oEPA
United States Environmental Protection Agency
Office of Water Regulations and Standards
Industrial Technology Division
Office of Water April 1990
Method 1613: Tetra- through
Octa- Chlorinated Dioxins and
Furans by Isotope Dilution
HRGC/HRMS
Revision A
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Introduction
Method 1613 was developed by the Industrial Technology
Division CITD) within the United States Environmental
Protection Agency's (USEPA) Office of Water Regulations and
Standards (OWRS) to provide improved precision and accuracy of
analysis of pollutants in aqueous and solid matrices. The ITD
is responsible for development and promulgation of nationwide
effluent limitation guidelines for pollutant levels in
industrial and municipal discharges.
Method 1613 is a high resolution capillary column gas
chromatography (HRGC)/high resolution mass spectrometry (HRMS)
method for analysis of tetra- through octa- chlorinated
dibenzo-p-dioxins and dibenzofurans using isotope dilution.
Specificity is provided for determination of the seventeen
2,3,7,8-substituted polychorinated dibenzo-p-dioxins (PCDO)
and polychlorinated dibenzofurans (PCDF).
Questions concerning the method or its application should be
addressed to:
U. A. Telliard, Chief
Analytical Methods Staff (UH-552)
USEPA Office of Water Regulations and Standards
401 M Street, S.W.
Washington, DC 20460
202/382-7120
OR
USEPA OWRS
Sample Control Center
P.O. Box 1407
Alexandria, Virginia 22313
703/557-5040
Revision A Publlcat ion Date April 1990; Updated - June 1990
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Method 1613 Revision A April 1990
Tetra- through Octa- Chlorinated Dioxins and Furans
by Isotope Dilution HRGC/HRMS
1 SCOPE AND APPLICATION
1.1 This method is designed to meet the survey
requirements of the USEPA ITD. The method
is used to determine the tetra- through
octa- chlorinated dibenzo-p-dioxins and
dibenzofurans associated with the Clean
Water Act (as amended 1987); the Resource
Conservation and Recovery Act (as amended
1986); and the Comprehensive Environmental
Response, Compensation and Liability Act
(as amended 1986); and other dioxin and
furan compounds amenable to high resolu-
tion capillary colum gas chromatography
(HRGC)/high resolution mass spectrometry
(HRMS). Specificity is provided for de-
termination of the 17 2,3,7,8-substituted
polychlorinated dibenzo-p-dioxins (PCDD)
and polychlorinated dibenzofurans (PCDF).
1.2 The method is based on EPA, industry, com-
mercial laboratory, and academic methods
(References 1-6).
1.3 The compounds listed in Table 1 may be
determined in waters, soils, sludges, and
other matrices by this method.
1.4 The detection limits of the method are
usually dependent on the level of inter-
ferences rather than instrumental limita-
tions. The levels in Table 2 typify the
minimum quantities that can be determined
in environmental samples using the method.
1.5 The GCMS portions of the method are for
use only by analysts experienced with
HRGC/HRMS or under the close supervision
of such qualified persons. Each labora-
tory that uses this method must demon-
strate the ability to generate acceptable
results using the procedure in Section
8.2.
2 SUMMARY OF METHOD
2.1 Stable isotopically labeled analogs of 15
of the PCDDs and PCDFs are added to each
sample prior to extraction. Samples con-
taining coarse solids are prepared for
extraction by grinding or homogenization.
Water samples are filtered and then
extracted with methylene chloride using
separatory funnel procedures; the particu-
lates from the water samples, soils, and
other finely divided solids are extracted
using a combined Soxhlet extraction/Oean-
Stark azeotropic distillation (Reference
7). Prior to cleanup and analysis, the
extracts of the filtered water and the
particulates are combined.
2.2 After extraction, '^Cl^-labeled 2,3,7,8-
TCDD is added to each extract to measure
the efficiency of the cleanup process.
Seniles cleanup may include back extrac-
tion with acid and/or base, and gel perme-
ation, alumina, silica gel, and activated
carbon chromatography. High performance
liquid chromatography (HPLC) can be used
for further isolation of the 2,3,7,8-
i sorters or other specific isomers or
congeners.
2.3 After cleanup, the extract is concentrated
to near dryness. Immediately prior to
injection, two internal standards are
added to each extract, and a 1 uL aliquot
of the extract is injected into the gas
chromatograph. The analytes are separated
by the GC and detected by a high resolu-
tion (>10,000) mass spectrometer. Two
exact masses (m/z's) are monitored for
each analyte. The isotopically labeled
compounds serve to correct for the
variability of the analytical technique.
2.4 Dioxins and furans are identified by
comparing GC retention times and the ion
abundance ratios of the m/z's with the
corresponding retention time ranges of
authentic standards and the theoretical
ion abundance ratios of the exact m/z's.
Isomers and congeners are identified when
the retention times and m/z abundance
ratios agree within pre-defined limits.
By using a GC column or coliBnns capable of
resolving the 2,3,7,8-substituted isomers
from all other tetra- isomers, the
2,3,7,8-substituted isomers are identified
when the retention time and m/z abundance
ratios agree within pre-defined limits of
the retention times and exact m/z ratios
of authentic standards.
2.5 Quantitative analysis is performed by GCMS
using selected ion current profile (SICP)
areas, in one of two ways.
2.5.1 For the 15 2,3,7,8-substituted isomers for
which labeled analogs are available (see
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Table 1), the GCMS system ts calibrated
and the compound concentration is deter-
mined using an isotope dilution technique.
Although a labeled analog of the octa-
chlorinated dibenzofuran (OCDF) is avail-
able, using high resolution mass spectrom-
etry it produces an m/z that may interfere
with the identification and quantitation
of the unlabeled octachlorinated dibenzo-
p-dioxin (OCOD). Therefore, this labeled
analog has not been included in the cali-
bration standards, and the unlabeled OCDF
is quantitated against the labeled OCDO.
