vvEPA
United States
Environmental Protection
Agency
Method 1668C
Chlorinated Biphenyl Congeners in Water, Soil,
Sediment, Biosolids, and Tissue by HRGC/HRMS
April 2010
-------
U.S. Environmental Protection Agency
Office of Water
Office of Science and Technology
Engineering and Analysis Division (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-820-R-10-005
EPA Method 1668C ii April 2010
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Method 1668C Chlorinated Biphenyl Congeners in Water, Soil,
Sediment, Biosolids, and Tissue by HRGC/HRMS April 2010
The Office of Science and Technology (OST) in EPA's Office of Water developed Method 1668C
(Method 1668C; the "Method") for use in Clean Water Act (CWA) programs. EPA is publishing this
Method for users who wish to measure PCBs as congeners now, and in 2010, EPA expects to publish a
proposal in the Federal Register for public comment to add this Method to other CWA Methods
published at 40 CFR Part 136.
This Method determines chlorinated biphenyl congeners in environmental samples by isotope dilution
and internal standard high-resolution gas chromatography/high-resolution mass spectrometry,
HRGC/HRMS. EPA developed this Method for use in wastewater, surface water, soil, sediment,
biosolids and tissue matrices. Other applications and matrices may be possible, which may or may not
require modifications of sample preparation, chromatography, etc.
EPA used the results of an interlaboratory validation study of Method 1668A, a peer review of that study,
user suggestions and additional interlaboratory data to write this version, 1668C, of Method 1668.
Method 1668C, the validation study report, Methodl668A Interlaboratory Validation Study Report (EPA-
821-08-021), and the addendum describing the revised QC acceptance criteria, Method 1668A Interlab
Study Report Addendum, are available at EPA's CWA methods website at
www.epa.gov/waterscience/methods.
This "C" version of Method 1668 revises the quality control (QC) acceptance criteria in EPA Method
1668B to allow the upper recovery limit for some congeners to be above 100 percent, to revise the
estimated method detection limits (EMDLs) and estimated minimum levels of quantitation (EMLs) to
MDLs and MLs, and to makes other changes summarized below. The QC acceptance criteria developed
in the interlaboratory method validation study of 1668A, and published in version B of the Method, did
not allow the upper recovery limit for some congeners to be above 100 percent. The criteria have been
revised based on data from the interlaboratory study and data from two laboratories with extensive
experience in use of Method 1668A. TestAmerica, Knoxville, Tennessee and AXYS Analytical Services,
Ltd., Sidney, British Columbia, Canada provided this new data. These two laboratories and Battelle-
Columbus provided MDLs for the congeners and congener groups, which EPA pooled and used to replace
the EMDLs and EMLs in Table 2 of Method 1668B with the MDLs and MLs in Method 1668C.
The detection limits and quantitation levels in this Method are usually dependent on the level of inter-
ferences and laboratory background levels rather than instrumental limitations. The method detection
limits (MDLs) and minimum levels of quantitation (MLs) in Table 2 are concentrations at which a
congener can be measured with no interferences present. In water, MDLs range from approximately 7 to
30 parts per quadrillion (picograms per liter, pg/L).
Interface, Inc. and CSC prepared this Method under EPA Contract EP-C-06-085. AXYS Analytical
provided the single-lab data in Method 1668A that was later replaced by multi-lab data from laboratories
that participated in EPA's inter-laboratory validation of 1668A (six labs for water and tissue, four for
biosolids).
Summary of changes between EPA Method 1668B (January 2009) and 1668C (April 2010)
Additional information on the concentration of extracts has been included in Section 4.2.
The following note has been added to Section 10.1, "RTs, RRTs, and RRT limits may differ slightly
from those in Table 2." This statement has also been added to the footnotes to Table 2.
EPA Method 1668C iii April 2010
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The note in Section 10.2.1 has been modified to inform the analyst that careful selection of the grade
and purity of PFK may help minimize interferences with the dichlorobiphenyl secondary quantitation
ion.
The diluted combined 209 congener solution is now used for calibration verification, in place of the
VER-3 solution. This allows all verification tests to be performed with a single solution.
Section 17.2.1 has been changed to clarify that concentrations of native compounds other than those
in the native toxics/LOC standard, in the labeled cleanup standard, and in the labeled injection
internal standard (except for labeled CB 178) should be determined using the response factors from
Section 10.5 or Section 15.4.2.3.
Section 17.6.5 has been added to provide information on the use of optional data qualifier flags for
reporting coeluting congeners.
Based on data from the interlab validation study and data from two laboratories, the QC acceptance
criteria in Table 6 have been revised to be consistent among tests for calibration verification (VER),
initial precision and recovery (IPR), on-going precision and recovery (OPR), and labeled compound
recovery from samples.
Reference 22 has been added to cite the Addendum to the interlaboratory validation study report.
Sections 1.3,4.1,4.6,9.1.2.1,9.5.2, 10.3.3, 17.6.1.4.1, 17.6.1.4.2, 17.6.1.4.3, and Table 2 been
revised to change estimated method detection limits (EMDLs) and estimated minimum levels of
quantitation (EMLs) to MDLs and MLs.
Reference 23 has been added to cite the MDL data from AXYS, TestAmerica-Knoxville, and
Battelle-Columbus, and to explain how these data were processed to produce the pooled MDLs in
Table 2.
A sentence was added to Section 11.4.2.1 to require weighing the sample bottle after emptying, and to
determine the volume using the density of water.
ML definition revised to cite the ML procedure.
A note was added to Section 10.3.3 to state that MDLs and MLs lower than those in Table 2 may be
established per Section 17.6.1.4.1.
Section 17.6.1.4.1 expanded to state how MDLs and MLs lower than those in Table 2 may be
established.
A footnote was added to Table 2 to cite Reference 23.
Summary of changes between EPA Method 1668A (8-20-03) and 1668B (January 2009)
(excluding typographical and grammatical error corrections, and section insertions or
deletions necessitated by the following changes).
Based on the interlaboratory validation study, single-laboratory QC acceptance criteria are replaced
with interlaboratory criteria (Table 6). A new footnote 1 to Table 6 references the EPA
interlaboratory study report, and the other footnote numbers are incremented.
EPA Method 1668C iv April 2010
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Section 1.5, the performance-based discussion, describes additional flexibility to modify CWA
Methods that is allowed by 40 CFR Part 136.6.
Section 2.5.2 now indicates that internal standards are the labeled congeners spiked into the sample.
Section 2.5.3 now indicates that injection internal standards are labeled compounds spiked into the
extract.
Section 5.4 is an added section on biohazards.
Section 7.8 notes that Method 1668A part numbers are valid for Method 1668B.
Section 8.1 allows use of alternate sample collection techniques, if documented.
Section 8.2 adds that one liter, or a larger or smaller volume of sample, may be collected.
Section 12.3 adds a note to indicate that SDS extraction may cause loss of some mono- through tri-
chloro congeners.
Section 12.5.6 states that a macro concentration device is to be used to concentrate extracts, and
deletes the requirement for collection of the extract in a round-bottom flask because any macro
concentration device may be used.
Section 16.2 requires an expert spectrometrist to determine analyte presence when an interference
precludes meeting the signal-to-noise requirement for dichloro-CB congeners.
Section 21 cites the validation studies, and that performance data are in the interlaboratory validation
study report.
Reference 1 was updated to the 2006 World Health Organization paper on toxicity equivalency
factors.
References 4 and 17 add titles to the papers in these references.
Reference 21 cites the Method 1668A Interlaboratory Validation Study Report.
Tables 2 and A-l revised the elution order for congeners 107-109.
Table 4 defines the solutions containing congeners 107, 108, and 109.
Table 6 contains revised QC acceptance criteria for performance tests, and footnote 1 to Table 6
references the Method 1668A Interlaboratory Validation Study Report.
Table 7 adds footnote 2 to require meeting the 10:1 signal-to-noise specification at the CS-2
calibration level.
EPA Method 1668C v April 2010
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Summary of corrections and changes to EPA Method 1668A as of August 20, 2003
(excluding typographical and grammatical error corrections, and section insertions or
deletions necessitated by the following changes).
Throughout: All references to IUPAC have been deleted. We have been informed that IUPAC does
not assign congener numbers. Therefore, all references to congeners by number are to "congener
number." The congener naming system given by Guitart, et al. (Guitart R., Puig P., Gomez-Catalan
J., Chemosphere 27 1451-1459, 1993) has been used in EPA Method 1668A since its inception and
continues in this version.
Sections 2.1.3, 12.4.2., 12.4.3, 12.4.5, and 12.4.9: Hexane has be deleted from the extraction solvent
for fish and other tissue to preclude loss of the more volatile CBs.
Section 7.7: A note has been added to reference the two known suppliers of labeled compounds.
Section 7.15: A statement has been added to include certified reference materials (CRMs) from the
National Resource Council of Canada.
Sections 8.2.3, 8.3.2, and 8.4.2: The preservation temperature for shipment of samples has been
changed to <6 °C to encompass the 4 ± 2 °C used by some organizations (e.g., USGS).
Section 8.2.3: The requirement to preserve aqueous samples with sulfuric acid has been deleted
because PCBs are stable in environmental samples, and the storage temperature for aqueous samples
has been changed to <6 °C.
Section 9.1.2.1: A statement has been added that a modification may be used routinely after it has
been demonstrated to meet the QC acceptance criteria of the performance tests, so long as the other
requirements in the Method are met (e.g., labeled compound recovery).
Section 10.1.2.3: The word "approximately" has been inserted in the requirement to meet the
retention times in Table 2 to reflect that slight changes in GC columns will produce slightly different
retention times.
Section 10.1.2.4: A statement has been added to indicate that the absolute and relative retention times
in Table 2 were obtained under the GC conditions given in Section 10.1.1.
Section 10.2.2: The text has been changed to clarify that the deviation between each monitored exact
m/z and the theoretical m/z (Table 7) must be less than 5 ppm.
Section 10.5: The text has been corrected to state that the diluted combined 209 congener solution
(Section 7.10.2.2 and Table 5) is used for single-point calibration of the Native Toxics/LOC CBs.
Section 12.4: A note has been added to allow use of a separate aliquot for percent lipid
determination.
Section 12.4.1: The minimum time required to dry the sample has been reduced from 12-24 hours to
30 minutes.
Section 15.6: A requirement has been added to analyze one or more aliquots of solvent after the OPR
if the CBs would be carried into the Method blank.
EPA Method 1668C vi April 2010
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Section 16.4: RRT QC limits may be based on the limits in Table 2 or limits developed from
calibration data.
Section 17.2.2: The units have been corrected to ng/mL
Section 17.4: A multiplier of 1000 has been inserted in the equation to convert ng in extract to pg in
sample.
Section 18.5: A section has been added to suggest that the carbon column should be used if
interferences preclude identification and/or quantitation of the Toxics.
Table 2: The relative retention times have been changed to correct errors and reference each
compound to the correct retention time and quantitation reference. The RT and RRT windows have
been adjusted to attempt to unambiguously identify each congener in the presence of other congeners.
Footnotes 7 and 8 have been revised to reflect this changes.
Table 3: Units for the diluted combined 209 congener solution have been corrected to ng/mL as have
the concentrations of the native compounds in the diluted combined 209 congener solution.
Table 6: The lower QC acceptance criteria limit for the labeled monochloro- and dichloro-CBs has
been lowered for the IPR, OPR, and recovery from samples to reflect that these compounds can be
lost by evaporation.
Table 7: Cl-3 scan descriptors have been added to Function 2 and the m/z types for the 13C12 Cl-4
PCBs have been corrected in Function 4.
Table 8: The m/z's forming the ratio, the ratio, and the QC limits have been corrected for
decachlorobiphenyl.
Table Al: The header has been corrected to delete reference to EMDLs and EMLs.
EPA Method 1668C vii April 2010
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Disclaimer
Mention of trade names or commercial products does not constitute endorsement or recommendation for
use.
Contact
Please address questions, comments, or suggestions to:
Richard Reding or Brian Englert
c/o The OST CWA Methods Team
Engineering and Analytical Support Branch
Engineering and Analysis Division (4303T)
Office of Science and Technology
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue
Washington, DC 20460
E-mail: OSTCWAMethods@epa.gov
EPA Method 1668C viii April 2010
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Method 1668C
Chlorinated Biphenyl Congeners in Water, Soil, Sediment, Biosolids,
and Tissue by HRGC/HRMS
April 2010
1.0 Scope and Application
1.1 Method 1668C (the Method) is for determination of chlorinated biphenyl congeners (CBs) in
wastewater and other matrices by high-resolution gas chromatography/high resolution mass
spectrometry (HRGC/HRMS).
1.1.1 The CBs that can be determined by this Method are the 12 poly chlorinated biphenyls
(PCBs) designated as toxic by the World Health Organization (WHO): congeners 77, 81,
105, 114, 118, 123, 126, 156, 157, 167, 169, and 189. The Method also determines the
remaining 197 CBs, approximately 125 of which are resolved adequately on an SPB-octyl
gas chromatographic column to be determined as individual congeners. The remaining
approximately 70 congeners are determined as mixtures of isomers (co-elutions).
1.1.2 The 12 PCBs designated as toxic by WHO (the "Toxics"; also known as dioxin-like PCBs;
DLPCBs), and the earliest and latest eluted congener at each level of chlorination are
determined by the isotope dilution quantitation technique; the remaining congeners are
determined by the internal standard quantitation technique.
1.1.3 This Method allows determination of the PCB toxicity equivalent (TEQPCB) for the Toxics
in a sample using toxicity equivalency factors (TEFs; Reference 1) and allows unique
determination of 19 of 21 CBs of interest to the National Oceanic and Atmospheric
Administration (NOAA; Reference 2). A second-column option is provided for resolution
of the two toxic PCB congeners (congener 156 and 157) that are not resolved on the SPB-
octyl column and for resolution of other CB congeners.
1.1.4 This Method also allows estimation of homolog totals by level of chlorination (LOG) and
estimation of total CBs in a sample by summation of the concentrations of the CB
congeners and congener groups.
1.1.5 The list of 209 CBs (Table 1) identifies the Toxics, the CBs of interest to NOAA, and the
LOG CBs.
1.2 EPA developed this Method for use in Clean Water Act (CWA) programs and for wastewater,
surface water, soil, sediment, biosolids and tissue matrices. Other applications and matrices may be
possible, which may or may not require modifications of sample preparation, chromatographic
conditions, etc. Method 1668C is a revision of previous versions of Method 1668 all of which are
based on a compilation of methods from the technical literature (References 3 and 4), and EPA's
dioxins and furans Method, Method 1613.
1.3 The detection limits and quantitation levels in this Method are usually dependent on the level of
interferences and laboratory background levels rather than instrumental limitations. The method
detection limits (MDLs; 40 CFR 136, appendix B) and minimum levels of quantitation (MLs; 68
FR 11790) in Table 2 are the levels at which the CBs can be determined with no interferences
present. The MDL for CB 126 in water is 16 pg/L (picograms-per-liter; parts-per-quadrillion).
EPA Method 1668C 1 April 2010
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1.4 The GC/MS portions of this Method are for use only by analysts experienced with HRGC/HRMS
or under the close supervision of such qualified persons. Each laboratory that uses this Method
must demonstrate the ability to generate acceptable results using the procedure in Section 9.2.
1.5 This Method is "performance-based," which means that you may make modifications without
additional EPA review to improve performance (e.g., overcome interferences, or improve the
sensitivity, accuracy or precision of the results) provided that you meet all performance criteria in
this Method. Requirements for establishing equivalency are in Section 9.1.2, and include 9.1.2.2.3
- explaining the reason for your modifications. For CWA uses, additional flexibility is described at
40 CFR 136.6. You must document changes in performance, sensitivity, selectivity, precision,
recovery, etc., that result from modifications within the scope of Part 136.6, and Section 9 of this
Method, and how these modifications compare to the specifications in this Method. Changes
outside the scope of Part 136.6 and Section 9 of this Method may require prior review or approval.
2.0 Summary of Method
Flow charts summarize procedures for sample preparation, extraction, and analysis for aqueous and
solid samples, multi-phase samples, and tissue samples (Figures 1, 2 and 3, respectively.)
2.1 Extraction
2.1.1 Aqueous samples (samples containing less than one percent solids) - Stable isotopically
labeled analogs of the Toxics and labeled LOC CBs are spiked into a 1-L sample. The
sample is extracted using solid-phase extraction (SPE), separatory funnel extraction (SFE),
or continuous liquid/liquid extraction (CLLE).
2.1.2 Solid, semi-solid, and multi-phase samples (excluding tissue) - The labeled compounds are
spiked into a sample containing 10 g (dry weight) of solids. Samples containing multiple
phases are pressure filtered and any aqueous liquid is discarded. Coarse solids are ground
or homogenized. Any non-aqueous liquid from multi-phase samples is combined with the
solids and extracted in a Soxhlet/Dean-Stark (SDS) extractor. The extract is concentrated
for cleanup.
2.1.3 Fish and other tissue - A 20-g aliquot of sample is homogenized, and a 10-g aliquot is
spiked with the labeled compounds. The sample is mixed with anhydrous sodium sulfate,
allowed to dry for 12 - 24 hours, and extracted for 18-24 hours using methylene chloride in
a Soxhlet extractor. The extract is evaporated to dryness, and the lipid content is
determined.
2.2 After extraction, a labeled cleanup standard is spiked into the extract which is then cleaned up using
back-extraction with sulfuric acid and/or base, and gel permeation, silica gel, or Florisil
chromatography. Activated carbon and high-performance liquid chromatography (HPLC) can be
used for further isolation of specific congener groups. Prior to the cleanup procedures cited above,
tissue extracts are cleaned up using an anthropogenic isolation column.
2.3 After cleanup, the extract is concentrated to 20 (iL. Immediately prior to injection, labeled injection
internal standards are added to each extract and an aliquot of the extract is injected into the gas
chromatograph (GC). The analytes are separated by the GC and detected by a high-resolution
(> 10,000) mass spectrometer. Two exact m/z's are monitored at each level of chlorination (LOC)
throughout a pre-determined retention time window.
EPA Method 1668C 2 April 2010
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2.4 An individual CB congener is identified by comparing the GC retention time and ion-abundance
ratio of two exact m/z's with the corresponding retention time of an authentic standard and the
theoretical or acquired ion-abundance ratio of the two exact m/z's. Isomer specificity for certain of
the CB congeners is achieved using GC columns that resolve these congeners.
2.5 Quantitative analysis is performed in one of two ways using selected ion current profile (SICP)
areas:
2.5.1 For the Toxics and the LOG CBs, the GC/MS is multi-point calibrated and the
concentration is determined using the isotope dilution technique.
2.5.2 For all congeners other than the Toxics and LOC CBs, the GC/MS is calibrated at a single
concentration and the concentrations are determined using the internal standard technique.
The internal standards are the labeled congeners spiked into the sample, thus affording
recovery correction for all congeners.
2.5.3 For the labeled Toxics, labeled LOC CBs, and the cleanup standards, the GC/MS is
calibrated using replicates at a single concentration and the concentrations of these labeled
compounds are determined using the internal standard technique. The labeled injection
internal standards are determined using the internal standard technique.
2.6 The quality of the analysis is assured through reproducible calibration and testing of the extraction,
cleanup, and HRGC/HRMS systems.
3.0 Definitions
Definitions are in the glossary at the end of this Method.
4.0 Contamination and interferences
4.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts, elevated
baselines, and/or lock-mass suppression causing misinterpretation of chromatograms. Specific
selection of reagents and purification of solvents by distillation in all-glass systems may be
required. Where possible, reagents are cleaned by extraction or solvent rinse. Environmentally
abundant CBs have been shown to be very difficult to completely eliminate from the laboratory at
levels lower than the MDLs in this Method (Table 2), and baking of glassware in a kiln or furnace
at 450 - 500 °C may be necessary to remove these and other contaminants.
4.2 Proper cleaning of glassware is extremely important, because glassware may not only contaminate
the samples but may also remove the analytes of interest by adsorption on the glass surface.
4.2.1 Glassware should be rinsed with solvent and washed with a detergent solution as soon after
use as is practical. Sonication of glassware containing a detergent solution for
approximately 30 seconds may aid in cleaning. Glassware with removable parts,
particularly separatory funnels with fluoropolymer stopcocks, must be disassembled prior
to detergent washing.
4.2.2 After detergent washing, glassware should be rinsed immediately, first with methanol, then
with hot tap water. The tap water rinse is followed by another methanol rinse, then
acetone, and then methylene chloride.
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4.2.3 Baking of glassware in a kiln or other high temperature furnace (300 - 500 °C) may be
warranted after particularly dirty samples are encountered. The kiln or furnace should be
vented to prevent laboratory contamination by CB vapors. Baking should be minimized, as
repeated baking of glassware may cause active sites on the glass surface that may
irreversibly adsorb CBs.
4.2.4 Immediately prior to use, the Soxhlet apparatus should be pre-extracted with toluene for
approximately 3 hours (see Sections 12.3.1-12.3.3). The extraction apparatus (Section 6.4)
should be rinsed with methylene chloride/toluene (80/20 mixture).
4.2.5 A separate set of glassware may to necessary to effectively preclude contamination when
low-level samples are analyzed.
4.2.6 Concentration of extracts by Kuderna-Danish (K-D) concentrator and/or final concentration
using nitrogen evaporation may help reduce levels of background PCBs in samples.
4.3 All materials used in the analysis must be demonstrated to be free from interferences by running
reference matrix Method blanks (Section 9.5) initially and with each sample batch (samples started
through the extraction process on a given 12-hour shift, to a maximum of 20 samples).
4.3.1 The reference matrix must simulate, as closely as possible, the sample matrix under test.
Ideally, the reference matrix should not contain the CBs in detectable amounts, but should
contain potential interferents in the concentrations expected to be found in the samples to
be analyzed.
4.3.2 When a reference matrix that simulates the sample matrix under test is not available,
reagent water (Section 7.6.1) can be used to simulate water samples; playground sand
(Section 7.6.2) or white quartz sand (Section 7.3.2) can be used to simulate soils; filter
paper (Section 7.6.3) can be used to simulate papers and similar materials; and corn oil
(Section 7.6.4) can be used to simulate tissues.
4.4 Interferences co-extracted from samples will vary considerably from source to source, depending
on the diversity of the site being sampled. Interfering compounds may be present at concentrations
several orders of magnitude higher than the CBs. The most frequently encountered interferences
are chlorinated dioxins and dibenzofurans, methoxy biphenyls, hydroxydiphenyl ethers,
benzylphenyl ethers, brominated diphenyl ethers, polynuclear aromatics, polychlorinated
naphthalenes, and pesticides. Because very low levels of CBs are measured by this Method,
elimination of interferences is essential. The cleanup steps given in Section 13 can be used to
reduce or eliminate these interferences and thereby permit reliable determination of the CBs at the
levels shown in Table 2.
4.5 Each piece of reusable glassware should be numbered to associate that glassware with the
processing of a particular sample. This will assist the laboratory in tracking possible sources of
contamination for individual samples, identifying glassware associated with highly contaminated
samples that may require extra cleaning, and determining when glassware should be discarded.
4.6 Contamination of calibration solutions - The MDLs and MLs in Table 2 are the levels that can be
achieved in the absence of laboratory backgrounds. Many of the MLs are greater than the
equivalent concentrations of the calibration solutions. To prevent contamination, calibration
solutions must be prepared in an area free from CB contamination using glassware free from
contamination. If these requirements cannot be met or are difficult to meet in the laboratory, the
EPA Method 1668C 4 April 2010
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laboratory should prepare the calibration solutions in a contamination-free facility or have a vendor
prepare the calibration standards and guarantee freedom from contamination.
4.7 Cleanup of tissue - The natural lipid content of tissue can interfere in the analysis of tissue samples
for the CBs. The lipid contents of different species and portions of tissue can vary widely. Lipids
are soluble to varying degrees in various organic solvents and may be present in sufficient quantity
to overwhelm the column chromatographic cleanup procedures used for cleanup of sample extracts.
Lipids must be removed by the anthropogenic isolation column procedure in Section 13.6, followed
by the gel permeation chromatography procedure in Section 13.2. Florisil (Section 13.7) is
recommended as an additional cleanup step.
4.8 If the laboratory air is a potential source of CB contamination, samples, reagents, glassware, and
other materials should be dried in a glove box or other area free from contamination.
5.0 Safety
5.1 The toxicity or carcinogenicity of each chemical used in this Method has not been precisely
determined; however, each compound should be treated as a potential health hazard. Exposure to
these compounds should be reduced to the lowest possible level.
5.1.1 PCBs have been tentatively classified as known or suspected human or mammalian
carcinogens. On the basis of the available toxicological and physical properties of the CBs,
pure standards should be handled only by highly trained personnel thoroughly familiar with
handling and cautionary procedures and the associated risks.
5.1.2 It is recommended that the laboratory purchase dilute standard solutions of the analytes in
this Method. However, if primary solutions are prepared, they must be prepared in a hood,
and a NIOSH/MESA approved toxic gas respirator must be worn when high concentrations
are handled.
5.2 The laboratory is responsible for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this Method. A reference file of material
safety data sheets (MSDSs) should also be made available to all personnel involved in these
analyses. It is also suggested that the laboratory perform personal hygiene monitoring of each
analyst who uses this Method and that the results of this monitoring be made available to the
analyst. Additional information on laboratory safety can be found in References 5-8. The
references and bibliography at the end of Reference 7 are particularly comprehensive in dealing
with the general subject of laboratory safety.
5.3 The pure CBs and samples suspected to contain these compounds are handled using essentially the
same techniques employed in handling radioactive or infectious materials. Well-ventilated,
controlled access laboratories are required. Assistance in evaluating the health hazards of particular
laboratory conditions may be obtained from certain consulting laboratories and from State
Departments of Health or Labor, many of which have an industrial health service. Each laboratory
must develop a strict safety program for handling these compounds. The practices in Reference 9
for handling chlorinated dibenzo-/?-dioxins and dibenzofurans (CDDs/CDFs) are also recommended
for handling the CBs.
5.3.1 Facility - When finely divided samples (dusts, soils, dry chemicals) are handled, all
operations (including removal of samples from sample containers, weighing, transferring,
and mixing) should be performed in a glove box demonstrated to be leak tight or in a fume
EPA Method 1668C 5 April 2010
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hood demonstrated to have adequate air flow. Gross losses to the laboratory ventilation
system must not be allowed. Handling of the dilute solutions normally used in analytical
and animal work presents no inhalation hazards except in the case of an accident.
5.3.2 Protective equipment - Disposable plastic gloves, apron or lab coat, safety glasses or mask,
and a glove box or fume hood adequate for radioactive work should be used. During
analytical operations that may give rise to aerosols or dusts, personnel should wear
respirators equipped with activated carbon filters. Eye protection (preferably full face
shields) must be worn while working with exposed samples or pure analytical standards.
Latex gloves are commonly used to reduce exposure of the hands. When handling samples
suspected or known to contain high concentrations of the CBs, an additional set of gloves
can also be worn beneath the latex gloves.
5.3.3 Training - Workers must be trained in the proper method of removing contaminated gloves
and clothing without contacting the exterior surfaces.
5.3.4 Personal hygiene - Hands and forearms should be washed thoroughly after each
manipulation and before breaks (coffee, lunch, and shift).
5.3.5 Confinement - Isolated work areas posted with signs, segregated glassware and tools, and
plastic absorbent paper on bench tops will aid in confining contamination.
5.3.6 Effluent vapors - The effluent of the sample splitter from the gas chromatograph (GC) and
from roughing pumps on the mass spectrometer (MS) should pass through either a column
of activated charcoal or be bubbled through a trap containing oil or high-boiling alcohols to
condense CB vapors.
5.3.7 Waste Handling - Good technique includes minimizing contaminated waste. Plastic bag
liners should be used in waste cans. Janitors and other personnel should be trained in the
safe handling of waste.
5.3.8 Decontamination
5.3.8.1 Decontamination of personnel - Use any mild soap with plenty of scrubbing
action.
5.3.8.2 Glassware, tools, and surfaces - Chlorothene NU Solvent is a less toxic solvent
that should be effective in removing CBs. Satisfactory cleaning may be
accomplished by rinsing with Chlorothene, then washing with any detergent and
water. If glassware is first rinsed with solvent, the wash water may be disposed
of in the sewer. Given the cost of disposal, it is prudent to minimize solvent
wastes.
5.3.9 Laundry - Clothing known to be contaminated should be collected in plastic bags. Persons
that convey the bags and launder the clothing should be advised of the hazard and trained in
proper handling. The clothing may be put into a washer without contact if the launderer
knows of the potential problem. The washer should be run through a cycle before being
used again for other clothing.
5.3.10 Wipe tests - A useful method of determining cleanliness of work surfaces and tools is to
perform a wipe test of the surface suspected of being contaminated.
EPA Method 1668C 6 April 2010
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5.3.10.1 Using a piece of filter paper moistened with Chlorothene or other solvent, wipe
an area approximately 10x10 cm.
5.3.10.2 Extract and analyze the wipe by GC with an electron capture detector (BCD) or
by this Method.
5.3.10.3 Using the area wiped (e.g., 10 x 10 cm = 0.01 m2), calculate the concentration in
(ig/m2. A concentration less than 1 (ig/m2 indicates acceptable cleanliness;
anything higher warrants further cleaning. More than 100 (ig/m2 constitutes an
acute hazard and requires prompt cleaning before further use of the equipment
or work space, and indicates that unacceptable work practices have been
employed.
5.4 Biosolids samples may contain high concentrations of biohazards, and must be handled with gloves
and opened in a hood or biological safety cabinet to prevent exposure. Laboratory staff should
know and observe the safety procedures required in a microbiology laboratory that handles
pathogenic organisms when handling biosolids samples.
6.0 Apparatus and materials
Note: Brand names, suppliers, and part numbers are for illustration purposes only and no endorsement
is implied. Equivalent performance may be achieved using apparatus and materials other than those
specified here. Meeting the performance requirements of this Method is the responsibility of the
laboratory.
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 containing 5 percent
solids or less) - Sample bottle, amber glass, 1.1-L minimum, with screw cap.
6.1.1.2 Solid samples (soils, sediments, sludges, paper pulps, filter cake, compost, and
similar materials that contain more than 5 percent solids) - Sample bottle, wide
mouth, amber glass, 500-mL minimum.
6.1.1.3 If amber bottles are not available, samples must be protected from light.
6.1.1.4 Bottle caps - Threaded to fit sample bottles. Caps must be lined with
fluoropolymer.
6.1.1.5 Cleaning
6.1.1.5.1 Bottles are detergent water washed, then solvent rinsed before use.
6.1.1.5.2 Liners are detergent water washed and rinsed with reagent water
(Section 7.6.1).
6.1.2 Compositing equipment - Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Only glass or fluoropolymer tub-
EPA Method 1668C 7 April 2010
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ing must be used. If the sampler uses a peristaltic pump, a minimum length of
compressible silicone rubber tubing may be used in the pump only. Before use, the tubing
must be thoroughly rinsed with methanol, followed by repeated rinsing with reagent water
to minimize sample contamination. An integrating flow meter is used to collect
proportional composite samples.
6.2 Equipment for glassware cleaning
Note: If blanks from bottles or other glassware or with fewer cleaning steps than required above show
no detectable CB contamination, unnecessary cleaning steps and equipment may be eliminated.
6.2.1 Laboratory sink with overhead fume hood
6.2.2 Kiln - Capable of reaching 450 °C within 2 hours and maintaining 450 - 500 °C within ±10
°C, with temperature controller and safety switch (Cress Manufacturing Co., Santa Fe
Springs, CA, B31H, X3 ITS, or equivalent). See the precautions in Section 4.2.3.
6.3 Equipment for sample preparation
6.3.1 Laboratory fume hood of sufficient size to contain the sample preparation equipment listed
below.
6.3.2 Glove box (optional)
6.3.3 Tissue homogenizer - VirTis Model 45 Macro homogenizer (American Scientific Products
H-3515, or equivalent) with stainless steel Macro-shaft and Turbo-shear blade.
6.3.4 Meat grinder - Hobart, or equivalent, with 3- to 5-mm holes in inner plate.
6.3.5 Equipment for determining percent moisture
6.3.5.1 Oven - Capable of maintaining a temperature of 110 ±5 °C
6.3.5.2 Desiccator
6.3.6 Balances
6.3.6.1 Analytical - Capable of weighing 0.1 mg
6.3.6.2 Top loading - Capable of weighing 10 mg
6.4 Extraction apparatus
6.4.1 Water samples
6.4.1.1 pH meter, with combination glass electrode
6.4.1.2 pH paper, wide range (Hydrion Papers, or equivalent)
6.4.1.3 Graduated cylinder, 1-L capacity
EPA Method 1668C 8 April 2010
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6.4.1.4 Liquid/liquid extraction - Separatory funnels, 250-, 500-, and 2000-mL, with
fluoropolymer stopcocks
6.4.1.5 Solid-phase extraction
6.4.1.5.1 1-L filtration apparatus, including glass funnel, frit support, clamp,
adapter, stopper, filtration flask, and vacuum tubing (Figure 4).
For wastewater samples, the apparatus should accept 90 or 144
mm disks. For drinking water or other samples containing low
solids, smaller disks may be used.
6.4.1.5.2 Vacuum source - Capable of maintaining 25 in. Hg, equipped with
shutoff valve and vacuum gauge
6.4.1.5.3 Glass-fiber filter - Whatman GMF 150 (or equivalent), 1 micron
pore size, to fit filtration apparatus in Section 6.4.1.5.1
6.4.1.5.4 Solid-phase extraction disk containing octadecyl (Ci8) bonded
silica uniformly enmeshed in an inert matrix - Fisher Scientific 14-
378F (or equivalent), to fit filtration apparatus in Section 6.4.1.5.1
6.4.1.6 Continuous liquid/liquid extraction (CLLE) - Fluoropolymer or glass
connecting joints and stopcocks without lubrication, 1.5-2 L capacity
(Hershberg-Wolf Extractor, Cal-Glass, Costa Mesa, California, 1000 mL or
2000 mL, or equivalent).
