United States
Environmental Protection
Agency
Office of Water
Engineering and Analysis Division (4303)
Washington, DC 20460
EPA821-B-94-005
October 1994
Revision B
Method 1613: Tetra-Through Octa-
Chlorinated Dioxins and Furans by
Isotope Dilution HRGC/HRMS
> Printed on Recycled Paper
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Acknowledgments
This method was prepared under the direction of William A. Telliard of the
Engineering and Analysis Division within the EPA Office of Water.
This document was prepared under EPA Contract No. 68-C3-0337 by
DynCorp Environmental Services Division with assistance
from its subcontractor Interface, Inc.
Disclaimer
This method has been reviewed by the Engineering and Analysis Division,
U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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Introduction
i
Method 1613 was developed by the United States Environmental Protection Agency's Office of
Science and Technology for isomer-specific determination of the 2,3,7,8-substituted, tetra- through
octa-chlorinated, dibenzo-p-dioxins and dibenzofurans in aqueous, solid, and tissue matrices by isotope
ution, high resolution capillary column gas chromatography (HRGQ/high resolution mass spectrom-
di
etry (HRMS).
i
Qi|estions concerning this method or its application should be addressed to:
W-A. Telliard
US EPA Office of Water '
Analytical Methods Staff j
Mail Code 4303
40 j M Street, SW
Washington, D.C. 20460 ''
202/260-7120
Requests for additional copies should be directed to:
i ,
Water Resource Center
Ma'il Code RC-4100
401 M Street, SW
Washington, D.C. 20460
202/260-7786 or 202/260-2814
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Method 1613, Revision B
Tetra- through Octa-Chlorinated Dioxins and Furans
by Isotope Dilution HRGC/HRMS
1.0 Scope and Application
1.1 This method is for determination of tetra- through octa-chlorinated dibenzo-p-dioxins (CDDs)
and dibenzofurans (CDFs) in water, soil, sediment, sludge, tissue, and other sample matrices by
high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). The
method is for use in EPA's data gathering and monitoring programs associated with the Clean
Water Act, the Resource Conservation and Recovery Act, the Comprehensive Environmental
Response, Compensation and Liability Act, and the Safe Drinking Water Act. The. method is
based on a compilation of EPA, industry, commercial laboratory, and academic methods
(References 1-6).
1.2 The seventeen 2,3,7,8-substituted CDDs/CDFs listed in Table 1 may be determined by this
method. Specifications are also provided for separate determination of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin (2,3,7,8-TCDD) and 2,3,7,8-tetrachloro-dibenzofuran (2,3,7,8-TCDF).
1.3 The detection limits and quantitation levels in this method are usually dependent on the level of
interferences rather than instrumental limitations. The minimum levels (MLs) in Table 2 are the
levels at which the CDDs/CDFs can be determined with no interferences present. The Method
Detection Limit (MDL) for 2,3,7,8-TCDD has been determined as 4.4 pg/L (parts-per-
quadrillion) using this method.
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". The analyst is permitted to modify the method to overcome
interferences or lower the cost of measurements, provided that all performance criteria in this
method are met. The requirements for establishing method equivalency are given in Section
9.1.2.
1.6 Any modification of this method, beyond those expressly permitted, shall be considered a major
modification subject to application and approval of alternate test procedures under 40 CFR 136.4
and 136.5.
2.0 Summary of Method
Flow charts that summarize procedures for sample preparation, extraction, and analysis are given
in Figure 1 for aqueous and solid samples, Figure 2 for multi-phase samples, and Figure 3 for
tissue samples.
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Method 1613
2.1
2.2
2.3
Extraction ;
2.1.1 Aqueous samples (samples containing less than one percent solids)—Stable isotopically
labeled analogs of 15 of the 2,3,7,8-substituted CDDs/CDFs are spiked into a one-liter
sample, and the sample is extracted by one of three procedures;
2.1.1.1 Samples containing no visible particles are extracted with rnethylene chloride in a
separatory funnel or by the solid-phase extraction technique summarized in
Section 2.1.1.3. The extract is concentrated for cleanup.
2.1.1.2 Samples containing visible particles are vacuum filtered through a glass-fiber
filter. The filter is extracted in a Soxhlet/Dean-Stark (SDS) extractor (Reference
7), and the filtrate is extracted with methylene chloride ;in a separatory funnel.
The methylene chloride extract is concentrated and combined with the SDS
extract prior to cleanup.
2.1.1.3 The sample is vacuum filtered through a glass-fiber filter on top of a solid-phase
extraction (SPE) disk. The filter and disk are extracted in an SDS extractor, and
the extract is concentrated for cleanup.
2.1.2 Solid, semi-solid, and multi-phase samples (but not 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 an SDS extractor. The extract is concentrated for cleanup.
2.1.3 Fish and other tissue—The sample is extracted by one of two procedures:
2.1.3.1 Soxhlet or SDS extraction—A 20-g aliquot of sample is homogenized, and a 10-g
aliquot is spiked with the labeled compounds. The sample is mixed with sodium
sulfate, allowed to dry for 12 - 24 hours, and extracted for 18-24 hours using
methylene chloride:hexane (1:1) in a Soxhlet extractor. The extract is evaporated
to dry ness, and the lipid content is determined. i
2.1.3.2 HC1 digestion—A 20-g aliquot is homogenized, and a 10-g aliquot is placed in a
bottle and spiked with the labeled compounds. After equilibration, 200 mL of
hydrochloric acid and 200 mL of methylene chloride:hexane (1:1) are added, and
the bottle is agitated for 12-24 hours. The extract is evaporated to dryness, and
the lipid content is determined.
After extraction, 37Q4-labeled 2,3,7,8-TCDD is added to each extract to measure the efficiency of
the cleanup process. Sample cleanups may include back-extraction with acid and/or base, and gel
permeation, alumina, silica gel, Florisil and activated carbon chromatography. High-performance
liquid chromatography (HPLC) can be used for further isolation of the 2,3,7,8- isomers or other
specific isomers or congeners. Prior to the cleanup procedures cited above, tissue extracts are
cleaned up using an anthropogenic isolation column, a batch silica gel adsorption, or sulfuric
acid and base back-extraction, depending on the tissue extraction procedure used.
After cleanup, the extract is concentrated to near dryness. Immediately prior to injection, internal
standards are added to each extract, and an aliquot of the extract is injescted into the gas
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Method 1613
chromatograph. The analytes are separated by the GC and detected by a high-resolution
(£10,000) mass spectrometer. Two exact m/z's are monitored for each analyte.
2.4 An individual CDD/CDF 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. The non-2,3,7,8 substituted
isomers and congeners are identified when retention times and ion-abundance ratios agree within
predefined limits. Isomer specificity for 2,3,7,8-TCDD and 2,3,7,8-TCDF is achieved using GC
columns that resolve these isomers from the other tetra-isomers.
2.5 Quantitative analysis is performed using selected ion current profile (SICP) areas, in one of three
ways:
2.5.1 For the fifteen 2,3,7,8-substituted CDDs/CDFs with labeled analogs (see Table 1), the
GC/MS system is calibrated, and the concentration of each compound is determined
using the isotope dilution technique.
2.5.2 For 1,2,3,7,8,9-HxCDD, QCDF, and the labeled compounds, the GC/MS system is
calibrated and the concentration of each compound is determined using the internal
standard technique.
2.5.3 For non-2,3,7,8-substituted isomers and for all isomers at a given level of chlorination
(i.e., total TCDD), concentrations are determined using response factors from calibration
of the CDDs/CDFs at the same level of chlorination.
2.6 The quality of the analysis is assured through reproducible calibration and testing of the
extraction, cleanup, and GC/MS systems.
3.0 Definitions
Definitions are given 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 and/or
elevated baselines causing misinterpretation of chromatograms (References 8-9). 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.
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.
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Method 1613
4.$
4.4
4.5i
4.61
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. ;
4.2.3 Do not bake reusable glassware in an oven as a routine part of cleaning. Baking may be
warranted after particularly dirty samples are encountered but should be minimized as
repeated baking of glassware may cause active sites on the glass surface that will '
irreversibly adsorb CDDs/CDFs.
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). Separatoty funnels should be shaken
with methylene chloride/toluene (80/20 mixture) for 2 minutes, drained, and then shaken
with pure methylene chloride for 2 minutes.
All materials used in the analysis shall be demonstrated to be free from interferences by running
reference matrix method blanks 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 CDDs/CDFs in detectable amounts
but should contain potential interferents in the concentrations expected to be found in the
samples to be analyzed. For example, a reference sample of human adipose tissue
containing pentachloronaphthalene can be used to exercise the cleanup systems when
samples containing pentachloronaphthalene are expected.
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.
Interferences coextracted 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 CDDs/CDFs. The most frequently
encountered interferences are chlorinated biphenyls, methoxy biphenyls, hydroxydiphenyl ethers
benzylphenyl ethers, polynuclear aromatics, and pesticides. Because very low levels of
CDDs/CDFs are measured by this method, the 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 CDDs/CDFs at the levels shown in Table 2.
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.
Cleanup of tissue—The natural lipid content of tissue can interfere in the analysis of tissue
samples for the CDDs/CDFs. 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
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Method 1613
for cleanup of sample extracts. Lipids must be removed by the lipid removal procedures in
Section 13.7, followed by alumina (Section 13.4) or Florisil (Section 13.8), and carbon (Section
13.5) as minimum additional cleanup steps. If chlorodiphenyl ethers are detected, as indicated by
the presence of peaks at the exact m/z's monitored for these interferents, alumina and/or Florisil
cleanup must be employed to eliminate these interferences.
5.0 Safety
5.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level.
5.1.1 The 2,3,7,8-TCDD isomer has been found to be acnegenic, carcinogenic, and teratogenic
in laboratory animal studies. It is soluble in water to approximately 200 ppt and in
organic solvents to 0.14%. On the basis of the available toxicological and physical
properties of 2,3,7,8-TCDD, all of the CDDs/CDFs 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 shall be prepared in a
hood, and a NIOSH/MESA approved toxic gas respirator shall 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 10-13. The
references and bibliography at the end of Reference 13 are particularly comprehensive in dealing
with the general subject of laboratory safety.
5.3 The CDDs/CDFs 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. The
CDDs/CDFs are extremely toxic to laboratory animals. Each laboratory must develop a strict
safety program for handling these compounds. The practices in References 2 and 14 are highly
recommended.
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 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
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Metnoa
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 equipment
(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
CDDs/CDFs, 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, i
,1
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 effluents of sample splitters 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 CDD/CDF 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 must 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 the least toxic solvent
shown to be effective. Satisfactory cleaning may be accomplished by rinsing with
.Chlorothene, then washing with any detergent and water. If glassware is first
rinsed with solvent, then the dish 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 who convey the bags and launder the clothing should be advised of the hazard
and trained in proper handling. The clothing may be put into a washer without contact if
the launderer knows of the potential problem. The washer should be run through a cycle
before being used again for other clothing.
Wipe tests—A useful method of determining cleanliness of work surfaces and tools is to
wipe the surface with a piece of filter paper. Extraction and analysis by GC with an
electron capture detector (BCD) can achieve a limit of detection of 0.1 ug per wipe;
analysis using this method can achieve an even lower detection limit. Less than 0.1 ng
per wipe indicates acceptable cleanliness; anything higher warrants further cleaning.
5.3.10
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Method 1613
More than 10 ug on a wipe 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.
5.3.11 Table or wrist-action shaker—The use of a table or wrist-action shaker for extraction of
tissues presents the possibility of breakage of the extraction bottle and spillage of acid
and flammable organic solvent. A secondary containment system around the shaker is
suggested to prevent the spread of acid and solvents in the event of such a breakage. The
speed and intensity of shaking action should also be adjusted to minimize the possibility
of breakage.
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 shall be protected from light.
6.1.1.4 Bottle caps—Threaded to fit sample bottles. Caps shall 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, rinsed with reagent water (Section
7.6.1) followed by solvent, and baked at approximately 200°C for a
minimum of 1 hour prior to use.
6.1.2 Compositing equipment—Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Only glass or fluoropolymer tub-
ing shall 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 shall 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—Laboratory sink with overhead fume hood.
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Melkod 1613
6.3
6.4
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 Dessicator.
6.3.6 Balances. '
6.3.6.1 Analytical—Capable of weighing O.I mg.
6.3.6.2 Top loading—Capable of weighing 10 mg. <
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.
6.4.1.4 Liquid/liquid extraction—Separatory funnels, 250-, 500v, and 2000-mL, with
fluoropolymer stopcocks.
6.4.1.5 Solid-phase extraction. j
6.4.1.5.1 1-L filtration apparatus, including glass funnel, glass 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 (Clg) 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.
6.4.2 Soxhlet/Dean-Stark (SDS) extractor (Figure 5) 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). ,
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Method 1613
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 Apparatus for extraction of tissue.
6.4.3.1 Bottle for extraction (if digestion/ extraction using HC1 is used)—500- to 600-mL
wide-mouth clear glass, with fluoropolymer-lined cap.
6.4.3.2 Bottle for back-extraction—100- to 200-mL narrow-mouth clear glass with
fluoropolymer-lined cap.
6.4.3.3 Mechanical shaker—Wrist-action or platform-type rotary shaker that produces
vigorous agitation (Sybron Thermolyne Model LE "Big Bill" rotator/shaker, or
equivalent).
6.4.3.4 Rack attached to shaker table to permit agitation of 4-9 samples simultaneously.
6.4.4 Beakers—400- to 500-mL.
6.4.5 Spatulas—Stainless steel.
6.5 Filtration apparatus.
6.5.1 Pyrex glass wool—Solvent-extracted by SDS for 3 hours minimum.
i
Note: Baking glass wool may cause active sites that will irreversibly adsorb CDDs/CDFs.
6.5.2 Glass funnel—125- to 250-mL.
6.5.3 Glass-fiber filter paper—Whatman GF/D (or equivalent), to fit glass funnel 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—to fit Buchner funnel in Section 6.5.5.
6.5.7 Filtration flasks—1.5- to 2.0-L, with side arm.
6.5.8 Pressure filtration apparatus—Millipore YT30 142 HW, 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.
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Method 1613
t i
6^7.1 Automated gel permeation chromatograph (Analytical Biochemical Labs, Inc, Columbia,
i MO, Model GPC Autoprep 1002, or equivalent). '|
: gj.1.1 Column—600-700 mm long x 25 mm ID, packed with 70 g of SX-3 Bio-beads
i (Bio-Rad Laboratories, Richmond, CA, or equivalent).
; 6.7.1.2 Syringe—10-mL, with Luer fitting. ;
i 6.7.1.3 Syringe filter holder—stainless steel, and glass- fiber or fluoropolymer filters
' (Gelman 4310, or equivalent). '
i " •'
; 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-uL micro-prep
I flow cell, 2-mm path; Pharmacia UV-1, 3-mm flow cell; LDC Milton-Roy UV-3,
j monitor #1203; or equivalent). ,i
61.7.2 Reverse-phase high-performance liquid chromatograph. !
i 6.7.2.1 Column oven and detector—Perkin-Elmer Model LC-65T (or equivalent)
i operated at 0.02 AUFS at 235 nm.
6.7.2.2 Injector—Rheodyne 7120 (or equivalent) with 50-uL sample loop.
> 6.7.2.3 Column—Two 6.2 mm x 250 mm Zorbax-ODS columns in series (DuPont
i Instruments Division, Wilmington, DE, or equivalent), operated at 50°C with 2.0
; mL/min methanol isocratic effluent.
! . 6.7.2.4 Pump—Altex 110A (or equivalent).
6.7.3 Pipets. ;
i 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, 10-mL (6-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
i 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 25-mm ID, with 300-mL reservoir and glass or fluoropolymer
: stopcock. i - '
16.7.5 Stirring apparatus for batch silica cleanup of tissue extracts. j
6.7.5.1 Mechanical stirrer—Corning Model 320, or equivalent. ,
6.7.5.2 Bottle—500- to 600-mL wide-mouth clear glass.
J6.7.6 Oven—For baking and storage of adsorbents, capable of maintaining a constant
I temperature (±5°C) in the range of 105-250°C. i
i
6.8 Concentration apparatus.
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Method 1613
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).