Because the labeled analog of 1,2,3,7,8,9-
HxCDD is used as an internal standard
(i.e., not added before extraction of the
sample), it cannot be used to quantitate
the unlabeled compound by strict isotope
dilution procedures. Therefore, the unla-
beled 1,2,3,7,8,9-HxCDD is quantitated
using the average of the responses of the
labeled analogs of the other two 2,3,7,8-
substituted HxCOD's, 1,2,3,4,7,8-HxCDD and
1,2,3,6,7,8-HxCDD. As a result, the con-
centration of the unlabeled 1,2,3,7,8,9-
HxCDD is corrected for the average
recovery of the other two HxCDD's.
2.5.2 For non-2,3,7,8-substituted isomers and
the total concentrations of all isomers
within a level of chlorination (i.e.,
total TCDD), concentrations are determined
using response factors from the calibra-
tion of labeled analogs at the same level
of chlorination.
2.6 The quality of the analysis is assured
through reproducible calibration and test-
ing of the extraction, cleanup, and GCMS
systems.
3 CONTAMINATION AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other
sample processing hardware may yield arti-
facts and/or elevated baselines causing
misinterpretation of chromatograms (Ref-
erences 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.
3.2 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.
3.2.1 Glassware should be rinsed with solvent
and washed with a detergent solution as
soon after use as is practical. Sonica-
tion of glassware containing a detergent
solution for approximately 30 seconds may
aid in cleaning. Glassware with removable
parts, particularly separatory funnels
with teflon stopcocks, must be
disassembled prior to detergent washing.
3.2.2 After detergent washing, glassware should
be immediately rinsed first with methanol,
then with hot tap water. The tap water
rinse is followed by another methanol
rinse, then acetone, and then methylene
chloride.
3.2.3 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
PCDDs/PCDFs.
3.2.4 Immediately prior to use, Soxhlet extrac-
tion glassware should be pre-extracted
with toluene for approximately 3 hours.
See Section 11.1.2.3. Separatory 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.
3.3 All materials used in the analysis shall
be demonstrated to be free from interfer-
ences by running reference matrix blanks
initially and with each sample set
(samples started through the extraction
process on a given 12-hour shift, to a
maximum of 20 samples). The reference
matrix blank must simulate, as closely as
possible, the sample matrix under test.
Reagent water (Section 6.6.1) is used to
simulate water samples; playground sand
(Section 6.6.2) or white quartz sand
(Section 6.3.2) can be used to simulate
soils; filter paper (Section 6.6.3) is
used to simulate papers and similar
materials; other materials (Section 6.6.4)
can be used to simulate other matrices.
3.4 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 PCDDs
and PCDFs. The most frequently encoun-
tered interferences are chlorinated-
biphenyls, methoxy biphenyls, hydroxy-
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diphenyl ethers, benzylphenyl ethers,
polynuclear aromatics, and pesticides.
Because very low levels of PCDDs and PCDFs
are measured by this method, the elimina-
tion of interferences is essential. The
cleanup steps given in Section 12 can be
used to reduce or eliminate these inter-
ferences and thereby permit reliable
determination of the PCDDs and PCDFs at
the levels shown in Table 2.
3.5 Each piece of reusable glassware should be
numbered in such a fashion that the
laboratory can associate all reusable
glassware with the processing of a parti-
cular sample. This will assist the
laboratory in: 1) tracking down possible
sources of contamination for individual
samples, 2) identifying glassware assoc-
iated with highly contaminated samples
that may require extra cleaning, and 3-)
determining when glassware should be
discarded.
4 SAFETY
4.1 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 conpounds should be
reduced to the lowest possible level.
4.1.1 The 2,3,7,8-TCDD isomer has been found to
be acnegenic, carcinogenic, and terato-
genic in laboratory animal studies. It is
soluble in water to approximately 200 ppt
and in organic solvents to 0.14 percent.
On the basis of the available
toxicological and physical properties of
2,3,7,8-TCDD, all of the PCDDs and PCDFs
should be handled only by highly trained
personnel thoroughly familiar with
handling and cautionary procedures, and
who understand the associated risks.
4.1.2 It is recommended that the laboratory pur-
chase 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/HESA
approved toxic gas respirator shall be
worn when high concentrations are handled.
4.2 The laboratory is responsible for main-
taining a current awareness file of OSHA
regulations regarding the safe handling of
the chemicals specified in this method. A
reference file of data handling sheets
should also be made available to all
personnel involved in these analyses.
Additional information on laboratory
safety can be found in References 10-13.
The references and bibliography at the end
of Reference 13 are particularly compre-
hensive in dealing with the general
subject of laboratory safety.
4.3 The PCDDs and PCDFs and samples suspected
to contain these compounds are handled
using essentially the same techniques
employed in handling radioactive or
infectious materials. Uell-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 PCDDs and PCDFs are extremely toxic to
laboratory animals. Each laboratory must
develop a strict safety program for
handling the PCDDs and PCDFs. The follow-
ing practices are recornnended (References
2 and 14).
4.3.1 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 demon-
strated to have adequate air flow. Gross
losses to the laboratory ventilation
system must not be allowed. Handling of
the dilute solutions normally used in
analytical and animal work presents no
inhalation hazards except in the case of
an accident.
4.3.2 Protective equipment -- Throwaway plastic
gloves, apron or lab coat, safety glasses
or mask, and a glove box or fume hood
adequate for radioactive work should be
utilized. During analytical operations
which 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 PCDDs
or PCDFs, an additional set of gloves can
also be worn beneath the latex gloves.
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4.3.3 Training -- Workers must be trained in the
proper method of removing contaminated
gloves and clothing without contacting the
exterior surfaces.
4.3.4 Personal hygiene -- Thorough washing of
hands and forearms after each manipulation
and before breaks (coffee, lunch, and
shift).