6.4.2 Soxhlet/Dean-Stark (SDS) extractor (Figure 5 and Reference 10) for filters and solid/sludge
samples
6.4.2.1 Soxhlet - 50-mm ID, 200-mL capacity with 500-mL flask (Cal-Glass LG-6900,
or equivalent, except substitute 500-mL round-bottom flask for 300-mL flat-
bottom flask)
6.4.2.2 Thimble - 43 x 123 to fit Soxhlet (Cal-Glass LG-6901-122, or equivalent)
6.4.2.3 Moisture trap - Dean Stark or Barret with fluoropolymer stopcock, to fit Soxhlet
6.4.2.4 Heating mantle - Hemispherical, to fit 500-mL round-bottom flask (Cal-Glass
LG-8801-112, or equivalent)
6.4.2.5 Variable transformer - Powerstat (or equivalent), 110-volt, 10-amp
6.4.3 Beakers - 400- to 500-mL
6.4.4 Spatulas - Stainless steel
6.5 Filtration apparatus
6.5.1 Pyrex glass wool - Solvent-extracted using a Soxhlet or SDS extractor for 3 hours
minimum
6.5.2 Glass funnel - 125- to 250-mL
EPA Method 1668C 9 April 2010
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6.5.3 Glass-fiber filter paper - Whatman GF/D (or equivalent), to fit glass runnel in Section
6.5.2.
6.5.4 Drying column - 15- to 20-mm ID Pyrex chromatographic column equipped with coarse-
glass frit or glass-wool plug
6.5.5 Buchner funnel - 15-cm
6.5.6 Glass-fiber filter paper for Buchner funnel above
6.5.7 Filtration flasks - 1.5- to 2.0-L, with side arm
6.5.8 Pressure filtration apparatus - Millipore YT30 142 FIW, or equivalent
6.6 Centrifuge apparatus
6.6.1 Centrifuge - Capable of rotating 500-mL centrifuge bottles or 15-mL centrifuge tubes at
5,000 rpm minimum
6.6.2 Centrifuge bottles - 500-mL, with screw-caps, to fit centrifuge
6.6.3 Centrifuge tubes - 12- to 15-mL, with screw-caps, to fit centrifuge
6.7 Cleanup apparatus
6.7.1 Automated gel permeation chromatograph (Analytical Biochemical Labs, Inc, Columbia,
MO, Model GPC Autoprep 1002, or equivalent)
6.7.1.1 Column - 600-700 mm long x 25 mm ID glass, packed with 70 g of 200-400
mesh SX-3 Bio-beads (Bio-Rad Laboratories, Richmond, CA, or equivalent)
6.7.1.2 Syringe - 10-mL, with Luer fitting
6.7.1.3 Syringe filter holder - stainless steel, and glass-fiber or fluoropolymer filters
(Gelman 4310, or equivalent)
6.7.1.4 UV detectors - 254-nm, preparative or semi-preparative flow cell (Isco, Inc.,
Type 6; Schmadzu, 5-mm path length; Beckman-Altex 152W, 8-(iL micro-prep
flow cell, 2-mm path; Pharmacia UV-1, 3-mm flow cell; LDC Milton-Roy UV-
3, monitor #1203; or equivalent).
6.7.2 Reverse-phase high-performance liquid chromatograph (Reference 4)
6.7.2.1 Pump - Perkin-Elmer Series 410, or equivalent
6.7.2.2 Injector - Perkin-Elmer ISS-100 Autosampler, or equivalent
6.7.2.3 6-Port switching valve - Valco N60, or equivalent
6.7.2.4 Column - Hypercarb, 100 x 4.6 mm, 5 (im particle size, Keystone Scientific, or
equivalent
EPA Method 1668C 10 April 2010
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6.7.2.5 Detector - Altex 110A (or equivalent) operated at 0.02 AUFS at 235 nm
6.7.2.6 Fraction collector - Isco Foxy II, or equivalent
6.7.3 Pipets
6.7.3.1 Disposable, Pasteur, 150-mm long x 5-mm ID (Fisher Scientific 13-678-6A, or
equivalent)
6.7.3.2 Disposable, serological, 50-mL (8- to 10- mm ID)
6.7.4 Glass chromatographic columns
6.7.4.1 150-mm long x 8-mm ID, (Kontes K-420155, or equivalent) with coarse-glass
frit or glass-wool plug and 250-mL reservoir
6.7.4.2 200-mm long x 15-mm ID, with coarse-glass frit or glass-wool plug and 250-
mL reservoir
6.7.4.3 300-mm long x 22-mm ID, with coarse-glass frit, 300-mL reservoir, and glass or
fluoropolymer stopcock
6.7.5 Oven - For baking and storage of adsorbents, capable of maintaining a constant
temperature ( ± 5 °C) in the range of 105-250 °C
6.8 Concentration apparatus
6.8.1 Rotary evaporator - Buchi/Brinkman-American Scientific No. E5045-10 or equivalent,
equipped with a variable temperature water bath
6.8.1.1 Vacuum source for rotary evaporator equipped with shutoff valve at the
evaporator and vacuum gauge
6.8.1.2 A recirculating water pump and chiller are recommended, as use of tap water for
cooling the evaporator wastes large volumes of water and can lead to
inconsistent performance as water temperatures and pressures vary.
6.8.1.3 Round-bottom flask - 100-mL and 500-mL or larger, with ground-glass fitting
compatible with the rotary evaporator
6.8.2 Kuderna-Danish (K-D) concentrator
6.8.2.1 Concentrator tube - 10-mL, graduated (Kontes K-570050-1025, or equivalent)
with calibration verified. Ground-glass stopper (size 19/22 joint) is used to
prevent evaporation of extracts.
6.8.2.2 Evaporation flask - 500-mL (Kontes K-570001-0500, or equivalent), attached to
concentrator tube with springs (Kontes K-662750-0012 or equivalent)
6.8.2.3 Snyder column - Three-ball macro (Kontes K-503000-0232, or equivalent)
EPA Method 1668C 11 April 2010
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6.8.2.4 Boiling chips
6.8.2.4.1 Glass or silicon carbide - Approximately 10/40 mesh, extracted
with methylene chloride and baked at 450 °C for one hour
minimum
6.8.2.4.2 Fluoropolymer (optional) - Extracted with methylene chloride
6.8.2.5 Water bath - Heated, with concentric ring cover, capable of maintaining a
temperature within ± 2 °C, installed in a fume hood
6.8.3 Nitrogen evaporation apparatus - Equipped with water bath controlled in the range of 30 -
60 °C (N-Evap, Organomation Associates, Inc., South Berlin, MA, or equivalent), installed
in a fume hood
6.8.4 Sample vials
6.8.4.1 Amber glass, 2- to 5-mL with fluoropolymer-lined screw-cap
6.8.4.2 Glass, 0.3-mL, conical, with fluoropolymer-lined screw or crimp cap
6.9 Gas chromatograph - Must have splitless or on-column injection port for capillary column,
temperature program with isothermal hold, and must meet all of the performance specifications in
Section 10.
6.9.1 GC column - Any GC column or column system (2 or more columns) that provides unique
resolution and identification of the Toxics for determination of a TEQPCB using TEFs
(Reference 1). Isomers may be unresolved so long as they have the same TEF and response
factor and so long as these unresolved isomers are uniquely resolved from all other
congeners. For example, the SPB-octyl column (Section 6.9.1.3) achieves unique GC
resolution of all Toxics except congeners with congener numbers 156 and 157. This
isomeric pair is uniquely resolved from all other congeners and these congeners have the
same TEF and response factor.
6.9.1.1 If an SPB-octyl column is used, it must meet the specification in Section 6.9.1
and the following additional specifications:
6.9.1.1.1 The retention time for decachlorobiphenyl (DeCB; PCB 209) must
be greater than 55 minutes.
6.9.1.1.2 The column must uniquely resolve congeners 34 from 23 and 187
from 182, and congeners 156 and 157 must co-elute within 2
seconds at the peak maximum. Unique resolution means a valley
height less than 40 percent of the shorter of the two peaks that
result when the Diluted combined 209 congener solution (Section
7.10.2.2) is analyzed (see Figures 6 and 7).
6.9.1.1.3 The column must be replaced when any of the criteria in Sections
6.9.1 -6.9.1.1.2 are not met.
6.9.1.2 If a column or column system alternate to the SPB-octyl column is used,
specifications similar to those for the SPB-octyl column (Sections 6.9.1 -
EPA Method 1668C 12 April 2010
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6.9.1.1.2) must be developed and be functionally equivalent to those
specifications.
6.9.1.3 Suggested column - 30 ± 5-m long x 0.25 ± 0.02-mm ID; 0.25-(im film SPB-
octyl (Supelco 2-4218, or equivalent). This column is capable of meeting the
requirements in Sections 6.9.1 - 6.9.1.1.2.
Note: The SPB-octyl column is subject to rapid degradation when exposed to oxygen. The analyst
should exclude oxygen from the carrier gas, should eliminate air leaks, and should cool the injector,
column, and transfer line before opening the column to the atmosphere. For further information on
precluding oxidation, contact the column manufacturer.
6.9.1.4 Column for resolution of additional congeners - See Appendix A for details on
the DB-1 column. The DB-1 column is optional and is capable of uniquely
resolving the congener pair with congener numbers 156 and 157. When used in
combination with the SPB-octyl column (Section 6.9.1.3), the two-column
system is capable of resolving a total of approximately 180 CB congeners.
6.10 Mass spectrometer - 28- to 40-eV electron impact ionization, must be capable of selectively
monitoring a minimum of 22 exact m/z's minimum at high resolution (> 10,000) during a period
less than 1.5 seconds, and must meet all of the performance specifications in Section 10.
6.11 GC/MS interface - The mass spectrometer (MS) must 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.
6.12 Data system - Capable of collecting, recording, storing, and processing MS data
6.12.1 Data acquisition - The signal at each exact m/z must be collected repetitively throughout
the monitoring period and stored on a mass storage device.
6.12.2 Response factors and multipoint calibrations - The data system must record and maintain
lists of response factors (response ratios for isotope dilution) and multipoint calibrations.
Computations of relative standard deviation (RSD) are be used to test calibration linearity.
Statistics on initial (Section 9.4) and ongoing (Section 15.5.4) performance should be
computed and maintained, either on the instrument data system, or on a separate computer
system.
7.0 Reagents and standards
7.1 pH adjustment and back-extraction
7.1.1 Potassium hydroxide - Dissolve 20 g reagent grade KOH in 100 mL reagent water.
7.1.2 Sulfuric acid - Reagent grade (specific gravity 1.84)
7.1.3 Hydrochloric acid - Reagent grade, 6N
7.1.4 Sodium chloride - Reagent grade, prepare at 5% (w/v) solution in reagent water
EPA Method 1668C 13 April 2010
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7.2 Solution drying and evaporation
7.2.1 Solution drying - Sodium sulfate, reagent grade, granular, anhydrous (Baker 3375, or
equivalent), rinsed with methylene chloride (20 mL/g), baked at 400 °C for 1 hour
minimum, cooled in a desiccator, and stored in a pre-cleaned glass bottle with screw-cap
that prevents moisture from entering. If, after heating, the sodium sulfate develops a
noticeable grayish cast (due to the presence of carbon in the crystal matrix), that batch of
reagent is not suitable for use and should be discarded. Extraction with methylene chloride
(as opposed to simple rinsing) and baking at a lower temperature may produce sodium
sulfate that is suitable for use.
7.2.2 Tissue drying - Sodium sulfate, reagent grade, powdered, treated and stored as in Section
7.2.1
7.2.3 Prepurified nitrogen
7.3 Extraction
7.3.1 Solvents - Acetone, toluene, cyclohexane, hexane, methanol, methylene chloride,
isooctane, and nonane; distilled in glass, pesticide quality, lot-certified to be free of
interferences
Note: Some solvents; e.g., isooctane and nonane, may need to be re-distilled to eliminate CB
backgrounds.
7.3.2 White quartz sand, 60/70 mesh - For Soxhlet/Dean-Stark extraction (Aldrich Chemical,
Cat. No. 27-437-9, or equivalent). Bake at 450 °C for 4 hour minimum.
7.4 GPC calibration solution - Prepare a solution containing 2.5 mg/mL corn oil, 0.05 mg/mL bis(2-
ethylhexyl) phthalate (BEHP), 0.01 mg/mL methoxychlor, 0.002 mg/mL perylene, and 0.008
mg/mL sulfur, or at concentrations appropriate to the response of the detector.
7.5 Adsorbents for sample cleanup
7.5.1 Silica gel
7.5.1.1 Activated silica gel - 100-200 mesh, Supelco 1-3651 (or equivalent), 100-200
mesh, rinsed with methylene chloride, baked at 180 °C for a minimum of 1 hour,
cooled in a desiccator, and stored in a precleaned glass bottle with screw-cap
that prevents moisture from entering.
7.5.1.2 Acid silica gel (30% w/w) - Thoroughly mix 44 g of concentrated sulfuric acid
with 100 g of activated silica gel in a clean container. Break up aggregates with
a stirring rod until a uniform mixture is obtained. Store in a screw-capped bottle
with fluoropolymer-lined cap.
7.5.1.3 Basic silica gel - Thoroughly mix 30 g of IN sodium hydroxide with 100 g of
activated silica gel in a clean container. Break up aggregates with a stirring rod
until a uniform mixture is obtained. Store in a screw-capped bottle with
fluoropolymer-lined cap.
EPA Method 1668C 14 April 2010
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7.5.1.4 Potassium silicate
7.5.1.4.1 Dissolve 56 g of high purity potassium hydroxide (Aldrich, or
equivalent) in 300 mL of methanol in a 750- to 1000-mL flat-
bottom flask.
7.5.1.4.2 Add 100 g of activated silica gel (Section 7.5.1.1) and a stirring
bar, and stir on an explosion-proof hot plate at 60-70 °C for 1-2
hours.
7.5.1.4.3 Decant the liquid and rinse the potassium silicate twice with 100-
mL portions of methanol, followed by a single rinse with 100 mL
of methylene chloride.
7.5.1.4.4 Spread the potassium silicate on solvent-rinsed aluminum foil and
dry for 2-4 hours in a hood. Observe the precaution in Section 4.8.
7.5.1.4.5 Activate overnight at 200-250 °C prior to use.
7.5.2 Carbon
7.5.2.1 Carbopak C - (Supelco 1-0258, or equivalent)
7.5.2.2 Celite 545 - (Supelco 2-0199, or equivalent)
7.5.2.3 Thoroughly mix 18.0 g Carbopak C and 18.0 g Celite 545 to produce a 50%
w/w mixture. Activate the mixture at 130 °C for a minimum of 6 hours. Store
in a desiccator.
Note: The carbon column has been included in this Method to allow separation of co-planar congeners
77, 126, and 169 from other congeners and interferences, should such separation be desired.
7.5.3 Anthropogenic isolation column - Pack the column in Section 6.7.4.3 from bottom to top
with the following:
7.5.3.1 2 g silica gel (Section 7.5.1.1)
7.5.3.2 2 g potassium silicate (Section 7.5.1.4)
7.5.3.3 2 g granular anhydrous sodium sulfate (Section 7.2.1)
7.5.3.4 10 g acid silica gel (Section 7.5.1.2)
7.5.3.5 2 g granular anhydrous sodium sulfate
7.5.4 Florisil column
7.5.4.1 Florisil - PR grade, 60-100 mesh (U.S. Silica Corp, Berkeley Springs, WV, or
equivalent). Alternatively, prepacked Florisil columns may be used. Use the
following procedure for Florisil activation and column packing.
EPA Method 1668C 15 April 2010
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7.5.4.1.1 Fill a clean 1- to 2-L bottle !/2 to 2/3 full with Florisil and place in
an oven at 130-150 °C for a minimum of three days to activate the
Florisil.
7.5.4.1.2 Immediately prior to use, dry pack a 300-mm x 22-mm ID glass
column (Section 6.7.4.3) bottom to top with 0.5-1.0 cm of warm
to hot anhydrous sodium sulfate (Section 7.2.1), 10-10.5 cm of
warm to hot activated Florisil (Section 7.5.4.1.1), and 1-2 cm of
warm to hot anhydrous sodium sulfate. Allow the column to cool
and wet immediately with 100 mL of n-hexane to prevent water
from entering.
7.5.4.2 Using the procedure in Section 13.7.3, establish the elution pattern for each
carton of Florisil or each lot of Florisil columns received.
7.6 Reference matrices - Matrices in which the CBs and interfering compounds are not detected by this
Method
7.6.1 Reagent water - Bottled water purchased locally, or prepared by passage through activated
carbon
7.6.2 High-solids reference matrix - Playground sand or similar material. Prepared by extraction
with methylene chloride and/or baking at 450 °C for a minimum of 4 hours.
7.6.3 Paper reference matrix - Glass-fiber filter, Gelman type A, or equivalent. Cut paper to
simulate the surface area of the paper sample being tested.
7.6.4 Tissue reference matrix - Corn or other vegetable oil.
7.6.5 Other matrices - This Method may be verified on any reference matrix by performing the
tests in Section 9.2. Ideally, the matrix should be free of the CBs, but in no case must the
background level of the CBs in the reference matrix exceed the minimum levels in Table 2.
If low background levels of the CBs are present in the reference matrix, the spike level of
the analytes used in Section 9.2 should be increased to provide a spike-to-background ratio
of approximately 5 (Reference 11).
7.7 Standard solutions - Prepare from materials of known purity and composition or purchase as solu-
tions or mixtures with certification to their purity, concentration, and authenticity. If the chemical
purity is 98 % or greater, the weight may be used without correction to calculate the concentration
of the standard. Observe the safety precautions in Section 5 and the recommendation in Section
5.1.2.
Note: Native PCB standards are available from several suppliers. 13C12-labeled congeners are
available from Cambridge Isotope Laboratories and Wellington Laboratories, and may be available from
other suppliers. Listing of these suppliers does not constitute a recommendation or endorsement for use.
Part numbers are for reference only.
7.7.1 For preparation of stock solutions from neat materials, dissolve an appropriate amount of
assayed reference material in solvent. For example, weigh 10 to 20 mg of PCB 126 to
three significant figures in a 10-mL ground-glass-stoppered volumetric flask and fill to the
mark with nonane. After the compound is completely dissolved, transfer the solution to a
clean 15-mL vial with fluoropolymer-lined cap.
EPA Method 1668C 16 April 2010
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7.7.2 When not being used, store standard solutions in the dark at room temperature in screw-
capped vials with fluoropolymer-lined caps. Place a mark on the vial at the level of the
solution so that solvent loss by evaporation can be detected. Replace the solution if solvent
loss has occurred.
7.8 Native (unlabeled) stock solutions
Note: Some of the part numbers for solutions described below contain the identifier "1668A. " These
part numbers remain valid for Method 1668C.
7.8.1 Native Toxics/LOC stock solution - Prepare to contain the native Toxics and LOG CBs at
the concentrations shown in Table 3, or purchase Accu-Standard M1668A-C-NT-LOC-
WD-GCPC, or equivalent. If additional CBs are to be determined by isotope dilution (e.g.,
170 and 180), include the additional native compounds in this stock solution.
7.8.2 Native 209 CB congener stock solutions - Solutions containing CB congeners to calibrate
the SPB-octyl column.
Note: If a column other than the SPB-octyl column is used, solutions that will allow separation of all
209 congeners on that column must be prepared.
7.8.2.1 Native congener mix stock solutions for separation of individual congeners on
the SPB-octyl column - Prepare the five solutions with the congeners listed in
Table 4 at the concentrations shown in Table 3 or purchase Accu-Standard M-
1668A-1, M-1668A-2, M-1668A-3, M-1668-4, and M-1668-5, or equivalent.
7.8.2.2 Combined 209 congener stock solution - Combine equal volumes of the
standards in Section 7.8.2.1 to form a stock solution containing all CB
congeners. This solution will be at 1/5 the concentration of the 5 individual
solutions.
7.8.3 Stock solutions should be checked for signs of degradation prior to preparation of
calibration or performance test standards. Reference standards that can be used to
determine the accuracy of standard solutions are available from several vendors.
7.9 Labeled compound stock solutions (Table 3)
7.9.1 Labeled Toxics/LOC/window-defming stock solution - Prepare in isooctane or nonane at
the concentrations in Table 3 or purchase Cambridge Isotope Laboratories (CIL) EC-4977,
or equivalent. If additional CBs are to be determined by isotope dilution (e.g., 170 and
180), include the additional labeled compounds in this stock solution.
7.9.2 Labeled cleanup standard stock solution - Prepare labeled CBs 28, 111, and 178 in iso-
octane or nonane at the concentration shown in Table 3 or purchase CIL EC-4978, or
equivalent.
7.9.3 Labeled injection internal standard stock solution - Prepare labeled CBs 9, 52, 101, 138,
and 194 in nonane or isooctane at the concentrations shown in Table 3, or purchase CIL
EC-4979, or equivalent.
EPA Method 1668C 17 April 2010
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7.10 Calibration standards
7.10.1 Calibration standards - Combine and dilute the solutions in Sections 7.8.1 and 7.9 to
produce the calibration solutions in Table 5 or purchase CIL EC-4976, or equivalent, for
the CS-1 to CS-5 set of calibration solutions. If a 6-point calibration is used, prepare the
CS-0.2 solution or purchase CIL EC-4976-0.2, or equivalent. These solutions permit the
relative response (labeled to native) and response factor to be measured as a function of
concentration. The CS-3 standard (CIL EC-4976-3, or equivalent) is used for calibration
verification (VER).
7.10.2 Solutions of congener mixes
7.10.2.1 Diluted individual solutions
7.10.2.1.1 The 5 individual solutions, when analyzed individually, allow
resolution of all 209 congeners on the SPB-octyl column, and are
used for establishing retention time and other data for each
congener. The elution order of the congeners present in each of
the 5 solutions (Section 7.8.2.1) is given in Table 4.
7.10.2.1.2 Individually combine an aliquot of each individual mix stock
solution (Section 7.8.2.1) with an aliquot of the Labeled
Toxics/LOC/window-defming stock solution (Section 7.9.1), the
Labeled cleanup standard stock solution (Section 7.9.2), and the
Labeled injection internal standard stock solution (7.9.3) to
produce concentrations of 100 ng/mL for the labeled compounds
and 25, 50, and 75 ng/mL for the MoCB-TrCB, TeCB-HpCB, and
OcCB-DeCB congeners, respectively, as shown in Table 3.
7.10.2.2 Diluted combined 209 congener solution
7.10.2.2.1 This solution combines the 5 individual mixes with the labeled
compounds to allow single-point calibration of the congeners not
included in the multi-point calibration, and establishes an average
response factor for the co-eluting isomeric congeners.
7.10.2.2.2 Combine an aliquot of the combined 209 congener solution
(Section 7.8.2.2) with an aliquot of the Labeled
Toxics/LOC/window-defming stock solution (Section 7.9.1), the
Labeled cleanup standard stock solution (Section 7.9.2), and the
Labeled injection internal standard stock solution (7.9.3) to
produce the same concentrations as in the diluted individual mix
solutions (Section 7.10.2.1.2 and Table 3).
7.11 Native Toxics/LOC standard spiking solution - Used for determining initial precision and recovery
(IPR; Section 9.2) and ongoing precision and recovery (OPR; Section 15.5). Dilute the Native
Toxics/LOC stock solution (Section 7.8.1) with acetone to produce a concentration of the Toxics at
1 ng/mL, as shown in Table 3. When 1 mL of this solution spiked into the IPR (Section 9.2.1) or
OPR (Section 15.5) and concentrated to a final volume of 20 (iL, the concentration in the final
volume will be 50 ng/mL (50 pg/(iL). Prepare only the amount necessary for each reference matrix
with each sample batch.
EPA Method 1668C 18 April 2010
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7.12 Labeled Toxics/LOC/window-defining standard spiking solution - This solution is spiked into each
sample (Section 9.3) and into the IPR (Section 9.2.1), OPR (Section 15.5), and blank (Section 9.5)
to measure recovery. Dilute the Labeled Toxics/LOC/window-defining stock solution (Section
7.9.1) with acetone to produce a concentration of the labeled compounds at 2 ng/mL, as shown in
Table 3. When 1 mL of this solution is spiked into an IPR, OPR, blank, or sample and concentrated
to a final extract volume of 20 (iL, the concentration in the final extract volume will be 100 ng/mL
(100 pg/(iL). Prepare only the amount necessary for each reference matrix with each sample batch.
7.13 Labeled cleanup standard spiking solution - This solution is spiked into each extract prior to
cleanup to measure the efficiency of the cleanup process. Dilute the Labeled cleanup standard
stock solution (Section 7.9.2) in methylene chloride to produce a concentration of the cleanup
standards at 2 ng/mL, as shown in Table 3. When 1 mL of this solution is spiked into a sample
extract and concentrated to a final volume of 20 (iL, the concentration in the final volume will be
100 ng/mL (100 pg/jiL).
7.14 Labeled injection internal standard spiking solution - This solution is added to each concentrated
extract prior to injection into the HRGC/HRMS. Dilute the Labeled injection internal standard
stock solution (Section 7.9.3) in nonane to produce a concentration of the injection internal
standards at 1000 ng/mL, as shown in Table 3. When 2 (iL of this solution is spiked into a 20 (iL
extract, the concentration of each injection internal standard will be nominally 100 ng/mL (100
pg/(iL).
Note: The addition of 2 ^L of the Labeled injection internal standard spiking solution to a 20-pL final
extract has the effect of diluting the concentration of the components in the extract by 10%. Provided all
calibration solutions and all extracts undergo this dilution as a result of adding the Labeled injection
internal standard spiking solution, the effect of the 10% solution is compensated, and correction for this
dilution should not be made.
7.15 QC Check Sample - A QC Check Sample should be obtained from a source independent of the
calibration standards. Ideally, this check sample would be a certified Standard Reference Material
(SRM) containing the CBs in known concentrations in a sample matrix similar to the matrix under
test. The National Institute of Standards and Technology (NIST) in Gaithersburg, Maryland has
SRMs, and the Institute for National Measurement Standards of the National Research Council of
Canada in Ottawa has certified reference materials (CRMs) for CBs in various matrices.
7.16 Stability of solutions - Standard solutions used for quantitative purposes (Sections 7.9 through
7.14) should be assayed periodically (e.g., every 6 months) against SRMs from NIST (if available),
or certified reference materials from a source that will attest to the authenticity and concentration,
to assure that the composition and concentrations have not changed.
8.0 Sample collection, preservation, storage, and holding times
8.1 Collect samples in amber glass containers following conventional sampling practices (Reference
12). Other sample collection techniques, or sample volumes may be used, if documented.
8.2 Aqueous samples
8.2.1 Samples that flow freely are collected as grab samples or in refrigerated bottles using
automatic sampling equipment. Collect one liter (or a larger or smaller volume) of sample
sufficient to meet project needs.
EPA Method 1668C 19 April 2010
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8.2.2 If residual chlorine is present, add 80 mg sodium thiosulfate per liter of water. EPA
Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 13).
8.2.3 Maintain aqueous samples in the dark at less than 6 °C from the time of collection until
receipt at the laboratory. If the sample will be frozen, allow room for expansion. Store in
the dark at less than 6 °C.
8.3 Solid, mixed-phase, semi-solid, and oily samples, excluding tissue.
8.3.1 Collect samples as grab samples using wide-mouth jars.
8.3.2 Maintain solid, semi-solid, oily, and mixed-phase samples in the dark at less than 6 °C from
the time of collection until receipt at the laboratory. Store solid, semi-solid, oily, and
mixed-phase samples in the dark at less than -10 °C.
8.4 Fish and other tissue samples
8.4.1 Fish may be cleaned, filleted, or processed in other ways in the field, such that the
laboratory may expect to receive whole fish, fish fillets, or other tissues for analysis.
8.4.2 Collect fish, wrap in aluminum foil, and maintain at less than 6 °C from the time of
collection until receipt at the laboratory, to a maximum time of 24 hours. If a longer
transport time is necessary, freeze the sample. Ideally, fish should be frozen upon
collection and shipped to the laboratory on dry ice.
8.4.3 Freeze tissue samples upon receipt at the laboratory and maintain them in the dark at less
than -10 °C until prepared. Maintain unused sample in the dark at less than -10 °C.
8.5 Holding times
8.5.1 There are no demonstrated maximum holding times associated with the CBs in aqueous,
solid, semi-solid, tissue, or other sample matrices. If stored in the dark at less than 6 °C,
aqueous samples may be stored for up to one year. Similarly, if stored in the dark at less
than -10 °C, solid, semi-solid, multi-phase, and tissue samples may be stored for up to one
year.
8.5.2 Store sample extracts in the dark at less than -10 °C until analyzed. If stored in the dark at
less than -10 °C, sample extracts may be stored for one year.
9.0 Quality assurance/quality control
9.1 Each laboratory that uses this Method is required to operate a formal quality assurance program
(Reference 14). The minimum requirements of this program consist of an initial demonstration of
laboratory 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
performance 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 to sample matrix other than water (e.g., soils, filter cake, compost,
tissue) the most appropriate alternate reference matrix (Sections 7.6.2 - 7.6.5 and 7.15) is sub-
stituted for the reagent water matrix (Section 7.6.1) in all performance tests.
EPA Method 1668C 20 April 2010
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9.1.1 The laboratory must make an initial demonstration of the ability to generate acceptable
precision and recovery with this Method. This demonstration is given in Section 9.2.
9.1.2 In recognition of advances that are occurring in analytical technology, and to overcome
matrix interferences, the laboratory is permitted certain options to improve separations or
lower the costs of measurements. These options include alternate extraction, concentration,
and cleanup procedures, and changes in sample volumes, columns and detectors. Alternate
determinative techniques, such as substitution of spectroscopic or immunoassay techniques
for HRGC/HRMS technology, and changes that degrade Method performance, are not
allowed without prior review and approval. If an analytical technique other than the
techniques specified in this Method is used, that technique must have a specificity equal to
or greater than the specificity of the techniques in this Method for the analytes of interest.
(Note: For additional flexibility to make modifications without prior EPA review see 40
CFR Part 136.6.)
9.1.2.1 Each time a modification is made to this Method, the laboratory is required to
repeat the procedure in Section 9.2. If MDLs would be affected by the change,
the laboratory is required to demonstrate that the MDLs (40 CFR Part 136,
Appendix B) are lower than one-third the regulatory compliance level or lower
than five times the MDLs in this Method, whichever are greater. If calibration
will be affected by the change, the instrument must be recalibrated per Section
10. Once the modification is demonstrated to produce results equivalent or
superior to results produced by this Method as written, that modification may be
used routinely thereafter, so long as the other requirements in this Method are
met (e.g., labeled compound recovery).
9.1.2.2 The laboratory is required to maintain records of modifications made to this
Method. These records include the following, at a minimum:
9.1.2.2.1 The names, titles, addresses, and telephone numbers of the
analyst(s) that performed the analyses and modification, and of the
quality control officer that witnessed and will verify the analyses
and modifications.
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 reason(s) for the modifications (see Section
1.5).
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 15.3).
c) Initial precision and recovery (Section 9.2).
d) Labeled compound recovery (Section 9.3).
e) Analysis of blanks (Section 9.5).
f) Accuracy assessment (Section 9.4).
EPA Method 1668C 21 April 2010
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9.1.2.2.5 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. These data are to include:
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 13).
g) Extract volume after each cleanup step (Section 13).
h) Final extract volume prior to injection (Section 14).
i) Injection volume (Section 14.3).
j) Dilution data, differentiating between dilution of a sample or
extract (Section 17.5).
k) Instrument and operating conditions.
1) Column (dimensions, liquid phase, solid support, film
thickness, etc).
m) Operating conditions (temperatures, temperature program, flow
rates).
n) Detector (type, operating conditions, etc).
o) Chromatograms, printer tapes, and other recordings of raw data.
p) Quantitation reports, data system outputs, and other data to link
the raw data to the results reported.
9.1.2.3 Alternate HRGC columns and column systems - See Sections 6.9.1. If a
column or column system alternate to those specified in this Method is used,
that column or column system must meet the requirements in Section 6.9.1 -
6.9.1.1.3.
9.1.3 Analyses of Method blanks are required to demonstrate freedom from contamination
(Section 4.3). The procedures and criteria for analysis of a Method blank are described in
Sections 9.5 and 15.6.
9.1.4 The laboratory must spike all samples with labeled compounds to monitor Method
performance. 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. Procedures for dilution are given in Section 17.5.
9.1.5 The laboratory must, on an ongoing basis, demonstrate through calibration verification and
the analysis of the ongoing precision and recovery standard (OPR) and blanks that the
analytical system is in control. These procedures are given in Sections 15.1 through 15.6.
9.1.6 The laboratory should maintain records to define the quality of data generated.
Development of accuracy statements is described in Section 9.4.
9.2 Initial precision and recovery (IPR) - To establish the ability to generate acceptable precision and
recovery, the laboratory must perform the following operations.
9.2.1 For low solids (aqueous) samples, extract, concentrate, and analyze four 1-L aliquots of
reagent water spiked with 1 mL each of the Native Toxics/LOC spiking solution (Section
7.11), the Labeled Toxics/LOC/window-defming standard spiking solution (Section 7.12),
EPA Method 1668C 22 April 2010
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and the Labeled cleanup standard spiking solution (Section 7.13), according to the
procedures in Sections 11 through 18. For an alternative sample matrix, four aliquots of the
alternative reference matrix (Section 7.6) are used. All sample processing steps that are to
be used for processing samples, including preparation (Section 11), extraction (Section 12),
and cleanup (Section 13), must be included in this test.
9.2.2 Using results of the set of four analyses, compute the average percent recovery (X) of the
extracts and the relative standard deviation (RSD) of the concentration for each compound,
by isotope dilution for CBs with a labeled analog, and by internal standard for CBs without
a labeled analog and for the labeled compounds.
9.2.3 For each CB and labeled compound, compare RSD and X with the corresponding limits for
initial precision and recovery in Table 6. If RSD 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 RSD exceeds the precision limit or any individual
X falls outside the range for recovery, system performance is unacceptable for that
compound. Correct the problem and repeat the test (Section 9.2).
9.3 To assess Method performance on the sample matrix, the laboratory must spike all samples with the
Labeled Toxics/LOC/window-defming standard spiking solution (Section 7.12) and all sample
extracts with the Labeled cleanup standard spiking solution (Section 7.13).
9.3.1 Analyze each sample according to the procedures in Sections 11 through 18.
9.3.2 Compute the percent recovery of the labeled Toxics/LOC/window-defming congeners and
the labeled cleanup congeners using the internal standard method (Section 17.2).
9.3.3 The recovery of each labeled compound must be within the limits in Table 6. If the
recovery of any compound falls outside of these limits, Method performance is
unacceptable for that compound in that sample. Additional cleanup procedures must then
be employed to attempt to bring the recovery within the normal range. If the recovery
cannot be brought within the normal range after all cleanup procedures have been
employed, water samples are diluted and smaller amounts of soils, sludges, sediments, and
other matrices are analyzed per Section 18.
9.4 It is suggested, but not required, that recovery of labeled compounds from samples be assessed and
records maintained.