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 blowdown 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—Shall have splitless or on-column injection port for capillary column,
temperature program with isothermal hold, and shall meet all of the performance specifications
in Section 10.
6.9,1 GC column for CDDs/CDFs and for isomer specificity for 2,3,7,8-TCDD—60±5-m long
x 0.32±0.02-mm ID; 0.25-um 5% phenyl, 94% methyl, 1% vinyl silicone bonded-phase
fused-silica capillary column (J & W DB-5, or equivalent).
6.9.2 GC column for isomer specificity for 2,3,7,8-TCDF—30±5-m long x 0.32±0.02-mm ID;
0.25-um bonded-phase fused-silica capillary column (J & W DB-225, or equivalent).
6.10 Mass spectrometer—28- to 40-eV electron impact ionization, shall be capable of repetitively
selectively monitoring 12 exact m/z's minimum at high resolution (>10,000) during a period of
approximately 1 second, and shall meet all of the performance specifications in Section 10.
11
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Method 1613
be interfaced
electron or ion beams. " ~" '" ' ™ °* ™ ^ S°UrCe but *«* not interce'p7th7
6.12| Data system-Capable of collecting, recording) ^ storing M, ^
7.0 \ Reagents and Standards
7.1 pH adjustment and back-extraction.
| 7.1.1 Potassium hydroxide—Dissolve 20 g
7.1.2 Sulfuric acid-Reagent grade (specific gravity&L84)!
7.1.3 Hydrochloric acid—Reagent grade, 6N.
7.2
i 7.2.3
7.3 Extraction.
7.4
7.5 Adsorbents for sample cleanup.
7.5.1 Silica gel.
-------
Method 1613
a stirring rod until a uniform mixture is obtained. Store in a bottle with a
fluoropolymer-lined screw-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 bottle with a fluoropolymer-lined
screw-cap.
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 silica gel and a stirring bar, and stir on a 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.
7.5.1.4.5 Activate overnight at 200-250°C.
7.5.2 Alumina—Either one of two types of alumina, acid or basic, may be used in the cleanup
of sample extracts, provided that the laboratory can meet the performance specifications
for the recovery of labeled compounds described in Section 9.3. The same type of
alumina must be used for all samples, including those used to demonstrate initial
precision and recovery (Section 9.2) and ongoing precision and recovery (Section 15.5).
7.5.2.1 Acid alumina—Supelco 19996-6C (or equivalent). Activate by heating to 130°C
for a minimum of 12 hours.
7.5.2.2 Basic alumina—Supelco 19944-6C (or equivalent). Activate by heating to 600°C
for a minimum of 24 hours. Alternatively, activate by heating in a tube furnace at
650 to 700°C under an air flow rate of approximately 400 cc/minute. Do not heat
over 700°C, as this can lead to reduced capacity for retaining the analytes. Store
at 130°C in a covered flask. Use within five days of baking.
7.5.3 Carbon.
7.5.3.1 Carbopak C—(Supelco 1-0258, or equivalent).
7.5.3.2 Celite 545—(Supelco 2-0199, or equivalent).
7.5.3.3 Thoroughly mix 9.0 g Carbopak C and 41.0 g Celite 545 to produce an 18% w/w
mixture. Activate the mixture at 130°C for a minimum of 6 hours. Store in a
dessicator.
7.5.4 Anthropogenic isolation column—Pack the column in Section 6.7.4.3 from bottom to top
with the following:
7.5.4.1 2 g silica gel (Section 7.5.1.1).
13
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Methofi 1613
7.6
7.7
7.8
7.5.4.2 2 g potassium silicate (Section 7.5.1.4).
7.5.4.3 2 g granular anhydrous sodium sulfate (Section 7.2.1).
7.5.4.4 10 g acid silica gel (Section 7.5.1.2).
7.5.4.5 2 g granular anhydrous sodium sulfate.
7.5.5 Florisil column
7.5.5.1 Florisil—60-100 mesh, Floridin Corp (or equivalent). Soxhlet extract in 500-g
portions for 24 hours.
7.5.5.2 Insert a glass wool plug into the tapered end of a graduated serological pipet
(Section 6.7.3.2). Pack with 1.5 g (approx 2 mL) of Florisil topped with approx 1
mL of sodium sulfate (Section 7.2.1) and a glass wool plug.
7.5.5.3 Activate in an oven at 130-150 °C for a minimum of 24 hours and cool for 30
minutes. Use within 90 minutes of cooling. ;
Reference matrices—Matrices in which the CDDs/CDFs 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. May be prepared by extraction
with methylene chloride. \
7.6.5 Other matrices—This method may be verified on any reference matrix by performing the
tests given in Section 9.2. Ideally, the matrix should be free of the CDDs/CDFs, but in
no case shall the background level of the CDDs/CDFs in the reference matrix exceed
three times the minimum levels in Table 2. If low background levels of the CDDs/CDFs
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 in the range of 1:1 to. 5:1
(Reference 15).
Standard solutions—Purchased as solutions or mixtures with certification to their purity,
concentration, and authenticity, or prepared from materials of known purity and composition. If
the chemical purity is 98% or greater, the weight may be used without correction to compute the
concentration of the standard. When not being used, standards are stored in the dark at room
temperature in screw-capped vials with fluoropolymer-lined caps. A mark is placed on the vial at
the level of the solution so that solvent loss by evaporation can be detected. If solvent loss has
occurred, the solution should be replaced.
Stock solutions.
14
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Method 1613
7.8.1 Preparation—Prepare in nonane per the steps below or purchase as dilute solutions
(Cambridge Isotope Laboratories (CIL), Wobum, MA, or equivalent). Observe the safety
precautions in Section 5, and the recommendation in Section 5.1.2.
7.8.2 Dissolve an appropriate amount of assayed reference material in solvent. For example,
weigh 1 to 2 mg of 2,3,7,8-TCDD to three significant figures in a 10-mL ground-glass-
stoppered volumetric flask and fill to the mark with nonane. After the TCDD is
completely dissolved, transfer the solution to a clean 15-mL vial with fluoropolymer-
lined cap.
7.8.3 Stock standard solutions should be checked for signs of degradation prior to the
preparation of calibration or performance test standards. Reference standards that can be '
used to determine the accuracy of calibration standards are available from CIL and may
be available from other vendors.
7.9 PAR stock solution.
7.9.1 All CDDs/CDFs—Using the solutions in Section 7.8, prepare the PAR stock solution to
contain the CDDs/CDFs at the concentrations shown in Table 3. When diluted, the
solution will become the PAR (Section 7.14).
7.9.2 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the PAR stock
solution to contain these compounds only.
7.10 Labeled-compound spiking solution.
7.10.1 All CDDs/CDFs—From stock solutions, or from purchased mixtures, prepare this
solution to contain the labeled compounds in nonane at the concentrations shown in
Table 3. This solution is diluted with acetone prior to use (Section 7.10.3).
7.10.2 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the labeled-
compound solution to contain these compounds only. This solution is diluted with
acetone prior to use (Section 7.10.3).
7.10.3 Dilute a sufficient volume of the labeled compound solution (Section 7.10.1 or 7.10.2) by
a factor of 50 with acetone to prepare a diluted spiking solution. Each sample requires
1.0 mL of the diluted solution, but no more solution should be prepared than can be used
in one day.
7.11 Cleanup standard—Prepare 37Cl4-2,3,7,8-TCDD in nonane at the concentration shown in Table 3.
The cleanup standard is added to all extracts prior to cleanup to measure the efficiency of the
cleanup process.
7.12 Internal standard(s).
7.12.1 All CDDs/CDFs—Prepare the internal standard solution to contain I3C12-1,2,3,4-TCDD
and I3C)2-l,2,3,7,8,9-HxCDD in nonane at the concentration shown in Table 3.
7.122 If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, prepare the internal
standard solution to contain 13C12-1,2,3,4-TCDD only.
7.13 Calibration standards (CS1 through CSS)—Combine the solutions in Sections 7.9-7.12 to
produce the five calibration solutions shown in Table 4 in nonane. These solutions permit the
relative response (labeled to native) and response factor to be measured as a function of concen-
15
-------
Method
tration. The CSS standard is used for calibration verification (VER). If only 2,3,7,8-TCDD and
2,3,7,8-TCDF are to be determined, combine the solutions appropriate to these compounds.
7.141 Precision and recovery (PAR) standard—Used for determination of initial (Section 9.2) and
| ongoing (Section 15.5) precision and recovery. Dilute 10 uL of the precision and recovery
I standard (Section 7.9.1 or 7.9.2) to 2.0 mL with acetone for each sample matrix for each sample
i batch. One mL each are required for the blank and OPR with each matrix in each batch.
7.15! GC retention time window defining solution and isomer specificity test standard Used to define
i the beginning and ending retention times for the dioxin and furan isorners and to demonstrate
isomer specificity of the GC columns employed for determination of 2,3,7,8-TCDD and 2,3,7,8-
I TCDF. The standard must contain the compounds listed in Table 5 (CIL EDF-4006, or
I equivalent), at a minimum. It is not necessary to monitor the window-defining compounds if
i only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined. In this case, an isomer-specificity
I test standard containing the most closely eluted isomers listed in Table 5 (CIL EDF-4033, or
j equivalent) may be used.
7.16 i 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 reference material
containing the CDDs/CDFs in known concentrations in a sample matrix similar to the matrix
under test.
7.17 Stability of solutions—Standard solutions used for quantitative purposes (Sections 7.9 through
| 7.15) should be analyzed periodically, and should be assayed against reference standards (Section
; 7.8.3) before further use.
i . "
8.01 Sample Collection, Preservation, Storage, and Holding Times.
I
8.1 i Collect samples in amber glass containers following conventional sampling practices (Reference
! 16). Aqueous samples that flow freely are collected in refrigerated bottles using automatic
I sampling equipment. Solid samples are collected as grab samples using wide-mouth jars.
8.2 ! Maintain aqueous samples in the dark at 0-4°C from the time of collection until receipt at the
I laboratory. If residual chlorine is present in aqueous samples, add 80 mg sodium thiosulfate per
i liter of water. EPA Methods 330.4 and 330.5 may be used to measure residual chlorine
; (Reference 17). If sample pH is greater than 9, adjust to pH 7-9 with sulfuric acid.
; Maintain solid, semi-solid, oily, and mixed-phase samples in the dark at <4°C from the time of
! collection until receipt at the laboratory. I
| Store aqueous samples in the dark at 0-4°C. Store solid, semi-solid, oily, mixed-phase, and tissue
samples in the dark at <-10°C.
8.3 I Fish and tissue samples.
8.3.1 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.
8.3.2 Fish collected in the field should be wrapped in aluminum foil, and must be maintained
at a temperature less than 4°C from the time of collection until receipt at the laboratory.
16
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Method 1613
8.3.3 Samples must be frozen upon receipt at the laboratory and maintained in the dark at <-
10°C until prepared. Maintain unused sample in the dark at <-10°C.
8.4 Holding times.
8.4.1 There are no demonstrated maximum holding times associated with CDDs/CDFs in
aqueous, solid, semi-solid, tissues, or other sample matrices. If stored in the dark at 0-
4°C and preserved as given above (if required), aqueous samples may be stored for up to
one year. Similarly, if stored in the dark at <-10°C, solid, semi-solid, multi-phase, and
tissue samples may be stored for up to one year.
8.4.2 Store sample extracts in the dark at <-10°C until analyzed. If stored in the dark at <-
10°C, sample extracts may be stored for up to 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 18). 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 matrix (Sections 7.6.2-7.6.5) is substituted for the reagent
water matrix (Section 7.6.1) in all performance tests.
9.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable
accuracy and precision with this method. This ability is established as described in
Section 9.2.
9.1.2 In recognition of advances that are occurring in analytical technology, and to allow the
analyst to overcome sample matrix interferences, the analyst is permitted certain options
to improve separations or lower the costs of measurements. These options include
alternate extraction, concentration, cleanup procedures, and changes in columns and
detectors. Alternate determinative techniques, such as the substitution of spectroscopic or
immuno-assay techniques, and changes that degrade method performance, are not
allowed. If an analytical technique other than the techniques specified in this method is
used, that technique must have a specificity equal to or better than the specificity of the
techniques in this method for the analytes of interest.
9.1.2.1 Each time a modification is made to this method, the analyst is required to repeat
the procedure in Section 9.2. If the detection limit of the method will be affected
by the change, the laboratory is required to demonstrate that the MDL (40 CFR
Part 136, Appendix B) is lower than one-third the regulatory compliance level cl-
one-third the ML in this method, whichever is higher. If calibration will be
affected by the change, the analyst must recalibrate the instrument per Section 10.
17
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Method 1613
18
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)
who performed the analyses and modification, and of the quality control
officer who 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.
9.1.2.2.4 Results from all quality control (QC) tests comparing the modified
method to this method, including:
a) Calibration (Section 10.5-10.7). j
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).
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.
9.1,3 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 shall spike all samples with labeled compounds to monitor method
performance. This test is described in Section 9.3. When results of these spikes indicate
-------
Method 1613
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 shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the ongoing precision and recovery aliquot that the analytical system
is in control. These procedures are described in Sections 15.1 through 15.5.
9.1.6 The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 9.4.
9.2 Initial precision and recovery (1PR)—To establish the ability to generate acceptable precision and
recovery, the analyst shall 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 the diluted labeled compound spiking solution (Section 7.10.3)
and the precision and recovery standard (Section 7.14) 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), shall be included in this test.
9.2.2 Using results of the set of four analyses, compute the average concentration (X) of the
extracts in ng/mL and the standard deviation of the concentration (s) in ng/mL for each
compound, by isotope dilution for CDDs/CDFs with a labeled analog, and by internal
standard for 1,2,3,7,8,9-HxCDD, OCDF, and the labeled compounds.
9.2.3 For each CDD/CDF and labeled compound, compare s and X with the corresponding
limits for initial precision and recovery in Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-
TCDF are to be determined, compare s and X with the corresponding limits for initial
precision and recovery in Table 6a. If s and X for all compounds meet the acceptance
criteria, system performance is acceptable and analysis of blanks and samples may begin.
If, however, any individual s exceeds the precision limit or any individual X falls outside
the range for accuracy, system performance is unacceptable for that compound. Correct
the problem and repeat the test (Section 9.2).
9.3 The laboratory shall spike all samples with the diluted labeled compound spiking solution
(Section 7.10.3) to assess method performance on the sample matrix.
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 compounds and the cleanup standard using
the internal standard method (Section 17.2).
9.3.3 The recovery of each labeled compound must be within the limits in Table 7 when all
2,3,7,8-substituted CDDs/CDFs are determined, and within the limits in Table 7a when
only 2,3,7,8-TCDD and 2,3,7,8-TCDF are determined. If the recovery of any compound
falls outside of these limits, method performance is unacceptable for that compound in
that sample. To overcome such difficulties, water samples are diluted and smaller
amounts of soils, sludges, sediments, and other matrices are reanalyzed per Section 18.4.
9.4 Recovery of labeled compounds from samples should be assessed and records should be
maintained.
19
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Method 1613
: 9.4.1 After the analysis of five 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
i 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 i Method blanks—Reference matrix method blanks are analyzed to demonstrate freedom from
I contamination (Section 4.3).
I 9.5.1 Prepare, extract, clean up, and concentrate a method blank with each sample batch
i (samples of the same matrix started through the extraction process on the same 12-hour
j shift, to a maximum of 20 samples). The matrix for the method blank shall be similar to
\ sample matrix for the batch, e.g., a 1-L reagent water blank (Section 7.6.1), high-solids
i 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). Analyze the blank
! immediately after analysis of the OPR (Section 15.5) to demonstrate freedom from
| contamination.
i 9.5.2 If any 2,3,7,8-substituted CDD/CDF (Table 1) is found in the blank at greater than the
minimum level (Table 2) or one-third the regulatory compliance level, whichever is
> greater; or if any potentially interfering compound is found in the blank at the minimum
! level for each level of chlorination given in Table 2 (assuming a response factor of 1
' relative to the I3C12-1,2,3,4-TCDD internal standard for compounds not listed in Table 1),
; analysis of samples is halted until the blank associated with the sample batch shows no
| evidence of contamination at this level. All samples must be associated with an
! uncontaminated method blank before the results for those samples may be reported for
i regulatory compliance purposes. ;
9.6 QC Check Sample—Analyze the QC Check Sample (Section 7.16) 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
i properly and then maintained in a calibrated state. The standards used for calibration (Section
i 10), calibration verification (Section 15.3), and for initial (Section 9.2) and ongoing (Section
i 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 the analyses of CDDs/CDFs 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.