4.3.5 Confinement -- Isolated work area, posted
with signs, segregated glassware and
tools, plastic absorbent paper on bench
tops.
4.3.6 Effluent vapors -- The effluents of sample
splitters for the gas chromatograph and
roughing pumps on the GC/HS should pass
through either a column of activated char-
coal or be bubbled through a trap contain-
ing oil or high-boiling alcohols.
4.3.7 waste Handling and Disposal
4 3.7.1 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.
4.3.7.2 Disposal
4.3.7.2.1 The PCDDs and PCDFs decompose above 800
°C. Low-level waste such as absorbent
paper, tissues, animal remains, and
plastic gloves may be burned in an appro-
priate incinerator. Gross quantities
(milligrams) should be packaged securely
and disposed through commercial or govern-
mental channels which are capable of
handling extremely toxic wastes.
4.3.7.2.2 Liquid or soluble waste should be dis-
solved in methanol or ethanol and
irradiated with ultraviolet light with a
wavelength greater than 290 run for several
days. (Use F 40 BL lamps or equivalent.)
Analyze liquid wastes and dispose of the
solutions when the PCDDs and PCDFs can no
longer be detected.
4.3.8 Decontamination
4.3.8.1
4.3.8.2
Personal decontamination -- Use any
soap with plenty of scrubbing action.
mi Id
Glassware, tools, and surfaces
Chlorothene NU Solvent (Trademark of the
Dow Chemical Company) is the least toxic
solvent shown to be effective. Satis-
factory cleaning may be accomplished by
4.3.9
4.3.10
4.3.11
5.1
rinsing with Chlorothene, then washing
with any detergent and water. If glass-
ware 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.
Laundry -- Clothing known to be contami-
nated 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 determin-
ing cleanliness of work surfaces and tools
is to wipe -the surface with a piece of
filter paper. Extraction and analysis by
GC can achieve a limit of detection of 0.1
ug per wipe. Less than 0.1 ug per wipe
indicates acceptable cleanliness; anything
higher warrants further cleaning. 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.
Accidents -- Remove contaminated clothing
immediately, taking precautions not to
contaminate skin or other articles. Wash
exposed skin vigorously and repeatedly
until medical attention is obtained.
APPARATUS AND MATERIALS
Sampling equipment
composite sampling.
for discrete or
5.1.1 Sample bottles and caps
5.1.1.1 Liquid samples (waters, sludges and simi-
lar materials containing five percent
solids or less) -- Sample bottle, amber
glass, 1.1 L minimum, with screw cap.
5.1.1.2 Solid samples (soils, sediments, sludges,
paper pulps, filter cake, compost, and
similar materials that contain more than
five percent solids) -- Sample bottle,
wide mouth, amber glass, 500 mL minimum.
5.1.1.3 If amber bottles are not available,
samples shall be protected from light.
5.1.1.4 Bottle caps -- Threaded to fit sample
bottles. Caps shall be lined with Teflon.
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5.1.1.5 Cleaning
5.1.1.5.1 Bottles are detergent water washed, then
solvent rinsed before use.
5.1.1.5.2 Liners are detergent water washed, then
rinsed with reagent water (Section 6.6.1)
and then solvent, and baked at
approximately 200 °C for one hour mininun
prior to use.
5.1.2 Compositing equipment -- Automatic or
manual compositing system incorporating
glass containers cleaned per bottle
cleaning procedure above. Glass or Teflon
tubing only shall be used. If the sampler
uses a peristaltic punp, a mini nun length
of compressible silicone rubber tubing may
be used in the purp only. Before use, the
tubing shall be thoroughly rinsed with
methanol, followed by repeated rinsings
with reagent water to minimize sample
contamination. An integrating flow meter
is used to collect proportional composite
samples.
5.2 Equipment for glassware cleaning
5.2.1 Laboratory sink with overhead fume hood
5.3 Equipment for sample preparation
5.3.1 Laboratory fume hood of sufficient size to
contain the sample preparation equipment
listed below
5.3.2 Glove box (optional)
5.3.3 Tissue homogenizer -- VirTis Model 45
Macro homogenizer (American Scientific
Products H-3515, or equivalent) with
stainless steel Macro-shaft and Turbo-
shear blade.
5.3.4 Meat grinder -- Hobart, or equivalent,
with 3-5 mm holes in inner plate.
5.3.5 Equipment for determining percent moisture
5.3.5.1 Oven, capable of maintaining a temperature
of 110 ±5 °C.
5.3.5.2 Oessicator
5.3.6 Balances
5.3.6.1 Analytical -- Capable of weighing 0.1 mg.
5.3.6.2 Top loading -- Capable of weighing 10 mg.
5.4 Extraction apparatus
5.4.1 Water samples
5.4.1.1 pH meter, with combination glass
electrode.
5.4.1.2 pH paper, wide range (Hydrion Papers, or
equivalent).
5.4.1.3 Graduated cylinder, 1 L capacity
5.4.1.4 1 L filtration flasks with side arm, for
use in vacuun filtration of water samples.
5.4.1.5 Separatory funnels -- 250, 500, and 2000
mL, with Teflon stop cocks.
5.4.2 Soxhlet/Dean-Stark (SDS) extractor
(Figure 1)
P
FIGURE 1 Soxhlet/Dean-Stark Extractor
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5.4.2.1 Soxhlet -- 50 mm i.d., 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).
5.4.2.2 Thimble -- 43 x 123 to fit Soxhlet (Cal-
Glass LG-6901-122, or equivalent).
5.4.2.3 Moisture trap -- Dean Stark or Barret with
Teflon stopcock, to fit Soxhlet.
5.4.2.4 Heating mantle -- Hemispherical, to fit
500 mL round bottom flask (Cal-Glass LG-
8801-112, or equivalent).
5.4.2.5 Variable transformer -- Powerstat (or
equivalent), 110 volt, 10 amp.