9.4.1 After the analysis of 30 samples of a given matrix type (water, soil, sludge, pulp, etc.) for
which the labeled compounds pass the tests in Section 9.3, compute the average percent
recovery (R) and the standard deviation of the percent recovery (SR) for the labeled
compounds only. Express the assessment as a percent recovery interval from R - 2SR to R
+ 2SR for each matrix. For example, if R = 90% and SR = 10% for five analyses of pulp,
the recovery interval is expressed as 70 to 110%.
9.4.2 Update the accuracy assessment for each labeled compound in each matrix on a regular
basis (e.g., after each five to ten new measurements).
9.5 Method blanks - A reference matrix Method blank is analyzed with each sample batch (Section
4.3) to demonstrate freedom from contamination. The matrix for the Method blank must be similar
to the sample matrix for the batch, e.g., a 1-L reagent water blank (Section 7.6.1), high-solids
EPA Method 1668C 23 April 2010
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reference matrix blank (Section 7.6.2), paper matrix blank (Section 7.6.3); tissue blank (Section
7.6.4), or alternative reference matrix blank (Section 7.6.5).
9.5.1 Spike 1.0 mL each of the Labeled Toxics/LOC/window-defming standard spiking solution
(Section 7.12), and the Labeled cleanup standard spiking solution (Section 7.13) into the
Method blank, according to the procedures in Sections 11 through 18. Prepare, extract,
clean up, and concentrate the Method blank. Analyze the blank immediately after analysis
of the OPR (Section 15.5) to demonstrate freedom from contamination.
9.5.2 If any CB (Table 1) is found in the blank at greater than two times the minimum level
(Table 2) or one-third the regulatory compliance limit, whichever is greater; or if any
potentially interfering compound is found in the blank at the minimum level for each CB
given in Table 2 (assuming a response factor of 1 relative to the quantitation reference in
Table 2 at that level of chlorination for a potentially interfering compound; i.e., a
compound not listed in this Method), analysis of samples must be halted until the sample
batch is re-extracted and the extracts re-analyzed, and the blank associated with the sample
batch shows no evidence of contamination at these levels. All samples must be associated
with an uncontaminated Method blank before the results for those samples may be reported
or used for permitting or regulatory compliance purposes.
9.6 QC Check Sample - Analyze the QC Check Sample (Section 7.15) periodically to assure the
accuracy of calibration standards and the overall reliability of the analytical process. It is suggested
that the QC Check Sample be analyzed at least quarterly.
9.7 The specifications contained in this Method can be met if the apparatus used is calibrated properly
and then maintained in a calibrated state. The standards used for calibration (Section 10),
calibration verification (Section 15.3), and for initial (Section 9.2) and ongoing (Section 15.5)
precision and recovery should be identical, so that the most precise results will be obtained. A
GC/MS instrument will provide the most reproducible results if dedicated to the settings and
conditions required for determination of CBs by this Method.
9.8 Depending on specific program requirements, field replicates may be collected to determine the
precision of the sampling technique, and spiked samples may be required to determine the accuracy
of the analysis when the internal standard method is used.
10.0 Calibration
10.1 Establish the operating conditions necessary to meet the retention times (RTs) and relative
retention times (RRTs) for the CBs in Table 2.
Note: RTs, RRTs, and RRT limits may differ slightly from those in Table 2.
10.1.1 Suggested GC operating conditions:
Inj ector temperature: 270 °C
Interface temperature: 290 °C
Initial temperature: 75 °C
Initial time: 2 minutes
Temperature program: 75-150 °C at 15 "C/minute
150-290 °C at 2.5 °C/minute
Final time: 1 minute
EPA Method 1668C 24 April 2010
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Note: All portions of the column that connect the GC to the ion source should remain at or above the
interface temperature specified above during analysis to preclude condensation of less volatile
compounds.
The GC conditions may be optimized for compound separation and sensitivity. Once optimized,
the same GC conditions must be used for the analysis of all standards, blanks, IPR and OPR
standards, and samples.
10.1.2 Retention time calibration for the CB congeners
10.1.2.1 Separately inject each of the diluted individual congener solutions (Section
7.10.2.1.2). Establish the beginning and ending retention times for the scan
descriptors in Table 7. Scan descriptors other than those listed in Table 7 may
be used provided the MLs in Table 2 are met. Store the retention time (RT) and
relative retention time (RRT) for each congener in the data system.
10.1.2.2 The absolute retention time of CB 209 must exceed 55 minutes on the SPB-
octyl column; otherwise, the GC temperature program must be adjusted and this
test repeated until the minimum retention time criterion is met. If a GC column
or column system alternate to the SPB-octyl column is used, a similar minimum
retention time specification must be established for the alternate column or
column systems so that interferences that may be encountered in environmental
samples will be resolved from the analytes of interest. This specification is
deemed to be met if the retention time of CB 209 is greater than 55 minutes on
such alternate column.
10.1.2.3 Inject the Diluted combined 209 congener solution (Section 7.10.2.2). Adjust
the chromatographic conditions and scan descriptors until the RT and RRT for
all congeners are approximately within the windows in Table 2 and the column
performance specifications in Sections 6.9.1 - 6.9.1.2 are met. If an alternate
column is used, adjust the conditions for that column. If column performance is
unacceptable, optimize the analysis conditions or replace the column and repeat
the performance tests. Confirm that the scan descriptor changes at times when
CBs do not elute.
10.1.2.4 After the column performance tests are passed (Section 10.1.2.2 - 10.1.2.3),
calculate and store the RT and RRT for the resolved congeners and the RT and
RRT for the isomeric congeners that co-elute. The windows in Table 2 were
developed based on the GC conditions given in Section 10.1.1.
10.2 Mass spectrometer (MS) resolution
10.2.1 Using perfluorokerosene (PFK) (or other reference substance) and a molecular leak, tune
the instrument to meet the minimum required resolving power of 10,000 (10% valley) at
m/z 330.9792 or any other significant PFK fragment in the range of 300 to 350. For each
descriptor (Table 7), monitor and record the resolution and exact m/z's of three to five
reference peaks covering the mass range of the descriptor. The level of PFK (or other
reference substance) metered into the HRMS during analyses should be adjusted so that the
amplitude of the most intense selected lock-mass m/z signal (regardless of the descriptor
number) does not exceed 10% of the full-scale deflection for a given set of detector
EPA Method 1668C 25 April 2010
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parameters. Under those conditions, sensitivity changes that might occur during the
analysis can be more effectively monitored.
Note: Different lots and types ofPFK can contain varying levels of contamination, and excessive PFK
(or other reference substance) may cause noise problems and contamination of the ion source
necessitating increased frequency of source cleaning. A minor PFK mass (223.9872) is known to
interfere with dichlorobiphenyl secondary quantitation ion (M+2). Careful selection of the grade and
purity of PFK and minimization of the amount of PFK bled into the HRMS has been shown to correct this
problem.
10.2.2 The analysis time for CBs may exceed the long-term mass stability of the mass
spectrometer. Because the instrument is operated in the high-resolution mode, mass drifts
of a few ppm (e.g., 5 ppm in mass) can have serious adverse effects on instrument
performance. Therefore, mass-drift correction is mandatory and a lock-mass m/z from PFK
or other reference substance is used for drift correction. The lock-mass m/z is dependent
on the exact m/z's monitored within each descriptor, as shown in Table 7. The deviation
between each monitored exact m/z and the theoretical m/z (Table 7) must be less than 5
ppm.
10.2.3 Obtain a selected ion current profile (SICP) at the two exact m/z's specified in Table 7 and
at > 10,000 resolving power at each LOG for the native congeners and congener groups and
for the labeled congeners. Because of the extensive mass range covered in each function, it
may not be possible to maintain 10,000 resolution throughout the mass range during the
function. Therefore, resolution must be >8,000 throughout the mass range and must be
>10,000 in the center of the mass range for each function.
10.2.4 If the HRMS has the capability to monitor resolution during the analysis, it is acceptable to
terminate the analysis when the resolution falls below the minimum (Section 10.2.3) to
save re-analysis time.
10.3 Ion abundance ratios, minimum levels, and signal-to-noise ratios. Choose an injection volume of
either 1 or 2 \\L, consistent with the capability of the HRGC/HRMS instrument. Inject a 1 or 2 \\L
aliquot of the CS-1 calibration solution (Table 5) using the GC conditions in Section 10.1.1.
10.3.1 Measure the SICP areas for each congener or congener group, and compute the ion
abundance ratios at the exact m/z's specified in Table 7. Compare the computed ratio to
the theoretical ratio given in Table 8.
10.3.1.1 The exact m/z's to be monitored in each descriptor are shown in Table 7. Each
group or descriptor must be monitored in succession as a function of GC
retention time to ensure that the CBs of interest are detected. Additional m/z's
may be monitored in each descriptor, and the m/z's may be divided among more
than the descriptors listed in Table 7, provided that the laboratory is able to
monitor the m/z's of all CBs that may elute from the GC in a given LOG
window. The laboratory must also monitor exact m/z's for congeners at higher
levels of chlorination to determine if fragments will compromise measurement
of congeners at lower levels of chlorination.
10.3.1.2 The mass spectrometer must be operated in a mass-drift correction mode, using
PFK (or other reference substance) to provide lock m/z's. The lock mass for
each group of m/z's is shown in Table 7. Each lock mass must be monitored
and must not vary by more than ± 20% throughout its respective retention time
EPA Method 1668C 26 April 2010
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window. Variations of lock mass by more than 20% indicate the presence of co-
eluting interferences that raise the source pressure and may significantly reduce
the sensitivity of the mass spectrometer. Re-injection of another aliquot of the
sample extract may not resolve the problem and additional cleanup of the
extract may be required to remove the interference. A lock mass interference or
suppression in a retention time region in which CBs and labeled compounds do
not elute may be ignored.
10.3.2 All CBs and labeled compounds in the CS-1 standard must be within the QC limits in Table
8 for their respective ion abundance ratios; otherwise, the mass spectrometer must be
adjusted and this test repeated until the m/z ratios fall within the limits specified. If the
adjustment alters the resolution of the mass spectrometer, resolution must be verified
(Section 10.2.3) prior to repeat of the test.
10.3.3 Verify that the HRGC/HRMS instrument achieves a minimum level (ML) for each
congener no greater than 2 times the MLs in Table 2. The peaks representing the CBs and
labeled compounds in the CS-1 calibration standard must have signal-to-noise ratios (S/N)
> 10; otherwise, the mass spectrometer must be adjusted and this test repeated until the
minimum levels in Table 2 are met.
Note: The MDLs and MLs in Table 2 are based on the levels of contamination normally found in
laboratories. Lower levels may be readily achievable if segregation and extensive cleaning of glassware
are employed. If lower levels are achievable, these lower levels must be established as described in
Section 17.6.1.4.1.
10.4 Calibration by isotope dilution - Isotope dilution is used for calibration of the Toxics/LOC CBs.
The reference compound for each native compound its labeled analog, as listed in Table 2. A 5- or
6-point calibration encompassing the concentration range is prepared for each native congener.
10.4.1 For the Toxics/LOC CBs determined by isotope dilution, the relative response (RR)
(labeled to native) vs. concentration in the calibration solutions (Table 5) is computed over
the calibration range according to the procedures described below. Five calibration points
are employed for less-sensitive HRMS instruments (e.g., VG 70); five or six points may be
employed for more-sensitive instruments (e.g., Micromass Autospec Ultima).
10.4.2 The response of each Toxics/LOC CB relative to its labeled analog is determined using the
area responses of both the primary and secondary exact m/z's specified in Table 7, for each
calibration standard, as follows:
RR=(Ak±A2JCL
(Al1 + A21)Cn
where:
Aln and A2n = The measured areas at the primary and secondary m/z's for the PCB
Ali and A2i = The measured areas at the primary and secondary m/z's for the labeled
compound
Ci = The concentration of the labeled compound in the calibration standard
(Table 4)
Cn = The concentration of the native compound in the calibration standard
(Table 4)
EPA Method 1668C 27 April 2010
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10.4.3 To calibrate the analytical system by isotope dilution, inject calibration standards CS-1
through CS-5 (Section 7.10 and Table 5) for a less sensitive instrument or CS-0.2 through
CS-5 for a more sensitive instrument. Use a volume identical to the volume chosen in
Section 10.3, the procedure in Section 14, and the conditions in Section 10.1.1. Compute
and store the relative response (RR) for each Native Toxics/LOC CB at each concentration.
Compute the average (mean) RR and the RSD of the 5 (or 6) RRs.
10.4.4 Linearity - If the RR for any Native Toxics/LOC CB is constant (less than 20% RSD), the
average RR may be used for that congener; otherwise, the complete calibration curve for
that congener must be used over the calibration range.
10.5 Calibration by internal standard - Internal standard calibration is applied to determination of the
native CBs for which a labeled compound is not available, determination of the Labeled
Toxics/LOC/window-defming congeners and Labeled cleanup congeners for performance tests and
intra-laboratory statistics (Sections 9.4 and 15.5.4), and determination of the Labeled injection
internal standards except for CB 178. The reference compound for each compound is listed in
Table 2. For the native congeners (other than the Native Toxics/LOC CBs), calibration is
performed at a single point using the Diluted combined 209 congener solution (Section 7.10.2.2 and
Table 5). For the labeled compounds, calibration is performed using data from the 5 (or 6) points in
the calibration for the Native Toxics/LOC CBs (Section 10.4).
10.5.1 Response factors - Internal standard calibration requires the determination of response
factors (RF) defined by the following equation:
pj (A1S + A2S)C1S
(A1IS + A2IS)CS
where:
Als and A2S = The measured areas at the primary and secondary m/z's for the PCB
A11S and A21S= The measured areas at the primary and secondary m/z's for the internal
standard
Cis = The concentration of the internal standard (Table 5)
Cs = The concentration of the compound in the calibration standard (Table 5)
10.5.2 To single-concentration calibrate the analytical system for native CBs other than the Native
Toxics/LOC CBs by internal standard, inject the Diluted combined 209 congener solution
(Section 7.10.2.2 and Table 3). Use a volume identical to the volume chosen in Section
10.3, the procedure in Section 14, and the conditions in Section 10.1.1.
10.5.3 Compute and store the response factor (RF) for all native CBs except the Native
Toxics/LOC CBs. Use the average (mean) response of the labeled compounds at each level
of chlorination (LOG) as the quantitation reference, to a maximum of 5 labeled congeners,
as shown in Table 2. For the combinations of isomeric congeners that co-elute, compute a
combined RF for the co-eluted group. For example, for congener 122, the areas at the two
exact m/z's for 104L, 105L, 114L, 118L, and 123L are summed and the total area is
divided by 5 (because there are 5 congeners in the quantitation reference).
Note: All labeled congeners at each LOC are used as reference to reduce the effect of an interference if
a single congener is used as reference. Other quantitation references and procedures may be used
provided that the results produced are as accurate as results produced by the quantitation references and
procedures described in this Section.
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10.5.4 Compute and store the response factor (RF) for the labeled compounds, except CB 138.
For the Labeled Toxics/LOC/window-defining compounds and the Labeled cleanup
standards, use the nearest eluted Labeled injection internal standard as the quantitation
reference, as given in Table 2. The Labeled injection internal standards are referenced to
CB 138, as shown in Table 2.
11.0 Sample preparation
11.1 Sample preparation involves modifying the physical form of the sample so that the CBs can be
extracted efficiently. In general, the samples must be in a liquid form or in the form of finely
divided solids in order for efficient extraction to take place. Table 9 lists the phases and suggested
quantities for extraction of various sample matrices.
For samples known or expected to contain high levels of the CBs, the smallest sample size
representative of the entire sample should be used (see Section 18). For all samples, the blank and
IPR/OPR aliquots must be processed through the same steps as the sample to check for
contamination and losses in the preparation processes.
11.1.1 For samples that contain particles, percent solids and particle size are determined using the
procedures in Sections 11.2 and 11.3, respectively.
11.1.2 Aqueous samples - Because CBs may be bound to suspended particles, the preparation of
aqueous samples is dependent on the solids content of the sample.
11.1.2.1 Aqueous samples containing one percent solids or less are prepared per Section
11.4 and extracted directly using one of the extraction techniques in Section
12.2.
11.1.2.2 For aqueous samples containing greater than one percent solids, a sample
aliquot sufficient to provide 10 g of dry solids is used, as described in Section
11.5.
11.1.3 Solid samples are prepared using the procedure described in Section 11.5 followed by
extraction using the SDS procedure in Section 12.3.
11.1.4 Multi-phase samples - The phase(s) containing the CBs is separated from the non-CB
phase using pressure filtration and centrifugation, as described in Section 11.6. The CBs
will be in the organic phase in a multi-phase sample in which an organic phase exists.
11.1.5 Procedures for grinding, homogenization, and blending of various sample phases are given
in Section 11.7.
11.1.6 Tissue samples - Preparation procedures for fish and other tissues are given in Section
11.8.
11.2 Determination of percent suspended solids
Note: This aliquot is used for determining solids content of the sample, not for determination of CBs.
EPA Method 1668C 29 April 2010
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11.2.1 Aqueous liquids and multi-phase samples consisting of mainly an aqueous phase
11.2.1.1 Desiccate and weigh a GF/D filter (Section 6.5.3) to three significant figures.
11.2.1.2 Filter 10.0 ± 0.02 mL of well-mixed sample through the filter.
11.2.1.3 Dry the filter a minimum of 12 hours at 110 ±5 °C and cool in a desiccator.
11.2.1.4 Calculate percent solids as follows:
weight of sample aliquot after drying (g) - weight of filter (g)
% solids = x 100
10 g
11.2.2 Non-aqueous liquids, solids, semi-solid samples, and multi-phase samples in which the
main phase is not aqueous; but not tissues
11.2.2.1 Weigh 5 to 10 g of sample to three significant figures in atared beaker.
11.2.2.2 Dry a minimum of 12 hours at 110 ± 5 °C, and cool in a desiccator.
11.2.2.3 Calculate percent solids as follows:
. ,. , weight of sample aliquot after drying (g)
% solids = x 100
weight of sample aliquot before drying (g)
11.3 Estimation of particle size
11.3.1 Spread the dried sample from Section 11.2.2.2 on apiece of filter paper or aluminum foil in
a fume hood or glove box.
11.3.2 Estimate the size of the particles in the sample. If the size of the largest particles is greater
than 1 mm, the particle size must be reduced to 1 mm or less prior to extraction using the
procedures in Section 11.7.
11.4 Preparation of aqueous samples containing one percent suspended solids or less
11.4.1 Aqueous samples containing one percent suspended solids or less are prepared using the
procedure below and extracted using the one of the extraction techniques in Section 12.2.
11.4.2 Preparation of sample and QC aliquots
11.4.2.1 Mark the original level of the sample on the sample bottle for reference. Weigh
the sample plus bottle to ± 1 g. After extraction (Section 12.2), re-weigh the
sample bottle and convert the weight to volume assuming a density of 1.00
g/mL.
11.4.2.2 Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard spiking
solution (Section 7.12) into the sample bottle. Cap the bottle and mix the
sample by careful shaking. Allow the sample to equilibrate for 1 to 2 hours,
with occasional shaking.
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11.4.2.3 For each sample or sample batch (to a maximum of 20 samples) to be extracted
during the same 12-hour shift, place two 1.0-L aliquots of reagent water in clean
sample bottles or flasks.
11.4.2.4 Spike 1.0 mL of the Labeled Toxics/LOC/window-defming standard spiking
solution (Section 7.12) into both reagent water aliquots. One of these aliquots
will serve as the Method blank.
11.4.2.5 Spike 1.0 mL of the Native Toxics/LOC standard spiking solution (Section
7.11) into the remaining reagent water aliquot. This aliquot will serve as the
OPR (Section 15.5).
11.4.2.6 For extraction using SPE, add 5 mL of methanol to the sample and QC aliquots.
Cap and shake the sample and QC aliquots to mix thoroughly, and proceed to
Section 12.2 for extraction.
11.5 Preparation of samples containing greater than one percent solids
11.5.1 Weigh a well-mixed aliquot of each sample (of the same matrix type) sufficient to provide
10 g of dry solids (based on the solids determination in Section 11.2) into a clean beaker or
glass jar.
11.5.2 Spike 1.0 mL of the Labeled Toxics/LOC/window-defming standard spiking solution
(Section 7.12) into the sample.
11.5.3 For each sample or sample batch (to a maximum of 20 samples) to be extracted during the
same 12 hour shift, weigh two 10-g aliquots of the appropriate reference matrix (Section
7.6) into clean beakers or glass jars.
11.5.4 Spike 1.0 mL of the Labeled Toxics/LOC/window-defming standard spiking solution
(Section 7.12) into both reference matrix aliquots. Spike 1.0 mL of the Native Toxics/LOC
standard spiking solution (Section 7.11) into one reference matrix aliquot. This aliquot will
serve as the OPR (Section 15.5). The other aliquot will serve as the Method blank.
11.5.5 Stir or tumble and equilibrate the aliquots for 1 to 2 hours.
11.5.6 Decant excess water. If necessary to remove water, filter the sample through a glass-fiber
filter and discard the aqueous liquid.
11.5.7 If particles >1 mm are present in the sample (as determined in Section 11.3.2), spread the
sample on clean aluminum foil in a hood. After the sample is dry, grind to reduce the
particle size (Section 11.7).
11.5.8 Extract the sample and QC aliquots using the SDS procedure in Section 12.3.
11.6 Multi-phase samples
11.6.1 Using the percent solids determined in Section 11.2.1 or 11.2.2, determine the volume of
sample that will provide 10 g of solids, up to 1 L of sample.
11.6.2 Spike 1.0 mL of the Labeled Toxics/LOC/window-defming standard spiking solution
(Section 7.12) into the amount of sample determined in Section 11.6.1, and into the OPR
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and blank. Spike 1.0 mL of the Native Toxics/LOC standard spiking solution (Section
7.11) into the OPR. Pressure filter the sample, blank, and OPR through Whatman GF/D
glass-fiber filter paper (Section 6.5.3). If necessary to separate the phases and/or settle the
solids, centrifuge these aliquots prior to filtration.
11.6.3 Discard any aqueous phase (if present). Remove any non-aqueous liquid present and
reserve the maximum amount filtered from the sample (Section 11.6.1) or 10 g, whichever
is less, for combination with the solid phase (Section 12.3.5).
11.6.4 If particles >1 mm are present in the sample (as determined in Section 11.3.2) and the
sample is capable of being dried, spread the sample and QC aliquots on clean aluminum
foil in a hood. Observe the precaution in Section 4.8.
11.6.5 After the aliquots are dry or if the sample cannot be dried, reduce the particle size using the
procedures in Section 11.7 and extract the reduced-size particles using the SDS procedure
in Section 12.3. If particles >1 mm are not present, extract the particles and filter in the
sample and QC aliquots directly using the SDS procedure in Section 12.3.
11.7 Sample grinding, homogenization, or blending - Samples with particle sizes greater than 1 mm (as
determined in Section 11.3.2) are subjected to grinding, homogenization, or blending. The method
of reducing particle size to less than 1 mm is matrix-dependent. In general, hard particles can be
reduced by grinding with a mortar and pestle. Softer particles can be reduced by grinding in a
Wiley mill or meat grinder, by homogenization, or in a blender.
11.7.1 Each size-reducing preparation procedure on each matrix must be verified by running the
tests in Section 9.2 before the procedure is employed routinely.
11.7.2 The grinding, homogenization, or blending procedures must be carried out in a glove box
or fume hood to prevent particles from contaminating the work environment.
11.7.3 Grinding - Certain papers and pulps, slurries, and amorphous solids can be ground in a
Wiley mill or heavy duty meat grinder. In some cases, reducing the temperature of the
sample to freezing or to dry ice or liquid nitrogen temperatures can aid in the grinding
process. Grind the sample aliquots from Sections 11.5.7 or 11.6.5 in a clean grinder. Do
not allow the sample temperature to exceed 50 °C. Grind the blank and reference matrix
aliquots using a clean grinder.
11.7.4 Homogenization or blending - Particles that are not ground effectively, or particles greater
than 1 mm in size after grinding, can often be reduced in size by high speed
homogenization or blending. Homogenize and/or blend the particles or filter from Sections
11.5.7 or 11.6.5 for the sample, blank, and OPR aliquots.
11.7.5 Extract the aliquots using the SDS procedure in Section 12.3.
11.8 Fish and other tissues - Prior to processing tissue samples, the laboratory must determine the exact
tissue to be analyzed. Common requests for analysis offish tissue include whole fish-skin on,
whole fish-skin removed, edible fish fillets (filleted in the field or by the laboratory), specific
organs, and other portions. Once the appropriate tissue has been determined, the sample must be
homogenized.
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11.8.1 Homogenization
11.8.1.1 Samples are homogenized while still frozen, where practical. If the laboratory
must dissect the whole fish to obtain the appropriate tissue for analysis, the
unused tissues may be rapidly refrozen and stored in a clean glass jar for
subsequent use.
11.8.1.2 Each analysis requires 10 g of tissue (wet weight). Therefore, the laboratory
should homogenize at least 20 g of tissue to allow for re-extraction of a second
aliquot of the same homogenized sample, if re-analysis is required. When
whole fish analysis is necessary, the entire fish is homogenized.
11.8.1.3 Homogenize the sample in a tissue homogenizer (Section 6.3.3) or grind in a
meat grinder (Section 6.3.4). Cut tissue that is too large to feed into the grinder
into smaller pieces. To assure homogeneity, grind three times.
11.8.1.4 Transfer approximately 10 g (wet weight) of homogenized tissue to a clean,
tared, 400- to 500-mL beaker.
11.8.1.5 Transfer the remaining homogenized tissue to a clean jar with a fluoropolymer-
lined lid. Seal the jar and store the tissue at less than -10 °C. Return any tissue
that was not homogenized to its original container and store at less than -10 °C.
11.8.2 QCaliquots
11.8.2.1 Prepare a Method blank by adding approximately 1-2 g of the oily liquid
reference matrix (Section 7.6.4) to a 400- to 500-mL beaker.
11.8.2.2 Prepare a precision and recovery aliquot by adding 1-2 g of the oily liquid
reference matrix (Section 7.6.4) to a separate 400- to 500-mL beaker. Record
the weight to the nearest 10 mg. If the initial precision and recovery test is to be
performed, use four aliquots; if the ongoing precision and recovery test is to be
performed, use a single aliquot.
11.8.3 Spiking
11.8.3.1 Spike 1.0 mL of the Labeled Toxics/LOC/window-defining standard spiking
solution (Section 7.12) into the sample, blank, and OPR aliquot.
11.8.3.2 Spike 1.0 mL of the Native Toxics/LOC standard spiking solution (Section
7.11) into the OPR aliquot.
11.8.4 Extract the aliquots using the procedures in Section 12.4.
12.0 Extraction and concentration
12.1 Extraction procedures include: solid-phase (Section 12.2.1), separatory funnel (Section 12.2.2), and
continuous liquid/liquid (Section 12.2.3) for aqueous liquids; Soxhlet/Dean-Stark (Section 12.3) for
solids and filters; and Soxhlet extraction (Section 12.4) for tissues. Acid/base back-extraction
(Section 12.5) is used for initial cleanup of extracts.
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Macro-concentration procedures include: rotary evaporation (Section 12.6.1), heating mantle
(Section 12.6.2), and Kuderna-Danish (K-D) evaporation (Section 12.6.3). Micro-concentration
uses nitrogen evaporation (Section 12.7).
12.2 Extraction of aqueous liquids
12.2.1 Solid-phase extraction of samples containing less than one percent solids
12.2.1.1 Disk preparation
12.2.1.1.1 Remove the test tube from the suction flask (Figure 4). Place an
SPE disk on the base of the filter holder and wet with methylene
chloride. While holding a GMF 150 filter above the SPE disk with
tweezers, wet the filter with methylene chloride and lay the filter
on the SPE disk, making sure that air is not trapped between the
filter and disk. Clamp the filter and SPE disk between the 1-L
glass reservoir and the vacuum filtration flask.
12.2.1.1.2 Rinse the sides of the reservoir with approx 15 mL of methylene
chloride using a squeeze bottle or pipet. Apply vacuum
momentarily until a few drops appear at the drip tip. Release the
vacuum and allow the filter/disk to soak for approx one minute.
Apply vacuum and draw all of the methylene chloride through the
filter/disk. Repeat the wash step with approx 15 mL of acetone
and allow the filter/disk to air dry.
12.2.1.2 Sample extraction
12.2.1.2.1 Pre-wetthe disk by adding approx 20 mLof methanol to the
reservoir. Pull most of the methanol through the filter/disk,
retaining a layer of methanol approx 2 mm thick on the filter. Do
not allow the filter/disk to go dry from this point until the
extraction is completed.
12.2.1.2.2 Add approx 20 mL of reagent water to the reservoir and pull most
through, leaving a layer approx 2 mm thick on the filter/disk.
12.2.1.2.3 Allow the sample (Section 11.4.2.6) to stand for 1-2 hours, if
necessary, to settle the suspended particles. Decant the clear layer
of the sample, the blank (Section 11.4.2.4), or IPR/OPR aliquot
(Section 11.4.2.5) into its respective reservoir and turn on the
vacuum to begin the extraction. Adjust the vacuum to complete
the extraction in no less than 10 minutes. For samples containing
a high concentration of particles (suspended solids), the extraction
time may be an hour or longer.
12.2.1.2.4 Before all of the sample has been pulled through the filter/disk, add
approx 50 mL of reagent water to the sample bottle, swirl to
suspend the solids (if present), and pour into the reservoir. Pull
through the filter/disk. Use additional reagent water rinses until all
solids are removed.
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12.2.1.2.5 Before all of the sample and rinses have been pulled through the
filter/disk, rinse the sides of the reservoir with small portions of
reagent water.
12.2.1.2.6 Partially dry the filter/disk under vacuum for approx 3 minutes.
12.2.1.3 Elution of the filter/disk
12.2.1.3.1 Release the vacuum, remove the entire filter/disk/reservoir
assembly from the vacuum flask, and empty the flask. Insert a test
tube for eluant collection into the flask. The test tube should have
sufficient capacity to contain the total volume of the elution
solvent (approx 50 mL) and should fit around the drip tip. The
drip tip should protrude into the test tube to preclude loss of
sample from spattering when vacuum is applied. Reassemble the
filter/disk/reservoir assembly on the vacuum flask.
12.2.1.3.2 Wet the filter/disk with 4-5 mL of acetone. Allow the acetone to
spread evenly across the disk and soak for 15-20 seconds. Pull the
acetone through the disk, releasing the vacuum when approx 1 mm
thickness remains on the filter.
12.2.1.3.3 Rinse the sample bottle with approx 20 mL of methylene chloride
and transfer to the reservoir. Pull approx half of the solvent
through the filter/disk and release the vacuum. Allow the
filter/disk to soak for approx 1 minute. Pull all of the solvent
through the disk. Repeat the bottle rinsing and elution step with
another 20 mL of methylene chloride. Pull all of the solvent
through the disk.
12.2.1.3.4 Release the vacuum, remove the filter/disk/reservoir assembly, and
remove the test tube containing the sample solution.
Quantitatively transfer the solution to a 250-mL separatory funnel
and proceed to Section 12.5 for back-extraction.
12.2.2 Separatory funnel extraction
12.2.2.1 Pour the spiked sample (Section 11.4.2.2) into a 2-L separatory funnel. Rinse
the bottle or flask twice with 5 mL of reagent water and add these rinses to the
separatory funnel.
12.2.2.2 Add 60 mL methylene chloride to the empty sample bottle. Seal the bottle and
shake 60 seconds to rinse the inner surface. Transfer the solvent to the separa-
tory funnel, and extract the sample by shaking the funnel for 2 minutes with
periodic venting. Allow the organic layer to separate from the aqueous phase
for a minimum of 10 minutes. If an emulsion forms and is more than one-third
the volume of the solvent layer, employ mechanical techniques to complete the
phase separation (see note below). Drain the methylene chloride extract
through a solvent-rinsed glass funnel approximately one-half full of granular
anhydrous sodium sulfate (Section 7.2.1) supported on clean glass-fiber paper
into a solvent-rinsed concentration device (Section 12.6).
EPA Method 1668C 35 April 2010
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Note: If an emulsion forms, the laboratory must employ mechanical techniques to complete the phase
separation. The optimum technique depends upon the sample, but may include stirring, filtration through
glass wool, use of phase separation paper, centrifugation, use of an ultrasonic bath with ice, addition of
NaCl, or other physical methods. Alternatively, solid-phase (Section 12.2.1), CLLE (Section 12.2.3), or
other extraction techniques may be used to prevent emulsion formation. Any alternative technique is
acceptable so long as the requirements in Section 9.2 are met.
12.2.2.3 Extract the water sample two more times with 60-mL portions of methylene
chloride. Drain each portion through the sodium sulfate into the concentrator.
After the third extraction, rinse the separatory runnel with at least 20 mL of
methylene chloride, and drain this rinse through the sodium sulfate into the
concentrator. Repeat this rinse at least twice.
12.2.2.4 Concentrate the extract using one of the macro-concentration procedures in
Section 12.6 and proceed to back extraction in Section 12.5. Set aside the
concentration device for use after back extraction or other cleanup.
12.2.3 Continuous liquid/liquid extraction
12.2.3.1 Place 100-150 mL methylene chloride in each continuous extractor and 200-300
mL in each distilling flask.
12.2.3.2 Pour the sample(s), blank, and QC aliquots into the extractors. Rinse the sample
containers with 50-100 mL methylene chloride and add to the respective
extractors. Include all solids in the extraction process.
12.2.3.3 Begin the extraction by heating the flask until the methylene chloride is boiling.
When properly adjusted, 1-2 drops of methylene chloride per second will fall
from the condenser tip into the water. Extract for 16-24 hours.
12.2.3.4 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 to
10 cm of granular anhydrous sodium sulfate into a 500-mL K-D evaporator
flask equipped with a 10-mL concentrator tube. Rinse the distilling flask with
30-50 mL of methylene chloride and pour through the drying column.
Concentrate and exchange to hexane per Section 12.6 and back extract per
Section 12.5. Set aside the concentration device for use after back extraction or
other cleanup.
12.3 SDS extraction of samples containing particles
Note: SDS extraction with toluene may cause loss of some of the mono- through tri- CB congeners. If
this loss is excessive, use Soxhlet extraction with methylene chloride (Section 12.4) and increase the
amount of powdered, anhydrous sodium sulfate as necessary to provide a free-flowing mixture.