20
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Method 1613
10.0 Calibration
10.1 Establish the operating conditions necessary to meet the minimum retention times for the internal
standards in Section 10.2.4 and the relative retention times for the CDDs/CDFs in Table 2.
10.1.1 Suggested GC operating conditions:
Injector temperature: 270°C
Interface temperature: 290°C
Initial temperature: 200°C
Initial time: 2 minutes
Temperature program: 200 to 220°C, at 5°C/minute
220°C for 16 minutes
220 to 235°C, at 5°C/minute
235°C for 7 minutes
235 to 330°C, at 5°C/minute
Note: All portions of the column that connect the GC to the ion source shall remain at or
above the interface temperature specified above during analysis to preclude condensation of less
volatile compounds.
Optimize GC conditions 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.
10.1.2 Mass spectrometer (MS) resolution—Obtain a selected ion current profile (SICP) of each
analyte in Table 3 at the two exact m/z's specified in Table 8 and at >10,000 resolving
power by injecting an authentic standard of the CDDs/CDFs either singly or as part of a
mixture in which there is no interference between closely eluted components.
10.1.2.1 The analysis time for CDDs/CDFs 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, a mass-drift correction is
mandatory and a lock-mass m/z from PFK 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 8. The level 6f PFK 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 parameters. Under those conditions,
sensitivity changes that might occur during the analysis can be more effectively
monitored.
Note: Excessive PFK (or any other reference substance) may cause noise problems and
contamination of the ion source necessitating increased frequency of source cleaning.
21
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] 10.1.2.2 If the HRMS has the capability to monitor resolution during the analysis, it is
| acceptable to terminate the analysis when the resolution falls below 10,000 to
I save reanalysis time.
! 10.1.2.3 Using a PFK molecular leak, tune the instrument to fneet the minimum required
i resolving power of 10,000 (10% valley) at rn/z 304.9824 (PFK) or any other
j reference signal close to m/z 304 (from TCDF). For each descriptor (Table 8),
| monitor and record the resolution and exact m/z's of three to five reference peaks
j covering the mass range of the descriptor. The resolution must be greater than or
\ . equal to 10,000, and the deviation between the exact rn/z and the theoretical m/z
I (Table 8) for each exact m/z monitored must be less than 5 ppm.
10.2 Ion abundance ratios, minimum levels, signal-to-noise ratios, and absolute retention times—
i Choose an injection volume of either 1 or 2 uL, consistent with the capability of the
HRGC/HRMS instrument. Inject a 1 or 2 jiL aliquot of the CS1 calibration solution (Table 4)
using the GC conditions from Section 10.1.1. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be
determined, the operating conditions and specifications below apply to analysis of those
compounds only.
10.2.1 Measure the SICP areas for each analyte, and compute the ion abundance ratios at the
exact m/z's specified in Table 8. Compare the computed ratio to the theoretical ratio
given in Table 9.
10.2.1.1 The exact m/z's to be monitored in each descriptor are shown in Table 8. Each
group or descriptor shall be monitored in succession as a function of GC
retention time to ensure that all CDDs/CDFs are detected. Additional m/z's may
be monitored in each descriptor, and the m/z's may be divided among more than
the five descriptors listed in Table 8, provided that the laboratory is able to
monitor the m/z's of all the CDDs/CDFs that may elute from the GC in a given
retention-time window. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be
determined, the descriptors may be modified to include only the exact m/z's for
the tetra- and penta-isomers, the diphenyl ethers, and the lock m/z's.
10.2.1.2 The mass spectrometer shall be operated in a mass-drift correction mode, using
perfluorokerosene (PFK) to provide lock m/z's. The lock-mass for each group of
m/z's is shown in Table 8. Each lock mass shall be monitored and shall not vary
by more than ±20% throughout its respective retention time window. Variations
of the lock mass by more than 20% indicate the presence of coeluting
interferences that may significantly reduce the sensitivity of the mass
spectrometer. Reinjection of another aliquot of the sample extract will not resolve
the problem. Additional cleanup of the extract may be required to remove the
interferences.
10.2.2 All CDDs/CDFs and labeled compounds in the CS1 standard shall be within the QC
limits in Table 9 for their respective ion abundance ratios; otherwise, the mass
spectrometer shall 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 shall be verified (Section 10.1.2) prior to repeat of the test.
22
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Method 1613
10.2.3 Verify that the HRGC/HRMS instrument meets the minimum levels in Table 2. The
peaks representing the CDDs/CDFs and labeled compounds in the CS1 calibration
standard must have signal-to-noise ratios (S/N) greater than or equal to 10.0. Otherwise,
the mass spectrometer shall be adjusted and this test repeated until the minimum levels in
Table 2 are met.
10.2.4 The absolute retention time of I3C,2-1,2,3,4-TCDD (Section 7.12) shall exceed 25.0
minutes on the DB-5 column, and the retention time of 13C12-1,2,3,4-TCDD shall exceed
15.0 minutes on the DB-225 column; otherwise, the GC temperature program shall be
adjusted and this test repeated until the above-stated minimum retention time criteria are
met.
10.3 Reicntion-time windows—Analyze the window defining mixtures (Section 7.15) using the
optimized temperature program in Section 10.1. Table 5 gives the elution order (first/last) of the
window-defining compounds. If 2,3,7,8-TCDD and 2,3,7,8-TCDF only are to be analyzed, this
test is not required.
10.4 Isomer specificity
10.4.1 Analyze the isomer specificity test standards (Section 7.15) using the procedure in
Section 14 and the optimized conditions for sample analysis (Section 10.1.1).
10.4.2 Compute the percent valley between the GC peaks that elute most closely to the 2,3,7,8-
TCDD and TCDF isomers, on their respective columns, per Figures 6 and 7.
10.4.3 Verify that the height of the valley between the most closely eluted isomers and the
2,3,7,8-substituted isomers is less than 25% (computed as 100 x/y in Figures 6 and 7). If
the valley exceeds 25%, adjust the analytical conditions and repeat the test or replace the
GC column and recalibrate (Sections 10.1.2 through 10.7).
10.5 Calibration by isotope dilution—Isotope dilution calibration is used for the fifteen 2,3,7,8-
substituted CDDs/CDFs for which labeled compounds are added to samples prior to extraction.
The reference compound for each CDD/CDF compound is shown in Table 2.
10.5.1 A calibration curve encompassing the concentration range is prepared for each compound
to be determined. The relative response (RR) (labeled to native) vs. concentration in
standard solutions is plotted or computed using a linear regression. Relative response is
determined according to the procedures described below. Five calibration points are
employed.
10.5.2 The response of each CDD/CDF relative to its labeled analog is determined using the
area responses of both the primary and secondary exact m/z's specified in Table 8, for
each calibration standard, as follows:
_ (Aln + A2n) C,
(41, + A2) Cn
Where:
Aln and A2n = The areas of the primary and secondary mfz!s for the CDDfCDF.
Alt and A2l = The areas of the primary and secondary m/z's for the labeled compound.
C{ - 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).
23
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Method 1613
10.6
10.5.3 To calibrate the analytical system by isotope dilution, inject a volume of calibration
standards CS1 through CSS (Section 7.13 and Table 4) identical to the volume chosen in
Section 10.2, using the procedure in Section 14 and the conditions in Section 10.1.1 and
Table 2. Compute the relative response (RR) at each concentration.
10.5.4 Linearity—If the relative response for any compound is constant (less than 20%
coefficient of variation) over the five-point calibration range, an averaged relative
response may be used for that compound; otherwise, the complete calibration curve for
that compound shall be used over the five-point calibration range.
Calibration by internal standard—The internal standard method is applied to determination of
1,2,3,7,8,9-HxCDD (Section 17.1.2), OCDF (Section 17.1.1), the non-2,3,7,8-substitute
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Method 1613
10.8 Data storage—MS data shall be collected, recorded, and stored.
10.8.1 Data acquisition—The signal at each exact m/z shall be collected repetitively throughout
the monitoring period and stored on a mass storage device.
10.8.2 Response factors and multipoint calibrations—The data system shall be used to record
and maintain lists of response factors (response ratios for isotope dilution) and multipoint
calibration curves. Computations of relative standard deviation (coefficient of variation)
shall be used to test calibration linearity. Statistics on initial performance (Section 9.2)
and ongoing performance (Section 15.5) should be computed and maintained, either on
the instrument data system, or on a separate computer system.
11.0 Sample Preparation
11.1 Sample preparation involves modifying the physical form of the sample so that the CDDs/CDFs
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 10 lists the phases and
suggested quantities for extraction of various sample matrices.
For samples known or expected to contain high levels of the CDDs/CDFs, the smallest sample
size representative of the entire sample should be used (see Section 17.5).
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.
t 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 CDDs/CDFs 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 visibly absent particles are prepared per Section 11.4 and
extracted directly using the separatory funnel or SPE techniques in Sections 12.1
or 12.2, respectively.
11.1.2.2 Aqueous samples containing visible particles and containing one percent
suspended solids or less are prepared using the procedure in Section 11.4. After
preparation, the sample is extracted directly using the SPE technique in 12.2 or
filtered per Section 11.4.3. After filtration, the particles and filter are extracted
using the SDS procedure in Section 12.3 and the filtrate is extracted using the
separatory funnel procedure in Section 12.1.
11.1 A3 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 via the SDS procedure in Section 12.3.
11.1.4 Multiphase samples—The phase(s) containing the CDDs/CDFs is separated from the non-
CDD/CDF phase using pressure filtration and centrifugation, as described in Section
11.6. The CDDs/CDFs will be in the organic phase in a multiphase sample in which an
organic phase exists.
25
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Method 1613
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 the solids content of the sample, not for
determination of CDDs/CDFs.
l
i
11.2.1 Aqueous liquids and multi-phase samples consisting of mainly an aqueous phase.
11.2.1.1 Dessicate 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 arid cool in a dessicator.
11.2.1.4 Calculate percent solids as follows:
,-j weight of sample aliquot after drying (g) - weight of filter (g) inn
% solids = — = —. x 1UU
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 a tared beaker.
11.2.2.2 Dry a minimum of 12 hours at 110 ± 5°C, and cool in a dessicator.
11.2.2.3 Calculate percent solids as follows: ;
i
_, ... weight of sample aliquot after drying
% so lias = —: —;— x
weight of sample aliquot before drying
11.3 Determination of particle size. ;
11.3.1 Spread the dried sample from Section 11.2.2.2 on a piece of filter paper or aluminum foil
i in a fume hood or glove box.
I 11.3.2 Estimate the size of the particles in the sample. If the size of the largest particles is
i greater than 1 mm, the particle size must be reduced to 1 mm or less prior to extraction
i 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 visibly absent particles are prepared per the procedure below and
extracted directly using the separatory funnel or SPE techniques in Sections 12.1 or 12.2,
respectively. Aqueous samples containing visible particles and one percent suspended
solids or less are prepared using the procedure below and extracted using either the SPE
-------
Method 1613
technique in Section 12.2 or further prepared using the filtration procedure in Section
11.4.3. The filtration procedure is followed by SDS extraction of the filter and particles
(Section 12.3) and separatory runnel extraction of the filtrate (Section 12.1). The SPE
procedure is followed by SDS extraction of the filter and disk.
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.
11.4.2.2- Spike 1.0 mL of the diluted labeled-compound spiking solution (Section 7.10.3)
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.
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 diluted labeled-compound spiking solution (Section 7.10.3)
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 PAR standard (Section 7.14) into the remaining reagent
water aliquot. This aliquot will serve as the OPR (Section 15.5).
11.4.2.6 If SPE is to be used, add 5 mL of methanol to the sample, cap and shake the
sample to mix thoroughly, and proceed to Section 12.2 for extraction. If SPE is
not to be used, and the sample is visibly absent particles, proceed to Section 12.1
for extraction. If SPE is not to be used and the sample contains visible particles,
proceed to the following section for filtration of particles.
11.4.3 Filtration of particles.
11.4.3.1 Assemble a Buchner funnel (Section 6.5.5) on top of a clean filtration flask.
Apply vacuum to the flask, and pour the entire contents of the sample bottle
through a glass-fiber filter (Section 6.5.6) in the Buchner funnel, swirling the
sample remaining in the bottle to suspend any particles.
11.4.3.2 Rinse the sample bottle twice with approximately 5-mL portions of reagent water
to transfer any remaining particles onto the filter.
11.4.3.3 Rinse any particles off the sides of the Buchner funnel with small quantities of
reagent water.
11.4.3.4 Weigh the empty sample bottle to ±1 g. Determine the weight of the sample by
difference. Save the bottle for further use.
11.4.3.5 Extract the filtrate using the separatory funnel procedure in Section 12.1.
11.4.3.6 Extract the filter containing the particles using the SDS procedure in Section
12.3.
11.5 Preparation of samples containing greater than one percent solids.
27
-------
\Method1613
11,6
11.7
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 diluted labeled compound spiking solution (Section 7.10.3) 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 diluted labeled compound spiking solution (Section 7.10.3) into each
reference matrix aliquot. One aliquot will serve as the method blank. Spike 1.0 mL of the.
PAR standard (Section 7.14) into the other reference matrix aliquot. This aliquot will
serve as the OPR (Section 15.5). (
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.
• Multiphase 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 Pressure filter the amount of sample determined in Section 11.6.1 through Whatman
GF/D glass-fiber filter paper (Section 6.5.3). Pressure filter the blank and OPR aliquots
through GF/D papers also. 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. 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 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.
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
28
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Meuioa
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 shall 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 shall 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 11.6.4 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 11.6.4 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 of fish 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.
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 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. For the alternate HC1 digestion/extraction, transfer
the tissue to a clean, tared 500- to 600-mL wide-mouth bottle. Record the weight
to the nearest 10 mg.
29
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Method 1613
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 <-10°C. Return any tissue that was
not homogenized to its original container and store at <-10°C.
11.8.2 QC aliquots. i
11.8.2.1 Prepare a method blank by adding approximately 10 g of the oily liquid reference
matrix (Section 7.6.4) to a 400- to 500-mL beaker. For the alternate HC1
digestion/extraction, add the reference matrix to a 500- to 600-mL wide-mouth
bottle. Record the weight to the nearest 10 mg.
11.8.2.2 Prepare a precision and recovery aliquot by adding approximately 10 g of the
oily liquid reference matrix (Section 7.6.4) to a separate 400- to 500-mL beaker ,
or wide-mouth bottle, depending on the extraction procedure to be used. 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 compound spiking solution (Section 7.10.3) into the
sample, blank, and OPR aliquot.
11.8.3.2 Spike 1.0 mL of the PAR standard (Section 7.14) into the OPR aliquot.
11.8.4 Extract the aliquots using the procedures in Section 12.4.
12.0 Extraction and Concentration
Extraction procedures include separatory funnel (Section 12.1) and solid phase (Section 12.2) for
aqueous liquids; Soxhlet/Dean-Stark (Section 12.3) for solids, filters, and SPE disks; and Soxhlet
sxtraction (Section 12.4.1) and HC1 digestion (Section 12.4.2) for tissues. Acid/base back-extraction
[Section 12.5) is used for initial cleanup of extracts.
Macro-concentration procedures include rotary evaporation (Section 12.6.1), heating mantle (Section
12.6.2), and Kudema-Danish (K-D) evaporation (Section 12.6.3). Micro-concentration uses nitrogen
jlowdown (Section 12.7).
12.1 Separatory funnel extraction of filtrates and of aqueous samples visibly absent particles.
12.1.1 Pour the spiked sample (Section 11.4.2.2) or filtrate (Section 11.4.3.5) 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.
