis, demonstrate through the analysis of the aqueous
performance standard (Section 7.14.5) mat the analysis system is in control This procedure
is described in Section 10.
9.1.5 The laboratory shall maintain records to define the quality of data that is generated.
9.2 Initial precision and accuracy—To establish the ability to generate acceptable precision and accuracy,
the analyst shall perform the following operations for compounds to be calibrated:
9.2.1 Analyze f our afiquots of the aqueous performance standard (Section 7.14 J) according to the
method beginning in Section 11.
9^2 Using results from Section 9.2.1, compute the average recovery (X) in percent of spike level
and the standard deviation of the recovery (s) in percent of the spike level for PEG-600 and
the surrogate.
9^3 For each compound, compares and Xwith the o)rrespondmg limits for initial precision and
accuracy found in Table 2. If s and X for all compounds meet the acceptance criteria, system
performance is acceptable and analysis of blanks and samples may begin. If, however, any
individual s exceeds the precision limit or any individual X falls outside the range for accu-
racy, system performance is unacceptable for that compound. This is an indication that the
analytical system is not performing properly for the compound(s) in question. In this event,
correct the problem and repeat the entire test (Section 92.1).
93 Blanks—Reagent water blanks are analyzed to demonstrate freedom from contamination.
9.3.1 With each sample batch (samples analyzed on the same 8-hour shift), a blank shall be carried
through the extraction and derivatization procedure and be analyzed immediately after anal-
ysis of the aqueous performance standard (Section 9.1.4) to demonstrate freedom from con-
tamination. If PEG-600 or any potentially interfering compound is found in a blank at
greater than 200 ug/L, analysis of samples is halted until the source of contamination is elim-
inated and a blank shows no evidence of contamination at this level.
122
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Method 1673
9.4 The specifications contained in this method can be met if the apparatus used is calibrated properly,
men maintained in a calibrated state. The standards used for calibration (Section 10.1), calibration
verification (Section 10.13.1) and for initial (Section 9.2) and ongoing (Section 9.1.4) precision and
accuracy should be identical, so that the most precise results will be obtained.
9.5 Depending on specific program requirements, field replicates may be collected to determine the pre-
cision of the sampling technique, and spiked samples may be required to determine the accuracy of
the analysis
10. Calibration
10.1 Establish liquid chromatographic operating parameters to produce a retention time equivalent to that
given in Section 12.2.1. Prepare derivatized calibration standards according to the procedure in Sec-
tion 10.1.1. Calibrate the chromatographic system using the external standard technique (Section
10.1.3).
10.1.1 Preparation of calibration standards. Prepare calibration standards by adding one mL of sur-
rogate standard (Section 7.143) to each of the secondary standards (Section 7.14.2).
10.12 Process each calibration standard solution through the extraction, concentration, and deriva-
tization procedures described in Section 11.
10.1.3 External standard calibration procedure. Analyze each derivatized calibration standard using
the chromatographic conditions specified in Section 12.1, and tabulate peak area against con-
centration injected. The results may be used to prepare calibration curves for PEG-600
10.1.3.1 The working calibration curve must be verified at the beginning of each 8-hour
shift by the measurement of one or more calibration standards. If the response for
any anaryte varies from the previously established responses by more than 10 per-
cent, the test must be repeated using a fresh calibration standard after it is verified
that the analytical system is in control Alternatively, a new calibration curve may
be prepared. If an autosampler is available, it is convenient to prepare a calibration
curve daily by analyzing standards along with test samples.
11. Sample Extraction and Defivatization
11.1 Extraction of samples and standards.
11.1.1 Place one liter of sample and 1 mL of surrogate standard (Section 7.143) or 1 L of calibra-
tion standard (Section 10.1.1) in the liquid-liquid extractor and extract with pesticide grade
dichloromethane for 18 hours.
11.1.2 Dry the dichloromethane solution over anhydrous sodium sulfate and evaporate off the sol-
vent using the Kudema-Danish procedure. Dry again over anhydrous sodium sulfate when
the volume reaches 10-25 mL and use a gentle stream of dry nitrogen to removemost of the
remaining solvent Quantitatively transfer the residue to a V-shaped reaction vial using
anhydrous dichloromethane or anhydrous tetrahydrofuran and remove the last of the solvent
with a stream of dry nitrogen.
11.2 Derivatization
112.1 After ensuring that the extract is free of water, add5 mL of derivatization solution (Section
7.13) to the vial and two drops of anhydrous pyridine (Section 7.12).
11.22 Heat the vial and contents in a sand bath at 60°C (±5°Q for 1 hour.
123
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Method 7673
11.2.3 Cool the vial and quantitatively transfer the contents to a 125-mL separatory funnel. Add 50
mL of diethyl ether (ether) and sequentially extract with two 25-mL portions of dilute hydro-
chloric acid (Section 7.9), then two 25-mL portions of reagent water, then two 25-mL por-
tions of sodium bicarbonate solution (Section 7.10) and finally with two 25-mL portions of
saturated sodium chloride solution (Section 7.11). Take care not to lose any ether solution
during the extraction procedure.
11.2.4 Place a small plug of glass wool in a funnel and add approximately 10 g of anhydrous
sodium sulfate to the funnel. Drain the ether solution through the sodium sulfate. If neces-
sary use a clean spatula to break up any lumps of sodium sulfate in the funnel, then rinse the
separatory funnel with two 10-mL portions of ether and drain through the anhydrous sodium
sulfate in the funnel.