12.3.1 Charge a clean extraction thimble (Section 6.4.2.2) with 5.0 g of 100/200 mesh silica
(Section 7.5.1.1) topped with 100 g of quartz sand (Section 7.3.2).
Note: Do not disturb the silica layer throughout the extraction process.
12.3.2 Place the thimble in a clean extractor. Place 30 to 40 mL of toluene in the receiver and 200
to 250 mL of toluene in the flask.
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12.3.3 Pre-extract the glassware by heating the flask until the toluene is boiling. When properly
adjusted, 1 to 2 drops of toluene will fall per second from the condenser tip into the
receiver. Extract the apparatus for a minimum of 3 hours.
12.3.4 After pre-extraction, cool and disassemble the apparatus. Rinse the thimble with toluene
and allow to air dry.
12.3.5 Load the wet sample and/or filter from Sections 11.5.8, 11.6.5, or 11.7.5 and any non-
aqueous liquid from Section 11.6.3 into the thimble and manually mix into the sand layer
with a clean metal spatula, carefully breaking up any large lumps of sample.
12.3.6 Reassemble the pre-extracted SDS apparatus, and add a fresh charge of toluene to the
receiver and reflux flask. Apply power to the heating mantle to begin re-fluxing. Adjust
the reflux rate to match the rate of percolation through the sand and silica beds until water
removal lessens the restriction to toluene flow. Frequently check the apparatus for foaming
during the first 2 hours of extraction. If foaming occurs, reduce the reflux rate until
foaming subsides.
12.3.7 Drain the water from the receiver at 1-2 hours and 8-9 hours, or sooner if the receiver fills
with water. Reflux the sample for a total of 16-24 hours. Cool and disassemble the
apparatus. Record the total volume of water collected.
12.3.8 Remove the distilling flask. Drain the water from the Dean-Stark receiver and add any
toluene in the receiver to the extract in the flask.
12.3.9 Concentrate the extracts from particles to approximately 10 mL using the rotary evaporator
(Section 12.6.1) or heating mantle (Section 12.6.2), transfer to a 250-mL separatory funnel,
and proceed with back-extraction (Section 12.5). Set aside the concentration device for use
after back-extraction or other cleanup.
12.4 Soxhlet extraction of tissue (References 3 and 15)
Note: This procedure includes determination of the lipid content of the sample (Sections 12.4.8 -
12.4.9), using the same sample extract that is analyzed by GC/MS. Alternatively, a separate sample
aliquot may be used for the lipid determination. If a separate aliquot is used, use nitrogen to evaporate
the main portion of the sample extract only to the extent necessary to effect the solvent exchange to n-
hexane, so that loss of low molecular weight CBs is avoided, i.e., it is not necessary to dry the main
portion of the sample to constant weight (Section 12.4.8).
12.4.1 Add 30 to 40 g of powdered anhydrous sodium sulfate (Section 7.2.2) to each of the
beakers (Section 11.8.4) and mix thoroughly. Cover the beakers with aluminum foil and
dry until the mixture becomes a free-flowing powder (30 minutes minimum). Remix prior
to extraction to prevent clumping.
12.4.2 Assemble and pre-extract the Soxhlet apparatus per Sections 12.3.1-12.3.4, except use
methylene chloride for the pre-extraction and rinsing and omit the quartz sand.
12.4.3 Reassemble the pre-extracted Soxhlet apparatus and add a fresh charge of methylene
chloride to the reflux flask.
12.4.4 Transfer the sample/sodium sulfate mixture (Section 12.4.1) to the Soxhlet thimble, and
install the thimble in the Soxhlet apparatus.
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1 2.4.5 Rinse the beaker with several portions of solvent and add to the thimble. Fill the
thimble/receiver with solvent. Extract for 18-24 hours.
12.4.6 After extraction, cool and disassemble the apparatus.
1 2.4.7 Quantitatively transfer the extract to a macro-concentration device (Section 12.6), and
concentrate to near dryness. Set aside the concentration apparatus for re-use.
12.4.8 Complete the removal of the solvent using the nitrogen blow evaporation procedure
(Section 12.7) and a water bath temperature of 60 °C. Weigh the receiver, record the
weight, and return the receiver to the blowdown apparatus, concentrating the residue until a
constant weight is obtained.
12.4.9 Percent lipid determination
12.4.9.1 Redissolve the residue in the receiver in hexane and spike 1.0 mL of the Labeled
cleanup standard spiking solution (Section 7.13) into the solution.
1 2.4.9.2 Transfer the residue/hexane to the anthropogenic isolation column (Section
13.6), retaining the boiling chips in the concentration apparatus. Use several
rinses to assure that all material is transferred. If necessary, sonicate or heat the
receiver slightly to assure that all material is re-dissolved. Allow the receiver to
dry. Weigh the receiver and boiling chips.
1 2.4.9.3 Calculate the lipid content to the nearest three significant figures as follows:
weight of tissue (g)
1 2.4.9.4 The laboratory should determine the lipid content of the blank, IPR, and OPR to
assure that the extraction system is working effectively.
1 2.5 Back-extraction with base and acid
12.5.1 Back-extraction may not be necessary for some samples. For some samples, the presence
of color in the extract may indicate that back-extraction is necessary. If back-extraction is
not necessary, spike 1.0 mL of the Labeled cleanup standard spiking solution (Section 7.13)
into the extract and concentrate the extract for cleanup or analysis (Sections 12.6 and 12.7).
If back-extraction is necessary, spike 1.0 mL of the Labeled cleanup standard spiking
solution (Section 7.13) into the separatory funnels containing the sample and QC extracts
from Section 12.2.3.4 or 12.3.9.
1 2.5.2 Partition the extract against 50 mL of potassium hydroxide solution (Section 7.1.1). Shake
for 2 minutes with periodic venting into a hood. Remove and discard the aqueous layer.
Repeat the base washing until no color is visible in the aqueous layer, to a maximum of
four washings. Minimize contact time between the extract and the base to prevent degrada-
tion of the CBs. Stronger potassium hydroxide solutions may be employed for back-
extraction, provided that the laboratory meets the specifications for labeled compound
recovery and demonstrates acceptable performance using the procedure in Section 9.2.
12.5.3 Partition the extract against 50 mL of sodium chloride solution (Section 7.1.4) in the same
way as with base. Discard the aqueous layer.
EPA Method 1 668C 38 April 2010
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12.5.4 Partition the extract against 50 mL of sulfuric acid (Section 7.1.2) in the same way as with
base. Repeat the acid washing until no color is visible in the aqueous layer, to a maximum
of four washings.
12.5.5 Repeat the partitioning against sodium chloride solution and discard the aqueous layer.
12.5.6 Pour each extract through a drying column containing 7 to 10 cm of granular anhydrous
sodium sulfate (Section 7.2.1) into a macro-concentration device (Section 12.6). If a
concentration device was set aside from extraction, that concentration device may be re-
used. Rinse the separatory funnel with 30 to 50 mL of solvent, and pour through the drying
column. Re-concentrate the sample and QC aliquots per Sections 12.6-12.7, and clean up
the samples and QC aliquots per Section 13.
12.6 Macro-concentration - Extracts in toluene are concentrated using a rotary evaporator or a heating
mantle; extracts in methylene chloride or hexane are concentrated using a rotary evaporator, heating
mantle, or Kuderna-Danish apparatus.
Note: In the concentration procedures below, the extract must not be allowed to concentrate to dryness
because the mono- through tri-chlorobiphenyls may be totally or partially lost.
12.6.1 Rotary evaporation - Concentrate the extracts in separate round-bottom flasks.
12.6.1.1 Assemble the rotary evaporator according to manufacturer's instructions, and
warm the water bath to 45 °C. On a daily basis, pre-clean the rotary evaporator
by concentrating 100 mL of clean extraction solvent through the system.
Archive both the concentrated solvent and the solvent in the catch flask for a
contamination check if necessary. Between samples, three 2- to 3- mL aliquots
of solvent should be rinsed down the feed tube into a waste beaker.
12.6.1.2 Attach the round-bottom flask containing the sample extract to the rotary
evaporator. Slowly apply vacuum to the system, and begin rotating the sample
flask.
12.6.1.3 Lower the flask into the water bath, and adjust the speed of rotation and the
temperature as required to complete concentration in 15 to 20 minutes. At the
proper rate of concentration, the flow of solvent into the receiving flask will be
steady, but no bumping or visible boiling of the extract will occur.
Note: If the rate of concentration is too fast, analyte loss may occur.
12.6.1.4 When the liquid in the concentration flask has reached an apparent volume of
approximately 2 mL, remove the flask from the water bath and stop the rotation.
Slowly and carefully admit air into the system. Be sure not to open the valve so
quickly that the sample is blown out of the flask. Rinse the feed tube with
approximately 2 mL of solvent.
12.6.1.5 Proceed to Section 12.6.4 for preparation for back-extraction or micro-
concentration and solvent exchange.
EPA Method 1668C 39 April 2010
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12.6.2 Heating mantle - Concentrate the extracts in separate round-bottom flasks.
12.6.2.1 Add one or two clean boiling chips to the round-bottom flask, and attach a
three-ball macro Snyder column. Prewet the column by adding approximately 1
mL of solvent through the top. Place the round-bottom flask in a heating
mantle, and apply heat 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.
12.6.2.2 When the liquid has reached an apparent volume of approximately 10 mL,
remove the round-bottom flask from the heating mantle and allow the solvent to
drain and cool for at least 10 minutes. Remove the Snyder column and rinse the
glass joint into the receiver with small portions of solvent.
12.6.2.3 Proceed to Section 12.6.4 for preparation for back-extraction or micro-
concentration and solvent exchange.
12.6.3 Kuderna-Danish (K-D) - Concentrate the extracts in separate 500-mL K-D flasks equipped
with 10-mL concentrator tubes. The K-D technique is used for solvents such as methylene
chloride and hexane. Toluene is difficult to concentrate using the K-D technique unless a
water bath fed by a steam generator is used.
12.6.3.1 Add 1 to 2 clean boiling chips to the receiver. Attach a three-ball macro Snyder
column. Prewet the column by adding approximately 1 mL of solvent 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.
12.6.3.2 Adjust the vertical position of the apparatus and the water temperature 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.
12.6.3.3 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 to 2 mL of solvent. A 5-mL syringe is
recommended for this operation.
12.6.3.4 Remove the three-ball Snyder column, add a fresh boiling chip, and attach a two
ball micro Snyder column to the concentrator tube. Prewet the column by
adding approximately 0.5 mL of solvent through the top. Place the apparatus in
the hot water bath.
12.6.3.5 Adjust the vertical position and the water temperature as required to complete
the concentration in 5 to 10 minutes. At the proper rate of distillation, the balls
of the column will actively chatter but the chambers will not flood.
12.6.3.6 When the liquid reaches an apparent volume of 0.5 mL, remove the apparatus
from the water bath and allow to drain and cool for at least 10 minutes.
12.6.3.7 Proceed to 12.6.4 for preparation for back-extraction or micro-concentration and
solvent exchange.
EPA Method 1668C 40 April 2010
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12.6.4 Preparation for back-extraction or micro-concentration and solvent exchange
12.6.4.1 For back-extraction (Section 12.5), transfer the extract to a 250-mL separatory
funnel. Rinse the concentration vessel with small portions of hexane, adjust the
hexane volume in the separatory funnel to 10 to 20 mL, and proceed to back-
extraction (Section 12.5).
12.6.4.2 For determination of the weight of residue in the extract, or for clean-up
procedures other than back-extraction, transfer the extract to a blowdown vial
using 2-3 rinses of solvent. Proceed with micro-concentration and solvent
exchange (Section 12.7).
12.7 Micro-concentration and solvent exchange
12.7.1 Extracts to be subjected to GPC cleanup are exchanged into methylene chloride. Extracts
to be cleaned up using silica gel, carbon, Florisil, and/or HPLC are exchanged into hexane.
12.7.2 Transfer the vial containing the sample extract to a nitrogen evaporation device. Adjust the
flow of nitrogen so that the surface of the solvent is just visibly disturbed.
Note: A large vortex in the solvent may cause analyte loss.
12.7.3 Lower the vial into a 45 °C water bath and continue concentrating.
12.7.3.1 If the extract or an aliquot of the extract is to be concentrated to dryness for
weight determination (Sections 12.4.8 and 13.6.4), blow dry until a constant
weight is obtained.
12.7.3.2 If the extract is to be concentrated for injection into the GC/MS or the solvent is
to be exchanged for extract cleanup, proceed as follows:
12.7.4 When the volume of the liquid is approximately 100 \\L, add 2 to 3 mL of the desired
solvent (methylene chloride for GPC and HPLC, or hexane for the other cleanups) and
continue concentration to approximately 100 \\L. Repeat the addition of solvent and
concentrate once more.
12.7.5 If the extract is to be cleaned up by GPC, adjust the volume of the extract to 5.0 mL with
methylene chloride. If the extract is to be cleaned up by HPLC, concentrate the extract to
1.0 mL. Proceed with GPC or HPLC cleanup (Section 13.2 or 13.5, respectively).
12.7.6 If the extract is to be cleaned up by column chromatography (silica gel, Carbopak/Celite, or
Florisil), bring the final volume to 1.0 mL with hexane. Proceed with column cleanup
(Sections 13.3, 13.4, or 13.7).
12.7.7 If the extract is to be concentrated for injection into the GC/MS (Section 14), quantitatively
transfer the extract to a 0.3-mL conical vial for final concentration, rinsing the larger vial
with hexane and adding the rinse to the conical vial. Reduce the volume to approximately
100 \\L. Add 20 \\L of nonane to the vial, and evaporate the solvent to the level of the
nonane. Seal the vial and label with the sample number. Store in the dark at room temper-
ature until ready for GC/MS analysis. If GC/MS analysis will not be performed on the
same day, store the vial at less than -10 °C.
EPA Method 1668C 41 April 2010
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13.0 Extract cleanup
13.1 Cleanup may not be necessary for relatively clean samples (e.g., treated effluents, groundwater,
drinking water). If particular circumstances require the use of a cleanup procedure, the laboratory
may use any or all of the procedures below or any other appropriate procedure. Before using a
cleanup procedure, the laboratory must demonstrate that the requirements of Section 9.2 can be met
using the cleanup procedure.
13.1.1 Gel permeation chromatography (Section 13.2) removes high molecular weight
interferences that cause GC column performance to degrade. It should be used for all soil
and sediment extracts. It may be used for water extracts that are expected to contain high
molecular weight organic compounds (e.g., polymeric materials, humic acids). It should
also be used for tissue extracts after initial cleanup on the anthropogenic isolation column
(Section 13.6).
13.1.2 Acid, neutral, and basic silica gel (Section 13.3) and Florisil (Section 13.7) are used to
remove non-polar and polar interferences.
13.1.3 Carbopak/Celite (Section 13.4) can be used to separate CBs 77, 126, and 169 from the
mono- and di- ortho-substituted CBs, if desired.
13.1.4 HPLC (Section 13.5) is used to provide specificity for certain congeners and congener
groups.
13.1.5 The anthropogenic isolation column (Section 13.6) is used for removal of lipids from tissue
samples.
13.2 Gel permeation chromatography (GPC)
13.2.1 Column packing
13.2.1.1 Place 70 to 75 g of SX-3 Bio-beads (Section 6.7.1.1) in a 400- to 500-mL
beaker.
13.2.1.2 Cover the beads with methylene chloride and allow to swell overnight (a
minimum of 12 hours).
13.2.1.3 Transfer the swelled beads to the column (Section 6.7.1.1) and pump solvent
through the column, from bottom to top, at 4.5 to 5.5 mL/minute prior to
connecting the column to the detector.
13.2.1.4 After purging the column with solvent for 1 to 2 hours, adjust the column head
pressure to 7 to 10 psig and purge for 4 to 5 hours to remove air. Maintain a
head pressure of 7 to 10 psig. Connect the column to the detector (Section
6.7.1.4).
13.2.2 Column calibration
13.2.2.1 Load 5 mL of the GPC calibration solution (Section 7.4) into the sample loop.
13.2.2.2 Inject the GPC calibration solution and record the signal from the detector. The
elution pattern will be corn oil, BEHP, methoxychlor, perylene, and sulfur.
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13.2.2.3 Set the "dump time" to allow >85% removal of BEHP and >85% collection of
methoxychlor.
13.2.2.4 Set the "collect time" to the time of the sulfur peak maximum.
13.2.2.5 Verify calibration with the GPC calibration solution after every 20 extracts.
Calibration is verified if the recovery of the methoxychlor is greater than 85%.
If calibration is not verified, the system must be recalibrated using the GPC
calibration solution, and the previous sample batch must be re-extracted and
cleaned up using the calibrated GPC system.
13.2.3 Extract cleanup - GPC requires that the column not be overloaded. The column specified
in this Method is designed to handle a maximum of 0.5 g of material from an aqueous, soil,
or mixed-phase sample in a 5-mL extract, and has been shown to handle 1.5 g of lipid from
a tissue sample in a 5-mL extract. If the extract is known or expected to contain more than
these amounts, the extract is split into aliquots for GPC, and the aliquots are combined after
elution from the column. The residue content of the extract may be obtained gravimetri-
cally by evaporating the solvent from a 50-(iL aliquot.
13.2.3.1 Filter the extract or load through the filter holder (Section 6.7.1.3) to remove
particles. Load the 5.0-mL extract onto the column.
13.2.3.2 Elute the extract using the calibration data determined in Section 13.2.2. Collect
the eluate in a clean 400- to 500-mL beaker. Allow the system to rinse for
additional 10 minutes before injecting the next sample.
13.2.3.3 Rinse the sample loading tube thoroughly with methylene chloride between
extracts to prepare for the next sample.
13.2.3.4 If an extract is encountered that could overload the GPC column to the extent
that carry-over could occur, a 5.0-mL methylene chloride blank must be run
through the system to check for carry-over.
13.2.3.5 Concentrate the eluate per Sections 12.6 and 12.7 for further cleanup or
injection into the GC/MS.
13.3 Silica gel cleanup
13.3.1 Place a glass-wool plug in a 15-mm ID chromatography column (Section 6.7.4.2). Pack
the column bottom to top with: 1 g silica gel (Section 7.5.1.1), 4 g basic silica gel (Section
7.5.1.3), 1 g silica gel, 8 g acid silica gel (Section 7.5.1.2), 2 g silica gel, and 4 g granular
anhydrous sodium sulfate (Section 7.2.1). Tap the column to settle the adsorbents.
13.3.2 Pre-elute the column with 50 to 100 mL of hexane. Close the stopcock when the hexane is
within 1 mm of the sodium sulfate. Discard the eluate. Check the column for channeling.
If channeling is present, discard the column and prepare another.
13.3.3 Apply the concentrated extract to the column. Open the stopcock until the extract is within
1 mm of the sodium sulfate.
13.3.4 Rinse the receiver twice with 1-mL portions of hexane, and apply separately to the column.
Elute the CBs with 25 mL of hexane and collect the eluate.
EPA Method 1668C 43 April 2010
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13.3.5 Concentrate the eluate per Section 12.6 and 12.7 for further cleanup or injection into the
HPLCorGC/MS.
13.3.6 For extracts of samples known to contain large quantities of other organic compounds, it
may be advisable to increase the capacity of the silica gel column. This may be
accomplished by increasing the strengths of the acid and basic silica gels. The acid silica
gel (Section 7.5.1.2) may be increased in strength to as much as 40% w/w (6.7 g sulfuric
acid added to 10 g silica gel). The basic silica gel (Section 7.5.1.3) may be increased in
strength to as much as 33% w/w (50 mL IN NaOH added to 100 g silica gel), or the
potassium silicate (Section 7.5.1.4) may be used.
Note: The use of stronger acid silica gel (44% w/w) may lead to charring of organic compounds in some
extracts. The charred material may retain some of the analytes and lead to lower recoveries of the CBs.
Increasing the strengths of the acid and basic silica gel may also require different volumes ofhexane than
those specified above to elute the analytes from the column. The performance of the Method after such
modifications must be verified by the procedure in Section 9.2.
13.4 Carbon column (Reference 16)
13.4.1 Cut both ends from a 50-mL disposable serological pipet (Section 6.7.3.2) to produce a 20-
cm column. Fire-polish both ends and flare both ends if desired. Insert a glass-wool plug
at one end, and pack the column with 3.6 g of Carbopak/Celite (Section 7.5.2.3) to form an
adsorbent bed 20 cm long. Insert a glass-wool plug on top of the bed to hold the adsorbent
in place.
13.4.2 Pre-elute the column with 20 mL each in succession of toluene, methylene chloride, and
hexane.
13.4.3 When the solvent is within 1 mm of the column packing, apply the n-hexane sample extract
to the column. Rinse the sample container twice with 1-mL portions ofhexane and apply
separately to the column. Apply 2 mL ofhexane to complete the transfer.
13.4.4 Elute the column with 25 mL of n-hexane and collect the eluate. This fraction will contain
the mono- and di-ortho CBs. If carbon particles are present in the eluate, filter through
glass-fiber filter paper.
13.4.5 Elute the column with 15 mL of methanol and discard the eluate. The fraction discarded
will contain residual lipids and other potential interferents, if present.
13.4.6 Elute the column with 15 mL of toluene and collect the eluate. This fraction will contain
CBs 77, 126, and 169. If carbon particles are present in the eluate, filter through glass-fiber
filter paper.
13.4.7 Concentrate the fractions per Section 12.6 and 12.7 for further cleanup or injection into the
HPLCorGC/MS.
EPA Method 1668C 44 April 2010
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13.5 HPLC (References 4 and 17)
13.5.1 Column calibration
13.5.1.1 Prepare a calibration standard containing the Toxics and other congeners of
interest at the concentrations of the stock solution in Table 3, or at a
concentration appropriate to the response of the detector.
13.5.1.2 Inject the calibration standard into the HPLC and record the signal from the
detector. Collect the eluant for reuse. Elution will be in the order of the di-
ortho, mono-ortho, and non-ortho congeners.
13.5.1.3 Establish the collection time for the congeners of interest. Following
calibration, flush the injection system with solvent to ensure that residual CBs
are removed from the system.
13.5.1.4 Verify the calibration with the calibration solution after every 20 extracts.
Calibration is verified if the recovery of the CBs is 75 to 125% compared to the
calibration (Section 13.5.1.1). If calibration is not verified, the system must be
recalibrated using the calibration solution, and the previous 20 samples must be
re-extracted and cleaned up using the calibrated system.
13.5.2 Extract cleanup - HPLC requires that the column not be overloaded. The column specified
in this Method is designed to handle a maximum of 5-50 (ig of a given CB, depending on
the congener (Reference 17). If the amount of material in the extract will overload the
column, split the extract into fractions and combine the fractions after elution from the
column.
13.5.2.1 Rinse the sides of the vial containing the sample and adjust to the volume
required for the sample loop for injection.
13.5.2.2 Inject the sample extract into the HPLC.
13.5.2.3 Elute the extract using the calibration data determined in Section 13.5.1. Collect
the fraction(s) in clean 20-mL concentrator tubes.
13.5.2.4 If an extract containing greater than 500 (ig of total CBs is encountered, a blank
must be run through the system to check for carry-over.
13.5.2.5 Concentrate the eluate per Section 12.7 for injection into the GC/MS.
13.6 Anthropogenic isolation column (Reference 3) - Used for removal of lipids from tissue extracts
13.6.1 Prepare the column as given in Section 7.5.3.
13.6.2 Pre-elute the column with 100 mL of hexane. Drain the hexane layer to the top of the
column, but do not expose the sodium sulfate.
13.6.3 Load the sample and rinses (Section 12.4.9.2) onto the column by draining each portion to
the top of the bed. Elute the CBs from the column into the apparatus used for concentration
(Section 12.4.7) using 200 mL of hexane.
EPA Method 1668C 45 April 2010
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13.6.4 Remove a small portion (e.g., 50 (iL) of the extract for determination of residue content.
Estimate the percent of the total that this portion represents. Concentrate the small portion
to constant weight per Section 12.7.3.1. Calculate the total amount of residue in the
extract. If more than 500 mg of material remains, repeat the cleanup using a fresh
anthropogenic isolation column.
13.6.5 If necessary, exchange the extract to a solvent suitable for the additional cleanups to be
used (Section 13.2-13.5 and 13.7).
13.6.6 Clean up the extract using the procedures in Sections 13.2-13.5 and 13.7. GPC (Section
13.2) and Florisil (Section 13.7) are recommended as minimum additional cleanup steps.
13.6.7 Following cleanup, concentrate the extract to 20 (iL as described in Section 12.7 and
proceed with the analysis in Section 14.
13.7 Florisil cleanup (Reference 18)
13.7.1 Begin to drain the n-hexane from the column (Section 7.5.4.1.2). Adjust the flow rate of
eluant to 4.5-5.0 mL/min.
13.7.2 When the n-hexane is within 1 mm of the sodium sulfate, apply the sample extract (in
hexane) to the column. Rinse the sample container twice with 1-mL portions of hexane and
apply to the column.
13.7.3 Elute the mono-ortho and di-ortho CBs with approx 165 mL of n-hexane and collect the
eluate. Elute the non-ortho co-planar CBs with approx 100 mL of 6% etherhexane and
collect the eluate. The exact volumes of solvents will need to be determined for each batch
of Florisil. If the mono/di-ortho CBs are not to be separated from the non-ortho co-planar
CBs, elute all CBs with 6% etherhexane.
13.7.4 Concentrate the eluate(s) per Sections 12.6-12.7 for further cleanup or for injection into the
HPLCorGC/MS.
14.0 HRGC/HRMS analysis
14.1 Establish the operating conditions given in Section 10.1.
14.2 Add 2 (iL of the labeled injection internal standard spiking solution (Section 7.14) to the 20 (iL
sample extract immediately prior to injection to minimize the possibility of loss by evaporation,
adsorption, or reaction. If an extract is to be reanalyzed and evaporation has occurred, do not add
more labeled injection internal standard spiking solution. Rather, bring the extract back to its
previous volume (e.g., 19 (iL) with pure nonane (18 (iL if 2 (iL injections are used).
14.3 Inject 1.0 or 2.0 (iL of the concentrated extract containing the Labeled injection internal standards
using on-column or splitless injection. The volume injected must be identical to the volume used
for calibration (Section 10.3).
14.3.1 Start the GC column initial isothermal hold upon injection. Start MS data collection after
the solvent peak elute s.
EPA Method 1668C 46 April 2010
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14.3.2 Monitor the exact m/z's at each LOC throughout the LOC retention time window. Where
warranted, monitor m/z's associated with congeners at higher levels of chlorination to
assure that fragments are not interfering with the m/z's for congeners at lower levels of
chlorination. Also where warranted, monitor m/z's associated with interferents expected
to be present.
14.3.3 Stop data collection after 13Ci2-DeCB has eluted. Return the column to the initial
temperature for analysis of the next extract or standard.
15.0 System and laboratory performance
15.1 At the beginning of each 12-hour shift during which analyses are performed, GC/MS system
performance and calibration are verified for all native CBs and labeled compounds. For these tests,
analyze the diluted combined 209 congener solution (Section 7.10.2.2) to verify all performance
criteria. Adjustment and/or recalibration (Section 10) must be performed until all performance
criteria are met. Only after all performance criteria are met may samples, blanks, IPRs, and OPRs
be analyzed.
15.2 MS resolution - Static resolving power checks must be performed at the beginning and at the end of
each shift per Section 10.2.1. If analyses are performed on successive shifts, only the beginning of
shift static resolving power check is required. If the requirement in Section 10.2.1 cannot be met,
the problem must be corrected before analyses can proceed. If any of the samples in the previous
shift may be affected by poor resolution, those samples must be re-analyzed.
15.3 Calibration verification
15.3.1 Inject and analyze the Diluted combined 209 congener solution (Section 7.10.2.2.2) using
the procedure in Section 14.
15.3.2 The m/z abundance ratios for each native CB and labeled compound in the VER standard
must be within the limits in Table 8; otherwise, the mass spectrometer must be adjusted
until the m/z abundance ratios fall within the limits specified when the verification test is be
repeated. If the adjustment alters the resolution of the mass spectrometer, resolution must
be verified (Section 10.2.1) prior to repeat of the verification test.
15.3.3 The GC peak representing each native CB and labeled compound in the VER standard must
be present with a S/N of at least 10; otherwise, the mass spectrometer must be adjusted and
the verification test repeated.
15.3.4 Compute the recovery of the Toxics/LOC CBs by isotope dilution (Section 17.1) and the
labeled compounds by internal standard (17.2). These recoveries are computed based on
the calibration data in Section 10.
15.3.5 For each compound, compare the recovery with the calibration verification limit in Table 6.
If all compounds meet the acceptance criteria, calibration has been verified and analysis of
standards and sample extracts may proceed. If, however, any compound fails its respective
limit, the measurement system is not performing properly. In this event, prepare a fresh
calibration standard or correct the problem and repeat the resolution (Section 15.2) and
verification (Section 15.3) tests, or recalibrate (Section 10). If recalibration is required,
recalibration for the 209 congeners (Section 10.5) must also be performed.
EPA Method 1668C 47 April 2010
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15.4 Retention times and GC resolution
15.4.1 Retention times
15.4.1.1 Absolute - The absolute retention times of the Labeled Toxics/LOC/window
defining standard congeners (Section 7.12) in the verification test (Section 15.3)
must be within ± 15 seconds of the respective retention times in the calibration
or, if an alternate column or column system is employed, within ±15 seconds of
the respective retention times in the calibration for the alternate column or
column system (Section 6.9.1.2).
15.4.1.2 Relative - The relative retention times of native CBs and labeled compounds in
the verification test (Section 15.3) must be within their respective RRT limits in
Table 2 or, if an alternate column or column system is employed, within their
respective RRT limits for the alternate column or column system (Section
6.9.1.2).
15.4.1.3 If the absolute or relative retention time of any compound is not within the
limits specified, the GC is not performing properly. In this event, adjust the GC
and repeat the verification test (Section 15.3) or recalibrate (Section 10), or
replace the GC column and either verify calibration or recalibrate.
15.4.2 GC resolution and minimum analysis time
15.4.2.1 As a final step in calibration verification, GC resolution and minimum analysis
time are verified and response factors for congeners other than the Toxics and
LOC CBs are updated.
15.4.2.2 The resolution and minimum analysis time specifications in Sections 6.9.1.1.2
and 6.9.1.1.1, respectively, must be met for the SPB-octyl column or, if an
alternate column or column system is employed, must be met as specified for
the alternate column or column system (Section 6.9.1.2). If these specifications
are not met, the GC analysis conditions must be adjusted until the specifications
are met, or the column must be replaced and the calibration verification tests
repeated Sections 15.4.1 through 15.4.2.2), or the system must be recalibrated
(Section 10).
15.4.2.3 After the resolution and minimum analysis time specifications are met, update
the retention times and relative retention times for all congeners, and response
factors for all congeners except the Toxics and LOC CBs. For the Toxics and
LOC CBs, the multi-point calibration data must be used ( Section 10.4) and
verified (Section 15.3.4).
15.5 Ongoing precision and recovery
15.5.1 Analyze the extract of the ongoing precision and recovery (OPR) aliquot (Section 11.4.2.5,
11.5.4, 11.6.2, or 11.8.3.2) prior to analysis of samples from the same batch.
15.5.2 Compute the percent recovery of the Toxics/LOC CBs by isotope dilution (Section 10.4).
Compute the percent recovery of each labeled compound by the internal standard method
(Section 10.5).
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15.5.3 For the Toxics/LOC CBs and labeled compounds, compare the recovery to the OPR limits
given in Table 6. If all compounds meet the acceptance criteria, system performance is
acceptable and analysis of blanks and samples may proceed. If, however, any individual
concentration falls outside of the range given, the extraction/concentration processes are
not being performed properly for that compound. In this event, correct the problem, re-
prepare, extract, and clean up the sample batch and repeat the ongoing precision and
recovery test (Section 15.5).
15.5.4 If desired, add results that pass the specifications in Section 15.5.3 to initial and previous
ongoing data for each compound in each matrix. Update QC charts to form a graphic
representation of continued laboratory performance. Develop a statement of laboratory
accuracy for each congener in each matrix type by calculating the average percent 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 accuracy is 85
to 105%.
15.6 Blank - Analyze the Method blank extracted with each sample batch immediately following
analysis of the OPR aliquot to demonstrate freedom from contamination and freedom from
carryover from the OPR analysis. If CBs will be carried from the OPR into the Method blank,
analyze one or more aliquots of solvent between the OPR and the Method blank. The results of the
analysis of the blank must meet the specifications in Section 9.5.2 before sample analyses may
proceed.
16.0 Qualitative determination
A CB or labeled compound is identified in a standard, blank, or sample when all of the criteria in
Sections 16.1 through 16.4 are met.
16.1 The signals for the two exact m/z's in Table 7 must be present and must maximize within the same
two scans.
16.2 The signal-to-noise ratio (S/N) for the GC peak at each exact m/z must be greater than or equal to
2.5 for each CB detected in a sample extract, and greater than or equal to 10 for all CBs in the
calibration and verification standards (Sections 10.3.3 and 15.3.3).
Note: An interference between DiCB m/z 223.9974 and PFKm/z 223.9872 may preclude meeting the
S/N requirement for the DiCB congeners. If identification is ambiguous, an experienced spectrometrist
(Section 1.4) must determine the presence or absence of the congener.
16.3 The ratio of the integrated areas of the two exact m/z's specified in Table 7 must be within the limit
in Table 8, or within ± 15 percent of the ratio in the midpoint (CS-3) calibration or calibration
verification (VER), whichever is most recent.
16.4 The relative retention time of the peak for a CB must be within the RRT QC limits specified in
Table 2 or within similar limits developed from calibration data (Section 10.1.2). If an alternate
column or column system is employed, the RRT for the CB must be within its respective RRT QC
limits for the alternate column or column system (Section 6.9.1.2).
EPA Method 1668C 49 April 2010
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Note: For native CBs determined by internal standard quantitation, a given CB congener may fall
within more than one RT window and be mis-identified unless the RRT windows are made very narrow,
as in Table 2. Therefore, consistency of the RT and RRT with other congeners and the labeled
compounds may be required for rigorous congener identification. Retention time regression analysis may
aid in this identification.
16.5 Because of congener overlap and the potential for interfering substances, it is possible that all of the
identification criteria (Sections 16.1-16.4) may not be met. It is also possible that loss of one or
more chlorines from a highly chlorinated congener may inflate or produce a false concentration for
a less-chlorinated congener that elutes at the same retention time (see Section 18.5). If
identification is ambiguous, an experienced spectrometrist (Section 1.4) must determine the
presence or absence of the congener.