12.1.2 Add 60 mL methylene chloride to the empty sample bottle (Section 12.1.1), seal, and
shake 60 seconds to rinse the inner surface. Transfer the solvent to the separatory 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
30
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Method 1613
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).
Note: If an emulsion forms, the analyst must employ mechanical techniques to complete the
phase separation. The optimum technique depends upon the sample, but may include stirring,
filtration through glass wool, use of phase separation paper, centrifugation, use of an ultrasonic
bath with ice, addition ofNaCl, or other physical methods. Alternatively, solid-phase or other
extraction techniques may be used to prevent emulsion formation. Any alternative technique
acceptable so long as the requirements in Section 9 are met.
is
Experience with aqueous samples high in dissolved organic materials (e.g., paper mill effluents)
has shown that acidification of the sample prior to extraction may reduce the formation of
emulsions. Paper industry methods suggest that the addition of up to 400 mL of ethanol to a 1-L
effluent sample may also reduce emulsion formation. However, studies by EPA suggest that the
effect may be a result of sample dilution, and that the addition of reagent water may serve the
same function. Mechanical techniques may still be necessary to complete the phase separation. If
either acidification or addition of ethanol is utilized, the laboratory must perform the startup tests
described in Section 9.2 using the same techniques.
12.1.3 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 funnel 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. Set aside the funnel with sodium sulfate if the extract is to be combined with the
extract from the particles.
12.1.4 Concentrate the extract using one of the macro-concentration procedures in Section 12.6.
12.1.4.1 If the extract is from a sample visibly absent particles (Section 11.1.2.1), adjust
the final volume of the concentrated extract to approximately 10 mL with hexane,
transfer to a 250-rnL separatory funnel, and back-extract using the procedure in
Section 12.5.
12.1.4.2 If the extract is from the aqueous filtrate (Section 11.4.3.5), set aside the
concentration apparatus for addition of the SDS extract from the particles
(Section 12.3.9.1.2).
12.2 SPE of samples containing less than one percent solids. (References 19-20).
12.2.1 Disk preparation.
12.2.1.1 Place an SPE disk on the base of the filter holder (Figure 4) and wet with
toluene. While holding a GMF 150 filter above the SPE disk with tweezers, wet
the filter with toluene 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.
12.2.1.2 Rinse the sides of the filtration flask with approx 15 mL of toluene using a
squeeze bottle or syringe. Apply vacuum momentarily until a few drops appear at
31
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Method 1613
12
the drip tip. Release the vacuum and allow the filter/disk to soak for approx one
minute. Apply vacuum and draw all of the toluene through the filter/disk. Repeat
the wash step with approx 15 mL of acetone and allow the filter/disk to air dry.
12.2.1.3 Re-wet the filter/disk with approximately 15 mL of rnethanol, allowing the
filter/disk to soak for approximately 1 minute. Pull the rnethanol through the
filter/disk using the vacuum, but retain a layer of methanol approximately 1 mm
thick on the filter. Do not allow the disk to go dry from this point until the end
of the extraction.
12.2.1.4 Rinse the filter/disk with two 50-mL portions of reagent water by adding the
water to the reservoir and pulling most through, leaving a layer of water on the
surface of the filter.
12.2.2 Extraction.
12.2.2.1 Pour the spiked sample (Section 11.4.2.2), blank (Section 11.4.2.4), or IPR/OPR
aliquot (Section 11.4.2.5) into the 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), filtration times may be 8 hours or longer.
12.2.2.2 Before all of the sample has been pulled through the filter/disk, rinse the sample
bottle with approximately 50 mL of reagent water to remove any solids, and pour
into the reservoir. Pull through the filter/disk. Use additional reagent water rinses
until all visible solids are removed. .[
12.2.2.3 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.
12.2.2.4 Allow the filter/disk to dry, then remove the filter and disk and place in a glass
Petri dish. Extract the filter and disk per Section 12.3.
,3 SDS extraction of samples containing particles, and of filters and/or disks.
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. <
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, filter, and/or disk from Sections 11.4.3.6, 11.5.8, 11.6.4, 11.7.3,
11.7.4, or 12.2.2,4 and any nonaqueous liquid from Section 11.6.3 into the thimble and
32
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Method 1613
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 refluxing. 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 to 2 hours and 8 to 9 hours, or sooner if the
receiver fills with water. Reflux the sample for a total of 16 to 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 extract using one of the macro-concentration procedures in Section 12.6
per the following:
12.3.9.1 Extracts from the particles in an aqueous sample containing less than one percent
solids (Section 11.4.3.6).
12.3.9.1.1 Concentrate the extract to approximately 5 mL using the rotary
evaporator or heating mantle procedures in Sections 12.6.1 or 12.6.2.
12.3.9.1.2 Quantitatively transfer the extract through the sodium sulfate (Section
12.1.3) into the apparatus that was set aside (Section 12.1.4.2) and
reconcentrate to the level of the toluene.
12.3.9.1.3 Adjust to approximately 10 mL with hexane, transfer to a 250-mL
separatory funnel, and proceed with back-extraction (Section 12.5).
12.3.9.2 Extracts from particles (Sections 11.5-11.6) or from the SPE filter and disk
(Section 12.2.2.4)—Concentrate to approximately 10 mL using the rotary
evaporator or heating mantle (Section 12.6.1 or 12.6.2), transfer to a 250-mL
separatory funnel, and proceed with back-extraction (Section 12.5).
12.4 Extraction of tissue—Two procedures are provided for tissue extraction.
12.4.1 Soxhlet extraction (Reference 21).
12.4.1.1 Add 30 to 40 g of powdered anhydrous sodium sulfate to each of the beakers
(Section 11.8.4) and mix thoroughly. Cover the beakers with aluminum foil and
allow to equilibrate for 12-24 hours. Remix prior to extraction to prevent
clumping.
12.4.1.2 Assemble and pre-extract the Soxhlet apparatus per Sections 12.3.1-12.3.4, except
use the methylene chloride:hexane (1:1) mixture for the pre-extraction and rinsing
and omit the quartz sand. The Dean-Stark moisture trap may also be omitted, if
desired.
12.4.1.3 Reassemble the pre-extracted Soxhlet apparatus and add a fresh charge of
methylene chloride:hexane to the reflux flask.
33
-------
Method 1613
12.4.1.4 Transfer the sample/sodium sulfate mixture (Section 12.4.1.1) to the Soxhlet
thimble, and install the thimble in the Soxhlet apparatus.
12.4.1.5 Rinse the beaker with several portions of solvent mixture and add to the thimble.
Fill the thimble/receiver with solvent. Extract for 18 to 24 hours.
12.4.1.6 After extraction, cool and disassemble the apparatus.
12.4.1.7 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.'
12.4.1.8 Complete the removal of the solvent using the nitrogen blowdown 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.
12.4.1.9 Percent lipid determination— The lipid content is determined by extraction of
tissue with the same solvent system (methylene chloriderhexane) that was used in
EPA's National Dioxin Study (Reference 22) so that lipid contents are consistent
with that study.
12.4.1.9.1 Redissolve the residue in the receiver in hexane and spike 1.0 mL of
the cleanup standard (Section 7.1 1) into the solution.
12.4.1.9.2 Transfer the residue/hexane to the anthropogenic isolation column
(Section 13.7.1) or bottle for the acidified silica gel batch cleanup
(Section 13.7.2), 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.
12.4.1.9.3 Calculate the lipid content to the nearest three significant figures as
follows:
Percent lipid = w^Sht of residue (g) x m
Weight of tissue (g)
12.4.1.9.4 It is not necessary to determine the lipid content of the blank, DPR, or
OPR aliquots.
12.4.2 HC1 digestion/extraction and concentration (References 23-2(5).
12.4.2.1 Add 200 mL of 6 N HC1 and 200 mL of methylene chloride:hexane (1:1) to the
sample and QC aliquots (Section 11.8.4).
12.4.2.2 Cap and shake each bottle 1 to 3 times. Loosen the cap in a hood to vent excess
pressure. Shake each bottle for 10 to 30 seconds and vent.
12.4.2.3 Tightly cap and place on shaker. Adjust the shaker action and speed so that the
acid, solvent, and tissue are in constant motion. However, take care to avoid such
-------
Method 1613
violent action that the bottle may be dislodged from the shaker. Shake for 12 to
24 hours.
12.4.2.4 After digestion, remove the bottles from the shaker. Allow the bottles to stand so
that the solvent and acid layers separate.
12.4.2.5 Decant the solvent through a glass funnel with glass-fiber filter (Sections 6.5.2-
6.5.3) containing approximately 10 grams of granular anhydrous sodium sulfate
(Section 7.2.1) into a macro-concentration apparatus (Section 12.6). Rinse the
contents of the bottle with two 25-mL portions of hexane and pour through the
sodium sulfate into the apparatus.
12.4.2.6 Concentrate the solvent to near dryness using a macro-concentration procedure
(Section 12.6).
12.4.2.7 Complete the removal of the solvent using the nitrogen blowdown apparatus
(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.
12.4.2.8 Percent lipid determination—The lipid content is determined in the same solvent
system [memylene chloride:hexane (1:1)] that was used in EPA's National Dioxin
Study (Reference 22) so that lipid contents are consistent with that study.
12.4.2.8.1 Redissolve the residue in the receiver in hexane and spike 1.0 mL of
the cleanup standard (Section 7.11) into the solution.
' 12.4.2.8.2 Transfer the residue/hexane to the narrow-mouth 100- to 200-mL bottle
retaining the boiling chips in the receiver. Use several rinses to assure
that all material is transferred, to a maximum hexane volume of
approximately 70 mL. Allow the receiver to dry. Weigh the receiver
and boiling chips.
12.4.2.8.3 Calculate the percent lipid per Section 12.4.1.9.3. It is not necessary to
determine the lipid content of the blank, IPR, or OPR aliquots.
12.4.2.9 Clean up the extract per Section 13.7.3.
12.5 Back-extraction with base and acid.
12,5.1 Spike 1.0 mL of the cleanup standard (Section 7.11) into the separately funnels
containing the sample and QC extracts from Section 12.1.4.1, 12.3.9.1.3, or 12.3.9.2.
12.5.2 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 degradation of the CDDs/CDFs. 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.
35
-------
Method 1613
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.
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). Rinse the separatory funnel with 30 to 50 mL of solvent,
and pour through the drying column. Collect each extract in a round-bottom flask. 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.
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, preclean 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: // 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. \
12.6.2 Heating mantle—Concentrate the extracts in separate round-bottom flasks.
36
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Method 1613
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 Kudema-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.
12.6.4 Preparation for back-extraction or micro-concentration and solvent exchange.
37
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Method 1613
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).
127 Micro-concentration and solvent exchange.
12.7.1 Extracts to be subjected to GPC or HPLC cleanup are exchanged into methylene
chloride. Extracts to be cleaned up using silica gel, alumina, carbon, and/or Florisil are
exchanged into hexane.
12.7.2 Transfer the vial containing the sample extract to a nitrogen blowdown 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 is to be concentrated to dry ness for weight determination (Sections
12.4.1.8, 12.4.2.7, and 13.7.1.4), 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 uL, 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 uL. 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, further concentrate
- the extract to 30 uL. Proceed with GPC or HPLC cleanup (Section 13.2 or 13.6,
respectively).
12.7.6 If the extract is to be cleaned up by column chromatography (alumina, silica gel,
Carbopak/Celite, or Florisil), bring the final volume to 1.0 mL with hexane. Proceed with
column cleanups (Sections 13.3 - 13.5 and 13.8).
12.7.7 If the extract is to be concentrated for injection into the GCfMS (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 yL. Add 10 uL 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 temperature until ready for GC/MS analysis. If GC/MS analysis will not be
performed on the same day, store the vial at <-10°C.
-------
Method 1613
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 analyst
may use any or all of the procedures below or any other appropriate procedure. Before using a
cleanup procedure, the analyst must demonstrate that the requirements of Section 9.2 can be met
using the cleanup procedure. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, the
cleanup procedures may be optimized for isolation of these two compounds.
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 and may be used for water extracts that are expected to contain
high molecular weight organic compounds (e.g., polymeric materials, humic acids).
13.1.2 Acid, neutral, and basic silica gel (Section 13.3), alumina (Section 13.4), and Florisil
(Section 13.8) are used to remove nonpolar and polar interferences. Alumina and Florisil
are used to remove chlorodiphenyl ethers.
13.1.3 Carbopak/Celite (Section 13.5) is used to remove nonpolar interferences.
13.1.4 HPLC (Section 13.6) is used to provide specificity for the 2,3,7,8-substituted and other
CDD and CDF isomers.
13.1.5 The anthropogenic isolation column (Section 13.7.1), acidified silica gel batch adsorption
procedure (Section 13.7.2), and sulfuric acid and base back-extraction (Section 13.7.3)
are 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 calibration solution (Section 7.4) into the sample loop.
13.2.2.2 Inject the calibration solution and record the signal from the detector. The elution
pattern will be corn oil, bis(2-ethyl hexyl)phthalate, pentachlorophenol, perylene,
and sulfur.
39
-------
•,
corn o,l and >85% collection
13.2
^3.3 Silica
r
13.3.1
13.3.2
13.3.3
13.3.4
13.3.5
13.2.2.3 Set the "dump time" to allow >85% removal of
of the phthalate. all°W >85% removal of
13.2.2.4 Set the "collect time" to the peak minimum between perylene and sulfur
13.2.2.5 Verify the calibration with the calibration solution after every 20 extracts
Calibration is verified if the recovery of the pentachlorophenol is greater than
85%. If calibration ,s not verified, the system shall be recalibrated using the
calibration solution, and the previous 20 samples shall be re-extracted and
cleaned up using the calibrated GPC system.
.3 Extract cleanup-GPC requires that the column not be overloaded. The column specified
m this method is designed to handle a maximum of 0.5 g of high molecular weiS
material in a 5-mL extract If the extract is known or expected to I^ 0.5
^ the extract ,. split into ahquots for GPC, and the aliquots are combined after elution
from the column. The residue content of the extract may be obtained gravimetrical'y by
evaporating the solvent from a 50-uL aliquot. metncai.y oy
13.2.3.1 Filter the extract or load through the filter holder (Section 6.7. 1 .3) to remove the
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-mJL beaker. j
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 a Particularly dirty extract is encountered, a 5.(jLmL methylene chloride blank
shall be run through the system to check for carry-over.
13.2.3.5 Concentrate the eluate per Section 12.6 and Section 12.7 for further cleanup or
injection into the GC/MS.
gel cleanup.
Place a glass-wool plug in a 15-mm ID chromatography column (Section 6.7 4 2) Pack
he column bottom to top with: 1 g silica gel (Section 7.5.1.1), 4 g basic silica gel
(Section 7.5. 13), 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.
Pre-elute the column with 50 to 100 mL of hexane. Close the stopcock when the hexane
is w,thm 1 mm of the sodium sulfate. Discard the eluate. Check the column for
channeling. If channeling is present, discard the column and prepare another.
Apply the concentrated extract to the column. Open the stopcock until the extract is
within 1 mm of the sodium sulfate.
Rinse the receiver twice with 1-mL portions of hexane, and apply separately to the
column. Elute the CDDs/CDFs with 100 mL hexane, and collect the eluate.
40
-------
13.3.6 For extracts of samples known to contain large quantities of other organic compounds
(such as paper mill effluents), 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 44% w/w (7.9 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 CDDs/CDFs. Increasing the strengths of the acid and basic silica gel may
also require different volumes of hexane than those specified above to elute the analytes off the
column. Therefore, the performance of the method after such modifications must be verified by
the procedure in Section 9.2.
13.4 Alumina cleanup.
13.4.1 Place a glass-wool plug in a 15-mm ID chromatography column (Section 6.7.4.2).
13.4.2 If using acid alumina, pack the column by adding 6 g acid alumina (Section 7.5.2.1). If
using basic alumina, substitute 6 g basic alumina (Section 7.5.2.2). Tap the column to
settle the adsorbents.
13.4.3 Pre-elute the column with 50 to 100 mL of hexane: Close the stopcock when the hexane
is within 1 mm of the alumina.
13.4.4 Discard the eluate. Check the column for channeling. If channeling is present, discard the
column and prepare another.