5.4.3 Beakers, 400-500 mL
5.4.4 Spatulas -- Stainless steel
5.5 Filtration apparatus
5.5.1 Pyrex glass wool -- Solvent extracted by
SOS for three hours imnirmjn. (NOTE:
Baking glass wool may cause active sites
that will irreversibly adsorb
PCDDs/PCDFs.)
5.5.2 Glass funnel -- 125-250 mL
5.5.3 Glass fiber filter paper (Whatman GF/0, or
equivalent)
5.5.4 Drying column -- 15 to 20 nm i.d. Pyrex
chromatographic column equipped with
coarse glass frit or glass wool plug.
5.5.5 Buchner funnel, 15 cm.
5.5.6 Glass fiber filter paper for above.
5.5.7 Pressure filtration apparatus -- Millipore
YT30 142 HU, or equivalent.
5.6 Centrifuge apparatus
5.6.1 Centrifuge -- Capable of rotating 500 mL
centrifuge bottles or 15 mL centrifuge
tubes at 5,000 rpm minimum
5.6.2 Centrifuge bottles -- 500 mL, with screw
caps, to fit centrifuge
5.6.3 Centrifuge tubes -- 12-15 mL, with screw
caps, to fit centrifuge
5.7
5.7.1
5.7.1.1
5.7.1.2
5.7.1.3
5.7.1.4
5.7.2
5.7.2.1
5.7.2.2
5.7.2.3
5.7.2.4
5.7.3
5.7.3.1
5.7.3.2
5.7.4
5.7.4.1
Cleanup apparatus
Automated gel permeation chromatograph
(Analytical Biochemical Labs, Inc,
Columbia, MO, Model GPC Autoprep 1002, or
equivalent).
Column -- 600-700 mm x 25 mm i.d., packed
with 70 g of SX-3 Bio-beads (Bio-Rad
Laboratories, Richmond, CA, or
equivalent).
Syringe, 10 mL, with Luer fitting.
Syringe filter holder, stainless steel,
and glass fiber or Teflon filters (Gelman
4310, or equivalent).
UV detectors -- 254-nm, preparative or
semi-prep flow cell: (Isco, Inc., Type 6;
Schmadzu, 5 mm path length; Beckman-Altex
152V, 8 uL micro-prep flow cell, 2 inn
path; Pharmacia UV-1, 3 mm flow cell; LDC
Milton-Roy UV-3, monitor #1203; or
equivalent).
Reverse phase high
chromatograph
performance liquid
Column oven and detector -- Perkin-Elmer
Model LC-65T (or equivalent) operated at
0.02 AUFS at 235 ran.
Injector -- Rheodyne 7120 (or equivalent)
with 50 uL sample loop.
Colunn -- Two 6.2 x 250 mm Zorbax-OOS
colums in series (DuPont Instruments
Division, Wilmington, DE, or equivalent),
operated at 50 °C with 2.0 mL/min methanol
isocratic effluent.
Pimp -- Altex 110A (or equivalent).
Pi pets
Disposable, Pasteur, 150 mm x 5 mm i.d.
(Fisher Scientific 13-678-6A, or
equivalent).
Disposable, serological, 10 mL (6 nm
1.d.).
Chromatographic colunns
150 nm x 8 mm i.d., (Kontes <-420155, or
equivalent) with coarse glass frit or
glass wool plug and 250 mL reservoir.
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5.7.4.2 200 mm x 15 mm i.d., with coarse glass
frit or glass wool plug and 250 ml
reservoir.
5.7.5 Oven -- For storage of adsorbents, capable
of maintaining a temperature of 130 ±5 °C.
5.8 Concentration apparatus
5.8.1 Rotary evaporator -- Buchi/Brinkman-
American Scientific No. E5045-10 or
equivalent, equipped with a variable
temperature water bath.
5.8.1.1 A vacuum source is required for use of the
rotary evaporator. It must be equipped
with a shutoff valve at the evaporator,
and preferably, have a vacuum gauge.
5.8.1.2 A recirculating water punp 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.
5.8.1.3 Round bottom flasks -- 100 mL and 500 mL
or larger, with ground glass fitting
compatible with the rotary evaporator.
5.8.2 Kuderna-Danish (K-D)
5.8.2.1 Concentrator tube--10mL, graduated (Kontes
K-570050-1025, or equivalent) with
calibration verified. Ground glass
stopper (size 19/22 joint) is used to
prevent evaporation of extracts.
5.8.2.2 Evaporation flask--500 mL (Kontes K-
570001-0500, or equivalent), attached to
concentrator tube with springs (Kontes K-
662750-0012).
5.8.2.3 Snyder column--three ball macro (Kontes K-
503000-0232, or equivalent).
5.8.2.4 Boiling chips
5.8.2.4.1 Glass or silicon carbide--approx 10/40
mesh, extracted with methylene chloride
and baked at 450 °C for one h minimum.
5.8.2.4.2 Teflon (optional)--extracted with
methylene chloride.
5.8.2.5 Water bath--heated, with concentric ring
cover, capable of maintaining a
temperature within +/- 2 °C, installed in
a fume hood.
5.8.3 Nitrogen blowdown apparatus -- Equipped
with water bath controlled at 35-40 °C (N-
Evap, Organomation Associates, Inc., South
Berlin, HA, or equivalent), installed in a
fune hood.
5.8.4 Sample vials -- Amber glass, 2-5 mL with
Teflon-lined screw cap.
5.9 Gas chromatograph -- Shall have splitless
or on-colunn injection port for capillary
column, temperature program with
isothermal hold, and shall meet all of the
performance specifications in Section 7.
5.9.1 GC Column for PCDDs and PCDFs and for
isomer specificity for 2,3,7,8-TCDD -- 60
±5 m x 0.32 ±0.02 mm i.d.; 0.25 un 5%
phenyl, 94X methyl, 1% vinyl silicone
bonded phase fused silica capillary column
(J & U DB-5, or equivalent).