16.6 If the criteria for identification in Sections 16.1-16.5 are not met, the CB has not been identified and
the result for that congener may not be reported or used for permitting or regulatory compliance
purposes. If interferences preclude identification, a new aliquot of sample must be extracted,
further cleaned up, and analyzed.
17.0 Quantitative determination
17.1 Isotope dilution quantitation
17.1.1 By adding a known amount of the Labeled Toxics/LOC/window-defining compounds to
every sample prior to extraction, correction for recovery of the CBs can be made because
the native compound and its labeled analog exhibit similar effects upon extraction,
concentration, and gas chromatography. Relative responses (RRs) are used in conjunction
with the calibration data in Section 10.4 to determine concentrations in the final extract, so
long as labeled compound spiking levels are constant.
17.1.2 Compute the concentrations in the extract of the Native Toxics/LOC CBs using the RRs
from the calibration data (Section 10.4) and following equation:
hA2!)RR
where:
Cex = concentration of the PCB in the extract (ng/mL) and the other terms are as
defined in Section 10.5.1
17.2 Internal standard quantitation and labeled compound recovery
17.2.1 Compute the concentrations in the extract of the labeled compounds (except labeled CB
178) and of the native compounds other than those in the Native Toxics/LOC standard
using the response factors determined from calibration (Section 10.5) or calibration
verification (Section 15.4.2.3) and the following equation:
EPA Method 1668C 50 April 2010
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(ng/mL) =---
"'B li, + A2i,)RF
where:
Cex = concentration of the native or labeled compound in the extract (ng/mL) and the
other terms are as defined in Section 10.5.1
17.2.2 Using the concentration in the extract determined above, compute the percent recovery of
the Labeled Toxics/LOC/window-defming CBs and the Labeled cleanup standard CBs
using the following equation:
_. ,,, Concentration found (ng/mL) ,_
Recovery (%) = x 100
Concentration spiked (ng/mL)
17.3 The concentration of a native CB in the solid phase of the sample is computed using the
concentration of the compound in the extract and the weight of the solids (Section 11.2.2.3), as
follows:
C V
Concentration in solid sample (ng/kg) =
where:
Cex = The concentration of the compound in the extract (ng/mL).
Vex = The extract volume in mL.
Ws = The sample weight (dry weight) in kg.
17.4 The concentration of a native CB in the aqueous phase of the sample is computed using the
concentration of the compound in the extract and the volume of water extracted (Section 11.4.2.1),
as follows:
C V
Concentration in aqueous sample (ng/L) = - x 1000
*s
where:
Cex = The concentration of the compound in the extract (pg/mL).
Vex = The extract volume in mL.
Vs = The sample volume in liters.
17.5 If the SICP area at either quantitation m/z for any congener exceeds the calibration range of the
system, dilute the sample extract by the factor necessary to bring the concentration within the
calibration range, adjust the concentration of the Labeled injection internal standard to 100 pg/(iL in
the extract, and analyze an aliquot of this diluted extract. If the CBs cannot be measured reliably by
isotope dilution, dilute and analyze an aqueous sample or analyze a smaller portion of a soil, tissue,
or mixed-phase sample. Adjust the CB congener concentrations, detection limits, and minimum
levels to account for the dilution.
17.6 Reporting of results - Results are reported to three significant figures for the CBs and labeled
compounds found in all standards, blanks, and samples.
17.6.1 Reporting units and levels
17.6.1.1 Aqueous samples - Report results in pg/L (parts-per-quadrillion).
EPA Method 1668C 51 April 2010
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17.6.1.2 Samples containing greaterthan 1% solids (soils, sediments, filter cake,
compost) - Report results in ng/kg based on the dry weight of the sample.
Report the percent solids so that the result may be converted to aqueous units.
17.6.1.3 Tissues - Report results in ng/kg of wet tissue, not on the basis of the lipid
content of the tissue. Report the percent lipid content, so that the data user can
calculate the concentration on a lipid basis if desired.
17.6.1.4 Reporting level
17.6.1.4.1 Report the result for each congener at or above the minimum level
of quantitation (ML; Table 2) for analyses of blanks, standards,
and samples. The MLs in Table 2 are the levels that can be
achieved in the presence of common laboratory contamination. A
laboratory may establish an ML for a CB congener lower than the
MLs in Table 2. MLs may be established as low as the lowest
calibration point (Table 5) provided that the concentration of the
congener in a minimum of 10 blanks for a sample medium (e.g.,
water, soil, sludge, tissue) is significantly below the ML in Table
2. "Significant" means that the ML for the congener is no less than
2 standard deviations above the mean (average) level in the
minimum of 10 blanks (Reference 19). The blanks must be
analyzed during the same period that samples are analyzed, ideally
over an approximately 1-month period.
17.6.1.4.2 Standards (VER, IPR, OPR) and samples - Report the result for
each congener at or above the ML (Table 2) to 3 significant
figures. Report results below the ML as
-------
17.6.4 If requested, the total concentration of all congeners at a given level of chlorination
(homolog; i.e., total TrCB, total PeCB, total HxCB) may be reported by summing the
concentrations of all congeners identified at that LOG, including both the Toxics and other
congeners. Also if requested, total CBs may be reported by summing all congeners
identified at all LOCs.
17.6.5 Reporting of coeluting PCB congeners-Optionally, Delaware River Basin Commission
(DRBC) data qualifier flags and conventions for reporting coeluting congeners (see
http://www.state.nj.us/drbc/PCB_info.htm), or other reporting convention agreed upon
between the laboratory and the discharger/permittee or regulatory/control authority, may be
used.
18.0 Analysis of complex samples
18.1 Some samples may contain high levels (>10 ng/L; >1000 ng/kg) of the compounds of interest,
interfering compounds, and/or polymeric materials. Some extracts may not concentrate to 20 (iL
(Section 12.7.7); others may overload the GC column and/or mass spectrometer. Fragment ions
from congeners at higher levels of chlorination may interfere with determination of congeners at
lower levels of chlorination.
18.2 Analyze a smaller aliquot of the sample (Section 17.5) when the extract will not concentrate to 20
(iL after all cleanup procedures have been exhausted. If a smaller aliquot of soils or mixed-phase
samples is analyzed, attempt to assure that the sample is representative.
18.3 Perform integration of peak areas and calculate concentrations manually when interferences
preclude computerized calculations.
18.4 Several laboratories have reported that backgrounds of many of the CB congeners are difficult to
eliminate, and that these backgrounds can interfere with the determination of the CBs in
environmental samples. Backgrounds of Toxics with congener numbers 105, 114, 118, 123, 156,
157, and 167 are common. The effects of contamination on results for these congeners should be
understood in order to make a reliable determination.
18.5 Interferences may pose a problem in the determination of congeners 81, 123, 126, and 169 in some
environmental samples. Loss of one or more chlorines from a highly chlorinated congener may
inflate or produce a false concentration for a less-chlorinated congener that elutes at the same
retention time. If, upon inspection of the chromatogram, the possibility of interferences is evident
(e.g., high concentrations of fragments from loss of one or two chlorines from higher chlorinated
closely eluting congeners), carbon column fractionation (Section 13.4) and analysis is
recommended.
18.6 Recovery of labeled compounds - In most samples, recoveries of the labeled compounds will be
similar to those from reagent water or from the alternate matrix (Section 7.6).
18.6.1 If the recovery of any of the labeled compounds is outside of the normal range (Table 6), a
diluted sample must be analyzed (Section 17.5).
18.6.2 If the recovery of any of the labeled compounds in the diluted sample is outside of normal
range, the Diluted combined 209 congener solution (Section 7.10.2.2.2) must be analyzed
and calibration verified (Section 15.3).
EPA Method 1668C 53 April 2010
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18.6.3 If the calibration cannot be verified, a new calibration must be performed and the original
sample extract reanalyzed.
18.6.4 If calibration is verified and the diluted sample does not meet the limits for labeled
compound recovery, the Method does not apply to the sample being analyzed and the result
may not be reported or used for permitting or regulatory compliance purposes. In this case,
alternate extraction and cleanup procedures in this Method or an alternate GC column must
be employed to resolve the interference. If all cleanup procedures in this Method and an
alternate GC column have been employed and labeled compound recovery remains outside
of the normal range, extraction and/or cleanup procedures that are beyond this scope of this
Method will be required to analyze the sample.
19.0 Pollution prevention
19.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity or toxicity
of waste at the point of generation. Many opportunities for pollution prevention exist in laboratory
operation. EPA has established a preferred hierarchy of environmental management techniques that
places pollution prevention as the management option of first choice. Whenever feasible,
laboratory personnel should use pollution prevention techniques to address waste generation. When
wastes cannot be reduced feasibly at the source, the Agency recommends recycling as the next best
option.
19.2 The CBs in this Method are used in extremely small amounts and pose little threat to the
environment when managed properly. Standards should be prepared in volumes consistent with
laboratory use to minimize the disposal of excess volumes of expired standards.
19.3 For information about pollution prevention that may be applied to laboratories and research
institutions, consult Less is Better: Laboratory Chemical Management for Waste Reduction,
available from the American Chemical Society's Department of Governmental Relations and
Science Policy, 1155 16th Street NW, Washington DC 20036, 202/872-4477.
20.0 Waste management
20.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing
waste management, particularly the hazardous waste identification rules and land disposal
restrictions, and to protect the air, water, and land by minimizing and controlling all releases from
fume hoods and bench operations. Compliance is also required with any sewage discharge permits
and regulations. An overview of requirements can be found in Environmental Management Guide
for Small Laboratories (EPA 233-B-98-001).
20.2 Samples containing HC1 or H2SO4 to pH <2 are hazardous and must be neutralized before being
poured down a drain or must be handled as hazardous waste.
20.3 The CBs decompose above 800 °C. Low-level waste such as absorbent paper, tissues, animal
remains, and plastic gloves may be burned in an appropriate incinerator. Gross quantities
(milligrams) should be packaged securely and disposed of through commercial or governmental
channels that are capable of handling extremely toxic wastes.
EPA Method 1668C 54 April 2010
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20.4 Liquid or soluble waste should be dissolved in methanol or ethanol and irradiated with ultraviolet
light with a wavelength shorter than 290 nm for several days. Use F40 BL or equivalent lamps.
Analyze liquid wastes, and dispose of the solutions when the CBs can no longer be detected.
20.5 For further information on waste management, consult The Waste Management Manual for
Laboratory 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 NW, Washington, DC 20036.
21.0 Method performance
The original version of Method 1668 was validated in single-laboratory studies at Pacific
Analytical, Inc., Carlsbad, California and AXYS Analytical Services, Ltd., Sidney, British
Columbia, Canada. The next version, Method 1668A, was validated and data were collected at
AXYS Analytical (Reference 20). Method 1668A was subjected to peer review in 1999, and
published in 2000. In 2003-2004, EPA conducted an interlaboratory method validation study of
Method 1668A (Reference 21), subjected the study to a peer review, and subsequently published
interlaboratory performance data in Method 1668B.
After release of Method 1668B, it was reported to EPA that some of the QC acceptance criteria in
Method 1668B did not allow excursions above 100 percent. As a result, the QC acceptance criteria
were re-developed using data from the interlaboratory study and data from AXYS Analytical and
TestAmerica-Knoxville, Tennessee. The revised QC acceptance criteria were published in
addendum to the Interlaboratory Study Report (Reference 22).
Subsequent to development of the revised QC acceptance criteria, AXYS Analytical, TestAmerica-
Knoxville, and Battelle-Columbus provided method detection limit (MDL) data to EPA. These
data were combined to produced pooled MDLs and MLs (Reference 23). Method 1668B was
revised to Method 1668C to incorporate the revised QC acceptance criteria and revised MDLs and
MLs.
Figure 8 is a chromatogram showing method performance at each level of chlorination.
22.0 References
1. Van den Berg, Martin, Linda S. Birnbaum, Michael Denison, Mike De Vito, William Farland, Mark
Feeley, Heidelore Fiedler, Helen Hakansson, Annika Hanberg, Laurie Haws, Martin Rose, Stephen
Safe, Dieter Schrenk, Chiharu Tohyama, Angelika Tritscher, Jouko Tuomisto, Mats Tysklind, Nigel
Walker, and Richard E. Peterson, 2006, "The 2005 World Health Organization Reevaluation of
Human and Mammalian Toxic Equivalency Factors for Dioxins and Dioxin-like Compounds,"
Toxicological Sciences 93(2): 223-241.
2. "Sampling and Analytical Methods of the National Status and Trends Program Mussel Watch Project:
1993-1996 Update," NOAA Technical Memorandum NOS ORCS 130, Coastal Monitoring and
Bioeffects Assessment Division, Office of Ocean Resources Conservation and Assessment, National
Ocean Service, National Oceanic and Atmospheric Administration, U.S. Department of Commerce,
N/ORCA2, SSMC4, 1305 East-West Highway, Silver Spring, MD 20910, p. 3, 1998.
3. Kuehl, D.W., B.C. Butterworth, J. Libal, and P. Marquis, "An Isotope Dilution High Resolution Gas
Chromatography-High Resolution Mass Spectrometric Method for the Determination of Coplanar
EPA Method 1668C 55 April 2010
-------
Poly chlorinated Biphenyls: Application to Fish and Marine Mammals," Chemosphere 22:9-10, 849-
858, 1991.
4. Echols, Kathy, Robert Gale, Donald E. Tillitt, Ted Schwartz, and Jerome O'Laughlin, "An Automated
HPLC Method for the Fractionation of Polychlorinated Biphenyls, Polychlorinated Dibenzo-p-
dioxins, and Polychlorinated Dibenzofurans in Fish Tissue on a Porous Graphitic Carbon Column,"
Environmental Toxicology and Chemistry 16:8 1590-1597, 1997.
5. "Working with Carcinogens," Department of Health, Education, & Welfare, Public Health Service,
Centers for Disease Control, NIOSH, Publication 77-206, August 1977, NTIS PB-277256.
6. "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910.
7. "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety, 1979.
8. "Standard Methods for the Examination of Water and Wastewater," 18th edition and later revisions,
American Public Health Association, 1015 15th St, NW, Washington, DC 20005, 1-35: Section 1090
(Safety), 1992.
9. "Method 613 - 2,3,7,8-Tetrachlorodibenzo-/>-dioxin," 40 CFR 136, Appendix A, Section 4.1.
10. Lamparski, L.L., andNestrick, T.J., "Novel Extraction Device for the Determination of Chlorinated
Dibenzo-p-dioxins (PCDDs) and Dibenzofurans (PCDFs) in Matrices Containing Water,"
Chemosphere, 19:27-31, 1989.
11. Provost, L.P., and Elder, R.S., "Interpretation of Percent Recovery Data" American Laboratory, 15:
56-83, 1983.
12. "Standard Practice for Sampling Water," ASTM Annual Book of Standards, ASTM, 1916 Race
Street, Philadelphia, PA 19103-1187, 1980.
13. "Methods 330.4 and 330.5 for Total Residual Chlorine," USEPA, EMSL, Cincinnati, OH 45268,
EPA 600/4-70-020, April 1979.
14. "Handbook of Analytical Quality Control in Water and Wastewater Laboratories," USEPA EMSL,
Cincinnati, OH 45268, EPA-600/4-79-019, April 1979.
15. "Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF in Fish",
U.S. Environmental Protection Agency, Environmental Research Laboratory, Duluth, MN 55804,
EPA/600/3-90/022, April 1990.
16. Storr-Hansen, E. and T. Cederberg, "Determination of Coplanar Polychlorinated Biphenyl (CB)
Congeners in Seal Tissues by Chromatography on Active Carbon, Dual-Column High Resolution
GC/ECD and High Resolution GC/High Resolution MS," Chemosphere 24:9, 1181-1196, 1992.
17. Echols, Kathy R., Robert W. Gale, Kevin Feltz, Jerome O'Laughlin, Donald E. Tillitt, and Ted R.
Schwartz, "Loading capacity and chromatographic behavior of a porous graphitic carbon column for
Polychlorinated biphenyls," J. Chromatog. A 811: 135-144, 1998.
18. Tessari, J.D., Personal communication with Dale Rushneck, available from U.S. Environmental
Protection Agency, Engineering and Analysis Division (4303T), 1200 Pennsylvania Avenue NW,
Washington, DC 20460.
EPA Method 1668C 56 April 2010
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19. Ferrario, J.C., C. Byrne, A.E. Dupuy, Jr., "Background Contamination by Coplanar Polychlorinated
Biphenyls (PCBs) in Trace Level High Resolution Gas Chromatography/High Resolution Mass
Spectrometry (HRGC/HRMS) Analytical Procedures" Chemosphere 34:11, 2451-2465, 1997.
20. "Development of a Full Congener Version of Method 1668 and Application to the Analysis of 209
PCB Congeners in Aroclors," AXYS Analytical Services, available from U.S. Environmental
Protection Agency, Engineering and Analysis Division (4303T), 1200 Pennsylvania Avenue NW,
Washington, DC 20460.
21. "Method 1668A Interlaboratory Validation Study Report," March 2010, EPA-820-R-10-004, U.S.
Environmental Protection Agency, Engineering and Analysis Division (4303T), 1200 Pennsylvania
Avenue NW, Washington, DC 20460.
22. "Method 1668A Interlaboratory Study Report Addendum," March 2010, EPA-820-R-10-003, U.S.
Environmental Protection Agency, Engineering and Analysis Division (4303T), 1200 Pennsylvania
Avenue NW, Washington, DC 20460.
23. "Development of Pooled Method Detection Limits (MDLs) and Minimum Levels of Quantitation
(MLs) for EPA Method 1668C," Brian Englert, USEPA, May 18 2010; available from U.S.
Environmental Protection Agency, Engineering and Analysis Division (4303T), 1200 Pennsylvania
Avenue NW, Washington, DC 20460.
EPA Method 1668C 57 April 2010
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23.0 Tables and Figures
Table 1. Names, Congener Numbers, and CAS Registry Numbers for Native and Labeled
Chlorinated Biphenyl (CB) Congeners Determined by Isotope Dilution and Internal
Standard HRGC/HRMS
CB congener name1
2-MoCB
3-MoCB
4-MoCB
2,2'-DiCB
2,3-DiCB
2,3'-DiCB
2,4-DiCB
2,4'-DiCB3
2,5-DiCB
2,6-DiCB
3,3'-DiCB
3,4-DiCB
3,4'-DiCB
3,5-DiCB
4,4'-DiCB
2,2',3-TrCB
2,2',4-TrCB
2,2',5-TrCB3
2,2',6-TrCB
2,3,3'-TrCB
2,3,4-TrCB
2,3,4'-TrCB
2,3,5-TrCB
2,3,6-TrCB
2,3',4-TrCB
2,3',5-TrCB
2,3',6-TrCB
2,4,4'-TrCB3
2,4,5-TrCB
2,4,6-TrCB
2,4',5-TrCB
2,4',6-TrCB
2',3,4-TrCB
2',3,5-TrCB
3,3',4-TrCB
3,3',5-TrCB
3,4,4'-TrCB
3,4,5-TrCB
3,4',5-TrCB
2,2',3,3'-TeCB
2,2',3,4-TeCB
2,2',3,4'-TeCB
2,2',3,5-TeCB
2,2',3,5'-TeCB3
Congener
number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
CAS Registry
number
2051-60-7
2051-61-8
2051-62-9
13029-08-8
16605-91-7
25569-80-6
33284-50-3
34883-43-7
34883-39-1
33146-45-1
2050-67-1
2974-92-7
2974-90-5
34883-41-5
2050-68-2
38444-78-9
37680-66-3
37680-65-2
38444-73-4
38444-84-7
55702-46-0
38444-85-8
55720-44-0
55702-45-9
55712-37-3
38444-81-4
38444-76-7
7012-37-5
15862-07-4
35693-92-6
16606-02-3
38444-77-8
38444-86-9
37680-68-5
37680-69-6
38444-87-0
38444-90-5
53555-66-1
38444-88-1
38444-93-8
52663-59-9
36559-22-5
70362-46-8
41464-39-5
Labeled analog name
13C12-2-MoCB2
13C12-4-MoCB2
13C12-2,2'-DiCB2
13C12-2,5-DiCB4
13C12-4,4'-DiCB2
13C12-2,2',6-TrCB2
13C12-2,4,4'-TriCB5
13C12-3,4,4'-TrCB2
Labeled
analog
congener
number
1L
3L
4L
9L
15L
19L
28L
37L
CAS Registry
number
234432-85-0
208263-77-8
234432-86-1
250694-89-4
208263-67-6
234432-87-2
208263-76-7
208263-79-0
EPA Method 1668C
58
April 2010
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Table 1. Names, Congener Numbers, and CAS Registry Numbers for Native and Labeled
Chlorinated Biphenyl (CB) Congeners Determined by Isotope Dilution and Internal
Standard HRGC/HRMS
CB congener name1
2,2',3,6-TeCB
2,2',3,6'-TeCB
2,2',4,4'-TeCB
2,2',4,5-TeCB
2,2',4,5'-TeCB
2,2',4,6-TeCB
2,2',4,6'-TeCB
2,2',5,5'-TeCB3
2,2',5,6'-TeCB
2,2',6,6'-TeCB
2,3,3',4'-TeCB
2,3,3',4'-TeCB
2,3,3',5-TeCB
2,3,3',5'-TeCB
2,3,3',6-TeCB
2,3,4,4'-TeCB
2,3,4,5-TeCB
2,3,4,6-TeCB
2,3,4',5-TeCB
2,3,4',6-TeCB
2,3,5,6-TeCB
2,3',4,4'-TeCB3
2,3',4,5-TeCB
2,3',455'-TeCB
2,3',4,6-TeCB
2,3',4',5-TeCB
2,3',4',6-TeCB
2,3',5,5'-TeCB
2,3',5',6-TeCB
2,4,4',5-TeCB
2,4,4',6-TeCB
2',3,4,5-TeCB
3,3',4,4'-TeCB3'6
3,3',4,5-TeCB
3,3',4,5'-TeCB
3,3',5,5'-TeCB
3,4,4',5-TeCB6
2,2',3,3',4-PeCB
2,2',3,3',5-PeCB
2,2',3,3',6-PeCB
2,2',3,4,4'-PeCB
2,2',3,4,5-PeCB
2,2',3A5'-PeCB
2,2',3,4,6-PeCB
2,2',35456'-PeCB
2,2',3,4',5-PeCB
Congener
number
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
CAS Registry
number
70362-45-7
41464-47-5
2437-79-8
70362-47-9
41464-40-8
62796-65-0
68194-04-7
35693-99-3
41464-41-9
15968-05-5
74338-24-2
41464-43-1
70424-67-8
41464-49-7
74472-33-6
33025-41-1
33284-53-6
54230-22-7
74472-34-7
52663-58-8
33284-54-7
32598-10-0
73575-53-8
73575-52-7
60233-24-1
32598-11-1
41464-46-4
41464-42-0
74338-23-1
32690-93-0
32598-12-2
70362-48-0
32598-13-3
70362-49-1
41464-48-6
33284-52-5
70362-50-4
52663-62-4
60145-20-2
52663-60-2
65510-45-4
55312-69-1
38380-02-8
55215-17-3
73575-57-2
68194-07-0
Labeled analog name
13C12-2,2',5,5'-TeCB4
13C12-2,2',6,6'-TeCB2
13C12-3,3',4,4'-TeCB2'7
13C12-3,4,4',5-TeCB7
Labeled
analog
congener
number
52L
54L
77L
81L
CAS Registry
number
208263-80-3
234432-88-3
105600-23-5
208461-24-9
EPA Method 1668C
59
April 2010
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Table 1. Names, Congener Numbers, and CAS Registry Numbers for Native and Labeled
Chlorinated Biphenyl (CB) Congeners Determined by Isotope Dilution and Internal
Standard HRGC/HRMS
CB congener name1
2,2',3,4',6-PeCB
2,2',35555'-PeCB
2,2',3,5,6-PeCB
2,2',3,5,6'-PeCB
2,2',3,5',6-PeCB
2,2',3,6,6'-PeCB
2,2',3',4,5-PeCB
2,2',3',4,6-PeCB
2,2',4,4',5-PeCB
2,2',4,4',6-PeCB
2,2',4,5,5'-PeCB3
2,2',4,5,6'-PeCB
2,2',4,5,'6-PeCB
2,2',4,6,6'-PeCB
2,3,3',4,4'-PeCB3'6
2,3,3',4,5-PeCB
2,3,3',4',5-PeCB
2,3,3',4,5'-PeCB
2,3,3',4,6-PeCB
2,3,3',4',6-PeCB
2,3,3',5,5'-PeCB
2,3,3',5,6-PeCB
2,3,3',5',6-PeCB
2,3,4,4',5-PeCB6
2,3,4,4',6-PeCB
2,3,4,5,6-PeCB
2,3,4',5,6-PeCB
2,3',4,4',5-PeCB3'6
253',4,4',6-PeCB
2,3',4,5,5'-PeCB
2,3',4,5,'6-PeCB
2',3,3',4,5-PeCB
2',3,4,4',5-PeCB6
2',3,4,5,5'-PeCB
2',3,4,5,6'-PeCB
3,3',4,4',5-PeCB3'6
3,3',4,5,5'-PeCB
2,2',3,3',4,4'-HxCB3
2,2',3,3',4,5-HxCB
2,2',3,3',4,5'-HxCB
2,2',3,3',4,6-HxCB
2,2',3,3',456'-HxCB
2,2',3,3',555'-HxCB
2,2',3,3',5,6-HxCB
2,2',3,3',556'-HxCB
2,2',3,3',656'-HxCB
Congener
number
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
CAS Registry
number
68194-05-8
52663-61-3
73575-56-1
73575-55-0
38379-99-6
73575-54-9
41464-51-1
60233-25-2
38380-01-7
39485-83-1
37680-73-2
68194-06-9
60145-21-3
56558-16-8
32598-14-4
70424-69-0
70424-68-9
70362-41-3
74472-35-8
38380-03-9
39635-32-0
74472-36-9
68194-10-5
74472-37-0
74472-38-1
18259-05-7
68194-11-6
31508-00-6
56558-17-9
68194-12-7
56558-18-0
76842-07-4
65510-44-3
70424-70-3
74472-39-2
57465-28-8
39635-33-1
38380-07-3
55215-18-4
52663-66-8
61798-70-7
38380-05-1
35694-04-3
52704-70-8
52744-13-5
38411-22-2
Labeled analog name
13C12-2,2',4,5,5'-PeCB4
13C12-2,2',4,6,6'-PeCB2
13C12-2,3,3',4,4'-PeCB7
13C12-2,3,3',5,5'-PeCB5
13C12-2,3,4,4',5-PeCB7
13C12-2,3',4,4',5-PeCB7
13C12-2',3,4,4',5-PeCB7
13C12-3,3',4,4',5-PeCB2'7
Labeled
analog
congener
number
101L
104L
105L
111L
114L
118L
123L
126L
CAS Registry
number
104130-39-4
234432-89-4
208263-62-1
235416-29-2
208263-63-2
104130-40-7
208263-64-3
208263-65-4
EPA Method 1668C
60
April 2010
-------
Table 1. Names, Congener Numbers, and CAS Registry Numbers for Native and Labeled
Chlorinated Biphenyl (CB) Congeners Determined by Isotope Dilution and Internal
Standard HRGC/HRMS
CB congener name1
2,2',3,4,4',5-HxCB
2,2',3,4,4',5'-HxCB3
2,2',3,4,4',6-HxCB
2,2',3,4,4',6'-HxCB
2,2',3,4,5,5'-HxCB
2,2',3,4,5,6-HxCB
2,2',3,4,5,6'-HxCB
2,2',3,4,5',6-HxCB
2,2',3,4,6,6'-HxCB
2,2',3,4',5,5'-HxCB
2,2',3,4',5,6-HxCB
2,2',3,4',5,6'-HxCB
2,2',3,4',5',6-HxCB
2,2',3,4',6,6'-HxCB
2,2',3,5,5',6-HxCB
2,2',3,5,6,6'-HxCB
2,2',4,4',5,5'-HxCB3
2,2',4,4',5',6-HxCB
2,2',4,4',6,6'-HxCB
2,3,3',4,4',5-HxCB6
2,3,3',4,4',5'-HxCB6
2,3,3',4,4',6-HxCB
2,3,3',4,5,5'-HxCB
2,3,3',4,5,6-HxCB
2,3,3',4,5',6-HxCB
2,353',4',5,5'-HxCB
2,3,3',4',5,6-HxCB
2,3,3',4',5',6-HxCB
2,3,3',5,5',6-HxCB
2,3,4,4',5,6-HxCB
2,3',4,4',5,5'-HxCB6
2,3',4,4',5',6-HxCB
3,3',4,4',5,5'-HxCB3'6
2,2',3,3',4,4',5-HpCB3
2,2'3,3',4,4',6-HpCB
2,2',3,3',4,5,5'-HpCB
2,2',3,3',4,5,6-HpCB
2,2',3,3',4,5,6'-HpCB
2,2',3,3',4,5',6-HpCB
2,2',3,3',4,6,6'-HpCB
2,2',3,3',4',5,6-HpCB
2,2',3,3',5,5',6-HpCB
2,2',3,3',55656'-HpCB
2,2',3,454',5,5'-HpCB3
2,2',3,4,4',5,6-HpCB
2,2',3,4,4',556'-HpCB
Congener
number
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
CAS Registry
number
35694-06-5
35065-28-2
56030-56-9
59291-64-4
52712-04-6
41411-61-4
68194-15-0
68194-14-9
74472-40-5
51908-16-8
68194-13-8
74472-41-6
38380-04-0
68194-08-1
52663-63-5
68194-09-2
35065-27-1
60145-22-4
33979-03-2
38380-08-4
69782-90-7
74472-42-7
39635-35-3
41411-62-5
74472-43-8
39635-34-2
74472-44-9
74472-45-0
74472-46-1
41411-63-6
52663-72-6
59291-65-5
32774-16-6
35065-30-6
52663-71-5
52663-74-8
68194-16-1
38411-25-5
40186-70-7
52663-65-7
52663-70-4
52663-67-9
52663-64-6
35065-29-3
74472-47-2
60145-23-5
Labeled analog name
13C12-2,2',3,454',5'-HxCB4
13C12-2,2',4,4',6,6'-HxCB2
13C12-2,3,3'A4',5-HxCB7
13C12-2,3,3',4,4',5'-HxCB7
13C12-2,3',4,4',5,5'-HxCB7
13C12-3,3',4,4',5,5'-HxCB2'7
13C12-2,2',3,3',4,4',5-HpCB
13C12-2,2',3,3',5,5',6-HpCB5
13C12-2,2',35454',5,5'-HpCB
Labeled
analog
congener
number
138L
155L
156L
157L
167L
169L
170L
178L
180L
CAS Registry
number
208263-66-5
234432-90-7
208263-68-7
235416-30-5
208263-69-8
208263-70-1
160901-80-4
232919-67-4
160901-82-6
EPA Method 1668C
61
April 2010
-------
Table 1. Names, Congener Numbers, and CAS Registry Numbers for Native and Labeled
Chlorinated Biphenyl (CB) Congeners Determined by Isotope Dilution and Internal
Standard HRGC/HRMS
CB congener name1
2,2',3,4,4',5',6-HpCB
2,2',3,4,4',656'-HpCB
2,2',3,4,5,5',6-HpCB
2,2',3,4,5,6,6'-HpCB
2,2',3,4',5,5',6-HpCB3
2,2',3,4',5,6,6'-HpCB
2,3,3',4,4',5,5'-HpCB6
2,3,3',4,4',5,6-HpCB
2,3,3',4,4',5',6-HpCB
2,3,3',4,5,5',6-HpCB
2,3,3',4',5,5',6-HpCB
2,2',3,3',4,4',5,5'-OcCB
2,2',3,3',4,4',5,6-OcCB3
2,2',3,3',4,4',5,6'-OcCB
2,2',3,3',4,4',6,6'-OcCB
2,2',3,3',4,5,5',6-OcCB
2,2',3,3',4,5,5',6'-OcCB
2,2',3,3',4555656'-OcCB
2,2',3,3',4,5',6,6'-OcCB
2,2',3,3',5,5',6,6'-OcCB
2,2',3,4,4',5,5',6-OcCB
2,2',3,4,4',55656'-OcCB
2,3,3',4,4',5,5',6-OcCB
2,2',353',4,4',5,5',6-NoCB3
2,2',353',4,4',5,6,6'-NoCB
2,2',353',4,5,5',6,6'-NoCB
DeCB3
Congener
number
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
CAS Registry
number
52663-69-1
74472-48-3
52712-05-7
74472-49-4
52663-68-0
74487-85-7
39635-31-9
41411-64-7
74472-50-7
74472-51-8
69782-91-8
35694-08-7
52663-78-2
42740-50-1
33091-17-7
68194-17-2
52663-75-9
52663-73-7
40186-71-8
2136-99-4
52663-76-0
74472-52-9
74472-53-0
40186-72-9
52663-79-3
52663-77-1
2051-24-3
Labeled analog name
13C12-2,2',3,4',5,6,6'-HpCB2
13C12-2,3,3',4,4',5,5'-HpCB2'7
13C12-2,2',3,3',4,4',5,5'-OcCB4
13C12-2,2',3,3'5555',6,6'-OcCB2
13C12-2,3,3',4,4',5,5',6-OcCB2
13C12-2,2',3,3',4,4',555',6-NoCB2
13C12-2,2',3,3'A5,5',6,6'-NoCB2
13C12-DeCB2
Labeled
analog
congener
number
188L
189L
194L
202L
205L
206L
208L
209L
CAS Registry
number
234432-91-8
208263-73-4
208263-74-5
105600-26-8
234446-64-1
208263-75-6
234432-92-9
105600-27-9
1. Abbreviations for chlorination levels
MoCB monochlorobiphenyl HxCB
DiCB dichlorobiphenyl HpCB
TrCB trichlorobiphenyl OcCB
TeCB tetrachlorobiphenyl NoCB
PeCB pentachlorobiphenyl DeCB
hexachlorobipheny 1
heptachlorobiphenyl
octachlorobiphenyl
nonachlorobiphenyl
decachlorobipheny 1
2. Labeled level of chlorination (LOG) window-defining congener
3. National Oceanic and Atmospheric Administration (NOAA) congener of interest
4. Labeled injection internal standard
5. Labeled clean-up standard
6. World Health Organization (WHO) toxic congener
7. Labeled analog of WHO toxic congener
EPA Method 1668C
62
April 2010
-------
Table 2. Retention times (RT), RT references, relative retention times (RRTs), method detection limits (MDLs), and minimum levels of quantitation
(MLs) for the 209 CB congeners on SPB-octyl.