13.4.5 Apply the concentrated extract to the column. Open the stopcock until the extract is
within 1 mm of the alumina.
13.4.6 Rinse the receiver twice with 1-mL portions of hexane and apply separately to the
column. Elute the interfering compounds with 100 mL hexane and discard the eluate.
13.4.7 The choice of eluting solvents will depend on the choice of alumina (acid or basic) made
in Section 13.4.2.
13.4.7.1 If using acid alumina, elute the CDDs/CDFs from the column with 20 mL
methylene chloride:hexane (20:80 v/v). Collect the eluate.
13.4.7.2 If using basic alumina, elute the CDDs/CDFs from the column with 20 mL
methylene chloride:hexane (50:50 v/v). Collect the eluate.
13.4.8 Concentrate the eluate per Section 12.6 and 12.7 for further cleanup or injection into the
HPLC or GC/MS.
13.5 Carbon column.
13.5.1 Cut both ends from a 10-mL disposable serological pipet (Section 6.7.3.2) to produce a
10-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 0.55 g of Carbopak/Celite (Section 7.5.3.3) to
41
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ml t T« T ene 0OWe y mL * me'Mene chloride:
m thanol.-toluene (5:4:1 v/v), 1 mL of methylene chloridexyclohexane (1:1 v/v), and 5
mL of hexane. If the flow rate of eluate exceeds 0.5 mL'minute, discard the column
13.5.3 When the solvent is within 1 mm of the column packing; apply the sample extract to the
column. R,nse the sample container twice with 1-mL portions of hexane and apply
separately to the column. Apply 2 mL of hexane to complete the transfer.
13.5.4 Elute the interfering compounds with two 3-mL portions of hexane, 2 mL of methylene
chlonde:cyclohexane (1:1 v/v), and 2 mL of methylene c}iloride:methanol:toluene (15-4-1
v/v). Discard the eluate. ; ' '
r
13.5.5 Invert the column, and elute the CDDs/CDFs with 20 mL of toluene. If carbon particles
are present in the eluate, filter through glass-fiber filter paper.
13.5.6 Conc^ntra^the eluate per Section 12.6 and ,2.7 for further cleanup or injection into the
113.6 HPLC (Reference 6),,
| 13.6.1 Column calibration.
13.6.1.1 Prepare a calibration standard containing the 2,3,7,,8-substituted isomers and/or
other isomers of interest at a concentration of approximately 500 pg/uL in
methylene chloride.
13.6.1.2 Inject 30 pL of the calibration solution into the HPLC and record the signal from
the detector. Collect the eluant for reuse. The elution order will be the tetra-
through octa- isomers.
13.6.1.3 Establish the collection time for the tetra-isomers and for the other isomers of
interest. Following calibration, flush the injection system with copious quantities
of methylene chloride, including a minimum of five 50-uL injections while the
detector is monitored, to ensure that residual CDDs/CDFs are removed from the
system.
13.6.1.4 Verify the calibration with the calibration solution after every 20 extracts
Calibration is verified if the recovery of the CDDs/CDFs from the calibration
standard (Section 13.6.1.1) is 75 to 125% compared to the calibration (Section
13.6.1.2). If calibration is not verified, the system shall be recalibrated using the
calibration solution, and the previous 20 samples shall be re-extracted and
cleaned up using the calibrated system.
13.6.2 Extract cieanup-HPLC requires that the column not be overloaded. The column
specified in this method is designed to handle a maximum of 30 uL of extract If the
extract cannot be concentrated to less than 30 ui,, it is split into fractions and the
fractions are combined after elution from the column.
13.6.2.1 Rinse the sides of the vial twice with 30 uL of methylene chloride and reduce to
30 uL with the evaporation apparatus (Section 12.7).
42
-------
Metnoa
13.6.2.2 Inject the 30 uL extract into the HPLC.
13.6.2.3 Elute the extract using the calibration data determined in Section 13.6.1. Collect
the fraction(s) in a clean 20-mL concentrator tube containing 5 mL of
hexane:acetone (1:1 v/v).
13.6.2.4 If an extract containing greater than 100 ng/mL of total CDD or CDF is encoun-
tered, a 30-uL methylene chloride blank shall be run through the system to check
for carry-over.
13.6.2.5 Concentrate the eluate per Section 12.7 for injection into the GC/MS.
13.7 Cleanup of tissue lipids—Lipids are removed from the Soxhlet extract using either the
anthropogenic isolation column (Section 13.7.1) or acidified silica gel (Section 13.7.2), or are
removed from the HC1 digested extract using sulfuric acid and base back-extraction (Section
13.7.3).
13.7.1 Anthropogenic isolation column (References 22 and 27)—Used for removal of lipids
from the Soxhlet/SDS extraction (Section 12.4.1).
13.7.1.1 Prepare the column as given in Section 7.5.4.
13.7.1.2 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.
13.7.1.3 Load the sample and rinses (Section 12.4.1.9.2) onto the column by draining each
portion to the top of the bed. Elute the CDDs/CDFs from the column into the
apparatus used for concentration (Section 12.4.1.7) using 200 mL of hexane.
13.7.1.4 Concentrate the cleaned up extract (Sections 12.6-12.7) to constant weight per
Section 12.7.3.1. If more than 500 mg of material remains, repeat the cleanup
using a fresh anthropogenic isolation column.
13.7.1.5 Redissolve the extract in a solvent suitable for the additional cleanups to be used
(Section 13.2-13.6 and 13.8).
13.7.1.6 Spike 1.0 mL of the cleanup standard (Section 7.11) into the residue/solvent.
13.7.1.7 Clean up the extract using the procedures in Sections 13.2-13.6 and 13.8.
Alumina (Section 13.4) or Florisil (Section 13.8) and carbon (Section 13.5) are
recommended as minimum additional cleanup steps.
13.7.1.8 Following cleanup, concentrate the extract to 10 uL as described in Section 12.7
and proceed with the analysis in Section 14.
13.7.2 Acidified silica gel (Reference 28)—Procedure alternate to the anthropogenic isolation
column (Section 13.7.1) that is used for removal of lipids from the Soxhlet/SDS
extraction (Section 12.4.1).
13.7.2.1 Adjust the volume of hexane in the bottle (Section 12.4.1.9.2) to approximately
200 mL.
13.7.2.2 Spike 1.0 mL of the cleanup standard (Section 7.11) into the residue/solvent.
13.7.2.3 Drop the stirring bar into the bottle, place the bottle on the stirring plate, and
begin stirring.
43
-------
13.7.2.4 Add 30-100 g of acid silica gel (Section 7.5.1.2) to the bottle while stirring,
keeping the silica gel in motion. Stir for 2 to 3 hours.
Note: 30 grams of silica gel should be adequate for most samples' and will minimize
contamination from this source.
13.7.2.5 After stirring, pour the extract through approximately 10 g of granular anhydrous
sodium sulfate (Section 7.2.1) contained in a funnel with glass-fiber filter into a
macro contration device (Section 12.6). Rinse the bottle and sodium sulfate with
hexane to complete the transfer.
13.7.2.6 Concentrate the extract per Sections 12.6-12.7 and clean up the extract using the'
procedures in Sections 13.2-13.6 and 13.8. Alumina (Section 13.4) or Florisil
(Section 13.8) and carbon (Section 13.5) are recommended as minimum
additional cleanup steps.
13.7.3 Sulfuric acid and base back-extraction—Used with HC1 digested extracts (Section
12.4.2).
13.7.3.1 Spike 1.0 mL of the cleanup standard (Section 7.1
1) into the residue/solvent
(Section 12.4.2.8.2).
13.7.3.2 Add 10 mL of concentrated sulfuric acid to the bottle. Immediately cap and
shake 1 to 3 times. Loosen cap in a hood to vent excess pressure. Cap and shake
the bottle so that the residue/solvent is exposed to the acid for a total time of
approximately 45 seconds.
13.7.3.3 Decant the hexane into a 250-mL separately funnel making sure that no acid is
transferred. Complete the quantitative transfer with several hexane rinses.
13.7.3.4 Back extract the solvent/residue with 50 mL of potassium hydroxide solution per
Section 12.5.2, followed by two reagent water rinses.
13.7.3.5 Drain the extract through a filter funnel containing approximately 10 g of
granular anhydrous sodium sulfate in a glass-fiber filter into a macro
concentration device (Section 12.6).
13.7.3.6 Concentrate the cleaned up extract to a volume suitable for the additional
cleanups given in Sections 13.2-13.6 and 13.8. Gel permeation chromatography
(Section 13.2), alumina (Section 13.4) or Florisil (Section 13.8), and
Carbopak/Celite (Section 13.5) are recommended as minimum additional cleanup steps.
13.7.3.7 Following cleanup, concentrate the extract to 10 uL as described in Section 12.7
and proceed with analysis per Section 14. i
13.8 Florisil cleanup (Reference 29).
13.8.1 Pre-elute the activated Florisil column (Section 7.5.3) with 10 mL of methylene chloride
followed by 10 mL of hexane:methylene chloride (98:2 v/v) and discard the solvents.
44
-------
Method 1613
13.8.2 When the solvent is within 1 mm of the packing, 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.
13.8.3 Elute the interfering compounds with 20 mL of hexane:methylene chloride (98:2) and
discard the eluate.
13.8.4 Elute the CDDs/CDFs with 35 mL of methylene chloride and collect the eluate.
Concentrate the eluate per Sections 12.6-12.7 for further cleanup or for injection into the
HPLC or GC/MS.
14.0 HRGC/HRMS Analysis
14.1 Establish the operating conditions given in Section 10.1.
14.2 Add IO uL of the appropriate internal standard solution (Section 7.12) to the 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
instrument internal standard solution. Rather, bring the extract back to its previous volume (e.g.,
19 pL) with pure nonane only (18 uL if 2 uL injections are used).
14.3 Inject 1.0 or 2.0 uL of the concentrated extract containing the internal standard solution, using
on-column or splitless injection. The volume injected must be identical to the volume used for
calibration (Section 10). Start the GC column initial isothermal hold upon injection. Start MS
data collection after the solvent peak elutes. Stop data collection after the OCDD and OCDF
have eluted. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, stop data collection
after elution of these compounds. 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 CDDs/CDFs and labeled compounds. For these
tests, analysis of the CS3 calibration verification (VER) standard (Section 7.13 and Table 4) and
the isomer specificity test standards (Section 7.15 and Table 5) shall be used to verify all
performance criteria. Adjustment and/or recalibration (Section 10) shall 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—A static resolving power of at least 10,000 (10% valley definition) must be
demonstrated at the appropriate m/z before any analysis is performed. Static resolving power
checks must be performed at the beginning and at the end of each 12-hour shift according to
procedures in Section 10.1.2. Corrective actions must be implemented whenever the resolving
power does not meet the requirement.
15.3 Calibration verification.
15.3.1 Inject the VER standard using the procedure in Section 14.
45
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Method 1613
15.3.2 The m/z abundance ratios for all CDDs/CDFs shall be within the limits in Table 9;
otherwise, the mass spectrometer shall be adjusted until the m/z abundance ratios fall
within the limits specified, and the verification test shall be repeated. If the adjustment
alters the resolution of the mass spectrometer, resolution shall be verified (Section 10.1.2)
prior to repeat of the verification test.
15.3.3 The peaks representing each CDD/CDF and labeled compound in the VER standard must
be present with S/N of at least 10; otherwise, the mass spectrometer shall be adjusted and
the verification test repeated.
15.3.4 Compute the concentration of each CDD/CDF compound by isotope dilution (Section
10.5) for those compounds that have labeled analogs (Table 1). Compute the concentra- '
tion of the labeled compounds by the internal standard method (Section 10.6). These
concentrations are computed based on the calibration data in Section 10.
15.3.5 For each compound, compare the concentration with the calibration verification limit in
Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, compare the
concentration to the limit in Table 6a. If all compounds meet the acceptance criteria,
calibration has been verified and analysis of standards and sample extracts may proceed.
i If, however, any compound fails its respective limit, the measurement system is not
| performing properly for that compound. In this event, prepare a fresh calibration standard
j or correct the problem causing the failure and repeat the resolution (Section 15.2) and
i verification (Section 15.3) tests, or recalibrate (Section 10).
15.4 Retention times and GC resolution. ;
; 15.4.1 Retention times. :
15.4.1.1 Absolute—The absolute retention times of the 13C,2-1,2,3,4-TCDD and I3C12-
1,2,3,7,8,9-HxCDD GCMS internal standards in the verification test (Section
15.3) shall be within ±15 seconds of the retention times obtained during
calibration (Sections 10.2.1 and 10.2.4).
15.4.1.2 Relative—The relative retention times of CDDs/CDFs and labeled compounds in
the verification test (Section 15.3) shall be within the limits given in Table 2.
15.4.2 GC resolution.
15.4.2.1 Inject the isomer specificity standards (Section 7.15) on their respective columns.
15.4.2.2 The valley height between 2,3,7,8-TCDD and the other tetra-dioxin isomers at
m/z 319.8965, and between 2,3,7,8-TCDF and the other tetra-furan isomers at
m/z 303.9016 shall not exceed 25% on their respective columns (Figures 6 and
7).
15.4.3 If the absolute retention time of any compound is not within the limits specified or if the
2,3,7,8-isomers are not resolved, 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.
Ongoing precision and recovery.
46
-------
Method 1613
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, 11.7.4, or 11.8.3.2) prior to analysis of samples from the same
batch.
15.5.2 Compute the concentration of each CDD/CDF by isotope dilution for those compounds
that have labeled analogs (Section 10.5). Compute the concentration of 1,2,3,7,8,9-
HxCDD, OCDF, and each labeled compound by the internal standard method (Section
10.6).
15.5.3 For each CDD/CDF and labeled compound, compare the concentration to the OPR limits
given in Table 6. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, compare
the concentration to the limits in Table 6a. 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 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 CDD/CDF 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. 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 CDD, CDF, 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 8 must be present and must maximize within the
same two seconds.
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 CDD or CDF detected in a sample extract, and greater than or equal to 10 for all
CDDs/CDFs in the calibration standard (Sections 10.2.3 and 15.3.3).
16.3 The ratio of the integrated areas of the two exact m/z's specified in Table 8 must be within the
limit in Table 9, or within ±10 percent of the ratio in the midpoint (CS3) calibration or
calibration verification (VER), whichever is most recent.
16.4 The relative retention time of the peak for a 2,3,7,8-substituted CDD or CDF must be within the
limit in Table 2. The retention time of peaks representing non-2,3,7,8-substituted CDDs/CDFs
must be within the retention time windows established in Section 10.3.
47
-------
Method 1613
1615 Confirmatory analysis—Isomer specificity for 2,3,7,8-TCDF cannot be achieved on the DB-5
I column. Therefore, any sample in which 2,3,7,8-TCDF is identified by analysis on a DB-5
1 column must have a confirmatory analysis performed on a DB-225, SP-2330, or equivalent GC
I column. The operating conditions in Section 10.1.1 may be adjusted to optimize the analysis on
i the second GC column, but the GC/MS must meet the mass resolution and calibration
: specifications in Section 10.
1616 If the criteria for identification in Sections 16.1-16.5 are not met, the CDD or CDF has not been
identified and the results may not be reported for 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—By adding a known amount of a labeled compound to every
sample prior to extraction, correction for recovery of the CDD/CDF can be made because the
CDD/CDF and its labeled analog exhibit similar effects upon extraction, concentration, and gas
chromatography. Relative response (RR) values are used in conjunction with the initial cali-
bration data described in Section 10.5 to determine concentrations directly, so long as labeled
compound spiking levels are constant, using the following equation:
(Aln + A2J C,
Ca (rig/ml) = —1 "-—I-
" (Al,+A2l)RR
where'.
Ca = The concentration of the CDD/CDF in the extract, and the
other terms are as defined in Section 10.5.2
i
•i
17.1.1 Because of a potential interference, the labeled analog of OCDF is not added to the
sample. Therefore, OCDF is quantitated against labeled OCDD. As a result, the
concentration of OCDF is corrected for the recovery of the labeled OCDD. In instances
where OCDD and OCDF behave differently during sample extraction, concentration, and
cleanup procedures, this may decrease the accuracy of the OCDF results. However, given
the low toxicity of this compound relative to the other dioxins and furans, the potential
decrease in accuracy is not considered significant.