5.9.2 GC Column for isomer specificity for
2,3,7,8-TCOF -- 30 ±5 m x 0.32 ±0.02 ran
i.d.; 0.25 urn bonded phase fused silica
capillary column (J & U DB-225, or
equivalent).
5.10 Mass spectrometer -- 28-40 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 7.
5.11 GCHS interface -- The mass spectrometer
(MS) shall be interfaced to the GC such
that the end of the capillary column
terminates within 1 cm of the ion source
but does not intercept the electron or ion
beams.
5.12 Data system -- Capable of collecting,
recording and storing MS data.
6 REAGENTS AND STANDARDS
6.1 pH adjustment and back extraction
6.1.1 Potassium hydroxide -- Dissolve 20 g
reagent grade KOH in 100 mL reagent water.
6.1.2 Sulfuric acid -- Reagent grade (specific
gravity 1.84).
6.1.3 Sodium chloride -- Reagent grade, prepare
a five percent (w/v) solution in reagent
water.
7
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6.2 Solution drying and evaporation
6.2.1 Solution drying -- Sodinn sulfate, reagent
grade, granular anhydrous (Baker 3375, or
equivalent), rinsed with methylene
chloride (20 mL/g), baked at 400 °C for
one hour mininun, cooled in a dessicator,
and stored in a pre-cleaned glass bottle
with screw cap that prevents moisture from
entering. If after heating the sodium
sulfate develops a noticeable grayish cast
(due to the presence of carbon in the
crystal matrix), that batch of reagent is
not suitable for use and should be
discarded. Extraction with methylene
chloride (as opposed to simple rinsing)
and baking at a lower temperature may
produce sodium sulfate that is suitable
for use.
6.2.2 Prepurified nitrogen
6.3 Extraction
6.3.1 Solvents -- Acetone, toluene, cyclohexane,
hexane, nonane, methanol, methylene
chloride, and nonane: distiI led-in-glass,
pesticide quality, lot certified to be
free of interferences.
6.3.2 White quartz sand, 60/70 mesh -- For
Soxhlet/Dean-Stark extraction, (Aldrich
Chemical Co, Milwaukee WI Cat No.
27,437-9, or equivalent). Bake at 450 °C
for four hours minimum.
6.4 GPC calibration solution -- Solution
containing 300 mg/ml corn oil, 15 mg/mL
bis(2-ethylhexyl) phthalate, 1.4 mg/ml
pentachlorophenol, 0.1 mg/mL perylene, and
0.5 mg/mL sulfur
6.5 Adsorbents for sample cleanup
6.5.1 Silica gel
6.5.1.1 Activated silica gel -- Bio-Sil A, 100-200
mesh (Bio-Rad 131-1340, or equivalent),
rinsed with methylene chloride, baked at
180 °C for one hour minimum, cooled in a
dessicator, and stored in a pre-cleaned
glass bottle with screw cap that prevents
moisture from entering.
6.5.1.2 Acid silica gel (30 percent w/w)
Thoroughly mix 44.0 g of concentrated
sulfuric acid with 100.0 g of activated
silica gel in a clean container. Break up
aggregates with a stirring rod until a
uniform mixture is obtained. Store in a
screw-capped bottle with Teflon-lined cap.
6.5.1.3 Basic silica gel -- Thoroughly mix 30 g of
1N sodium hydroxide with 100 g of
activated silica gel in a clean container.
Break up aggregates with a stirring rod
until a uniform mixture is obtained.
Store in a screw-capped bottle with
Teflon-lined cap.
6.5.2 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 8.3. The
same type of alumina must be used for all
samples, including those used to
demonstrate initial precision and accuracy
(Section 8.2) and ongoing precision and
accuracy (Section 14.5).
6.5.2.1 Acid alumina -- Bio-Rad Laboratories 132-
1340 Acid Alumina AG 4 (or equivalent).
Activate by heating to 130 °C for 12 hours
mininun.
6.5.2.2 Basic alumina -- Bio-Rad Laboratories 132-
1240 Basic Alumina AG 10 (or equivalent).
Activate by heating to 600 °C for 24 hours
mininun. Alternatively, activate by
heating alumina in a tube furnace at 650-
700 °C under an air flow of approximately
400 cc/min. 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.
6.5.3 AX-21/Celite
6.5.3.1 Activated carbon -- AX-21 (Anderson
Development Company, Adrian, HI, or
equivalent). Prewash with methanol and
dry in vacuo at 110 °C.
6.5.3.2
6.5.3.3
Celite 545
equivalent).
(Supelco 2-0199, or
6.6
6.6.1
Thoroughly mix 5.35 g AX-21 and 62.0 g
Celite 545 to produce a 7.9X w/w mixture.
Activate the mixture at 130 °C for six
hours minimum. Store in a dessicator.
Reference matrices
Reagent water -- Water in which the PCDDs
and PCDFs and interfering compounds are
not detected by this method.
8
-------
6.6.2 High solids reference matrix -- Playground
sand or similar material in which the
PCDDs and PCDFs and interfering compounds
are not detected by this method. May be
prepared by extraction with methylene
chloride and/or baking at 450 °C for four
hours minimum.
6.6.3 Filter paper -- Gel man type A (or equiva-
lent) glass fiber filter paper in which
the PCDDs and PCDFs and interfering com-
pounds are not detected by this method.
Cut the paper to simulate the surface area
of the paper sample being tested.
6.6.4 Other matrices -- This method may be
verified on any matrix by performing the
tests given in Section 8.2. Ideally, the
matrix should be free of the PCDDs and
PCDFs, but in no case shall the background
level of the PCDDs and PCDFs in the
reference matrix exceed three times the
minimum levels given in Table 2. If low
background levels of the PCDDs and PCDFs
are present in the reference matrix, the
spike level of the analytes used in
Section 8.2 should be increased to provide
a spike-to-background ratio in the range
of 1/1 to 5/1 (Reference 15).