Detection limits and minimum levels -
Matrix and concentration10
Cl Window
iNo.1 Congener No. 2'3 ! RTRef4 | RT5 j RRT6 i RRT limits7 | (sec)8 \
Compounds using 9L (13Ci2-2,5-DiCB) as Labeled injection internal standard
CB congener
Quantitation reference9
Water
(pg/L)
i MDL \ ML |
Other
(ng/kg)
MDL j ML i
Extract
(pg/nL)
ML
; Monochlorobiphenyls
1
1
1
1
2
3
1L
3L
3L
13:44 1.0012
16:08 0.9878
16:21 1.0010
0.9988-1.0036
0.9847-0.9908
0.9990-1.0031
-1+3 .
6
-1+3
1L
1L/3L
3L
10
7
11
20 *
20
50
1.0
0.7
1.1
2
2
5
1
1
2.5
Dichlorobiphenyls
2
2
2
2
2
2
2
2
2
2
2
2
2
4
10
9
7
6
5
8
14
11
13
12
13/12
15
4L
4L
4L
4L
4L
4L
i 4L
15L
15L
15L
15L
15L
15L
16:40 1.0010
16:53 1.0140
18:55 1.1361
19:07 1.1481
19:26 1.1672
19:48 : 1.1892
\ 19:56 i 1.1972 \
21:42 0.9267
22:42 0.9694
23:03 0.9843
23:06 0.9865
23:04 0.9851
23:26 1.0007
0.9990-1.0030
1.0110-1.0170
1.1331-1.1391
1.1451-1.1512
1.1642-1.1702
1.1862-1.1922
1.1942-1.2002
0.9246-0.9288
0.9673-0.9715
0.9822-0.9865
0.9843-0.9886
0.9829-0.9872
0.9993-1.0021
-1+3
6
6
6
6
6
6 I
6
6
6
6
6
-1+3
4L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
4L/15L
15L
13
13
7
8
! 7
8
: 15
8
34
19
16
50
50
20
20
20
, 20
i 50 i
20
100
50
50
1.3
1.3
0.7
0.8
0.7
0.8
1.5
0.8
3.4
1.9
1.6
5
5
2
2
: 2
, 2
i 5 :
2
10
5
5
2.5
2.5
1
1
1
1
2.5
1
5
2.5
2.5
Trichlorobiphenyls
3
' 3
3
19
30
18
19L
19L
19L
20:19 1.0008
22:15 1.0961
22:23 1.1026
0.9992-1.0025
1.0936-1.0985
1.1002-1.1051
-1+3
6
6
19L
19L/37L
19L/37L
8
i 16
20
50 i
0.8
1.6
2
5
1
2.5
EPA Method 1668C
63
April 2010
-------
Cl
No.1
3
3
3
~~1T
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1
1
2
2
3
3
Congener No. 2'3
30/18
17
27
24
16
32
34
23
29
26
29/26
25
31
28
20
28/20
21
33
21/33
22
36
39
38
35
37
Labeled Compoi
1L
3L
4L
15L
19L
37L
RTRef4
19L
19L
19L
19L
19L
19L
19L
19L
19L
19L
19L
37L
37L
37L
37L
37L
37L
37L
37L
37L
37L
37L
37L
37L
37L
i
9L
9L
9L
9L
9L
52L
RT5
22:19
22:49
23:06
23:14
23:25
24:57
25:17
25:26
[ 25:47
25:48
25:48
26:04
26:25
26:44
26:49
26:47
26:58
27:01
26:59
27:29
29:05
29:30
30:10
30:42
31:11
13:43
16:20
16:39
23:25
20:18
31:10
RRT6
1.0993
1.1240
1.1379
1.1445
1.1535
1.2291
1.2455
1.2529
1.2701 I
1.2709
1.2709
0.8364
0.8476
0.8578
0.8604
0.8594
0.8652
0.8668
0.8658
0.8818 !
0.9332
0.9465
0.9679
0.9850
1.0005
0.7257
0.8642
0.8810
1.2390
1.0741 I
1.0841
Window
RRT limits7 (sec)8
1.0969-1.1018 6
1.1215-1.1264 6
1.1355-1.1404 6
1.1420-11470 6
1.1511-1.1560 6
1.2266-1.2315 6
1.2430-1.2479 6
1.2504-1.2553 6
1.2660-1.2742 10
1.2668-1.2750 10
1.2668-1.2750 10
0.8348-0.8380 6
0.8460-0.8492 6
0.8551-0.8604 10
0.8578-0.8631 10
0.8567-0.8620 10
0.8626-0.8679 10
0.8642-0.8695; 10
0.8631-0.8684 10
0.8802-0.8834 6
0.9316-0.9348 6
0.9449-0.9481 6
0.9663-0.9695 6
0.9834-0.9866 6
0.9995-1.0011 -1+3
0.7125-0.7390 30
0.8510-0.8774 30
0.8677-0.894Z 30
1.2302-1.2478 20
1.0608-1.0873 30
1.0754-1.0928 30
Quantitation reference9
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
19L/37L
37L
9L
9L
9L
9L
9L
52L
Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
(pg/L): (ng/kg) (pg/uL)
MDL ML MDL ML ML
10
9
8
7
7
12
18
22
21
9
7
9
10
20
20
20
20
20
20
20
50
20
50
50
50
20
20
20
20
20
20
0.9
0.8
1.0
0.9
0.8
0.7
0.7
1.2
0.8
1.8
2.2
2.1
0.9
0.8
0.8
0.7
0.9
1.0
1
1
III
1
1
1
1
2.5
2.5
2.5
2.5
1
1
1
1
1
EPA Method 1668C
64
April 2010
-------
! i =
Cl . ! ; ; .
No.1 Congener No. 23 RTRef4 RT5 RRT6
Compounds using 52L (13C12-2,2',5,5'-TeCB) as Labeled
CB congener
^T
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
Tetrachlorobiphenyls
54 54L
50
53
50/53
45
51
45/51
46
52
73
43
69
49
69/49
48
65
47
44
65/47/44
62
75
59
62/75/59
42
41
71
40
41/71/40
64
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
54L
"23-5T
26:07
26:09
26:08
26:55
26:58
26:57
27:18
28:45
28:52
28:58
29:08
29:16
29:12
29:33
29:49
29:50
29:53
29:50
30:06
30:08
30:12
30:09
30:26
30:52
30:58
31:01
30:58
31:12
L0007
1.0958
1.0972
1.0965
1.1294
1.1315 i
1.1308
1.1455
1.2063
1.2112
1.2154
1.2224
1.2280
1.2252
1.2399
1.2510
1.2517 i
1.2538
1.2517
1.2629
1.2643
1.2671
1.2650
1.2769
1.2951
1.2993
1.3014
1.2993 i
1.3091
Window:
RRT limits7 (sec)8 i Quantitation reference9
injection internal standard
0~9993-L0021 -1+3
1.0923-1.0993 10
1.0937-1.1007 10
1.0930-1.1000 10
1.1259-1.1329 10
1.1280-1.1350 10
1.1273-1.1343 10
1.1434-1.1476 6
1.2042-1.2084 6
1.2091-1.2133 6
1.2133-1.2175 6
1.2189-1.2259 10
1.2245-1.2315 10
1.2217-1.2287 10
1.2378-1.2420 6
1.2476-1.2545 10
1.2483-1.2552 10
1.2503-1.2573 10
1.2483-1.2552 10
1.2594-1.2664 10
1.2608-1.2678 10
1.2636-1.2706 10
1.2615-1.2685 10
1.2748-1.2790 6
1.2916-1.2986 10
1.2958-1.3028 10
1.2979-1.3049 10
1.2958-1.3028 10
1.3070-1.3112 6
54L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
Detection limits .
Matrix and
Water
(pg/L) :
MDL ML
14 50
25 100
22 50 |
10 20
15 50
14 50
14 50
26 100
14 50
40 l 100 !
37 100
16 50
42 100
13 50
ind minimum levels -
concentration10
Other Extract
(ng/kg) (pg/uL)
MDL ML ML
1.4
2.5
2.2
1.0
1.5
1.4
1.4
2.6
1.4
4.0 l
3.7
1.6
4.2
1.3
5 2.5
10 5
5 2.5
2 1
5 2.5
5 2.5
5 2.5
10 5
5 2.5
10 ' 5
10 5
5 2.5
10 5
5 2.5
EPA Method 1668C
65
April 2010
-------
Cl
No.1
4
4
4
__
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
Congener No. 2'3
72
68
57
58
67
63
61
70
76
74
61/70/76/74
66
55
56
60
80
79
78
81
77
Labeled compounds
54L
81L
77L
RTRef4
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
81L
77L
52L
52L
52L
Compounds using 101L (13C12-2,2',4,5,5
CB congener
RT5
31:59
32:18
32:46
33:05
33:13
33:30
33:46
33:53
33:55
33:57
33:55
34:15
34:28
35:03
35:16
35:32
37:16
37:52
38:23
39:02
23:50
38:22
39:01
RRT6 RRT limits7
0.8336
0.8419
0.8540
0.8623 ~~~
0.8658
0.8732
0.8801
0.8831
0.8840 1
0.8849
0.8840
0.8927
0.8983
0.9136
0.9192
0.9262
0.9713
0.9870
1.0004
1.0004 1
0.8290
1.3345
1.3571
'-PeCB) as Labeled
0.8323-0.8349
0.8406-0.8432
0.8527-0.8553
_________^
0.8645-0.8671
0.8719-0.8745
0.8775-0.8827
0.8805-0.8858
0.8814-0.8866
0.8827-0.8871
0.8814-0.8866
0.8914-0.8940
0.8970-0.8997
0.9123-0.9149
(X9T7W).9205
0.9248-0.9275
0.9700-0.9726
0.9857-0.9883
0.9996-1.0013
0.9996-1.0013
0.8232-0.8348
1.3287-1.3403
1.3513-1.3629
Window
(sec)8
6
6
6
6
6
6
12
12
12
10
12
6
6
6
6
6
6
6
-1+3
-1+3
20
20
20
Quantitation reference9
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
54L/81L/77L
81L
77L
52L
52L
52L
Detection limits and minimum
Matrix and concentration
Water
(pg/L) :
MDL ML
13 50
14 50
11 50
14 50
12 50
12 50
59 200 |
17 50
12 50
15 50
14 50
11 50
13 50
16 50 I
18 50
14 50 |
Other
(ng/kg) .
MDL ML
1.3
1.4
1.1
1.4
1.2
1.2
5.9
1.7
1.2
1.5
1.4
1.1
1.3
1.6
1.8
1.4
5
5
5
5
5
5
20 I
5
5
5
5
5
5
5
5
5 1
levels -
10
Extract
(pg/nL)
ML
2.5
2.5
2.5
_
2.5
2.5
10
2.5
2.5
2.5
2~5
2.5
2.5
2.5
2.5
2.5
injection internal standard
Pentachlorobiphenyls
5
5
5
5
5
104
96
103
94
95
104L
104L
104L
104L
104L
29:46
30:17
32:11
32:29
33:00
1.0000
1.0174
1.0812
1.0913 1
1.1086
0.9994-1.0017
1.0146-1.0202
1.0795-1.0829
1.0896-1.0929
1.1058-1.1114
-1+3
10
6
6
10
104L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
14 50
15 50
11 50
13 50 |
77 200
1.4
1.5
1.1
1.3
7.7
5
5
5
5 1
20
2.5
2.5
2.5
2.5
10
EPA Method 1668C
66
April 2010
-------
Cl
No.1
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Congener No. 2'3
100
93
102
98
95/100/93/102/98
88
91
88/91
84 |
89
121
92
113
90
101
113/90/101
83
99
83/99
112 |
119
109
86
97
125
87
119/109/86/97/125/87
117
116
85
117/116/85 |
110
RTRef4
104L
104L
104L
104L
104L
104L
104L
104L
104L
104L
104L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
RT5
33:06
33:14
33:21
33:26
33:13
33:48
33:55
33:52
| 34:14
34:44
34:57
35:26
36:01
36:03
36:04
36:03
36:39
36:41
36:40
| 36:51
37:12
37:12
37:17
37:17
37:21
37:25
37:19
37:57
38:02
38:05
| 38:00
38:16
RRT6
1.1120
1.1165
1.1204
1.1232
1.1159
1.1355
1.1394
1.1377
1.1501 |
1.1669
1.1741
0.8639
0.8781
0.8789
0.8793
0.8789
0.8935
0.8944
0.8939
0.8984
0.9069
0.9069
0.9090
0.9090
0.9106
0.9122
0.9098
0.9252
0.9273
0.9285
0.9265 |
0.9330
RRT limits7
1.1092-1.1148
1.1137-1.1193
1.1176-1.1232
1.1204-1.1260
1.1131-1.1187
1.1321-1.1389
1.1366-1.1422
1.1344-1.1411
1.1484-1.1517
1.1652-1.1685
1.1725-1.1758
0.8627-0.8651
0.8761-0.8801
0.8769-0.8809
0.8773-0.8813
0.8769-0.8809
0.8911-0.8960
0.8923-0.8964
0.8915-0.8964
0.8972-0.8996
0.9037-0.9102
0.9037-0.9102
0.9057-0.9122
0.9057-0.9122
0.9074-0.9139
0.9102-0.9143
0.9065-0.9130
0.9228-0.9277
0.9248-0.9297
0.9265-0.9305
0.9240-0.9289
0.9309-0.9350
Window
(sec)8
10
10
10
10
15
12
10
12
6
6
6
6
10
10
10
10
12
10
12
6
16
16
16
16
16
10
16
12
12
10
12
10
Quantitation reference9
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
(pg/L) : (ng/kg) (pg/uL)
MDL ML MDL ML ML
22
47
29
14
74
50
200
100
50
200
2.2
4.7
2.9
1.4
7.4
20
10
20
2.5
11
13
12
13
1 20 1
50 ;
50
50
1.1
1.3
1.2
1.3
1 2 1
5
5
5
1
2.5
2.5
2.5
10
2.5
10
38 100 3.8 10 5
39 100 3.9 10 5
EPA Method 1668C
67
April 2010
-------
Cl
No.1
5
5
5
__
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Congener No. 2'3
115
110/115
82
Ill
120
108
124
108/124
107
123
106
118
122
114
To5
127
126
Labeled compounds
104L
123L
118L
114L
105L
126L
RTRef4
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
123L
118L
118L
114L
105L
105L
126L
101L
101L
101L
101L
101L
101L
RT5
38:18
38:17
38:40
38:52
39:21
40:39
40:40
40:39
40:54
41:02
41:10
41:22
41:49
41:58
42:43
44:09
45:58
29:46
41:01
41:21
41:57
42:44
45:57
RRT6
0.9338
0.9334
0.9427
0.9476
0.9594
0.9911
0.9915
0.9911
0.9972 1
1.0004
1.0037
1.0004
1.0113
1.0004
0.9996
1.0332
1.0004
0.8257
1.1378 l
1.1470
1.1637
1.1854
1.2746
RRT limits7
0.9317-0.9358
0.9313-0.9354
0.9415-0.9439
^9464M)!9488
0.9581-0.9606
0.9890-0.9931
0.9894-0.9935
0.9890-0.9931
0.9959-0.9984
0.9996-1.0012
1.0024-1.0049
0.9996-1.0012
1.0101-1.0125
0.9999-1.0012
1X9996^1.0012
1.0320-1.0343
0.9996-1.0011
0.8211-0.8303
1.1331-1.1424
1.1424-1.1516
1.1590-1.1683
1.1808-1.1900
1.2700-1.2792
Window
(sec)8
10
10
6
6
6
10
10
10
6
-1+3
6
-1+3
6
-1+3
-2+3
6
-1+3
20
20
20
20
20
20
Quantitation reference9
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
^^^^^^ ..__.._._.
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
104L/123L/1 14L/1 18L/105L
123L
104L/123L/1 14L/1 18L/105L
118L
104L/123L/1 14L/1 18L/105L
114L
105L
104L/123L/1 14L/1 18L/105L
126L
101L
101L
101L
101L
101L
101L
Detection limits and minimum
Matrix and concentration
Water
(pg/L) :
MDL ML
15 50
14 50
13 50
29 100 I
17 50 |
17 50
17 50
30 100
12 50
15 50
17 50
14 50
16 50
Other
(ng/kg) .
MDL ML
1.5
1.4
1.3
2.9
1.7
1.7
1.7
3.0
1.2
1.5
1.7
1.4
1.6
5
5
5
10
5
5
5
10
5
5
5
5
5
levels -
10
Extract
(pg/nL)
ML
2.5
_
2.5
5
2.5
2.5
2.5
5
2.5
2.5
2~5
2.5
2.5
Compounds using 138L (13Ci2-2,2',3,4,4',5'-HxCB) as Labeled injection internal standard
CB congener
Hexachlorobiphenyls
6
6
6
6
6
155
152
150
136
145
155L
155L
155L
155L
155L
35:44
36:07
36:15
36:44
37:00
1.0000
1.0107
1.0145
1.0280 1
1.0354
0.9995-1.0014
1.0093-1.0121
1.0131-1.0159
1.0266-1.0294
1.0340-1.0368
-1+3
6
6
6
6
155L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
14 50
14 50
15 50
16 50 |
16 50
1.4
1.4
1.5
1.6
1.6
5
5
5
5 1
5
2.5
2.5
2.5
2.5
2.5
EPA Method 1668C
68
April 2010
-------
Cl
No.1
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Congener No. 2'3
148
151
135
154
151/135/154
144
147
149
147/149
134
143
134/143
139
140
139/140
131
142
132
133
165
146
161
153
168
153/168
141
130
137
164
138
163
129
RTRef4
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
155L
167L
167L
167L
167L
167L
167L
167L
167L
167L
167L
167L
167L
167L
RT5
34:26
39:10
39:17
39:21
39:15
39:47
40:09
40:12
40:10
40:27
40:30
40:29
40:47
40:48
40:47
41:03
41:13
41:36
41:57
42:23
42:38
42:47
43:17
43:21
43:19
43:34
44:01
44:14
44:22
44:42
44:42
44:47
RRT6
1.0756
1.0961
1.0993
1.1012
1.0984
1.1133
1.1236
1.1250
1.1241 |
1.1320
1.1334
1.1329
1.1413
1.1418
1.1413
1.1488
1.1535
1.1642
1.1740
0.8864 !
0.8916
0.8947
0.9052
0.9066
0.9059
0.9111
0.9205
0.9251
0.9278
0.9348
0.9348 |
0.9366
RRT limits7
1.0742-1.0770
1.0938-1.0984
1.0970-1.1017
1.0989-1.1035
1.0961-1.1007
1.1119-1.1147
1.1213-1.1259
1.1227-1.1273
1.1217-1.1264
1.1297-1.1343
1.1311-1.1357
1.1306-1.1353
1.1390-1.1437
1.1395-1.1441
1.1390-1.1437
1.1474-1.1502
1.1521-1.1549
1.1618-1.1665
1.1726-1.1754
0.8853-0.8874
0.8906-0.8926
0.8937-0.8958
0.9035-0.9069
0.9048-0.9083
0.9041-0.9076
0.9101-0.9122
0.9195-0.9216
0.9240-0.9261
0.9268-0.9289
0.9324-0.9373
0.9324-0.9373
0.9341-0.9390
Window
(sec)8
6
10
10
10
10
6
10
10
10
10
10
10
10
10
10
6
6
10
6
6
6
6
10
10
10
6
6
6
6
14
14
14
Quantitation reference9
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
(pg/L) : (ng/kg) (pg/uL)
MDL ML MDL ML ML
14 50 1.4 5 2.5
33
100
3.3
46 100 4.6 10
15 50 1.5 5 2.5
35 100 3.5 10
10
29
17
17
16
12
13
14
13
30
11
13
15
15
100
50
50
50
50
50 |
50 *
50
100
50
50
50
50
2.9
1.7
1.7
1.6
1.2
1.3
1.4
1.3
3.0
1.7
1.3
1.5
1.5
10
5
5
: 5
5
5 !
5
5
10
5
5
5
5
5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
5
2~5
2.5
2.5
2.5
63
200
6.3
20
10
EPA Method 1668C
69
April 2010
-------
Cl
No.1
6
6
6
6
6
6
6
6
6
6
6
6
6
Congener No. 2'3
160
138/163/129/160
158
166
128
128/166
159
162
167
156
157
156/157
169
RTRef4 RT5
167L 44:53
167L 44:47
167L 45:05
167L 45:59
167L 46:09
167L 46:04
167L 46:59
167L 47:18
167L 47:49
156L/157L 49:05
156L/157L 49:09
156L/157L 49:07
169L 52:31
RRT6
0.9387
0.9366
0.9428
0.9617
0.9651
0.9634
0.9826
0.9892
1.0000 \
0.9993
1.0007
1.0000
1.0003
Window
RRT limits7 (sec)8
0.9369-0.9404 10
0.9341-0.9390 14
0.9418-0.9439 6
0.9599-0.9634 10
0.9634-0.9669 10
0.9617-0.9651 10
0.9815-0.9836 6
0.9881-0.9902 6
0.9997-1.0010 -1+3
0.9983-1.0003 6
0.9990-1.0024 10
0.9990-1.1010 6
0.9997-1.0010 -1+3
Quantitation reference9
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
155L/156L/157L/167L/169L
167L
156L/157L
156L/157L
156L/157L
169L
Detection limits and minimum
Matrix and concentration
Water
(pg/L) :
MDL ML
16 50
29 100
14 50
13 50
13 | 50 |
23 100
15 50
Other
(ng/kg) .
MDL ML
1.6 5
2.9 10
1.4 5
1.3 5
1.3 5 I
2.3 10
1.5 5
levels -
10
Extract
(pg/uL)
ML
2.5
5
2.5
2.5
2.5
5
2.5
Labeled compounds
6
6
6
6
6
6
155L
167L
156L
157L
156L/157L
169L
138L 35:44
138L 47:49
138L 49:05
138L 49:08
138L 49:07
1 138L ; 52:30
0.7997
1.0701
1.0985
1.0996
1.0992
1.1749 !
Compounds using 194L(13C12-2,2',3,3',4,4',5,5'-OcCB) as
CB congener
0.7960-0.8034 20
1.0664-1.0739 20
1.0974-1.0996 20
1.0959-1.1033 20
1.0981-1.1003 20
1.1738-1.1761! 20
138L
138L
138L
138L
138L
138L
Labeled injection internal standard
Heptachlorobiphenyls
7
7
7
7
7
7
7
7
7
188
179
184
176
186
178
175
187
182
188L 41:51
188L 42:19
188L 42:45
188L 43:15
188L 43:45
188L 45:06
188L 45:46
| 188L \ 46:02
188L 46:14
1.0000
1.0112
1.0215
1.0335
1.0454
1.0777
1.0936
1.1000 I
1.1047
0.9996-1.0012 -1+3
1.0100-1.0123 6
1.0203-1.0227 6
1.0323-1.0346 6
1.0442-1.0466 6
1.0765-1.0789 6
1.0924-1.0948 6
1.0988-1.1012) 6
1.1035-1.1059 6
188L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
15 50
14 50
14 50
12 50
15 50
14 50
14 50
17 I 50 |
13 50
1.5 5
1.4 5
1.4 5
1.2 5
1.5 5
1.4 5
1.4 5
1.7 I 5
1.3 5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
EPA Method 1668C
70
April 2010
-------
Cl
No.1
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
Congener No. 2'3
183
185
183/185
174
177
181
171
173
171/173
172
192
193
180
193/180
191
170
190
189
RTRef4
188L
188L
188L
188L
188L
188L
188L
188L
188L
189L
189L
189L
189L11
189L
189L
189L11
189L
189L
RT5
46:42
46:53
46:47
47:02
47:30
47:52
48:10
48:11
48:10
49:47
50:06
50:26
50:27
50:26
50:51
51:54
52:26
55:07
RRT6
1.1159
1.1203
1.1179
1.1239
1.1350
1.1438
1.1509
1.1513
1.1509 |
0.9035
0.9093
0.9153
0.9156
0.9153
0.9229
0.9419
0.9516
1.0003
Window
RRT limits7 (sec)8
1.1147-1.1171 6
1.1191-1.1215 6
1.1167-1.1191 6
L1227-LT251 6
1.1338-1.1362 6
1.1426-1.1450 6
1. 1489-1. 1529; 10
1.1501-1.1525 6
1.1489-1.1529 6
0.9026-0.9044 6
0.9083-0.9102 6
0.9144-0.9162 6
0.9147-0.9165 6
0.9144-0.9162 6
0.9220-0.9238 6
0.9410-0.9428 6
0.9507-0.9525 6
0.9997-1.0009; -1+3
Quantitation reference9
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L
188L/189L11
188L/189L
188L/189L
188L/189L11
188L/189L
189L
Octachlorobiphenyls
8
8
8
8
8
8
8
8
8
8
8
8
8
202
201
204
197
200
197/200
198
199
198/199
196
203
195
194
202L
202L
202L
202L
202L
202L
202L
202L
202L
205L
205L
205L
205L
47:32
48:31
49:11
49:27
49:40
49:33
52:30
52:32
52:31
53:13
53:26
54:55
57:19
1.0004 !
1.0210
1.0351
1.0407
1.0452
1.0428
1.1049
1.1056
1.1052
0.9207
0.9245
0.9501 |
0.9916
0.9996-1.0011 -1+3
1.0193-1.0228 10
1.0340-1.0361 6
1.0396-1.0417 6
1.0442-1.0463 6
1.0417-1.0438 6
L103T-LT066 10
1.1045-1.1066 6
1.1035-1.1070 10
0.9198-0.9216; 6
0.9236-0.9253 6
0.9493-0.9510 6
0.9908-0.9925 6
202L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
202L/205L
Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
(pg/L) : (ng/kg) (pg/uL)
MDL ML MDL ML ML
28
""is""
11
13
30
13
13
13
12
14
13
24
20
21
43
20
18
22
18
100
50
50
50
100
50
50
100
50
50
50
50
100
50
50
100
100
50
50
50
50
1.5
1.1
1.3
3.0
1.3
1.3
3.0
1.3
1.2
1.4
1.3
2.4
2.0
2.1
4.3
3.7
2.0
1.8
2.2
1.8
10
5
5
5
10
5
5
10
5
5
5
5
10
5
5
10
10
5
5
5
5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
5
2.5
2.5
2.5
2.5
2.5
2.5
EPA Method 1668C
71
April 2010
-------
Cl
iNo.1
8
Congener No. 2'3
205
RTRef4
205L
RT5
57:49
RRT6
1.0003
Window:
RRT limits7 (sec)8 i
0.9997-1
.0009
-1+3
(
Quantitation reference9 ; MD1
205L
15
Nonachlorobiphenyls
9
9
9
208
207
206
208L
208L
206L
54:33
55:32
59:37
1.0003
1.0183
1.0003
0.9997-1
1.0174-1
0.9997-1
.0009
.0193
.0008
-1+3
6
-1+3
208L
208L/206L
206L
16
19
16
Decachlorobiphenyl
' 10
209
209L
61:15
1.0003
0.9997-1
.0008
-1+3
209L
16
Labeled compounds
I 7
7
7
7
8
8
9
9
10
188L
180L
170L
189L
202L
205L
208L
206L
209L
| 194L \
194L
194L
194L
194L
194L
194L
194L
194L
41:51 1
50:27 i
51:53
55:06
47:31
57:48
54:32
59:36
61:14
0.7304 I
0.8805
0.9055
0.9616
0.8293
1.0087
0.9517
1.0401
1.0686
0.7275-0
0.8775-0
0.9026-0
0.9587-0
0.8264-0
1.0044-1
0.9488-0
1.0358-1
1.0643-1
.7333J
.8834
.9084
.9645
.8322
.0131
.9546
.0445
.0730
20 1
20 i
20
20
20
30
20
30
30
194L
194L
194L
194L
194L
194L
194L
194L
194L
Labeled clean-up standards
3
I 5
7
28L
111L
178L
52L
| 101L \
138L
26:44
38:51 I
45:05
0.9266
1.0777 \
1.0090
0.9209-0
1.0730-1
1.0052-1
.9324
.0823)
.0127
20
20 I
20
52L
101L
138L
Labeled injection internal standards
2
4
5
6
8
1.
2.
o
J.
4.
9L
52L
101L
138L
194L
Number of chlorines
138L
138L
138L
138L
138L
on congener.
18:54
28:45
36:03
44:41
57:18
0.4230
0.6434
0.8068
1.0000
1.2824
0.4183-0.4276
0.6388-0
0.8021-0
0.9996-1
1.2777-1
.6481
.8115
.0011
.2870
25
25
25
100
25
Suffix "L" indicates labeled compound.
Multiple congeners in a box indicates congeners that co-elute or may not be adequately
Retention time (RT) reference used to locate target congener.
138L
138L
138L
138L
138L
resolved on a 30-m
SPB-octyl column.
Detection limits and minimum levels -
Matrix and concentration10
Water Other Extract
(ng/kg) (pg/uL)
ML MDL ML ML
50 1.5 5 2.5
50
50
50
50
1.6
1.9
1.6
1.6
5. Retention time of target congener.
EPA Method 1668C
2.5
__
2.5
2.5
72
April 2010
-------
6. Relative retention time (RRT) between the RT for the congener and RT for the reference.
7. RRT limits based on RT window. RTs, RRTs, and RRT limits may differ slightly from those in Table 2.
8. RT window width necessary to attempt to unambiguously identify the congener in the presence of other congeners.
9. Labeled congeners that form the quantitation reference. Areas from the exact m/z's of the congeners listed in the quantitation reference are summed, and divided by the
number of congeners in the quantitation reference. For example, for congener 10, the areas at the exact m/z's for 4L and 15L are summed and the sum is divided by 2
(because there are 2 congeners in the quantitation reference).
10. MDLs for water pooled from data from AXYS Analytical, TestAmerica-Knoxville, and Battelle-Columbus (see Reference 24). MLs for water per ML procedure at 68 FR
11790. MDLs and MLs for "Other" and "Extract" calculated from sample amount and extract volume.
11. If congeners 170L and 180L are included in the calibration and spiking solutions, these congeners should be used as RT and quantitation references.
EPA Method 1668C 73 April 2010
-------
Table 3. Concentrations of Native and Labeled Chlorinated Biphenyls in Stock Solutions
Spiking Solutions
CB Congener
Solution Concentrations
Stock (jig/mL)
Spiking (ng/mL)
Extract (ng/mL)
Native toxics/LOC1
1
3
4
15
19
37
54
77
81
104
105
114
118
123
126
155
156
157
167
169
188
189
202
205
206
208
209
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
Native congener mix stock solutions2
MoCB thru TrCB
TeCBthruHpCB
OcCB thru DeCB
2.5
5.0
7.5
Labeled toxics/LOC/window-defining3
1L
3L
4L
15L
19L
37L
54L
77L
81L
104L
105L
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
100
100
100
100
100
100
100
100
100
100
100
EPA Method 1668C
74
April 2010
-------
Table 3. Concentrations of Native and Labeled Chlorinated Biphenyls in Stock Solutions
Spiking Solutions
CB Congener
114L
118L
123L
126L
155L
156L
157L
167L
169L
188L
189L
202L
205L
206L
208L
209L
Solution Concentrations
Stock (jig/mL)
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Spiking (ng/mL)
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
.2.0
2.0
2.0
2.0
2.0
Extract (ng/mL)
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Labeled clean-up4
28L
111L
178L
1.0
1.0
1.0
2.0
2.0
2.0
100
100
100
Labeled injection internal5
9L
52L
101L
138L
194L
Diluted combined 209 congener6
Standard
Native congeners
MoCB thru TrCB
TeCB thru HpCB
OcCB thru DeCB
Labeled toxics/LOC/window-defining
Labeled cleanup
Labeled injection internal
5.0
5.0
5.0
5.0
5.0
1000
1000
1000
1000
1000
100
100
100
100
100
Solution Concentration (ng/mL)
Native Labeled
25
50
75
100
100
100
1. Stock solution: Section 7.8.1; Spiking solution: Section 7.11
2. Section 7.8.2.1
3. Stock solution: Section 7.9.1; Spiking solution: Section 7.12
4. Stock solution: Section 7.9.2; Spiking solution: Section 7.13
5. Stock solution: Section 7.9.3; Spiking solution: Section 7.14
6. Section 7.10.2.2.2
EPA Method 1668C
75
April 2010
-------
Table 4. Composition of Individual Native CB Congener Solutions1
Solution Identifier
A2
B2
C2
D2
E2
Accu-Standard part number
M-1668A-1
2
10
9
6
8
14
11
30
27
32
34
26
31
33
36
38
35
50
45
52
49
75
41
72
57
63
66
79
78
81
96
103
95
88
89
92
113
83
119
87
85
82
120
124
106
M-1668A-2
7
5
12
18
24
23
28
22
39
53
51
73
48
62
71
68
58
61
55
60
94
100
91
121
90
99
109
117
111
108
118
114
150
145
135
149
139
132
165
168
137
160
128
162
157
M-1668A-3
13
17
29
20
46
65
59
40
67
76
80
93
84
101
112
86
116
107
154
147
140
146
141
164
158
182
174
173
193
M-1668A-4
25
21
69
47
42
a64
70
102
97
115
123
134
131
163
180
M-1668A-5
1
3
4
15
19
16
37
54
43
44
74
56
77
104
98
125
110
126
155
138
169
188
189
202
205
208
206
209
EPA Method 1668C
76
April 2010
-------
Table 4. Composition of Individual Native CB Congener Solutions1
Solution Identifier
A2
B2
C2
D2
E2
Accu-Standard part number
M-1668A-1
122
105
127
152
136
148
151
144
143
142
133
161
153
130
129
166
159
167
156
179
176
178
175
183
177
171
172
191
170
190
201/200
204
200/199
198
196
195
194
207
Total number
of congeners
83
M-1668A-2
184
186
187
185
181
192
197
199/201
203
54
M-1668A-3
29
M-1668A-4
15
M-1668A-5
28
1. Congeners present in each standard solution are listed in elution order for each
level of chlorination. Congener number (Table 1) listed first; BZ number
listed second, where ambiguous. See Table 3 for concentrations of congeners
in stock solutions and Table 5 for concentrations in calibration standards.