17.1.2 Because 13C12-l,2,3,7,8,9-HxCDD is used as an instrument internal standard (i.e., not
added before extraction of the sample), it cannot be used to quantitate the 1,2,3,7,8,9-
HxCDD by strict isotope dilution procedures. Therefore, 1,2,3,7,8,9-HxCDD is
quantitated using the averaged response of the labeled analogs of the other two 2,3,7,8-
substituted HxCDD's: 1,2,3,4,7,8-HxCDD and 1,2,3,6,7,8-HxCDD. As a result, the
concentration of 1,2,3,7,8,9-HxCDD is corrected for the average recovery of the other
two HxCDD's.
48
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Method 1613
17.1.3 Any peaks representing non-2,3,7,8-substituted CDDs/CDFs are quantitated using an
average of the response factors from all of the labeled 2,3,7,8- isomers at the same level
of chlorination.
17.2 Internal standard quantitation and labeled compound recovery
17.2.1 Compute the concentrations of 1,2,3,7,8,9-HxCDD, OCDF, the l3C-Iabeled analogs and
the 37C-labeled cleanup standard in the extract using the response factors determined from
the initial calibration data (Section 10.6) and the following equation:
(41, + A2J Ch
C (ng/ml) = —-1 ——
" ' *' (41 u - A2J RF
where:
C^ = The concentration of the compound in the extract.
The other terms are defined in Section 10.6.1
Note: There is only one m/z. for the 37Cl-labeled standard.
17.2.2 Using the concentration in the extract determined above, compute the percent recovery of
the I3C-Iabeled compounds and the 37C-labeled cleanup standard using the following
equation:
, o /a^ Concentration found (wr/mL) 1An
Recovery (%) = — ——J ™ /x 100
Concentration spiked (/jg/mL)
17.3 The concentration of a CDD/CDF 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 11.5.1), as
follows:
Concentration in solid (ng/kg) =
where:
Ca - The concentration of the compound in the extract.
Va - The extract volume in mL.
Ws - The sample weight (dry weight) in kg.
49
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Method 1613
17.4 The concentration of a CDD/CDF 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 or
11.5), as follows:
(C x V )
Concentration in aqueous phase (pg/L) = —— —
where:
Ca = The concentration of the compound in the extract.
Va = The extract volume in mL. \
; Vs - The sample volume in liters.
i
i
i ;
i
17.5 If the SICP area at either quantitation m/z for any compound exceeds the calibration range of the
! system, a smaller sample aliquot is extracted. i
i 17.5.1 For aqueous samples containing 1% solids or less, dilute 100 mL, 10 mL, etc., of sample
I to 1 L with reagent water and re-prepare, extract, clean up, and analyze per Sections 11-
! 14.
!
i 17.5.2 For samples containing greater than 1% solids, extract an amount of sample equal to
1/10, 1/100, etc., of the amount used in Section 11.5.1. Re-prepare, extract, clean up, and
analyze per Sections 11-14. :
| . 17.5.3 If a smaller sample size will not be representative of the entire sample, dilute the sample
•• extract by a factor of 10, adjust the concentration of the instrument internal standard to
• 100 pg/uL in the extract, and analyze an aliquot of this diluted extract by the internal
standard method.
[ j
17|.6 Results are reported to three significant figures for the CDDs/CDFs and labeled compounds
i found in all standards, Blanks, and samples.
j 17.6.1 Reporting units and levels.
!
| - 17.6.1.1 Aqueous samples—Report results in pg/L (parts-per-quadrillion).
| 17.6.1.2 Samples containing greater than 1% solids (soils, sediments, filter cake,
J compost)—Report results in ng/kg based on the dry weight of the sample. Report
i the percent solids so that the result may be corrected.
; 17.6.1.3 Tissues—Report results in ng/kg of wet tissue, not on the basis of the lipid
content of the sample. 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 Standards (VER, IPR, OPR) and samples—Report results at or above
the minimum level (Table 2). Report results below the minimum level
as not detected or as required by the regulatory authority.
17.6.1.4.2 Blanks—Report results above one-third the ML.
50
-------
Method 1613
17.6.2 Results for CDDs/CDFs in samples that have been diluted are reported at the least dilute
level at which the areas at the quantitation m/z's are within the calibration range (Section
17.5).
17.6.3 For CDDs/CDFs having a labeled analog, results are reported at the least dilute level at
which the area at the quantitation m/z is within the calibration range (Section 17.5) and
the labeled compound recovery is within the normal range for the method (Section 9.3
and Tables 6, 6a, 7, and 7a).
17.6.4 Additionally, if requested, the total concentration of all isomers in an individual level of
chlorination (i.e., total TCDD, total TCDF, total PeCDD, etc.) may be reported by
summing the concentrations of all isomers identified in that level of chlorination,
including both 2,3,7,8-substituted and non-2,3,7,8-substituted isomers.
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 will not concentrate to 10 uL
(Section 12.7); others may overload the GC column and/or mass spectrometer.
18.2 Analyze a smaller aliquot of the sample (Section 17.5) when the extract will not concentrate to
10 uL after all cleanup procedures have been exhausted.
18.3 Chlorodiphenyl ethers—If chromatographic peaks are detected at the retention time of any
CDDs/CDFs in any of the m/z channels being monitored for the chlorodiphenyl ethers (Table 8),
cleanup procedures must be employed until these interferences are removed. Alumina (Section
13.4) and Florisil (Section 13.8) are recommended for removal of chlorodiphenyl ethers.
18.4 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).
18.4.1 If the recovery of any of the labeled compounds is outside of the normal range (Table 7),
a diluted sample shall be analyzed (Section 17.5).
18.4.2 If the recovery of any of the labeled compounds in the diluted sample is outside of
normal range, the calibration verification standard (Section 7.13) shall be analyzed and
calibration verified (Section 15.3).
18.4.3 If the calibration cannot be verified, a new calibration must be performed and the
original sample extract reanalyzed.
18.4.4 If the calibration is verified and die 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 for regulatory compliance purposes. In this case, alternate
extraction and cleanup procedures in this method must be employed to resolve the
interference. If all cleanup procedures in this method 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 these
samples.
51
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Method 1613
Pollution Prevention
I
1J9.1 The solvents used in this method pose little threat to the environment when managed properly.
The solvent evaporation techniques used in this method are amenable to solvent recovery, and it
is recommended that the laboratory recover solvents wherever feasible.
i
19.2 Standards should be prepared in volumes consistent with laboratory use to minimize disposal of
i standards. [
i
:
20.0 Waste Management
2&.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations
governing waste management, particularly the hazardous waste identification rules and land
disposal 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.
2ID.2 Samples containing HC1 to pH <2 are hazardous and must be neutralized before being poured
i down a drain or must be handled as hazardous waste.
20.3 The CDDs/CDFs 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.
2d.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 CDDs/CDFs 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 Mianagement for Waste
Reduction," available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.
21.0 Method Performance
Method performance was validated and performance specifications were developed using data from
EPA's international interlaboratory validation study (References 30-31) and the EPA/paper industry
Long-Term Variability Study of discharges from the pulp and paper industry (58 FR 66078).
22.0
References
1 Tondeur, Yves, "Method 8290: Analytical Procedures and Quality Assurance for Multimedia
Analysis of Polychlorinated Dibenzo-p-dioxins and Dibenzofurans by High Resolution Gas
Chromatography/High Resolution Mass Spectrometry," USEPA EMSL, Las Vegas, Nevada, June
1987.
52
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Method 1613
2 "Measurement of 2,3,7,8-Tetrachlorinated Dibenzo-p-dioxin (TCDD) and 2,3,7,8-TetrachIorinated
Dibenzofuran (TCDF) in Pulp, Sludges, Process Samples and Wastewaters from Pulp and Paper
Mills," Wright State University, Dayton, OH 45435, June 1988.
3 "NCASI Procedures for the Preparation and Isomer Specific Analysis of Pulp and Paper Industry
Samples for 2,3,7,8-TCDD and 2,3,7,8-TCDF," National Council of the Paper Industry for Air
and Stream Improvement Inc., 260 Madison Avenue, New York, NY 10016, Technical Bulletin
No. 551, Pre-Release Copy, July 1988.
4 "Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF in
Fish," USEPA, Environmental Research Laboratory, 6201 Congdon Boulevard, Duluth, MN
55804, April 1988.
5 Tondeur, Yves, "Proposed GC/MS Methodology for the Analysis of PCDDs and PCDFs in
Special Analytical Services Samples," Triangle Laboratories, Inc., 801-10 Capitola Dr, Research
Triangle Park, NC 27713, January 1988; updated by personal communication September 1988.
6 Lamparski, L.L., and Nestrick, T.J., "Determination of Tetra-, Hexa-, Hepta-, and
Octachlorodibenzo-p-dioxin Isomers in Paniculate Samples at Parts per Trillion Levels,"
Analytical Chemistry, 52: 2045-2054, 1980.
7 Lamparski, L.L., and Nestrick, 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.
8 Patterson, D.G., et. al. "Control of Interferences in the Analysis of Human Adipose Tissue for
2,3,7,8-Tetrachlorodibenzo-p-dioxin," Environmental lexicological Chemistry, 5: 355-360, 1986.
9 Stanley, John S., and Sack, Thomas M., "Protocol for the Analysis of 2,3,7,8-
Tetrachlorodibenzo-p-dioxin by High Resolution Gas Chromatography/High Resolution Mass
Spectrometry," USEPA EMSL, Las Vegas, Nevada 89114, EPA 600/4-86-004, January 1986.
10 "Working with Carcinogens," Department of Health, Education, & Welfare, Public Health
Service, Centers for Disease Control, NIOSH, Publication 77-206, August 1977, NTIS PB-
277256.
11 "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910.
12 "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety, 1979.
13 "Standard Methods for the Examination of Water and Wastewater," 18th edition and later
revisions, American Public Health Association, 1015 15th St, N.W., Washington, DC 20005, 1-
35: Section 1090 (Safety), 1992.
14 "Method 613—2,3,7,8-Tetrachlorodibenzo-/7-dioxin," 40 CFR 136 (49 FR 43234), October 26,
1984, Section 4.1.
15 Provost, L.P., and Elder, R.S., "Interpretation of Percent Recovery Data," American Laboratory,
15: 56-83, 1983.
16 "Standard Practice for Sampling Water," ASTM Annual Book of Standards, ASTM, 1916 Race
Street, Philadelphia, PA 19103-1187, 1980.
-------
Method 1613
j!7 "Methods 330.4 and 330.5 for Total Residual Chlorine," USEPA, EMSL, Cincinnati, OH 45268,
| EPA 600/4-79-020, March 1979. ;
18 "Handbook of Analytical Quality Control in Water and Wastewater Laboratories," USEPA
; EMSL, Cincinnati, OH 45268, EPA-600/4-79-019, March 1979.
JI9 Williams, Rick, letter to Bill Telliard, June 4, 1993, available from the EPA Sample Control
> Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria VA 22314, 703-519-1140.
120 Barkowski, Sarah, Fax to Sue Price, August 6, 1992, available from the EPA Sample Control
Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria VA 22314, 703-519-1140.
! . . '
21 "Analysis of Multi-media, Multi-concentration Samples for Dioxins and Furans, PCDD/PCDF
Analyses Data Package", Narrative for Episode 4419, MRI Project No. 3091-A, op.cit. February
12, 1993, Available from the EPA Sample Control Center operated by DynCorp Viar Inc, 300 N
Lee St, Alexandria, VA 22314 (703-519-1140). I
[ '
22 "Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF in
I Fish", U.S. Environmental Protection Agency, Environmental Research Laboratory, Duluth MN
i 55804, EPA/600/3-90/022, March 1990.
23 Afghan, B.K., Carron, J., Goulden, P.D., Lawrence, J., Leger, D., Onuska, F., Sherry, J., and
Wilkenson, R.J., "Recent Advances in Ultratrace Analysis of Dioxins and Related Halogenated
Hydrocarbons", Can J. Chem., 65: 1086-1097, 1987.
i
J24 Sherry, J.P., and Tse, H., "A Procedure for the Determination of Polychlorinated Dibenzo-p-
! dioxins in Fish", Chemosphere, 20: 865-872, 1990. \
!25 • "Preliminary Fish Tissue Study", Results of Episode 4419, available from the EPA Sample
Control Center operated by DynCorp Viar, Inc., 300 N Lee St, Alexandria, VA 22314, 703-519-
1140. ;
26 Nestrick, Terry L., DOW Chemical Co., personal communication with D.R. Rushneck, April 8,
1993. Details available from the U.S. Environmental Protection Agency Sample Control Center
operated by DynCorp Viar Inc, 300 N Lee St, Alexandria, VA 22314, 703-519-1140.
27 Barnstadt, Michael, "Big Fish Column", Triangle Laboratories of RIP, Inc., SOP 129-90, 27
March 27, 1992.
28 "Determination of Polychlorinated Dibenzo-/?-Dioxins (PCDD) and Dibenzofurans (PCDF) in
Environmental Samples Using EPA Method 1613", Chemical Sciences Department, Midwest
Research Institute, 425 Volker Boulevard, Kansas City, MO 44110-2299, Standard Operating
Procedure No. CS-153, January 15, 1992.
;29 Ryan, John J., Raymonde Lizotte and William H. Newsome, J. Chwmatog. 303 (1984) 351-360.
J30 Telliard, William A., Harry B. McCarty, and Lynn S. Riddick, "Results of the Interlaboratory
i Validation Study of USEPA Method 1613 for the Analysis of Tetrai- through Octachlorinated
Dioxins and Furans by Isotope Dilution GC/MS," Chemosphere, 27, 41-46 (1993).