6.7 Standard solutions -- Purchased as solu-
tions or mixtures with certification to
their purity, concentration, and authen-
ticity, or prepared from materials of
known purity and composition. If compound
purity is 98 percent 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 Teflon-lined caps. A
mark is placed on the vial at the level of
the solution so that solvent evaporation
loss can be detected. If solvent loss has
occurred, the solution should be replaced.
6.8 Stock solutions
6.8.1 Preparation -- Prepare in nonane per the
steps below or purchase as dilute solu-
tions (Cambridge Isotope Laboratories,
Cambridge, HA, or equivalent). Observe
the safety precautions in Section 4, and
the recommendation in Section 4.1.2.
6.8.2 Dissolve an appropriate amount of assayed
reference material in solvent. For
example, weigh 1-2 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
Teflon-lined cap.
6.8.3 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
Cambridge Isotope Laboratories.
6.9 Secondary standard -- Using stock solu-
tions (Section 6.8), prepare secondary
standard solutions containing the com-
pounds and concentrations shown in Table 4
in nonane.
6.10 Labeled compound stock standard -- From
stock standard solutions prepared as
above, or from purchased mixtures, prepare
this standard to contain the labeled com-
pounds at the concentrations shown in
Table 4 in nonane. This solution is
diluted with acetone prior to use (Section
10.3.2).
6.11 Cleanup standard - Prepare ^Cl^-2,3,7,8-
TCDD at the concentration shown in Table 4
in nonane.
6.12 Internal standard -- Prepare at the con-
centration shown in Table 4 in nonane.
6.13 Calibration standards (CS1 through CS5) --
Combine the solutions in Sections 6.9,
6.10, 6.11, and 6.12 to produce the five
calibration solutions shown in Table 4 in
nonane. These solutions permit the rela-
tive response (labeled to unlabeled) and
response factor to be measured as a func-
tion of concentration. The CS3 standard
is used for calibration verification
(VER).
6.14 Precision and recovery standard (PAR) --
Used for determination of initial (Section
8.2) and ongoing (Section 14.5) precision
and accuracy. This solution contains the
analytes and labeled compounds at the con-
centrations listed in Table 4 in nonane.
This solution is diluted with acetone
prior to use (Section 10.3.4).
6.15 GC retention time window defining solu-
tions -- Used to define the beginning and
ending retention times for the dioxin and
furan isomers.
9
-------
6.15.1 DB-5 colunn window defining standards --
Cambridge Isotope Laboratories ED-1732-A
(dioxins) and ED-1731-A (furans), or
equivalent, containing the compounds
listed in Table 5.
6.16 Isomer specificity test standards -- Used
to demonstrate isomer specificity for the
2,3,7,8-tetra- isomers of dioxin and
furan.
6.16.1 Standards for the D8-5 colunn -- Cambridge
Isotope Laboratories ED-908, ED-908-C, or
ED-935, or equivalent, containing the
compounds listed in Table 5.
6.16.2 Standards for the DB-225 column
Cambridge Isotope Laboratories EF-937 or
EF-938, or equivalent, containing the
compounds listed in Table 5.
6.17 Stability of solutions -- Standard
solutions used for quantitative purposes
(Sections 6.9-6.14) shall be analyzed
within 48 hours of preparation and on a
monthly basis thereafter for signs of
degradation. Standards will remain
acceptable if the peak area at the
quantitation m/z remains within ±15
percent of the area obtained in the
initial analysis of the standard. Any
standards failing to meet this criterion
should be assayed against reference
standards, as in Section 6.8.3., before
further use.
7 CALIBRATION
7.1 Assemble the GCMS and establish the
operating conditions necessary to meet the
relative retention time specifications in
Table 2.
7.1.1 The following GC operating conditions may
be used for guidance and adjusted as
needed to meet the relative retention time
specifications in Table 2:
Injector temp: 270 "C
Interface temp: 290 °C
Initial temp and time: 200 °C, 2 min
Temp Program: 200-220 °C at 5 "C/min
220 "C for 16 min
220-235 °C at 5 °C/min
235 °C for 7 min
235-330 °C at 5 °C/min
NOTE: All portions of the column which
connect the GC to the ion source shall
remain at the interface temperature
specified above during analysis, to
preclude condensation of less volatile
compounds.
7.1.2 Mass spectrometer (MS) resolution
Obtain a selected ion current profile
(S1CP) of each analyte in Table 4 at the
two exact masses specified in Table 3 and
at >10,000 resolving power by injecting an
authentic standard of the PCDDs and PCDFs
either singly or as part of a mixture in
which there is no interference between
closely eluted components, using the
procedure in Section 13.
7.1.2.1 The analysis time for PCDDs and PCDFs 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
instrunent performance. Therefore, a
mass-drift correction is mandatory. A
lock-mass ion from the reference compound
(PFK) is used for tuning the mass
spectrometer. The lock-mass ion is
dependent on the masses of the ions
monitored within each descriptor, as shown
in Table 3. The level of the reference
compound (PFK) metered into the ion
chamber during HRGC/HRMS analyses should
be adjusted so that the amplitude of the
most intense selected lock-mass ion signal
(regardless of the descriptor number) does
not exceed 10 percent 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 resulting in an increase in
time lost in cleaning the source.
7.1.2.2 By using a PFK molecular leak, tune the
instrunent to meet the minimum required
resolving power of 10,000 (10 percent
valley) at m/z 304.9824 (PFK) or any other
reference signal close to m/z 303.9016
(from TCDF). By using the peak matching
unit and the PFK reference peak, verify
that the exact mass of m/z 380.9760 (PFK)
is within 5 ppm of the required value.