EPA Method 1668C
77
April 2010
-------
Table 5. Concentration of Congeners in Calibration and Calibration Verification Standards
Congener Name
Congener
No.1
Solution Concentration (ng/mL)
CS-0.2 (Hi sens)2
CS-1
CS-2
CS-3 (VER)
CS-4
CS-5
Native toxics/LOC
2-MoCB
4-MoCB
2,2'-DiCB
4,4'-DiCB
2,2',6'-TrCB
3,4,4'-TrCB
2,2',6,6'-TeCB
3,3',4,4'-TeCB
3,4,4',5-TeCB
2,2',4,6,6'-PeCB
2,3,3',4,4'-PeCB
2,3,4,4',5-PeCB
2,3',4,4',5-PeCB
2',3,4,4',5-PeCB
3,3',4,4',5-PeCB
2,2',4,4',6,6'-HxCB
2,3,3',4,4',5-HxCB
2,3,3',4,4',5'-HxCB
2,3',4,4',5,5'-HxCB
3,3',4,4',5,5'-HxCB
2,2',3,4',55656'-HpCB
2,3,3',4,4',5,5'-HpCB
2,2',3,3',5,5',6,6'-OcCB
2,3,3',4,4',5,5',6-OcCB
2,2',3,3',4,4',5,5',6-NoCB
2,2',3,3',4',5,5',6,6'-NoCB
DeCB
1
3
4
15
19
37
54
77
81
104
105
114
118
123
126
155
156
157
167
169
188
189
202
205
206
208
209
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
2000
Labeled toxics/LOC/window-defining
13C12-2-MoCB
13C12-4-MoCB
13C12-2,2'-DiCB
13C12-4,4'-DiCB
13C12-2,2',6'-TrCB
13C12-3,4,4'-TrCB
13C12-2,2',6,6'-TeCB
13C12-3,3',4,4'-TeCB
13C12-3,4,4',5-TeCB
13C12-2,2',4,6,6'-PeCB
13C12-2,3,3'A4'-PeCB
13C12-2,3,4,4',5-PeCB
13C12-2,3',4,4',5-PeCB
13C12-2',3A4',5-PeCB
13C12-3,3',4,4',5-PeCB
13C12-2,2',4,4',6,6'-HxCB
13C12-2,3,3',4,4',5-HxCB
13C12-2,3,3',4,4',5'-HxCB
13C12-2,3',4,4',5,5'-HxCB
13C12-3,3',4,4',5,5'-HxCB
1L
3L
4L
15L
19L
37L
54L
77L
81L
104L
105L
114L
118L
123L
126L
155L
156L
157L
167L
169L
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
EPA Method 1668C
78
April 2010
-------
Table 5. Concentration of Congeners in Calibration and Calibration Verification Standards
Congener Name
13C12-2,2',3,4',5,6,6'-HpCB
13C12-2,3,3',4,4',5,5'-HpCB
13C12-2,2',3,3',5,5',6,6'-OcCB
13C12-2,353',4,4',5,5',6-OcCB
13C12-2,2',3,3',4,4',5,5',6-NoCB
13C12-2,2',3,3',4',5,5',6,6'-NoCB
13C12-DeCB
Congener
No.1
188L
189L
202L
205L
206L
208L
209L
Solution Concentration (ng/mL)
CS-0.2 (Hi sens)2
100
100
100
100
100
100
100
CS-1
100
100
100
100
100
100
100
CS-2
100
100
100
100
100
100
100
CS-3 (VER)
100
100
100
100
100
100
100
CS-4
100
100
100
100
100
100
100
CS-5
100
100
100
100
100
100
100
Labeled clean-up
13C12-2A4'-TrCB
13C12-2,3,3',5,5'-PeCB
13C12-2,2',3,3',5,5',6-HpCB
28L
111L
178L
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Labeled injection internal
13C12-2,5-DiCB
13C12-2,2',5,5'-TeCB
13C12-2,2',4',555'-PeCB
13C12-2,2',3',4,4',5'-HxCB
13C12-2,2',3,3',4,4',5,5'-OcCB
9L
52L
101L
138L
194L
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
1. Suffix "L" indicates labeled compound.
2. Additional concentration used for calibration of high sensitivity HRGC/HRMS systems. If the ion abundance ratio (Table 8)
cannot be achieved at this level (see Section 10.3.3), a calibration point at 0.4 or 0.5 ng/mL may be used.
EPA Method 1668C
79
April 2010
-------
Table 6. QC Acceptance Criteria for VER, IPR, OPR, and Labeled Compounds in Samples
1,2
Congener Name
2-MoCB
4-MoCB
2,2'-DiCB
4,4'-DiCB
2,2'6-TrCB
3,4,4'-TrCB
2,2'6,6'TeCB
3,3',4,4'-TeCB
3,4,4',5-TeCB
2,2',4,6,6'-PeCB
2,3,3',4,4'-PeCB
2,3,4,4',5-PeCB
2,3',4,4',5-PeCB
2',3,4,4',5-PeCB
3,3',4,4',5-PeCB
2,2',4,4',6,6'-HxCB
2,3,3',4,4',5-HxCB 6
2,3,3',4,4',5'-HxCB 6
2,3',4,4',5,5'-HxCB
3,3',4,4',5,5'-HxCB
2,2',3,4',5,6,6'-HpCB
2,3,3',4,4',555'-HpCB
2,2',3,3',5,5'56,6'-OcCB
2,3,3',4,4',5,5',6-OcCB
2,2',3,3',4,4',5,5',6-NoCB
2,2',3,3,'4,5,5',6,6'-NoCB
DeCB
13C12-2-MoCB
13C12-4-MoCB
13C12-2,2'-DiCB
13C12-4,4'-DiCB
13C12-2,2',6-TrCB
13C12-3,4,4'-TrCB
13C12-2,2',6,6'-TeCB
Congener
No.3
1
3
4
15
19
37
54
77
81
104
105
114
118
123
126
155
156
157
167
169
188
189
202
205
206
208
209
1L
3L
4L
15L
19L
37L
54L
Test Cone. (ng/mL)4
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
100
100
100
100
100
100
100
VER (%)s
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
75 - 125
50-145
50-145
50-145
50-145
50-145
50-145
50-145
RSD (%)
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
70
70
70
70
70
70
70
IPR
Mean Recovery (%)
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
20-135
20-135
20-135
20-135
20-135
20-135
20-135
OPR Recovery (%)
60-135
60-135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60-135
60 - 135
60-135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
60 - 135
15 - 145
15 - 145
15 - 145
15 - 145
15 - 145
15 - 145
15 - 145
Labeled Compound
Recovery in Samples (%)
NA
5-145
5-145
5-145
5-145
5-145
5-145
5-145
EPA Method 1668C
April 2010
-------
Table 6. QC Acceptance Criteria for VER, IPR, OPR, and Labeled Compounds in Samples
1,2
Congener Name
13C12-3,3',4,4'-TeCB
13C12-3,4,4',5-TeCB
13C12-2,2',4,6,6'-PeCB
13C12-2,3,3',4,4'-PeCB
13C12-2,3,4,4',5-PeCB
13C12-2,3',4,4',5-PeCB
13C12-2',3,4,4',5-PeCB
13C12-3,3',4,4',5-PeCB
13C12-2,2',4,4',6,6'-HxCB
13Ci2-2,3,3',4,4',5-HxCB6
13C12-2,3,3',4,4',5'-HxCB 6
13C12-2,3',4,4',5,5'-HxCB
13C12-3,3',4,4',5,5'-HxCB
13C12-2,2',3,4',5,6,6'-HpCB
13C12-2',3,3',4,4',5,5'-HpCB
13C12-2,2',3,3',5,5',6,6'-OcCB
13C12-2,3,3',4,4',5,5',6-OcCB
13C12-2,2',3,3',4,4',5,5',6-NoCB
13C12-2,2',3,3',4,5,5',6,6'-NoCB
13C12-2,2',3,3',4,4',5,5',6,6'-DeCB
Cleanup standards
13C12-2,4,4'-TrCB
13C12-2,3,3',5,5'-PeCB
13C12-2,2',3,3',5,5',6-HpCB
Congener
No.3
77L
81L
104L
105L
114L
118L
123L
126L
155L
156L
157L
167L
169L
188L
189L
202L
205L
206L
208L
209L
28L
111L
178L
Test Cone. (ng/mL)4
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
VER (%)s
50-145
50-145
50 - 145
50 - 145
50-145
50 - 145
50-145
50 - 145
50-145
50-145
50-145
50-145
50-145
50-145
50-145
50-145
50 - 145
50-145
50 - 145
50 - 145
65-135
75 - 125
75 - 125
RSD (%)
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
70
50
50
IPR
Mean Recovery (%)
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
45-135
20-135
45-135
45-135
OPR Recovery (%)
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
40 - 145
15 - 145
40 - 145
40 - 145
Labeled Compound
Recovery in Samples (%)
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
10-145
5-145
10-145
10-145
1. Reference 22 describes how interlaboratory results were pooled from analyses of wastewater, biosolids, and fish tissue samples.
2. QC acceptance criteria for IPR, OPR, and samples based on a 20-|iL extract final volume
3. Suffix "L" indicates labeled compound.
4. See Table 5.
5. Section 15.3.
6. CBs 156/157 and 156L/157L are tested as the sum of the two congeners
NA = Not applicable
EPA Method 1668C
81
April 2010
-------
Table 7. Scan Descriptors, Levels of Chlorination, m/z Information, and Substances Monitored by
HRGC/HRMS
Function and Chlorine Level
Fn-1; Cl-1
Fn-2; Cl-2, 3
Fn-3; Cl-3, 4, 5
Fn-4; Cl-4, 5, 6
m/z1
188.0393
190.0363
200.0795
202.0766
218.9856
222.0003
223.9974 (2)
225.9944
234.0406
236.0376
242.9856
255.9613
257.9584
268.0016
269.9986
255.9613
257.9584
259.9554
268.0016
269.9986
280.9825
289.9224
291.9194
293.9165
301.9626
303.9597
323.8834
325.8804
327.8775
337.9207
339.9178
289.9224
291.9194
293.9165
301.9626
303.9597
323.8834
325.8804
327.8775
330.9792
337.9207
339.9178
m/z Type
M
M+2
M
M+2
lock
M
M+2
M+4
M
M+2
lock
M
M+2
M
M+2
M
M+2
M+4
M
M+2
lock
M
M+2
M+4
M
M+2
M
M+2
M+4
M+2
M+4
M
M+2
M+4
M
M+2
M
M+2
M+4
lock
M+2
M+4
m/z Formula
12C12H935C1
12C12H937C1
13C12H935C1
13C12H937C1
C4F9
12C12H835C12
12C12H835C137C1
12C12H837C12
13C12H835C12
13C12H835C137C1
C6F9
12C12H735C13
12C12H735C1237C1
13C12H735C13
13C12H735C1237C1
12p TT 35r,
C12H7 C13
12C12H735C1237C1
12C12H735C137C12
13C12H735C13
13C12H735C1237C1
C6Fn
12C12H635C14
12C12H635C1337C1
12C12H635C1237C12
13C12H635C14
13C12H635C1337C1
13C12H535C15
12C12H535C1437C1
12C12H535C1337C12
13C12H535C1437C1
13C12H535C1337C12
Ci2H6 C14
12C12H635C1337C1
12C12H635C1237C12
13C12H635C1337C1
13C12H635C1237C12
12C12H535C15
12C12H535C1437C1
12C12H535C1337C12
C7F15
13C12H535C1437C1
13C12H535C1337C12
Substance
Cl-1 CB
Cl-1 CB
13C12 Cl-1 CB
13C12 Cl-1 CB
PFK
Cl-2 PCB
Cl-2 PCB
Cl-2 PCB
13C12 Cl-2 PCB
13C12 Cl-2 PCB
PFK
Cl-3 PCB
Cl-3 PCB
13C12 Cl-3 PCB
13C12 Cl-3 PCB
Cl-3 PCB
Cl-3 PCB
Cl-3 PCB
13C12 Cl-3 PCB
13C12 Cl-3 PCB
PFK
Cl-4 PCB
Cl-4 PCB
Cl-4 PCB
13C12 Cl-4 PCB
13C12 Cl-4 PCB
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
13C12 Cl-5 PCB
13C12 Cl-5 PCB
Cl-4 PCB
Cl-4 PCB
Cl-4 PCB
13C12 Cl-4 PCB
13C12 Cl-4 PCB
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
PFK
13C12 Cl-5 PCB
13C12 Cl-5 PCB
EPA Method 1668C
82
April 2010
-------
Table 7. Scan Descriptors, Levels of Chlorination, m/z Information, and Substances Monitored by
HRGC/HRMS
Function and Chlorine Level
Fn-4; Cl-4, 5, 6
Fn-5; Cl-5, 6, 7
Fn-6; Cl-7, 8, 9, 10
m/z1
359.8415
361.8385
363.8356
371.8817
373.8788
323.8834
325.8804
327.8775
337.9207
339.9178
354.9792
359.8415
361.8385
363.8356
371.8817
373.8788
393.8025
395.7995
397.7966
405.8428
407.8398
454.9728
393.8025
395.7995
397.7966
405.8428
407.8398
427.7635
429.7606
431.7576
439.8038
441.8008
442.9728
454.9728
461.7246
463.7216
465.7187
473.7648
475.7619
495.6856
497.6826
499.6797
m/z Type
M+2
M+4
M+6
M+2
M+4
M
M+2
M+4
M+2
M+4
lock
M+2
M+4
M+6
M+2
M+4
M+2
M+4
M+6
M+2
M+4
QC
M+2
M+4
M+6
M+2
M+4
M+2
M+4
M+6
M+2
M+4
QC
lock
M+2
M+4
M+6
M+2
M+4
M+2
M+4
M+6
m/z Formula
12C12H435C1537C1
12C12H435C1437C12
12C12H435C1337C13
13C12H435C1537C1
13C12H435C1437C12
12C12H535C15
12C12H535C1437C1
12C12H535C1337C12
13C12H535C1437C1
13C12H535C1337C12
C9F13
12C12H435C1537C1
12C12H435C1437C12
12C12H435C1337C13
13C12H435C1537C1
13C12H435C1437C12
12C12H335C1637C1
12C12H335C1537C12
12C12H335C1437C13
13C12H335C1637C1
13C12H335C1537C12
CnF17
12C12H335C1637C1
12C12H335C1537C12
12C12H335C1437C13
13C12H335C1637C1
13C12H335C1537C12
12C12H235C1737C1
12C12H235C1637C12
12C12H235C1537C13
13C12H235C1737C1
13C12H235C1637C12
CioFi3
Cn FIS
uCuUi35Cls31Cl
''QzHj^CV'Clz
^CizH^Cls^Cls
"dzHj^Clg^Cl
"CizHj^CV'Clz
12C12H435C1937C1
12C12 35C18 37C12
12C1235C1737C13
Substance
Cl-6 PCB
Cl-6 PCB
Cl-6 PCB
13C12 Cl-6 PCB
13C12 Cl-6 PCB
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
13C12 Cl-5 PCB
13C12 Cl-5 PCB
PFK
Cl-6 PCB
Cl-6 PCB
Cl-6 PCB
13C12 Cl-6 PCB
13C12 Cl-6 PCB
Cl-7 PCB
Cl-7 PCB
Cl-7 PCB
13C12 Cl-7 PCB
13C12 Cl-7 PCB
PFK
Cl-7 PCB
Cl-7 PCB
Cl-7 PCB
13C12 Cl-7 PCB
13C12 Cl-7 PCB
Cl-8 PCB
Cl-8 PCB
Cl-8 PCB
13C12 Cl-8 PCB
13C12 Cl-8 PCB
PFK
PFK
Cl-9 PCB
Cl-9 PCB
Cl-9 PCB
13C12 Cl-9 PCB
13C12 Cl-9 PCB
Cl-10 PCB
Cl-10 PCB
Cl-10 PCB
EPA Method 1668C
83
April 2010
-------
Table 7. Scan Descriptors, Levels of Chlorination, m/z Information, and Substances Monitored by
HRGC/HRMS
Function and Chlorine Level
Fn-6; Cl-7, 8, 9, 10
m/z1
507.7258
509.7229
511.7199
m/z Type
M+2
M+4
M+6
m/z Formula
13C12 35C19 37C1
13C1235C1837C12
13C12 35C17 37C13
Substance
13C12 C1-10PCB
13C12 C1-10PCB
13C12 C1-10PCB
1. Isotopic masses used for accurate mass calculation
'H
12C
13C
35C1
37C1
19F
1.0078
12.0000
13.0034
34.9689
36.9659
18.9984
2. An interference with PFK m/z 223.9872 may preclude meeting 10:1 S/N for the DiCB congeners at the CS-0.2
andCS-1 calibration levels (Section 10.3.3 andTableS). If this interferences occurs, 10:1 S/N must be met at
the CS-2 level. See the note at Section 10.2.1 for information on how to minimize this interference.
EPA Method 1668C
84
April 2010
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Table 8. Theoretical Ion Abundance Ratios and QC Limits
Chlorine Atoms
1
2
3
4
5
6
7
8
9
10
m/z's Forming Ratio
M/(M+2)
M/(M+2)
M/(M+2)
M/(M+2)
(M+2)/(M+4)
(M+2)/(M+4)
(M+2)/(M+4)
(M+2)/(M+4)
(M+2)/(M+4)
(M+4)(M+6)
Theoretical Ratio
3.13
1.56
1.04
0.77
1.55
1.24
1.05
0.89
0.77
1.16
Lower QC Limit
2.66
1.33
0.88
0.65
1.32
1.05
0.89
0.76
0.65
0.99
Upper QC Limit
3.60
1.79
1.20
0.89
1.78
1.43
1.21
1.02
0.89
1.33
EPA Method 1668C
85
April 2010
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Table 9. Suggested Sample Quantities to be Extracted for Various Matrices1
Sample Matrix2
Example
Percent Solids
Phase
Quantity Extracted
Single-phase
Aqueous
Solid
Organic
Tissue
Drinking water
Groundwater
Treated wastewater
Dry soil
Compost
Ash
Waste solvent
Waste oil
Organic polymer
Fish
Human adipose
<1
>20
<1
3
Solid
Organic
Organic
1000 mL
10 g
10 g
10 g
Multi-phase - Liquid/Solid
Aqueous/Solid
Organic/solid
Wet soil
Untreated effluent
Digested municipal sludge
Filter cake
Paper pulp
Industrial sludge
Oily waste
1-30
1-100
Solid
Both
10 g
10 g
Multi-phase - Liquid/Liquid
Aqueous/organic
In-process effluent
Untreated effluent
Drum waste
<1
Organic
10 g
Multi-phase - Liquid/Liquid/Solid
Aqueous/organic/solid
Untreated effluent
Drum waste
>1
Organic
and solid
10 g
1. The quantity of sample to be extracted is adjusted to provide 10 g of solids (dry weight). One liter of aqueous
samples containing one percent solids will contain 10 grams of solids. For aqueous samples containing greater
than one percent solids, a lesser volume is used so that 10 grams of solids (dry weight) will be extracted. Other
sample volumes may be used to meet project needs.
2. The sample matrix may be amorphous for some samples. In general, when the CBs are in contact with a multi-
phase system in which one of the phases is water, they will be preferentially dispersed in or adsorbed on the
alternate phase because of their low solubility in water.
3. Aqueous samples are filtered after spiking with the labeled compounds. The filtrate and the materials trapped on
the filter are extracted separately, and the extracts are combined for cleanup and analysis.
EPA Method 1668C
April 2010
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Determine % solids
§11.2
Determine particle see
§ 11.3
Prep per § 11.5
Spike Labeled Toxics/LOC
window-deirters per
511.5.2.2
Particle
size > 1 mm'
(from §11.3)
SDS extraction
per §12.3
Prep per § 11.4
Spike Labeled Toxics/LOC
window-definers per
§11.4.2.2
Spike Cleanup standard per
§12.5.1
Back extract pec
§12.5
Transfer firauqh
Na2S04 per §12.5.6
Extract per §12.2.1
§ 12.2.2, or §12.2.3
Concentrate per
§12.6-§12.7
Clean up per
§13.2-§13.5, or §13.7
Concentrate per
§12.6-§12.7
Spike injection internal
standard per § 14.2
Analyze per
§14-§18
Figure 1 Flow Chart for Analysis of Aqueous and Solid Samples
EPA Method 1668C
87
April 2010
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Aqueous
Discard
Determine % solids
per§1?-2
Determine partide size
per§ 11.3
Spike Labeled ToxicsfLOC
window-definets per §
11.6.2
Figure 2 Flow Chart for Analysis of Multi-Phase Samples
Spike Geamip standard per
612.5.1
Clean up per
§13.2-§13.5, §13.7
Spike injection internal
standard per § 14.2
EPA Method 1668C
April 2010
-------
Homogenize tissue
per § 11.8.1
Remove 10 g
per§ 11.6.1.4
Spike Labeled ToxiesfLOC
win
-------
1000
750
500
^50
90-mmGMF ISO Filler
1-Liter Suction Flask
Figure 4. Solid-phase Extraction Apparatus
EPA Method 1668C
90
April 2010
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Figure 5. Soxhlet/Dean Stark Extractor
EPA Method 1668C
91
April 2010
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3
,
9
;
21
8
17
; 1
!
|
M
is a
i
i
y
i
I \
34
^ t
\
A
/ X
2H1
a
1 f
"
1
|
TJ
3
25
I I]
im
i
j
1
a
3
21
|
( F
3
33
f
f
J
3<
J
i 3!
i
t
f
ij
) 31
I
'A/
3
* 37
i
l^
Figiu'e 6 Octyl column resolution test #1: Separation of Cl-3 congeners 34 and 23 with valley <40% (i.e.
100x/y<40%)
EPA Method 1668C
92
April 2010
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ITS Itfi
ISS 1B4 I
174 mi n
175 Va !« J77
171 187 133 181
/tv
..A
WW1SO
I7Z
190
191 170
189
M AA
"K'< ,,,,...,
Tx
Figm'e 7 Octyl column resolution test #2: Separation of Cl-7 congeners 187 and 182 with valley < 40%
(i.e. 100x/y<40%)
EPA Method 1668C
93
April 2010
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C110
C19
C18
C17
C16
CIS
14
C13
C12
Cll
II
ill
Liiiiii
i I I i i i i i r
10 15 20 25 30 35 40 45 50 55
Figure S. CB congeners at each level of chlorination on the SPB-octyl colunui
EPA Method 1668C
94
April 2010
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24.0 Glossary
These definitions and purposes are specific to this method, but have been conformed to common usage to
the extent possible.
24.1 Units of weight and measure and their abbreviations
24.1.1 Symbols
°C degrees Celsius
(iL microliter
(im micrometer
< less than
> greater than
% percent
24.1.2 Alphabetical abbreviations
cm centimeter
g gram
h hour
ID inside diameter
in. inch
L liter
M molecular ion
m meter
mg milligram
min minute
mL milliliter
mm millimeter
m/z mass-to-charge ratio
N normal; gram molecular weight of solute divided by hydrogen equivalent of
solute, per liter of solution
OD outside diameter
pg picogram
ppb part-per-billion
ppm part-per-million
ppq part-per-quadrillion
ppt part-per-trillion
psig pound-per-square-inch gauge
v/v volume per unit volume
w/v weight per unit volume
24.2 Definitions and acronyms (in alphabetical order)
Analyte - A CB tested for by this method. The analytes are listed in Table 1.
Calibration standard (CAL) - A solution prepared from a secondary standard and/or stock
solutions and used to calibrate the response of the HRGC/HRMS instrument.
Calibration verification standard (VER) - The mid-point calibration standard (CS-3) that is used
to verify calibration. See Table 5.
EPA Method 1668C 95 April 2010
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CB - Chlorinated biphenyl congener. One of the 209 individual chlorinated biphenyl congeners
determined using this method. The 209 CBs are listed in Table 1.
CS-0.2, CS-1, CS-2, CS-3, CS-4, CS-5 - See Calibration standards and Table 5
DeCB - Decachlorobiphenyl (PCB 209)
DiCB - Dichlorobiphenyl
Field blank - An aliquot of reagent water or other reference matrix that is placed in a sample
container in the laboratory or the field, and treated as a sample in all respects, including exposure to
sampling site conditions, storage, preservation, and all analytical procedures. The purpose of the
field blank is to determine if the field or sample transporting procedures and environments have
contaminated the sample.
GC - Gas chromatograph or gas chromatography
GPC - Gel permeation chromatograph or gel permeation chromatography
HpCB - Heptachlorobiphenyl
HPLC - High performance liquid chromatograph or high performance liquid chromatography
HRGC - High resolution GC
HRMS - High resolution MS
HxCB - Hexachlorobiphenyl
Labeled injection internal standard - All five, or any one of the five, 13Ci2-labeled CB congeners
spiked into the concentrated extract immediately prior to injection of an aliquot of the extract into
the HRGC/HRMS. The five Labeled injection internal standards in this method are CBs with
congener numbers 9L, 52L, 101L, 138L, and 194L.
Internal standard - a labeled compound used as a reference for quantitation of other labeled
compounds and for quantitation of native CB congeners other than the congener of which it is a
labeled analog. See Internal standard quantitation.
Internal standard quantitation - A means of determining the concentration of (1) a naturally
occurring (native) compound by reference to a compound other than its labeled analog and (2) a
labeled compound by reference to another labeled compound
IPR - Initial precision and recovery; four aliquots of a reference matrix spiked with the analytes of
interest and labeled compounds and analyzed to establish the ability of the laboratory to generate
acceptable precision and recovery. An IPR is performed prior to the first time this method is used
and any time the method or instrumentation is modified.
Isotope dilution quantitation - A means of determining a naturally occurring (native) compound
by reference to the same compound in which one or more atoms has been isotopically enriched. In
this method, all 12 carbon atoms in the biphenyl molecule are enriched with carbon-13 to produce
13Ci2- labeled analogs of the chlorinated biphenyls. The 13Ci2-labeled CBs are spiked into each
EPA Method 1668C 96 April 2010
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sample and allow identification and correction of the concentration of the native compounds in the
analytical process.
K-D - Kuderna-Danish concentrator; a device used to concentrate the analytes in a solvent
Laboratory blank - See Method blank
Laboratory control sample (LCS) - See Ongoing precision and recovery standard (OPR)
Laboratory reagent blank - See Method blank
May - This action, activity, or procedural step is neither required nor prohibited.
May not - This action, activity, or procedural step is prohibited.
Method blank - An aliquot of reagent water that is treated exactly as a sample including exposure
to all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with
samples. The method blank is used to determine if analytes or interferences are present in the
laboratory environment, the reagents, or the apparatus.
Method Detection Limit - The minimum concentration of a substance that can be measured and
reported with 99% confidence that the analyte concentration is greater than zero (40 CFR 136,
Appendix B)
Minimum level of quantitation (ML) - The lowest level at which the entire analytical system
must give a recognizable signal and acceptable calibration point for the analyte. The ML represents
the lowest concentration at which an analyte can be measured with a known level of confidence. It
may be equivalent to the concentration of the lowest calibration standard, assuming that all method-
specified sample weights, volumes, and cleanup procedures have been employed. The ML is
calculated by multiplying the MDL (pooled or unpooled, as appropriate) by 3.18 and rounding the
result to the number nearest to 1, 2, or 5 x 10 n, where n is zero or an integer (see 68 FR 11790).
MoCB - Monochlorobiphenyl
MS - Mass spectrometer or mass spectrometry
Must - This action, activity, or procedural step is required.
NoCB - Nonachlorobiphenyl
OcCB - Octachlorobiphenyl
OPR - Ongoing precision and recovery standard (OPR); a method blank spiked with known
quantities of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the
results produced by the laboratory remain within the limits specified in this method for precision
and recovery.
Perfluorokerosene (PFK) - A mixture of compounds used to calibrate the exact m/z scale in the
HRMS
Preparation blank - See Method blank
EPA Method 1668C 97 April 2010
-------
Quality control check sample (QCS) - A sample containing all or a subset of the analytes at
known concentrations. The QCS is obtained from a source external to the laboratory or is prepared
from a source of standards different from the source of calibration standards. It is used to check
laboratory performance with test materials prepared external to the normal preparation process.
PeCB - Pentachlorobiphenyl
PCB - Polychlorinated biphenyl
Reagent water - Water demonstrated to be free from the analytes of interest and potentially
interfering substances at the method detection limit for the analyte.
Relative standard deviation (RSD) - The standard deviation times 100 divided by the mean. Also
termed "coefficient of variation."
RF - Response factor. See Section 10.5.
RR - Relative response. See Section 10.4.
SDS - Soxhlet/Dean-Stark extractor; an extraction device applied to the extraction of solid and
semi-solid materials (Reference 11 and Figure 5)
Signal-to-noise ratio (S/N) - The height of the signal as measured from the mean (average) of the
noise to the peak maximum divided by the width of the noise
Should - This action, activity, or procedural step is suggested but not required.
SICP - Selected ion current profile; the line described by the signal at an exact m/z
SPE - Solid-phase extraction; an extraction technique in which an analyte is extracted from an
aqueous sample by passage over or through a material capable of reversibly adsorbing the analyte.
Also termed liquid-solid extraction.
Stock solution - A solution containing an analyte that is prepared using a reference material
traceable to EPA, the National Institute of Science and Technology (NIST), or a source that will
attest to the purity and authenticity of the reference material.
TeCB - Tetrachlorobiphenyl
TEF - Toxicity equivalency factor; an estimate of the toxicity of a specific congener relative to
2,3,7,8-tetrachlorodibenzo-p-dioxin
TEQ - The toxicity equivalent concentration in an environmental sample. It is the sum of the
concentrations of each individual toxic PCB and each individual 2,3,7,8-substituted, tetra-through
octa-chlorinated, dibenzo-p-dioxin and dibenzofuran multiplied by their respective TEFs
(Reference 1).
IB - The portion of the TEQ attributable to the toxic PCBs
TrCB - Trichlorobiphenyl
EPA Method 1668C 98 April 2010
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Unique GC resolution or uniquely resolved - Two adjacent chromatographic peaks in which the
height of the valley is less than 40 percent of the height of the shorter peak. See Section 6.9.1.1.2
and Figures 6 and 7 for unique resolution specific to the SPB-octyl column.
VER - See Calibration verification
EPA Method 1668C 99 April 2010
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Appendix A - Preliminary Information for Determination of 209 CBs
on the DB-1 Column
1.0 Column and Conditions
1.1 Column - 30 ± 5-m long x 0.25 ± 0.02-mm ID; 0.25 (im film DB-1 (J&W, or equivalent).
1.2 Suggested GC operating conditions:
Inj ector temperature: 270 °C
Interface temperature: 290 °C
Initial temperature: 75 °C
Initial time: 2 minutes
Temperature program: 75-150 °C at 15 "C/minute
150-270 °C at 2.5 °C/minute
Final time: 7 minutes
Carrier gas velocity: 40 cm/sec at 200 °C
Note: The GC conditions may be optimized for compound separation and sensitivity. Once optimized, the
same GC conditions must be used for the analysis of all standards, blanks, IPR and OPR aliquots, and
samples.
2.0 Operating Information
2.1 Congener solutions - Mixes of individual congeners that will allow separation of all 209 congeners
on the DB-1 column had not been developed when writing Method 1668C.
2.2 Elution order data - The congener mixes developed for the SPB-octyl column (Table 4 of Method
1668C) were run on the DB-1 column. Although some congeners in these mixes co-elute, the mixes
allow determination of retention times for many congeners on the DB-1 column. These retention
times are shown in Appendix Table A-l.
2.3 Window-defining congeners - The beginning and ending congeners at each level of chlorination are
the same as for the SPB-octyl column. See Table 2 in Method 1668C.