11.2.5 Quantitatively transfer the ether solution to a clean Kudema-Danish apparatus and evaporate
most of the solvent (alternatively, dry nitrogen can be used to evaporate the ether while
warming on a steam bath).
11.2.6 Perform a solvent exchange with 40% acetonitrile in water, adjust the volume to 2 mL and
filter, if necessary, for analysis.
12. High-Pressure Liquid Chromatography
12.1 Chromatographic conditions.
Column: Betasil C18,250 mm long x 4.6 mm ID, 5-|jm particle size (Keystone 255-701, or equiva-
lent).
Mobile Phase: 40% acetonitrile/water to 100% acetonitrile over a period of 20 minutes.
Flow Rate: 2.0 mL/min
UV Detector: 254 nm
Injection Vol.: 50 \iL
12.2 Analysis.
12.2.1 Analyze samples by HPLC, using conditions described in Section 12.1. The retention time
of the PEG-600 derivative relative to the surrogate derivative is centered about 0.63. The ML
achievable in reagent water is 1 mg/L. Other HPLC columns, chromatographic conditions,
or detectors may be used if the requirements of Section 9 are met.
12.2.2 Because PEG-600 is a mixture of poly(ethylene glycol) oligomers, the exact nature of PEG-
600 samples from various manufacturers and different batches from a single manufacturer,
may vary. For this reason, concentrations of PEG-600 in a specific waste stream are best
determined when standards are prepared using the same batch of PEG-600 in use by the
pharmaceutical manufacturer at the time of discharge of the waste stream under analysis.
Where it is not possible to obtain such a sample, adequate results can be obtained by the use
of a PEG-600 product as a standard that is unrelated to the one in use by the pharmaceutical
manufacturer, and careful definition of an "elution range" for derivatized PEG-600 in both
the external standards and the samples. (See Section 12.2.3)
12.2.3 An "elution range" or retention time window is defined as a characteristic period of time dur-
ing which the derivatized PEG-600 elutes from the chromatographic column. This range
should encompass at least 90 percent of the PEG-600 derivative in both the standard and the
sample. The width of the retention time window used for quantitation should be based upon
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measurements of actual retention time variations of standards over the course of a day.
Three times the standard deviation of the retention time for a compound can be used to cal-
culate a suggested window size; however, the experience of the analyst should weigh heavily
in the interpretation of chromatograms.
12.2.4 If the peak area exceeds the linear range of the calibration curve, a smaller sample volume
may be injected. Alternatively, the final solution (Section 11.2.6) may be diluted with 40%
acetonitrile/water, as appropriate, and reanalyzed.
12.3 Calculations.
12.3.1 Calculate each response factor (RF) as follows (mean value based on 5 points):
concentration of standard
Rr = -
area of the signal
5
mean RF = RF =
12.3.2 Calculate the concentration of PEG-600 as follows:
mg/mL = RF x area of signal x concentration factor
where:
final volume of extract
concentratwn factor = initial sample volume
13. Method Performance
13.1 The ML for PEG-600 was obtained using reagent water.
13.2 This method has been tested for linearity of recovery from spiked reagent water and has been demon-
strated to be applicable over the range from the ML to 30 times the ML.
13.3 A representative chromatogram is presented as Figure 1 .
14. Waste Management
14.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations governing
waste management, particularly the hazardous waste identification rules and land disposal restric-
tions. In addition it is the laboratory's responsibility to protect air, water, and land resources by min-
imizing and controlling all releases from fume hoods and bench operations. Also, compliance is
required with any sewage discharge permits and regulations.
14.2 Samples containing acids at a pH of less than 2 are hazardous and must be neutralized before being
poured down a drain or must be handled as hazardous waste.
14.3 For further information on waste management, consult "The Waste Management Manual for Labora-
tory Personnel," available from the American Chemical Society's Department of Government Rela-
tions and Science Policy, 115 16th Street, NW, Washington, DC 20036
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References
1. "Working with Carcinogens," DHEW, PHS, NIOSH, Publication 77-206 (1977).
2. "OSHA Safety and Health Standards, General Industry," 29 CFR 1910, OSHA 2206, (1976).
3. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication, Committee
on Chemical Safety (1979).
4. "Handbook of Analytical Quality Control in Water and Wastewater Laboratories," USEPA, EMSL
Cincinnati, OH 45268, EPA-4-79-019 (March 1979).
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Table LPMIAnalyte
PMIAnalyte CASRN1 Minimum Level2
Polyethylene glycol)-600 25322-68-3 1 mg/L
(PEG-600)
1 Chemical Abstracts Service Registry Number.
2 This is the minimum level at which the entire analytical system shall give a recognizable signal and an acceptable calibration
point.
Table 2. Requirements for Initial Precision and Accuracy
Percent
Spike Average Percent Standard Deviation
PMI Analyte and Surrogate Level (ug/L) Recovery (X) (s)
PEG-600 1058 22-79 28
Di(ethylene glycol) monohexyl ether 215 d-102 53
d = Detected (analyte must be detected)
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2.00 -,
Reverse Phase Column
40% ACN/HOH to 100% ACN
Calibration Std.
1 ppm w/surrogate
1.50 -
1.00 -
0.50 -
Rgure 1. Chromatogram of the Aqueous Performance Standard
62425-18
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