7.2 Ion abundance ratios, minimum levels,
signal-to-noise ratios, and absolute
retention times -- Inject the CS1
10
-------
calibration solution (Table 4) per the
procedure in Section 13 and the conditions
in Table 2.
7.2.1 Measure the SICP areas for each analyte
and compute the ion abundance ratios
specified in Table 3A. Compare the
computed ratio to the theoretical ratio
given in Table 3A.
7.2.1.1 The groups of m/z's to be monitored are
shown in Table 3. Each group or
descriptor shall be monitored in succes-
sion as a function of GC retention time to
ensure that all PCDOs and PCDFs are
detected. The theoretical abundance
ratios for the m/z's are given in Table
3A, along with the control limits of each
ratio.
7.2.1.2 The mass spectrometer shall be operated in
a mass drift correction mode, using per-
fluorokerosene (PFK) to provide lock
masses. The lock mass for each group of
m/z's is shown in Table 3. Each lock mass
shall be monitored and shall not vary by
more than ±10 percent throughout its
respective retention time window. Varia-
tions of the lock mass by more than 10
percent indicate the presence of coeluting
interferences that may significantly
reduce the sensitivity of the mass
spectrometer. Re-injection of another
aliquot of the sample extract will not
resolve the problem. Additional cleanup
of the extract may be required to remove
the interferences.
7.2.2 All PCODs and PCDFs shall be within their
respective 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 7.1) prior to repeat of
the test.
7.2.3 Verify that the HRGC/HRMS instrument meets
the minimum levels in Table 2. The peaks
representing both unlabeled and labeled
analytes in the calibration standards must
have a signal-to-noise ratio (S/N) greater
than or equal to 10; otherwise, the mass
spectrometer shall be adjusted and this
test repeated until the minimum levels in
Table 2 are met.
7.2.4 The absolute retention time of C^-
1,2,3,4-TCDD (Section 6.12) shall exceed
25.0 minutes on the DB-5 column, and the
retention time of C12~^ ,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.
7.3 Retention time windows -- Analyze the
window defining mixtures (Section 6.15)
using the procedure in Section 13 (Figures
2A-2D). Table 5 gives the elution order
(first/last) of the compound pairs.
7.4 Isomer specificity
7.4.1 Analyze the isomer specificity test
standards (Section 6.16) using the
procedure in Section 13.
7.4.2 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 Figure 3.
7.4.3 Verify that the height of the valley
between the most closely eluted isomers
and the 2,3,7,8- isomers is less than 25
percent (computed as 100 x/y in Figure 3).
If the valley exceeds 25 percent, adjust
the analytical conditions and repeat the
test or replace the GC column and recali-
brate (Section 7.2 through 7.4).
7.5 Calibration with isotope dilution
Isotope dilution is used for the 15
2,3,7,8-substituted PCDDs and PCDFs with
labeled compounds added to the samples
prior to extraction, and for 1,2,3,7,8,9-
HxCDD and OCDF (see Section 16.1). The
reference compound for each unlabeled
compound is shown in Table 6.
7.5.1 A calibration curve encompassing the
concentration range is prepared for each
compound to be determined. The relative
response (RR) (unlabeled to labeled) vs.
concentration in standard solutions is
plotted or computed using a linear regres-
sion. Relative response is determined
according to the procedures described
below. A minimum of five data points are
employed for calibration.
7.5.2 The relative response of each unlabeled
PCDD/PCDF and its labeled analog is
determined using the area responses of
11
-------
S-MAY-88 Sir. Voltage 705 Sys: DB5US
Sample 1 Injection 1 Group 2 Mass 303.9016
100
80
Norm:
3044
60-
40
20
1,3,6,8-TCDF
1,2,8,9-TCDF
L—Btl
r-
25:20 26:40 28:00 29:20 30:40 32:00 33:20 34:40 36:00 37:20 38:40
6-MAY-88 Sir: Voltage 705 Sys: DB5US
Sample 1 Injection 1 Group 2 Mass 319.8965
Norm-
481
100
1,3,6,8-TCDD
1,2,8.9-TCDD
80-
2,3,7,8-TCDD
60-
1,2,3,7/1.2,3,8-TCDD
1,2,3,4-TCDD
1,2,3,9-TCDD
40-
20-
25:20 26:40 28:00 29:20 30:40- 32:00 /33:20 34:40 36:00 37:20 38:40
FIGURE 2A First and Last Eluted Tetra- Dioxin and Furan Isomers
12
-------
6-MAY-88 Sir: Voltage 705 Sys: OB5US
Sample 1 Injection 1 Group 2 Mass 339.8597
100
Norm: 652
1.3,4.6,8-PeCDF
1,2,3,8,9-PeCDF
i ¦ ¦ * i
29:20 30:40 32:00 33:20 34:40 36:00 37:20 38:40
6-MAY-88 Sir- Voltage 705 Sys: DB5US
Sample 1 Injection 1 Group 2 Mass 355.8546
100i
80
60
40
20
1,2,4,7,9-PeCDD
A
Norm: 503
1,2,3,8,9-PeCDD
\
29:20 30:40 32:00 33:20 34:40 36:00 37:20 38:40
FIGURE 2B First and Last Eluted Penta- Dioxin and Furan Isomers
13
-------
6-MAY-88 Sir: Voltage 705 Sys: DB5US
Sample 1 Injection 1 Group 3 Mass 373.8208
100
80
60
40
20
1,2,3,4,6,8-HxCDF
Norm: 560
1,2,3,4,8,9-HxCDF
v_
39:30 40:00 40:30 41:00 41:30 42:00 42:30 43:00 43:30 44:00 44:30
6-MAY-88 Sir: Voltage 705 Sys: DB5US
Sample 1 Injection 1 Group 3 Mass 389.8156
100
00
60
40
20
1.2,4,6,7,9/1,2,4,6,8,9-HxCDD
Norm: 384
XL
1,2.3.4,6,7-HxCDD
39:30 40:00 40:30 41:00 41:30 42:00 42:30 43:00 43:30 44:00 44:30
FIGURE 2C First and Last Eluted Hexa- Dioxin and Furan Isomers
14
-------
6-MAY-88 Sir: Voltage 705 Sys: DB5US
Sample 1 Injection 1 Group 4 Mass 407.7818
100
80
60
40
20
1,2,3.4,6.7,8-HpCDF
Norm: 336
k.