2.4 Scan descriptors - The 6-function scan descriptors are shown in Appendix Table A-2.
EPA Method 1668C 100 April 2010
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Table A-l. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Labeled or Native CB1
13C12-2-MoCB4
2-MoCB
3-MoCB
13C12-4-MoCB4'5
4-MoCB
13C12-2,2'-DiCB4
2,2'-DiCB
2,6-DiCB
2,5-DiCB
2,4-DiCB
2,3'-DiCB
2,4'-DiCB6
2,3-DiCB
13C12-2,2',6-TrCB4
3,5-DiCB
2,4,6-TrCB
3,3'-DiCB
3,4'-DiCB
3,4-DiCB
2,2',5-TrCB6
13C12-4,4'-DiCB4'5
4,4'-DiCB
2,2',4-TrCB
2,3',6-TrCB
2,3,6-TrCB
2,2',3-TrCB
2,4',6-TrCB
13C12-2,2',6,6'-TeCB4
2,2',6,6'-TeCB
2',3,5-TrCB
2,3,5-TrCB
2,4,5-TrCB
Congener
No.2
1L
1
2
3L
o
J
4L
4
10
9
7
6
8
5
19L
14
30
11
13
12
18
15L
15
17
27
24
16
32
54L
54
34
23
29
Retention Time and Quantitation
References
13C12-4-MoCB4'5
13C12-2-MoCB4
13C12-4-MoCB4'5
13C12-2,2',5,5'-TeCB7
13C12-4-MoCB4'5
13C12-4,4'-DiCB4'5
13C12-2,2'-DiCB4
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-2,4,4'-TrCB5
13C12-4,4'-DiCB4'5
13C12-2,4,4'-TrCB5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-4,4'-DiCB4'5
13C12-2,4,4'-TrCB5
13C12-2,2',5,5'-TeCB7
13C12-4,4'-DiCB4'5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,2',6,6'-TeCB4
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
Congener
No.2
3L
1L
3L
52L
3L
15L
4L
15L
15L
15L
15L
15L
15L
28L
15L
28L
15L
15L
15L
28L
52L
15L
28L
28L
28L
28L
28L
77L
54L
28L
28L
28L
RT
09:17
09:17
10:22
10:29
10:29
11:08
11:08
11:10
12:08
12:09
12:31
12:43
12:46
13:31
13:36
14:06
14:11
14:26
14:27
14:36
14:40
14:40
14:43
15:06
15:06
15:26
15:29
16:02
16:02
16:03
16:07
16:18
RRT
0.8855
1.0000
0.9889
0.5561
1.0000
0.7591
1.0000
0.7614
0.8273
0.8284
0.8534
0.8670
0.8705
0.7990
0.9273
0.8335
0.9670
0.9841
0.9852
0.8631
0.7781
1.0000
0.8700
0.8926
0.8926
0.9123
0.9153
0.6139
1.0000
0.9488
0.9527
0.9635
RRT QC Limits3
0.8776-0.8935
0.9964-1.0072
0.9809-0.9968
0.5473-0.5650
0.9968-1.0064
0.7477-0.7705
0.9925-1.0075
0.7500-0.7727
0.8216-0.8330
0.8227-0.8341
0.8477-0.8591
0.8614-0.8727
0.8648-0.8761
0.7892-0.8089
0.9216-0.9330
0.8286-0.8384
0.9614-0.9727
0.9784-0.9898
0.9795-0.9909
0.8581-0.8680
0.7692-0.7869
0.9977-1.0043
0.8650-0.8749
0.8877-0.8975
0.8877-0.8975
0.9074-0.9172
0.9103-0.9202
0.6075-0.6203
0.9979-1.0042
0.9438-0.9537
0.9478-0.9576
0.9586-0.9685
EPA Method 1668C
101
April 2010
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Table A-l. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Labeled or Native CB1
2,3',5-TrCB
2,3',4-TrCB
2,4',5-TrCB
13C12-2,4,4'-TrCB5
2,4,4'-TrCB6
2,2',4,6-TeCB
2,3,4-TrCB
2,2',5,6'-TeCB
2,3,3'-TrCB
2',3,4-TrCB
2,2',4,6'-TeCB
2,3,4'-TrCB
2,2',3,6-TeCB
3,3',5-TrCB
2,2',3,6'-TeCB
3,4',5-TrCB
13C12-2,2',5,5'-TeCB7
2,2',5,5'-TeCB6
2,3',4,6-TeCB
2,3',5',6-TeCB
2,2',4,5'-TeCB
2,2',3,5-TeCB
3,4,5-TrCB
2,2',4,4'-TeCB
2,4,4',6-TeCB
2,2',4,5-TeCB
2,3,5,6-TeCB
2,3,4,6-TeCB
3,3',4-TrCB
13C12-2,2',4,6,6'-PeCB4
2,2',4,6,6'-PeCB
2,2',3,5'-TeCB6
Congener
No.2
26
25
31
28L
28
50
21
53
20
33
51
22
45
36
46
39
52L
52
69
73
49
43
38
47
75
48
65
62
35
104L
104
44
Retention Time and Quantitation
References
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-2,2',5,5'-TeCB7
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13C12-2,2',5,5'-TeCB7
13d -3,3',4,4'-TeCB4'5'9
13d -3,3',4,4'-TeCB4'5'9
13d -3,3',4,4'-TeCB4'5'9
13d -3,3',4,4'-TeCB4'5'9
13d -3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13d -3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13d2-3,3',4,4'-TeCB4'5'9
13d2-3,3',4,4'-TeCB4'5'9
13d2-3,3',4,4'-TeCB4'5'9
13C12-2,4,4'-TrCB5
13d2-2,3',4,4',5-PeCB5'9
13C12-2,2',4,6,6'-PeCB4
13d2-3,3',4,4'-TeCB4'5'9
Congener
No.2
28L
28L
28L
52L
28L
77L
28L
77L
28L
28L
77L
28L
77L
28L
77L
28L
52L
77L
77L
77L
77L
77L
28L
77L
77L
77L
77L
77L
28L
118L
104L
77L
RT
16:29
16:36
16:52
16:55
16:55
16:55
17:21
17:26
17:22
17:24
17:42
17:43
18:00
18:16
18:24
18:37
18:51
18:51
18:52
18:57
19:00
19:04
19:12
19:15
19:20
19:20
19:31
19:36
19:41
19:45
19:45
19:55
RRT
0.9744
0.9813
0.9970
0.8974
1.0000
0.6477
1.0256
0.6675
1.0266
1.0286
0.6777
1.0473
0.6892
1.0798
0.7045
1.1005
1.0000
0.7218
0.7224
0.7256
0.7275
0.7301
1.1350
0.7371
0.7403
0.7403
0.7473
0.7505
1.1635
0.7037
1.0000
0.7626
RRT QC Limits3
0.9695-0.9793
0.9764-0.9862
0.9921-1.0020
0.8930-0.9019
0.9980-1.0039
0.6414-0.6541
1.0207-1.0305
0.6611-0.6739
1.0217-1.0315
1.0236-1.0335
0.6713-0.6841
1.0424-1.0522
0.6828-0.6956
1.0749-1.0847
0.6981-0.7109
1.0956-1.1054
0.9956-1.0044
0.7154-0.7281
0.7160-0.7288
0.7192-0.7320
0.7211-0.7339
0.7237-0.7364
1.1300-1.1399
0.7307-0.7435
0.7339-0.7466
0.7339-0.7466
0.7409-0.7537
0.7441-0.7569
1.1586-1.1685
0.6977-0.7096
0.9983-1.0034
0.7562-0.7690
EPA Method 1668C
102
April 2010
-------
Table A-l. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Labeled or Native CB1
13C12-3,4,4'-TrCB4
3,4,4'-TrCB
2,3,3',6-TeCB
2,2',3,4'-TeCB
2,3',5,5'-TeCB
2,3',4',6-TeCB
2,3,4',6-TeCB
2,2',3,4-TeCB
2,2',3,6,6'-PeCB
2,3',4,5'-TeCB
2,2',3,3'-TeCB
2,3,3',5-TeCB
2,2',4,5,'6-PeCB
2,3',4,5-TeCB
2,2',4,4',6-PeCB
2,3,3',5'-TeCB
2,3,4',5-TeCB
2,2',3,5,6'-PeCB
2,4,4',5-TeCB
2,3,4,5-TeCB
2,3',4',5-TeCB
2',3,4,5-TeCB
2,2',3',4,6-PeCB
2,3',4,4'-TeCB6
2,2',4,5,6'-PeCB
2,2',3,5',6-PeCB
2,2',3,5,6-PeCB
3,3',5,5'-TeCB
2,2',3,4,6-PeCB
2,2',3,4',6-PeCB
2,3,3',4'-TeCB
2,3',4,5,'6-PeCB
Congener
No.2
37L
37
59
42
72
71
64
41
96
68
40
57
103
67
100
58
63
94
74
61
70
76
98
66
102
95
93
80
88
91
55
121
Retention Time and Quantitation
References
13C12-2,4,4'-TrCB5
13C12-3,4,4'-TrCB4
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
Congener
No.2
28L
37L
77L
77L
77L
77L
77L
77L
118L
77L
77L
77L
118L
77L
118L
77L
77L
118L
77L
77L
77L
77L
118L
77L
118L
118L
118L
77L
118L
118L
77L
118L
RT
20:03
20:03
20:05
20:07
20:36
20:36
20:37
20:39
20:48
20:52
20:58
21:21
21:22
21:38
21:41
21:43
21:51
22:05
22:07
22:11
22:20
22:25
22:28
22:29
22:32
22:34
22:36
22:45
22:49
22:55
22:57
23:04
RRT
1.1852
1.0000
0.7690
0.7703
0.7888
0.7888
0.7894
0.7907
0.7411
0.7990
0.8028
0.8175
0.7613
0.8283
0.7726
0.8315
0.8366
0.7868
0.8468
0.8494
0.8551
0.8583
0.8005
0.8609
0.8029
0.8040
0.8052
0.8711
0.8129
0.8165
0.8787
0.8219
RRT QC Limits3
1.1803-1.1901
0.9983-1.0033
0.7626-0.7754
0.7639-0.7766
0.7824-0.7951
0.7824-0.7951
0.7830-0.7958
0.7843-0.7971
0.7352-0.7470
0.7926-0.8054
0.7996-0.8060
0.8143-0.8207
0.7553-0.7672
0.8251-0.8315
0.7666-0.7785
0.8283-0.8347
0.8334-0.8398
0.7809-0.7928
0.8437-0.8500
0.8462-0.8526
0.8519-0.8583
0.8551-0.8615
0.7975-0.8034
0.8577-0.8641
0.7999-0.8058
0.8011-0.8070
0.8023-0.8082
0.8679-0.8743
0.8100-0.8159
0.8135-0.8195
0.8756-0.8819
0.8189-0.8248
EPA Method 1668C
103
April 2010
-------
Table A-l. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Labeled or Native CB1
2,3,3',4'-TeCB
2,3,4,4'-TeCB
13C12-2,2',4,4',6,6'-HxCB4
2,2',4,4',6,6'-HxCB
2,2',3,3',6-PeCB
2,2',3,5,5'-PeCB
2,2',3,4,6'-PeCB
2,2',3,4',5-PeCB
13C12-2,2',4,5,5'-PeCB7
2,2',4,5,5'-PeCB6
2,3,3',5',6-PeCB
3,3',4,5'-TeCB
2,2',4,4',5-PeCB
2,2',3,4',6,6'-HxCB
2,3',4,4',6-PeCB
2,3,3',5,6-PeCB
2,3,3',4,6-PeCB
2,2',3,5,6,6'-HxCB
2,2',3,3',5-PeCB
2,2',3',4,5-PeCB
2,2',3,4,5-PeCB
13C12-3,4,4',5-TeCB9
3,4,4',5-TeCB10
2',3,4,5,6'-PeCB
2,3,4',5,6-PeCB
2,2',3,4,5'-PeCB
3,3',4,5-TeCB
2,2',3,4,6,6'-HxCB
2,3,4,4',6-PeCB
13C12-2,3,3',5,5'-PeCB8
2,3,3',5,5'-PeCB
2,2',3,4,4'-PeCB
Congener
No.2
56
60
155L
155
84
92
89
90
101L
101
113
79
99
150
119
112
109
152
83
97
86
81L
81
125
117
87
78
145
115
111L
111
85
Retention Time and Quantitation
References
13C12-3,3',4,4'-TeCB4'5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,2',4,4',6,6'-HxCB4
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,2',5,5'-TeCB7
13C12-3,4,4',5-TeCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5-PeCB5'9
Congener
No.2
77L
77L
167L
155L
118L
118L
118L
118L
101L
118L
118L
77L
118L
167L
118L
118L
118L
167L
118L
118L
118L
52L
77L
118L
118L
118L
77L
167L
118L
101L
118L
118L
RT
23:24
23:24
23:43
23:43
23:44
23:50
23:53
24:07
24:11
24:11
24:23
24:27
24:28
24:52
24:54
25:00
25:09
25:17
25:20
25:22
25:27
25:32
25:32
25:36
25:37
25:38
25:40
25:42
25:44
25:51
25:51
25:51
RRT
0.8960
0.8960
0.7104
1.0000
0.8456
0.8492
0.8510
0.8593
1.0000
0.8616
0.8688
0.9362
0.8717
0.7449
0.8872
0.8907
0.8961
0.7574
0.8919
0.9038
0.9068
1.3546
1.0000
0.9121
0.9127
0.9133
0.9598
0.7698
0.9169
1.0689
0.9210
0.9210
RRT QC Limits3
0.8928-0.8992
0.8928-0.8992
0.7054-0.7154
0.9986-1.0028
0.8426-0.8486
0.8462-0.8521
0.8480-0.8539
0.8563-0.8622
0.9966-1.0034
0.8587-0.8646
0.8658-0.8717
0.9330-0.9394
0.8688-0.8747
0.7399-0.7499
0.8842-0.8901
0.8878-0.8937
0.8931-0.8990
0.7524-0.7624
0.8890-0.8949
0.9008-0.9068
0.9038-0.9097
1.3457-1.3634
0.9987-1.0026
0.9091-0.9151
0.9097-0.9157
0.9103-0.9163
0.9566-0.9630
0.7649-0.7748
0.9139-0.9198
1.0655-1.0724
0.9181-0.9240
0.9181-0.9240
EPA Method 1668C
104
April 2010
-------
Table A-l. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Labeled or Native CB1
2,3,4,5,6-PeCB
13C12-3,3',4,4'-TeCB4'5'9
3,3',4,4'-TeCB6'10
2,2',3,3',6,6'-HxCB
2,3',4,5,5'-PeCB
2,2',3,4',5,6'-HxCB
2,3,3',4',6-PeCB
2,2',4,4',5,6'-HxCB
2,2',3,3',4-PeCB
2,2',3,5,5',6-HxCB
2,2',3,3',5,6'-HxCB
2',3,4,5,5'-PeCB
2,2',3,4,5',6-HxCB
2,3,3',4,5'-PeCB
2,2',3,4',5,6-HxCB
2,3,3',4',5-PeCB
2,2',3,4',5',6-HxCB
2,2',3,3',5,6-HxCB
2,2',3,4,5,6'-HxCB
13C12-2',3,4,4',5-PeCB9
2',3,4,4',5-PeCB10
2,2',3,4,4',6-HxCB
2,3,3',4,5-PeCB
13C12-2,3',4,4',5-PeCB5'9
2,3',4,4',5-PeCB6'10
2,2',3,4,4',6'-HxCB
13C12-2,3,4,4',5-PeCB9
2,3,4,4',5-PeCB10
2',3,3',4,5-PeCB
2,2',3,3',4,6-HxCB
2,2',3,4,5,6-HxCB
2,2',3,3',5,5'-HxCB
Congener
No.2
116
77L
77
136
120
148
110
154
82
151
135
124
144
108
147
107
149
134
143
123L
123
139
106
118L
118
140
114L
114
122
131
142
133
Retention Time and Quantitation
References
13C12-2,3',4,4',5-PeCB5'9
13C12-2,2',5,5'-TeCB7
13C12-3,3',4,4'-TeCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-2',3,4,4',5-PeCB9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-2,3,4,4',5-PeCB9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
Congener
No.2
118L
52L
77L
167L
118L
167L
118L
167L
118L
167L
167L
118L
167L
118L
167L
118L
167L
167L
167L
101L
123L
167L
118L
101L
118L
167L
101L
114L
118L
167L
167L
167L
RT
25:48
26:07
26:07
26:10
26:12
26:14
26:16
26:44
26:48
27:18
27:31
27:36
27:38
27:40
27:44
27:45
28:01
28:35
28:34
27:53
27:53
28:01
28:04
28:04
28:04
28:12
28:38
28:38
28:48
28:52
28:59
28:59
RRT
0.9192
1.3855
1.0000
0.7793
0.9335
0.7858
0.9359
0.8008
0.9549
0.8178
0.8243
0.9834
0.8278
0.9857
0.8308
0.9887
0.8392
0.8562
0.8557
1.1530
1.0000
0.8392
1.0000
1.1606
1.0000
0.8447
1.1840
1.0000
1.0261
0.8647
0.8682
0.8682
RRT QC Limits3
0.9163-0.9222
1.3767-1.3943
0.9987-1.0026
0.7743-0.7843
0.9305-0.9365
0.7808-0.7908
0.9329-0.9388
0.7983-0.8033
0.9519-0.9578
0.8153-0.8203
0.8218-0.8268
0.9804-0.9863
0.8253-0.8303
0.9828-0.9887
0.8283-0.8333
0.9857-0.9917
0.8367-0.8417
0.8537-0.8587
0.8532-0.8582
1.1496-1.1564
0.9988-1.0024
0.8367-0.8417
0.9970-1.0030
1.1571-1.1640
0.9988-1.0024
0.8422-0.8472
1.1806-1.1875
0.9988-1.0023
1.0232-1.0291
0.8622-0.8672
0.8657-0.8707
0.8657-0.8707
EPA Method 1668C
105
April 2010
-------
Table A-l. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Labeled or Native CB1
2,2',3,3',4,6'-HxCB
2,3,3',5,5',6-HxCB
13C12-2,2',3,4',5,6,6'-HpCB4
2,2',3,4',5,6,6'-HpCB
2,2',3,4',5,5'-HxCB
13C12-2,3,3',4,4'-PeCB9
2,3,3',4,4'-PeCB6'10
2 3 3' 4 5' 6-HxCB
2,2',4,4',5,5'-HxCB6
2,2',3,4,4',6,6'-HpCB
3,3',4,5,5'-PeCB
2,3',4,4',5',6-HxCB
2,2',3,4,5,5'-HxCB
2,2',3,3',5,6,6'-HpCB
2,2',3,4,4',5-HxCB
2,2',3,3',4,5'-HxCB
2,2',3,3',4,6,6'-HpCB
13C12-2,2',3,4,4',5'-HxCB7
2,2',3,4,4',5'-HxCB6
2,3,3',4',5',6-HxCB
2,3,3',4',5,6-HxCB
2,3,3',4,5,6-HxCB
2 3 3' 4 4' 6-HxCB
2,2',3,4,5,6,6'-HpCB
2,2',3,3',4,5-HxCB
13C12-3,3',4,4',5-PeCB4'9
3,3',4,4',5-PeCB6'10
2,3,4,4',5,6-HxCB
13C12-2,2',3,3',5,5',6-HpCB7
2 2' 3 3' 5 5' 6-HpCB
2 2' 3 3' 4 5' 6-HpCB
2,3,3',4,5,5'-HxCB
Congener
No.2
132
165
188L
188
146
105L
105
161
153
184
127
168
141
179
137
130
176
138L
138
164
163
160
158
186
129
126L
126
166
178L
178
175
159
Retention Time and Quantitation
References
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,2',3,4',5,6,6'-HpCB4
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-2,3,3',4,4'-PeCB9
13C12-2 3' 4 4' 5 5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,3',4,4',5-PeCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,2',3,4,4',5'-HxCB7
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2 3' 4 4' 5 5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,2',4,5,5'-PeCB7
13C12-3,3',4,4',5-PeCB4'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,2',3,3',5,5',6-HpCB7
13C12-2' 3 3' 4 4' 5 5'-HpCB4'5'9
13C12-2' 3 3' 4 4' 5 5'-HpCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
Congener
No.2
167L
167L
189L
188L
167L
101L
105L
167L
167L
189L
118L
167L
167L
189L
167L
167L
189L
138L
167L
167L
167L
167L
167L
189L
167L
101L
126L
167L
178L
189L
189L
167L
RT
29:32
29:21
29:22
29:22
29:24
29:30
29:30
29-32
29:48
29:49
29:57
29:59
30:31
30:33
30:51
30:57
31:01
31:20
31:20
31:22
31:28
31:33
31-35
31:36
31:48
31:49
31:49
32:13
32:14
32-14
32-33
32:43
RRT
0.8847
0.8792
0.9511
1.0000
0.8807
1.2198
1.0000
08847
0.8927
0.7482
1.0671
0.8982
0.9141
0.7666
0.9241
0.9271
0.7783
1.0000
0.9386
0.9396
0.9426
0.9451
09461
0.7930
0.9526
1.3156
1.0000
0.9651
1.0000
08089
08168
0.9800
RRT QC Limits3
0.8822-0.8872
0.8767-0.8817
0.7327-0.7411
0.9989-1.0023
0.8782-0.8832
1.2130-1.2267
0.9989-1.0023
0 8822-0 8872
0.8902-0.8952
0.7440-0.7524
1.0641-1.0701
0.8957-0.9006
0.9116-0.9166
0.7624-0.7708
0.9216-0.9266
0.9246-0.9296
0.7742-0.7825
0.9973-1.0027
0.9361-0.9411
0.9371-0.9421
0.9401-0.9451
0.9426-0.9476
0 9436-0 9486
0.7888-0.7972
0.9501-0.9551
1.3088-1.3225
0.9990-1.0021
0.9626-0.9675
0.9974-1.0026
08068-08110
0 8147-0 8189
0.9775-0.9825
EPA Method 1668C
106
April 2010
-------
Table A-l. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Labeled or Native CB1
2,2',3,4',5,5',6-HpCB6
2,2',3,4,4',5,6'-HpCB
2,2',3,3',4,4'-HxCB6
2,3,3',4',5,5'-HxCB
2,2',3,4,4',5',6-HpCB
13C12-2,3',4,4',5,5'-HxCB5'9
2 3' 4 4' 5 5'-HxCB10
2,2',3,4,5,5',6-HpCB
2,2',3,3',4,5,6'-HpCB
2,2',3,4,4',5,6-HpCB
2,2',3,3',4',5,6-HpCB
2,2'3,3',4,4',6-HpCB
13C12-2,3,3',4,4',5 -HxCB9
2,3,3',4,4',5-HxCB10
13C12-2,2',3,3',5,5',6,6'-OcCB4
2,2',3,3',5,5',6,6'-OcCB
13C12-2,3,3',4,4',5'-HxCB9
2,3,3',4,4',5'-HxCB10
2,2',3,3',4,5,6-HpCB
2,2',3,3',4,5',6,6'-OcCB
2,2',3,4,4',5,6,6'-OcCB
2,2',3,3',4,5,5'-HpCB
2,3,3',4,5,5',6-HpCB
2,2',3,3',4,4',6,6'-OcCB
2,2',3,4,4',5,5'-HpCB6
2,3,3',4',5,5',6-HpCB
2,3,3',4,4',5',6-HpCB
2,2',3,3',4,5,6,6'-OcCB
13C12-3,3',4,4',5,5'-HxCB4'9
3,3',4,4',5,5'-HxCB6'10
2,2',3,3',4,4',5-HpCB6
2,3,3',4,4',5,6-HpCB
Congener
No.2
187
182
128
162
183
167L
167
185
174
181
177
171
156L
156
202L
202
157L
157
173
201
204
172
192
197
180
193
191
200
169L
169
170
190
Retention Time and Quantitation
References
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2,3',4,4',5,5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,2',3,4,4',5'-HxCB7
13C12-2 3' 4 4' 5 5'-HxCB5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2,2',3,4,4',5'-HxCB7
13C12-2,3,3',4,4',5 -HxCB9
13C12-C18-PCB-1945
13C12-2,2',3,3',5,5',6,6'-OcCB4
13C12-2,2',3,4,4',5'-HxCB7
13C12-2,3,3',4,4',5'-HxCB9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-C18-PCB-1945
13C12-C18-PCB-1945
13f-< ^i o oi A /li c ci TJV^D 4,5,9
C12-2 ,3,3 ,4,4 ,5,5 -HpCB
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-C18-PCB-1945
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-C18-PCB-1945
13C12-2,2',3,4,4',5'-HxCB7
13C12-3,3',4,4',5,5'-HxCB4'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
Congener
No.2
189L
189L
167L
167L
189L
138L
167L
189L
189L
189L
189L
189L
138L
156L
194L
202L
138L
157L
189L
194L
194L
189L
189L
194L
189L
189L
189L
194L
138L
169L
189L
189L
RT
32:46
32:47
32:52
33:00
33:06
33:23
33-23
33:43
34:07
34:11
34:22
34:40
34:40
34:40
34:56
34:56
34:57
34:57
35:04
35:25
35:36
35:41
35:51
35:55
36:07
36:20
36:34
36:49
37:19
37:19
37:44
37:56
RRT
0.8223
0.8227
0.9845
0.9885
0.8306
1.0654
10000
0.8461
0.8561
0.8578
0.8624
0.8699
1.1064
1.0000
0.8265
1.0000
1.1154
1.0000
0.8800
0.8379
0.8423
0.8954
0.8996
0.8498
0.9063
0.9118
0.9176
0.8711
1.1910
1.0000
0.9469
0.9519
RRT QC Limits3
0.8202-0.8243
0.8206-0.8248
0.9820-0.9870
0.9860-0.9910
0.8285-0.8327
1.0628-1.0681
0 9990-1 0020
0.8440-0.8482
0.8540-0.8582
0.8557-0.8599
0.8603-0.8645
0.8678-0.8720
1.1037-1.1090
0.9990-1.0019
0.8245-0.8285
0.9990-1.0019
1.1128-1.1181
0.9990-1.0019
0.8779-0.8821
0.8360-0.8399
0.8403-0.8442
0.8934-0.8975
0.8975-0.9017
0.8478-0.8517
0.9042-0.9084
0.9097-0.9138
0.9155-0.9197
0.8691-0.8730
1.1883-1.1936
0.9991-1.0018
0.9448-0.9490
0.9498-0.9540
EPA Method 1668C
107
April 2010
-------
Table A-l. Retention time (RT) References, Quantitation References, and Relative Retention Times (RRTs) for CB Congeners using a
DB-1 Column
Labeled or Native CB1
2,2',3,3',4,5,5',6-OcCB
2,2',3,3',4,5,5',6'-OcCB
2,2',3,3',4,4',5,6'-OcCB
2 2' 3 4 4' 5 5' 6-OcCB
13C12-2',3,3',4,4',5,5'-HpCB4'5'9
2 3 3' 4 4' 5 5'-HpCB10
2,2',3,3',4,4',5,6-OcCB6
13C12-2,2',3,3',4,5,5',6,6'-NoCB4
2,2',3,3',4,5,5',6,6'-NoCB
2,2',3,3',4,4',5,6,6'-NoCB
13C12-2,2',3,3',4,4',5,5'-OcCB5
2,2',3,3',4,4',5,5'-OcCB
13C12-2,3,3',4,4',5,5',6-OcCB4
2,3,3',4,4',5,5',6-OcCB
13C12-2,2',3,3',4,4',5,5',6-NoCB4'5
2,2',3,3',4,4',5,5',6-NoCB6
13C12-2,2',3,3',4,4',5,5',6,6'-DeCB4'5
2,2',3,3',4,4',5,5',6,6'-DeCB6
Congener
No.2
198
199
196
203
189L
189
195
208L
208
207
194L
194
205L
205
206L
206
209L
209
Retention Time and Quantitation
References
13C12-C18-PCB-1945
13C12-C18-PCB-1945
13C12-C18-PCB-1945
13C12-C18-PCB-1945
13C12-2,2',3,3',5,5',6-HpCB7
13C12-2' 3 3' 4 4' 5 5'-HpCB4'5'9
13C12-C18-PCB-1945
13C12-C19-PCB-2064'5
13C12-2,2',3,3',4,5,5',6,6'-NoCB4
13C12-C19-PCB-2064'5
13C12-2,2',3,3',5,5',6-HpCB7
13C12-C18-PCB-1945
13C12-C18-PCB-1945
13C12-2,3,3',4,4',5,5',6-OcCB4
13C12-2,2',3,3',5,5',6-HpCB7
13C12-C19-PCB-2064'5
13C12-2,2',3,3',5,5',6-HpCB7
13C12-C110-PCB-2094'5
Congener
No.2
194L
194L
194L
194L
178L
189L
194L
206L
208L
206L
178L
194L
194L
205L
178L
206L
178L
209L
RT
38:34
38:43
39:05
39-05
39:51
39-51
40:45
41:03
41:03
41:32
42:16
42:16
42:44
42:44
44:52
44:52
46:55
46:55
RRT
0.9125
0.9160
0.9247
09247
1.2363
10000
0.9641
0.9149
1.0000
0.9257
1.3113
1.0000
1.0110
1.0000
1.3919
1.0000
1.4555
1.0000
RRT QC Limits3
0.9105-0.9144
0.9140-0.9180
0.9227-0.9267
0 9227-0 9267
1.2311-1.2415
0 9992-1 0017
0.9621-0.9661
0.9131-0.9168
0.9992-1.0016
0.9238-0.9276
1.3061-1.3164
0.9992-1.0016
1.0091-1.0130
0.9992-1.0016
1.3868-1.3971
0.9993-1.0015
1.4504-1.4607
0.9993-1.0014
1. Abbreviations for chlorination levels
MoCB
DiCB
TrCB
TeCB
PeCB
monochlorobiphenyl
dichlorobiphenyl
trichlorobiphenyl
tetrachlorobiphenyl
pentachlorobiphenyl
HxCB
HpCB
OcCB
NoCB
DeCB
hexachlorobiphenyl
heptachlorobiphenyl
octachlorobiphenyl
nonachlorobiphenyl
decachlorobiphenyl
2. Suffix "L" indicates labeled compound
3. For native CBs determined by isotope dilution quantitation, RRT QC limits were constructed using -2 to +4
seconds around the retention time for the labeled analog. For native CBs determined by internal standard
quantitation, RRT QC limits were constructed using a ± 2 percent window around the retention time for
retention times in the range of 0.8-1.2 and a ± 4 percent window around the retention time for retention times
<0.8 and >1.2. These windows may not be adequate for analyte identification (See the note in Section 16.4)
4. Labeled level of chlorination (LOG) window-
defining congener
5. Labeled level of chlorination (LOG) quantitation
congener
6. National Oceanic and Atmospheric Administration
(NOAA) congener of interest
7. Instrument internal standard
8. Clean-up standard
9. Labeled internal standard for World Health
Organization (WHO) toxic congener
10. WHO toxic congener
EPA Method 1668C
108
April 2010
-------
Table A-2. Scan Descriptors, Levels of Chlorination, m/z Information, and Substances Monitored
by HRGC/HRMS
Function and Chlorine Level
Fn-1 Cl-1
Fn-2 Cl-2,3
Fn-3 Cl-3,4,5
Fn-4 Cl-4,5,6
m/z
188.0393
190.0363
200.0795
202.0766
218.9856
222.0003
223.9974
225.9944
234.0406
236.0376
242.9856
255.9613
257.9584
255.9613
257.9584
259.9554
268.0016
269.9986
280.9825
289.9224
291.9194
293.9165
301.9626
303.9597
323.8834
325.8804
327.8775
337.9207
339.9178
289.9224
291.9194
293.9165
301.9626
303.9597
323.8834
325.8804
327.8775
330.9792
337.9207
339.9178
359.8415
361.8385
363.8356
371.8817
373.8788
m/z Type
M
M+2
M
M+2
lock
M
M+2
M+4
M
M+2
lock
M
M+2
M
M+2
M+4
M
M+2
lock
M
M+2
M+4
M
M+2
M
M+2
M+4
M+2
M+4
M
M+2
M+4
M+2
M+4
M
M+2
M+4
lock
M+2
M+4
M+2
M+4
M+6
M+2
M+4
m/z Formula
12C12H935C1
12C12H937C1
13C12H935C1
13C12H937C1
C4F9
12C12H835C12
12C12H835C137C1
12C12H837C12
13C12H835C12
13C12H835C137C1
C6F9
12C12H735C13
12C12H735C1237C1
12C12H735C13
12C12H735C1237C1
12C12H735C137C12
13C12H735C13
13C12H735C1237C1
CsFn
12C12H635C14
12C12H635C1337C1
12C12H635C1237C12
13C12H635C14
13C12H635C1337C1
12C12H535C15
12C12H535C1437C1
12C12H535C1337C12
13C12H535C1437C1
13C12H535C1337C12
12C12H635C14
12C12H635C1337C1
12C12H635C1237C12
13C12H635C1337C1
13C12H635C1237C12
12C12H535C15
12C12H535C1437C1
12C12H535C1337C12
C7F15
13C12H535C1437C1
13C12H535C1337C12
13C12H435C1537C1
13C12H435C1437C12
13C12H435C1337C12
13C12H435C1537C1
13C12H435C1437C12
Substance
Cl-1 PCB
C1-1P CB
13C12 Cl-1 PCB
13C12 Cl-1 PCB
PFK
Cl-2 PCB
Cl-2 PCB
Cl-2 PCB
13C12 Cl-2 PCB
13C12 Cl-2 PCB
PFK
Cl-3 PCB
Cl-3 PCB
Cl-3 PCB
Cl-3 PCB
Cl-3 PCB
13C12 Cl-3 PCB
13C12 Cl-3 PCB
PFK
Cl-4 PCB
Cl-4 PCB
Cl-4 PCB
13C12 Cl-4 PCB
13C12 Cl-4 PCB
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
13C12 Cl-5 PCB
13C12 Cl-5 PCB
Cl-4 PCB
Cl-4 PCB
Cl-4 PCB
13C12 Cl-4 PCB
13C12 Cl-4 PCB
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
PFK
13C12 Cl-5 PCB
13C12 Cl-5 PCB
Cl-6 PCB
Cl-6 PCB
Cl-6 PCB
13C12 Cl-6 PCB
13C12 Cl-6 PCB
EPA Method 1668C
109
April 2010
-------
Table A-2. Scan Descriptors, Levels of Chlorination, m/z Information, and Substances Monitored
by HRGC/HRMS
Function and Chlorine Level
Fn-5 Cl-5,6,7,8
Fn-6 Cl-8,9,10
m/z
323.8834
325.8804
327.8775
337.9207
339.9178
354.9792
359.8415
361.8385
363.8356
371.8817
373.8788
393.8025
395.7995
397.7966
405.8428
407.8398
427.7635
429.7606
431.7576
439.8038
441.8008
454.9728
427.7635
429.7606
431.7576
439.8038
441.8008
442.9728
454.9728
461.7246
463.7216
465.7187
473.7648
475.7619
495.6856
499.6797
501.6767
507.7258
509.7229
511.7199
m/z Type
M
M+2
M+4
M+2
M+4
lock
M+2
M+4
M+6
M+2
M+4
M+2
M+4
M+6
M+2
M+4
M+2
M+4
M+6
M+2
M+4
QC
M+2
M+4
M+6
M+2
M+4
QC
lock
M+2
M+4
M+6
M+2
M+4
M+2
M+4
M+6
M+2
M+4
M+6
m/z Formula
12C12H535C15
12C12H535C1437C1
12C12H535C1337C12
13C12H535C1437C1
13C12H535C1337C12
C9F13
12C12H435C1537C1
12C12H435C1437C12
12C12H435C1337C13
13C12H435C1537C1
13C12H435C1437C12
12C12H335C1637C1
12C12H335C1537C12
12C12H335C1437C13
13C12H335C1637C1
13C12H335C1537C12
12C12H235C1737C1
12C12H235C1637C12
12C12H235C1537C13
13C12H235C1737C1
13C12H235C1637C12
CnF17
12C12H235C1737C1
12C12H235C1637C12
12C12H235C1537C13
13C12H235C1737C1
13C12H235C1637C12
CioFi3
CnF13
^CizHj^dg^Cl
^CizHj^CV'Clz
uCutti35Cl631Cl3
"CizHj^dg^Cl
"C^Hj^Clv^Clj
13C12H435C1937C1
12C1235C1737C13
12C1235C1637C14
13C12H435C1937C1
13C12H435C1837C12
13C12H435C1837C14
Substance
Cl-5 PCB
Cl-5 PCB
Cl-5 PCB
13C12 Cl-5 PCB
13C12 Cl-5 PCB
PFK
Cl-6 PCB
Cl-6 PCB
Cl-6 PCB
13C12 Cl-6 PCB
13C12 Cl-6 PCB
Cl-7 PCB
Cl-7 PCB
Cl-7 PCB
13C12 Cl-7 PCB
13C12 Cl-7 PCB
Cl-8 PCB
Cl-8 PCB
Cl-8 PCB
13C12 Cl-8 PCB
13C12 Cl-8 PCB
PFK
Cl-8 PCB
Cl-8 PCB
Cl-8 PCB
13C12 Cl-8 PCB
13C12 Cl-8 PCB
PFK
PFK
Cl-9 PCB
Cl-9 PCB
Cl-9 PCB
13C12 Cl-9 PCB
13C12 Cl-9 PCB
Cl-10 PCB
Cl-10 PCB
Cl-10 PCB
13C12 Cl-10 PCB
13C12 Cl-10 PCB
13C12 Cl-10 PCB
Isotopic masses used for accurate mass calculation
'H 1.0078 37C1 36.9659
12.0000
13.0034
19F
35C1
18.9984
34.9689
EPA Method 1668C
110
April 2010
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