|31 "Results of the International Interlaboratory Validation Study of USEPA Method 1613", October
! 1994, available from the EPA Sample Control Center operated by DynCorp Viar, Inc., 300 N
1 LeeSt, Alexandria, VA 22314,703-519-1140. i
54
-------
Method 1613
23.0 Tables and Figures
Table 1: Chlorinated Dibenzo-p-dioxins and Furans Determined by Isotope Dilution
and Internal Standard High Resolution Gas Chromatography (HRGC)/High
Resolution Mass Spectrometry (HRMS)
CDDs/CDFs1 CAS
2,3,7,8-TCDD
Total TCDD
2.3,7.8-TCDF
Tolal-TCDF
1.2,3.7.8-PeCDD
Tolal-PeCDD
1,2,3,7,8-PeCDF
2.3,4,7,8-PeCDF
Total-PeCDF
1.2,3,4,7.8-HxCDD
1,2.3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
Tolal-HxCDD
1,2.3,4,7,8-HxCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7,8-HxCDF
Total-HxCDF
1,2,3,4,6,7,8-HpCDD
Total-HpCDD
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
Total-HpCDF
OCDD
OCDF
Registry
1746-01-6
41903-57-5
51207-31-9
55722-27-5
40321-76-4
36088-22-9
57117-41-6
57117-31-4
30402-15-4
39227-28-6
57653-85-7
19408-74-3
34465-46-8
70648-26-9
57117-44-9
72918-21-9
60851-34-5
55684-94-1
35822-46-9
37871-00-4
67562-39-4
55673-89-7
38998-75-3
3268-87-9
39001-02-0
1 . Chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans
TCDD s Tetrachlorodibenzo-p-dioxin
PeCDD s Pentachlorodibenzo-p-dioxin
HxCDD = Hexachlorodibenzo-p-dioxin
HpCDD » Heptachlorodibenzo-pKlioxin
OCDD = Octachlorodibenzo-pKlioxin
TCDF
PeCDF
HxCDF
HpCDF
OCDF
Labeled analog
13C12-2,3,7,8-TCDD
37CI4-2,3,7,8-TCDD
13C,2-2,3,7,8-TCDF
'3C12-1,2,3,7,8-PeCDD
"Cl2-1,2,3,7,8-PeCDF
13Cl2-2,3,4,7,8-PeCDF
"0,2-1,2,3,4,7,8-HxCDD
13C12-1,2,3,6,7,8-HxCDD
"C,2-1,2,3,7,8,9-HxCDD
13C12-1,2,3,4,7,8-HxCDF
"C,2-1,2,3,6.7,8-HxCDF
13C12-1,2,3,7,8,9-HxCDF
13C12-2,3,4,6l7,8-HxCDF
'3C,2-1,2,3,4,6,7,8-HpCDD
"0^1,2,3,4,6,7,8-HpCDF
13C12-1l2l3,4l7,8,9-HpCDF
"C12-OCDD
not used
Tetrachlorodibenzofuran
Pentachlorodibenzofuran
Hexachlorodibenzofuran
Heptachlorodibenzofuran
Octachlorodibenzofuran
CAS Registry
76523-40-5
85508-50-5
89059-46-1
109719-79-1
109719-77-9
116843-02-8
109719-80-4
109719-81-5
109719-82-6
114423-98-2
116843-03-9
116843-04-0
116843-05-1
109719-83-7
109719-84-8
109719-94-0
114423-97-1
55
-------
Method 1613
Table 2: Retention Time References, Quantitation References,
Times, and Minimum Levels for CDDs and CDFs
Relative Retention
Minimum level1
Retention time Relative
and quantitation retention
CJDD/CDF reference time
Compounds using "0^-1 ,2,3,4-TCDD as the injection internal standard
2,3,7,8-TCDF "C,2-2,3,7,8-TCDF 0.999-1.003
2,3,7,8-TCDD 13C,2-2,3,7,8-TCDD 0.999-1.002
1,2,3,7,8-PeCDF "C,2-1,2,3,7,8-PeCDF 0.999-1.002
2,3,4,7,8-PeCDF 13C12-2,3,4,7,8-PeCDF 0.999-1.002 I
1,2,3,7,8-PeCDD "Cl2-1,2,3,7,8-PeCDD 0.999-1.002
13Cl2-2,3,7,8-TCDF "C12-1 ,2,3,4-TCDD 0.923-1.103 \
"C12-2,3,7,8-TCDD "C,2-1 ,2,3,4-TCDD 0.976-1.043
37CI4-2,3,7,8-TCDD 13C12-1 ,2,3,4-TCDD 0.989-1.052
"{V1,2,3,7,8-PeCDF "0,2-1 ,2,3,4-TCDD 1.000-1.425 '
13012-2,3,4,7,8-PeCDF 13C,2-1,2,3,4-TCDD 1.011-1.526
13fe12-1,2,3,7,8-PeCDD "C,2-1 ,2,3,4-TCDD 1.000-1.567 ;
Compounds using °C1f 1,2,3, 7,8,9-HxCDD as the injection internal standard ;
1,2,3,4,7,8-HxCDF "C,2-1,2,3,4,7,8-HxCDF 0.999-1.001
1,J2,3,6,7,8-HxCDF 13C,2-1 ,2,3,6,7,8-HxCDF 0.997-1.005
1,2,3,7,8,9-HxCDF "C,2-1 ,2,3,7,8,9-HxCDF 0.999-1.001
2,p,4,6,7,8-HxCDF "C12-2,3,4,6,7,8,-HxCDF 0.999-1.001
1,2,3,4,7,8-HxCDD "0,2-1 ,2,3,4,7,8-HxCDD 0.999-1.001
1,2,3,6,7,8-HxCDD "C,j-1 ,2,3,6,7,8,-HxCDD 0.998-1.004
1,|2,3,7,8,9-HxCDD — 2 1.000-1.019
1,2,3,4,6,7,8-HpCDF "C,2-1 ,2.3,4,6,7,8-HpCDF 0.999-1.001
1,b,3,4,7,8,9-HpCDF "C,2-1 ,2,3,4,7,8,9-HpCDF 0.999-1.001
1,b,3,4,6,7,8-HpCDD "C,2-1 ,2,3,4,6,7,8-HpCDD 0.999-1.001
obDF "0,2-OCDD 0.999-1.008 \
OCDD "0,2-OCDD 0.999-1.001 ;
"0,2-1 ,2,3,4,7,8-HxCDF "0,2-1 ,2,3,7,8,9-HxCDD 0.944-0.970
"0,2-1 ,2,3,6,7,8-HxCDF "C,2-1 ,2,3,7,8,9-HxCDD 0.949-0.975
"0,2-1 ,2,3,7,8,9-HxCDF "0,2-1 ,2,3,7,8,9-HxCDD 0.977-1.047 :
"0,2-2,3,4,6,7,8,-HxCDF "C,2-1 ,2,3,7,8,9-HxCDD 0.959-1.021
"0,2-1 ,2,3,4,7,8-HxCDD "C,2-1,2,3,7.8,9-HxCDD 0.977-1.000
"0,2-1 ,2,3,6,7,8-HxCDD "C,2-1 ,2,3,7,8,9-HxCDD 0.981-1.003
13£,2-1,2,3,4,6,7,8-HpCDF "C,2-1 ,2,3,7,8,9-HxCDD 1.043-1.085
"C,2-1,2,3,4,7,8,9-HpCDF "C,2-1 ,2,3.7,8,9-HxCDD 1.057-1.151 j
"0,2-1, 2,3,4,6,7,8-HpCDD "C,,-1 ,2,3,7,8,9-HxCDD 1.086-1.110
"JVOCDD "C,2-1,2,3,7.8,9-HxCDD T.032-1.311
Water
(P9/L;
ppq)
10
10
50
50
50
50
50
50
50
50
50
50
50
50
50
100
100
Solid
(ng/kg;
Ppt)
1
1
5
5
5
5
5
5
5
5
5
5
5
5
5
10
10
Extract
(pg/^L;
ppb)
0.5
0.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
5.0
5.0
I., The Minimum Level (ML) for each anaiyte is defined as the level at which the entire analytical system must give a
| recognizable signal and acceptable calibration point. It is equivalent to tie concentration of the lowest calibration
• standard, assuming that all method-specified sample weights, volumes, and cleanup procedures have been employed.
2.1 The retention time reference for 1,2,3,7,8,9-HxCDD is 13C,2-1,2,3,6,7,8-HxCDD, and 1,2,3,7,8,9-HxCDD is quantified
: using the averaged responses for "C,2-1,2,3,4,7,8-HxCDD and "C,2-1,2,3,6,7,8-HxCDD.
5(5 i
-------
Method 1613
spiking
solution2
(ng/mL) (ng/mL)
"C,i!-1,2,3,4,6l7,8-HpCDF
"Cl2-1,2,3,4,7,8,9-HpCDF
"C12-OCDD
100
100
100
100
100
100
100
100
100
100
100
100
100
100
200
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
PAR stock PAR spiking
solution3 solution4
(ng/mL)
40
40
200
200
. 200
200
200
Table 3: Concentration of Stock and Spiking Solutions Containing CDDs/CDFs and
Labeled Compounds
Labeled Labeled
compound compound
stock
solution1
CDD/CDF
2,3,7,8-TCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDD
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD - - 200
1,2,3,4,7,8-HxCDF - - 200
1,2,3,6,7,8-HxCDF - - 200
1,2,3,7,8,9-HxCDF - - 200
2,3,4,6,7,8-HxCDF - - 200
1,2,3,4,6,7,8-HpCDD - - 200
1,2,3,4,6,7,8-HpCDF - - 200
1,2,3,4,7,8,9-HpCDF - - 200
OCDD — — 400
OCDF - - 400
"0,2-2,3,7,8-1000
"0,.,-2,3,7,8-TCDF
13C12-U3,7,8-PeCDD
\3C12-1,2,3,7,8-PeCDF
uC12-2,3,4,7,8-PeCDF
"0,2-1,2,3,4,7,8-HxCDD
1JC12-1,2l3l6,7l8-HxCDD
13C,2-1,2,3l4,7,8-HxCDF
"C,2-1,2,3,6,7l8-HxCDF
"C,2-1l2,3,7,8l9-HxCDF
"C12-213,4,61718-HxCDF
(ng/mL)
0.8
0.8
4
4
4
4
4
4
4
4
4
4
4
4
4
8
8
Cleanup Standard
3TCI,-2,317,8-TCOD
Internal Standard^
"0,2-1,2,3,4-1000
"C,j-1,2,3,7,8,9-HxCDD
Concentration
(ng/ml)
0.8
200
200
1.Section 7.10—
2.Section 7.10.3—
S.Section 7.9—
4.Section 7.14—
5.Section7.11—
B.Section 7.12—
prepared jn nonane and diluted to prepare spiking solution.
prepared in acetone from stock solution daily.
prepared in nonane and diluted to prepare spiking solution.
prepared in acetone from stock solution daily.
prepared jn nonane and added to extract prior to cleanup.
prepared in nonane and added to the concentrated extract immediately prior to injection into the GC
(Section 14.2).
57
-------
Method 1613
I
Table 4: Concentration of CDDs/CDFs in Calibration and Calibration Verification
i Solutions
'
CbD/CDF
2,3,7,8-TCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDD
1,2,^,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,13,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
1,2,J3,4,7>HxCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,!t,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDD
1,2,|3,4,6,7,8-HpCDF
1,2,j3,4,7,8,9-HpCDF
OCDD
OCDF
"C12-2,3,7,8-TCDD
"0,2-2,3,7,8-TCDF
"C,2-1,2,3,7,8-PeCDD
"0,2-PeCDF
"0,2-2,3,4,7,8-PeCDF
"C,2-1,2,3,4,7,8-HxCDD
"0,2-1 ,2,3,6,7,8-HxCDD
"0,2-1 ,2,3,4,7,8-HxCDF
"0,2-1 ,2,3,6,7,8-HxCDF
"0,12-1,2,3,7,8,9-HxCDF
"C,!2-1,2,3,4,6,7,8-HpCDD
"C,2-1 ,2,3,4,6,7,8-HpCDF
13C,2-1,2,3,4,7,8,9-HpCDF
"CJj-OCDD
Clebnup Standard
37CI4-2,3,7,8-TCDD
Internal Standards
"C^-l ,2,3,4-TCDD
"C,L-1,2,3(7,8,9-HxCDD
CS1
(ng/mL)
0.5
0.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
5.0
5.0
100
100
100
100
100
100
100
100
100
100
100
100
100
200
0.5
100
100
CS2
(ng/mL)
2
2
10
10
10
10
10
10
10
10
10
10
10
10
10
20
20
100
100
100
100
100
100
100
100
100
100
100
100
100
200
2
100
100
VER1
CS3
(ng/mL)
10
10
50
50
50
50
50
50
50
50
50
50
50
50
50
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
200
10
100
100
CS4
(ng/mL)
40
40
200
200
200
200
200
: 200
200
200
200
200
200
200
200
400
400
100
100
100
: 100
100
; 100
100
100
100
100
100
!• 100
100
200
; 40
i
100
! 100
CSS
(ng/mL)
200
200
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
2000
2000
100
100
100
100
100
100
100
100
100
100
100
100
100
200
200
100
100
1. Section 15.3, calibration verification solution.
581
-------
Table 5: GC Retention Time Window Defining Solution and Isomer Specificity Test
Standard (Section 7.15)
DB-5 Column GC Retention-Time Window Defining Solution
CDD/CDF
TCDF
TCDD
PeCDF
PeCOD
HxCDF
HxCDD
HpCDF
HpCDD
First eluted
1,3,6,8-
1,3,6,8-
1,3,4,6,8-
1,2,4,7.9-
1,2,3,4,6,8-
1,2,4,6,7,9-
1,2,3,4,6,7.8-
1,2,3,4,6,7,9-
Last eluted
1,2,8,9-
1,2,8,9-
1,2,3,8,9-
1,2,3,8,9-
1,2,3,4,8,9-
1,2,3,4,6,7-
1,2,3,4,7,8,9-
1,2,3,4,6,7,8-
DB-5 Column TCDD Specificity Test Standard
1,2,3,7+1,2,3,8-TCDD
2.3.7,8-TCDD
1,2,3.9-TCDD
DB-225 Column TCDF Isomer Specificity Test Standard
2,3,4.7-TCDF
2,3,7,8-TCDF
1,2,3,9-TCDF
-------
Method 1613
Table 6: Acceptance
Tested1
CDP/CDF
2,3,7,8-TCDD
2,3,7,b-TCDF
1,2,3J7,8-PeCDD
1,2,3,k8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,k8,9-HxCDD
1,2,3,4,7,8-HxCDF
1,2,3,6,7,8-HxCDF
1,2,3,17,8,9-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDD
1,2,3,4,6,7,8-HpCDF
1,2,3,'4,7,,8,9-HpCDF
OCDD
OCDF
13C12-2,3,7,8-TCDD
13C12-S,3,7,8-TCDF
'3C12-il,2,3,7,8-PeCDD
l3C12-1,2,3,7,8-PeCDF
13C12-2,3,4,718-PeCDF
13C12-1,2,3,4,7,8-HxCDD
13C12-1,2,3,6,7,8-HxCDD
13C,2-1,2,3,4,7,8-HxCDF
13C,2-1,2,3,6,7,8-HxCDF
13C12jl.2,3,7,8,9-HxCDF
13C12-2,3,4,617,8,-HxCDF
13C12-1,2,3,4,6,7,8-HpCDD
13C12-1,2,3,4,6,7,8-HpCDF
13C12-1,2,3,4l7,8,9-HpCDF
13C12bCDD
37CI«^,3,7,8-TCDD
1. All specifications are given
Criteria for Performance Tests When
Test
I V9H •
cone
(ng/mL)
10
10
50
50
50
50
50
50
50
50
50
50
50
50
50
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
200
10
All CDDs/CDFs
are
.,-
IPR2-3
s
(ng/mL)
2.8
2.0
7.5
7.5
8.6
9.4
7.7
11.1
8.7
6.7
6.4
7.4
7.7
6.3
8.1
19
27
37
35
39
34
38
41
38
43
35
40
37
35
41
40
95
3.6
as concentration in the final extract
X
(ng/mL)
8.3-12.9
8.7-13.7
38-66
43-62
36-75
39-76
42-62
37-71
41-59
46-60
42-61
37-74
38-65
45-56
43-63
89-127
74-146
28-134
31-113
27-184
27-156
16-279
29-147
34-122
27-152
30-122
24-157
29-136
34-129
32-110
28-141
41-276
3.9-15.4
!
OPR
(ng/mL)
6.7-15.8
7.5-15.8
. 35-71
40-67
; 34-80
35-82
38-67
! 32-81
36-67
42-65
39-65
35-78
' 35-70
' 41-61
; 39-69
78-144
63-170
20-175
22-152
| 21-227
! 21-1 92
13-328
21-193
25-163
19-202
21-159
I
* 17-205
'• 22-1 76
i 26-1 66
| 21-158
20-186
. 26-397
'3.1-19.1
VER
(ng/mL)
7.8-12.9
8.4-12.0
39-65
41-60 ;>
41-61
39-64
39-64
41-61
45-56
44-57
45-56
44-57
43-58
45-55
43-58
79-126
63-159
82-121
71-140
62-160
76-130
77-130
85-117
85-118
76-131
70-143
74-135
73-137
72-138
78-129
77-129
96-415
7.9-12.7
assuming a 20-^L volume.
2. is = standard deviation of the concentration
3. X = average concentration.
60
-------
Method 1613
Table 6a. Acceptance Criteria for Performance Tests When Only Tetra Compounds
are Tested1
CDD/CDF
2,3,7,8-TCDD
2,3,7,8-TCDF
"0,2-2,3,7.8-7000
"C12-2,3,7,8-TCDF
"CI4-2,3,7,8-TCDD
1. All specifications are given as concentration in the final extract, assuming a 20-nL volume.
2. s = standard deviation of the concentration
3. X = average concentration
Test
cone
(ng/mL)
10
10
100
100
10
IF
(ng/rnL)
2.7
2.0
35
34
3.4
'FT-
X
(ng/mL)
8.7-12.4
9.1-13.1
32-115
35-99
4.5-13.4
OPR
(ng/mL)
7.3-14.6
8.0-14.7
25-141
26-126
3.7-15.8
VER
(ng/mL)
8.2-12.3
8
6-11.6
85-117
76-131
8.3-12.1
61
-------
Table 7: Labeled Compound Recovery in Samples When All
Compound
13Cl2-2,3,7,8-TCDD ,
)3C,2-2,3,7,8-TCDF
13C,2-1,2,3,7,8-PeCDD
faC«-1,2,3>7,8-PeCDF
'3C,2-2,3,4,7,8-PeCDF
13C«-1,2,3l4,7,8-HxCDO
"0,2-1,2,3,6,7,8,-HxCDD
13Cl2-1,2,3,4,7,8-HxCDF
13C12-1,2,3,6l7,8-HxCDF
'3C,2-1,2,3,7,8l9-HxCDF
13C12-2,3,4,6,7,8,-HxCDF
13C,2-1,2,3,4,6l7,8-HpCDD
'3C,2-1,2,3,4,6,7,8-HpCDF
13C«-1,2,3,4,7I8l9-HpCOF
13C,2-OCDD
37CI4-2,3,7,8-TCDD
Labeled compound
Test cone Recovery
(ng/mL) (ng/mL)1
1.
Soe
100
100
100
100
100
100
100
100
100
100
100
100
100
100
200
10
25-164
24-169
25-181
24-185
21-178
32-141
28-130
26-152
26-123
29-147
28-136
23-140
28-143
26-138
34-313
3.5-19.7
25-164
24-169
25-181
24-185
21-178
32-141
28-130
26-152
26-123
29-147
28-136
23-140
28-143
26-138
17-157
35-197
Specification given as concentration in the final extract, assuming a 20-nL volume.
Compound
13C12-2,3,7,8-TCDD
13C12-2,3,7,8-TCDF
37CI4-2,3,7,8-TCDD
Ra°°V"y '" Samp'eS When Onl* Tetra Compounds
Test cone
Labeled compound
recovery
* 1 ._ .
_ icwx/very
(ng/mL) (ng/mL)1 (%)
I
1. Specification given as concentration in the final extract, assuming a 20-ML volume.
i
i
100
100
10
31-137
29-140
4.2-16.4
31-137
29-140
! 42-164
62
-------
Method 1613
Table 8: Descriptors, Exact
CDDs and CDFs
Descriptor Exact m/z1
1 292.9825
303.9016
305.8987
315.9419
317.9389
319.8965
321.8936
327.8847
330.9792
331.9368
333.9339
375.8364
2 339.8597
341.8567
351.9000
353.8970
354.9792
355.8546
357.8516
367.8949
369.8919
409.7974
3 373.8208
375.8178
383.8639
385.8610
389.8157
391.8127
392.9760
401.8559
403.8529
430.9729
445.7555
m/z's, m/z Types, and Elemental Compositions of the
m/z type
Lock
M
M+2
M
M+2
M
M+2
M
QC
M
M+2
M+2
M+2
M+4
M+2
M+4
Lock
M+2
M+4
M+2
M+4
M+2
M+2
M+4
M
M+2
M+2
M+4
Lock
M+2
M+4
QC
M+4
Elemental composition
CjF,,
CttH^O
C)2H43SCI337CIO
13C,2H4MCI40
'3C,2H4M(V7CIO
CK.H^Q,
C12H435CI337CI02
C,2H437CI402
C7F13
XH.^CI.O,
13C12 H4 "Clj 37CI 02
C,2H435CI537CIO
CnHt*CAttrQO
C12HIS8CI1SIC!20
"C^Hj^CI/'CIO
XHs'VCIjO
C9F13
C^Hj^CI^CIOj
C12H3MCI337CI202
XHa^CI^CIOj
XH.^'CI.Q,
C^Hj^Clg^CIO
C12H235CI537CIO
C^H^^CIjO
XHj^O
"C^HZ^IJ^CIO
C,2H235CIS37CI02
C12H2MCI437CI202
C,F15
"CvU^C^ClOt
»C12H235CI437CI202
C,F17
CuHj^CI.^O
Substance2
PFK
TCDF
TCDF
TCDF3
TCDF3
TCDD
TCDD
TCDD4
PFK
TCDD3
TCDD3
HxCDPE
PeCDF
PeCDF
PeCDF
PeCDF3
PFK
PeCDD
PeCDD
PeCDD3
PeCDD3
HpCDPE
HxCDF
HxCDF
HxCDF3
HxCDF3
HxCDD
HxCDD
PFK
HxCDD3
HxCDD3
PFK
OCDPE
63
-------
* ' 1. '
Method 1613
Table 8: Descriptors, Exact m/z's, m/z
CDDs and CDFs (continued)
Descriptor Exact m/z1 m/z type
4 407.7818 M+2
409.7789 M+4
417.8253 M
419.8220 M+2
; 423.7766 M+2
425.7737 M+4
430.9729 Lock
435.8169 M+2
437.8140 M+4
479.7165 M+4
5 441.7428 M+2
442.9728 Lock
443.7399 M+4
457.7377 M+2
459.7348 M+4
469.7779 M+2
471.7750 M+4
513.6775 M+4
1. Nuclidic masses used:
H = 1.007825 C = 12.00000
0 = 15.994915 *C\ = 34.968853
2. TCDD = Tetrachlorodibenzo-/xlioxin
PeCDD = Pentachlorodibenzo-p-dioxin
HxCDD = Hexachlorodibenzo-p-dioxin
HpCDD = Heptachlorodibenzo-p-dioxin
OCDD = Octachlorodibenzo-pKlioxin
HxCDPE = Hexachlorodiphenyl ether
OCDPE = Octachlorodiphenyl ether
DCDPE = Decachlorodiphenyl ether
Types, and Elemental Compositions of the
Elemental composition Substance2
C18 H "CIs 37CI 0 HpCDF
C12 H MCI5 "CI, 0 HpCDF
XH^O ! HpCDF3
13C12 H ^Clg 37CI 0 HpCDF3
C,2 H "Gig 37CI 02 HpCDD
CttH^^O, HpCDD
C, F17 PFK
"C,2 H "Clg 37CI 02 : HpCDD3
1'C1,Ha8CI537Clj-Of HpCDD3
C,2 H "Clj 37Clj 0 NCDPE
^"C^CIO OCDF
C10F17 : PFK
Cu^Clg^CljO OCDF
C12MCL37CI02 OCDD
Ct^Clg^O, OCDD
^^"CIO, i OCDD3
13C12 ^Cl, "CIj O2 OCDD3
C12 ^Cl, "Clj 0 , DCDPE
1
13C = 13.003355 I F = 18.9984
37CI = 36.965903
TCDF = Tetrachlorodibenzofuran
PeCDF = Pentachlorodibenzofuran
HxCDF = Hexachlorodibenzofuran
HpCDF = Heptachlorodibenzofuran
OCDF = Octachlcfodibenzofuran
HpCDPE = Heptachlorodiphenyl efrier
NCDPE = Nonachlorodiphenyl ether
PFK = Perfluorcikerosene
3. ; Labeled compound
4. j There is only one m/z for 37CI4-2,3,7,8,-TCDD (cleanup standard).
64
-------
Method 1613
Table 9: Theoretical Ion Abundance Ratios and QC Limits
Number of
chlorine atoms
4a
5
6
6J
7
f
8
1. QC limits represent ±15% windows around the theoretical ion abundance ratios.
2. Does not apply to "CI^.S.T.e-TCDD (cleanup standard).
3. Used for "C12-HxCDF only.
4. Used for "C,2-HpCDF only.
m/z's
forming ratio
M/(M+2)
(M+2)/(M+4)
(M+2)/(M+4)
M/(M+2)
(M+2)/(M+4)
M/(M+2)
(M+2)/(M+4)
Theoretical
ratio
0.77
1.55
1.24
0.51
1.05
0.44
0.89
XIV 1
Lower
0.65
1.32
1.05
0.43
0.88
0.37
0.76
IIDII
Upper
0.89
1.78
1.43
0.59
1.20
0.51
1.02
-------
Method 1613
Table 10: Suggested Sample Quantities to be Extracted for Various Matrices1
Sample matrix2
Single-phase
Aqueous
Solid
i
j
l
Organic
Tissue
Multi-phase
Liquid/Solid
Aqueous/Solid
Organic/solid
i
Liquid/Liquid
Aqueous/organic
Aqilieous/organic/solid
Example
Drinking water
Groundwater
Treated wastewater
Dry soil
Compost
Ash
Waste solvent
Waste oil
Organic polymer
Fish
Human adipose
Wet soil
Untreated effluent
Digested municipal sludge
Filter cake
Paper pulp
Industrial sludge
Oily waste
In-process effluent
Untreated effluent
Drum waste
Untreated effluent
Drum waste
Percent
solids
Phase
>20
Solid
Organic
Organic
1-30
Solid
1-100
>1
Both
Organic
Organic & solid
Quantity
extracted
1000 ml
10g
10g
10 g
10 g
10g
10 g
10 g
1. i The quantity of sample to be extracted is adjusted to provide 10 g of solids (dry weight). One liter of aqueous samples
i 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.
2. : The sample matrix may be amorphous for some samples. In general, when the CDDs/CDFs are in contact with a
| multiphase 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
j are extracted separately, and the extracts are combined for cleanup and analysis.
66\
-------
Method 1613
Determine % solids
§11.2
Determine particle size
§11.3
Prep per §11,5
WillSPE
Visible
<
/Par
'size>
\Hrom
tele \ No
1mm?>— -,
§11. Y
Yes
>
Grind per §11. 7
SDS extraction
per §12.3
Concentrate
per §12,6.1 or
§12.6.2'
l
\ be used? /
JYes
SPE extraction per §12.2
i
SDSextracfon
ofSPEdisk
per §12.3
>
Concentrate
per §12.6.1 or
§12.6.2*
N. particles? /
jMfes
Filter per §11. 4.3
1
>
AND
i
SDS extraction
of filter per
§12.3
\
Concentrate
per §12.6.1 or
§12.6.2'
1
Transfer
through
sodium sutfate
Sep. funnel
extraction of
filtrate per §12.1
i
Sep. funnel
extraction per
§12.1
Concentrate per
§12.6.1, §12.6.2
or §12.6.3
1
Mix sep. funnel
extract & SDS
extract together
per §12.3.9.1 .2
\
Back-c
per§
sxtract
12.5
Concentrate per
§12.6.1, §12.62
or §12.6.3
' The K-D concentration procedure in §12.6.3 can be
used if the water bath is fed by a steam generator.
Rgure 1. Flow Chart for Analysis of Aqueous and Solid Samples
52-028-1A
67
-------
Method 1613 .
Aqueous
Discard
Determine %
solids per §11,2
Determine particle size
per§11.3
Pressure filter
aliquot per §11.6.2
Macro-concentrate per
§12.6.1- §12.6.2*
Reserve 10g
or maximum
amount from 1 -L
sample,
whichever is less
Particle \ yes
size < 1mm?
* The' K-D concentration procedure in §12 6 3
can|be used if the water bath is fed by a steam
generator.
Concentrate
per §12.6-§12.7
Clean up per
§13.2-§13.6, §13.8
Reconcentrate
per §12.6-§12.7
Back-extract per §12.5
Transfer thru
Analysis
per§14-§i8
Figure 2. Flow Chart for Analysis of Multi-Phase Samples
52-028-2A
-------
* I-
Method 1613
Homogenize
tissue per §11.8
Remove 10g
Soxhlel Extraction
OR
Mix Na2S04
Extract per §12.4.1
Decant thru
Na2S04
Concentrate to dryness
per §12.6-§12.7
Determine % liptds per
§12.4.1.9
Column Cleanup |
Batch Cleanup
Remove Kpids
per §13.7.1
OR
_L
Redissolve and spike
cleanup std
Concentrate per
§12.6-§12.7
Remove liptds per
§13.72
Redissolve and spke
cleanup std
Transfer thai
Concentrate per
§12.6-§12.7
Ctean-up per §13.2-
§13.6, §13.8
Concentrate per
§12.6-§12.7
Analyze per §14-§18
HC! Digestion
HCI digest per §12.4.2
Decant thru Na £0t
Macro-concentrate
per §12.6
Micro-concentrate
to dryness per §12.7
Determine % lipids
per §12.4.2.8
Redissolve and spike
cleanup std
Back-extract with
H2S04 per§13.7.3
Back-extract per
§12.5.2
Transfer thru Na2S04
Concentrate per
§12.6-§12.7
52-028-3A
Figure 3. Flow Chart for Analysis of Tissue Samples
-------
Method 1613
70
90-mmGMF 150 Filter
Figure 4. Solid-Phase Extraction Apparatus
52-027-1A
-------
Method 1613
52-027-2A
Figure 5. Soxhlet/Dean-Stark Extractor
71
-------
(QIJ
3B DBS Column
100n
80
60
40-
20-
24:00
25:30
Retention Time (minutes)
27:00
Figure 6. Isomer-Specific Separation of 2,3,7,8?TCDD on DB-5 Column
52-027-03
72
-------
Method 1613
6-May-88 Sir: Voltage 705 Sys: DB5US
Sample 11njection 1 Group 1 Mass 305.8987
Text: Column Performance
100-n
80-
60-
40-
20-
0-L-T
2,3,4,8-TCDF
Norm: 3466
1,2,3,9-TCDF
16:10 16:20 16:30 16:40 16:50 17:00 17:10 17:20 17:30 17:40 17:40 18:00
Retention Time (minutes)
Figure 7. Isomer-Specific Separation of 2,3,7,8-TCDF on DB-5 Column
52-027-4A
73
-------
Method 1613
24.0 Glossary of Definitions and Purposes
These definitions and purposes are specific to this method but have been conformed to
common usage as much as possible.
!
24.1 |Units of weight and measure and their abbreviations
24J1.1 Symbols
i°C degrees Celsius
microliter
t
L
.m
24.h.2
micrometer
less than
greater than
percent
Alphabetical abbreviations
ampere
centimeter ;
gram
hour
inside diameter
inch
liter
Molecular ion ,
meter
milligram
minute
milliliter
millimeter !
mass-to-charge ratio
normal; gram molecular weight of solute divided by hydrogen equivalent of solute,
per liter of solution
outside diameter
picogram
part-per-billion
part-per-million
part-per-quadrillion
part-per-trillion
pounds-per-square inch gauge
volume per unit volume
weight per unit volume
24.2 Definitions and acronyms (in alphabetical order).
i
Analyte—A CDD or CDF tested for by this method. The analytes are listed in Table 1.
I
Calibration standard (CAL)—A solution prepared from a secondary standard and/or stock
solutions and used to calibrate the response of the instrument with respect to analyte
concentration. ;
;m
3
i
D
n.
M
Tl
Tig
nin
mL
mm
(n/z
N
|DD
pg
Ppb
ppm
jjpq
ppt
psig
y/v
j/v/v
74
-------
Method 1613
Calibration verification standard (VER)—The mid-point calibration standard (CS3) that is
used in to verify calibration. See Table 4.
ODD—Chlorinated dibenzo-p-dioxin. The isomers and congeners of tetra- through octa-
chlorodibenzo-p-dioxin.
CDF—Chlorinated dibenzofuran. The isomers and congeners of tetra- through octa-
chlorodibenzofuran.
CS1, CS2, CSS, CS4, CSS—See Calibration standards and Table 4.
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.
HPLC—High performance liquid chromatograph or high performance liquid chromatography.
HRGC—High resolution GC.
HRMS—High resolution MS.
t
IPR—initial precision and recovery; four aliquots of the diluted PAR standard analyzed to
establish the ability to generate acceptable precision and accuracy. An IPR is performed
prior to the first time this method is used and any time the method or instrumentation is
modified.
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.
75
-------
Method 1613
Minimum level (ML) — The level at which the entire analytical system must give a
recognizable signal and acceptable calibration point for the analyte. It is equivalent to the
concentration of the lowest calibration standard, assuming that all method-specified sample
weights, volumes, and cleanup procedures have been employed. :
i
MS — Mass spectrometer or mass spectrometry.
i
i
Must— This action, activity, or procedural step is required.
-------
Method 1613
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.
TCDD—Tetrachlorodibenzo-p-dioxin.
TCDF—Tetrachlorodibenzofuran.
VER—See Calibration verification standard.
77
------- |