1,2,3,4,7,8,9-HpCDF
45:20 46:40 48:00 49:20 50:40 52:00 53.20 54:40 56:00 57:20
6-MAY-88 Sir: Voltage 705 Sys: DB5US
Sample 1 Injection 1 Group 4 Mass 423 7766
1.2,3,4,6.7.9-HpCDO
100, .* Norm 282
80-
60
40
20
1,2,3,4,6,7,8-HpCDD
45:20 46:40 48:00 49 20 50:40 52:00 53 20 54:40 56:00 57:20
6-MAY-88 Sir: Voltage 705 Sys: DB5US
Sample 1 Injection 1 Group 4 Mass 441 7428
100
80
60
40
20
Norm:
13
OCDF
45:20 46:40 48:00 49 20 50:40 52:00 53:20 54.40 56 00 57:20
6-MAY-88 Sir: Voltage 705 Sys- DB5US
Sample 1 Injection 1 Group 4 Mass 457.7377
100
80
60
40
20
OCDD
Norm:
45:20 46:40 48:00 49:20 50:40 52:00 53 20 54:40 56 00 57:20
FIGURE 2D First and Last Eluted Hepta- Dioxin and Furan Isomers
15
-------
3A DB225 Column
21-APR-88 Sir Voltage 705 Sys* DB225
Sample 1 Injection 1 Group 1 Mass 305 8987
Text: COLUMN PERFORMANCE
2,3,7,8-TCDF
100
Norm.
1,2,3,9-TCDF
3466
2,3,4,7-TCDF
16-10 16:20 16.30 16 40 16:50 17 00 17:10 17:20 17:30 17 40 17:50 18.00
3B DB5 Column
100i
j3
ad
OJ
22:30 24:00 25:30
Time
27.00
FIGURE 3 Valley between 2,3,7,8- Tetra Dioxin and Furan Isomers and Other Closely Eluted Isomers
16
-------
both the primary and secondary m/z's
specified in Table 3, for each calibration
standard, as follows:
RR =
(A,
1
(A,
«„2>
'I
Al2)
to be calibrated,
only one m/z for
See Table 3.)
3f
NOTE: There is
Cl4-2,3,7,8-TCDD.
A.
is
and A. are the areas of the primary
secondary
and secondary m/z's
internal standard.
for the GCMS
where,
A^ and An^ are the areas of the primary
and secondary m/z's for the unlabeled
compound.
Aj^ and A^ are the areas of the primary
and secondary m/z's for the labeled
compound.
is the concentration of the labeled
compound in the calibration standard.
Cn is the concentration of the unlabeled
compound in the calibration standard.
7.5.3 To calibrate the analytical system by
isotope dilution, inject a 1.0 uL aliquot
of calibration standards CS1 through CSS
(Section 6.13 and Table 4) using the
procedure in Section 13 and the conditions
in Table 2. Compute the relative response
(RR) at each concentration.
7.5.4 Linearity -- If the relative response for
any compound is constant (less than 20
percent coefficient of variation) over the
5-point calibration range, an averaged
relative response may be used for that
compound; otherwise, the complete calibra-
tion curve for that compound shall be used
over the 5-point calibration range.
7.6 Calibration by internal standard -- The
internal standard method is applied to
determination of non-2,3,7,8-substituted
compounds having no labeled analog in this
method, and to measurement of labeled
compounds for intralaboratory statistics
(Sections 8.4 and 14.5.4).
7.6.1 Response factors -- Calibration requires
the determination of response factors (RF)
defined by the following equation:
RF = Cis
(A. 1 + A 2) C
IS - IS s
C. is the concentration of the GCMS
1 s
internal standard (Section 6.12 and
Table 4).
Cg is the concentration of the compound in
the calibration standard.
7.6.2 To calibrate the analytical system by
internal standard, inject a 1.0 uL aliquot
of calibration standards CS1 through CS5
(Section 6.13 and Table 4) using the
procedure in Section 13 and the conditions
in Table 2. Compute the response factor
(RF) at each concentration.
7.6.3 Linearity -- If the response factor (RF)
for any compound is constant (less than 35
percent coefficient of variation) over the
5-point calibration range, an averaged
response factor may be used for that
compound; otherwise, the complete calibra-
tion curve for that compound shall be used
over the 5-point range.
7.7 Combined calibration -- By using calibra-
tion solutions (Section 6.13 and Table 4)
containing the unlabeled and labeled
compounds, and the internal standards, a
single set of analyses can be used to
produce calibration curves for the isotope
dilution and internal standard methods.
These curves are verified each shift
(Section 14.3) by analyzing the calibra-
tion verification standard (VER, Table 4).
Recalibration is required if calibration
verification criteria (Section 14.3.4)
cannot be met.
7.8
7.8.1
7.8.2
Data storage -- MS data shall
collected, recorded, and stored.
be
where,
Ag^ and Ag2 are the areas of the primary
and secondary m/z's for the compound
Data acquisition -- The signal at each
exact m/z shall be collected repetitively
throughout the monitoring period and
stored on a mass storage device.
Response factors and multipoint
calibrations -- The data system shall be
used to record and maintain lists of
response factors (response ratios for
17
-------
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 8.2) and
ongoing performance (Section 14.5) shall
be computed and maintained.
8 QUALITY ASSURANCE/QUALITY CONTROL
8.1 Each laboratory that uses this method is
required to operate a formal quality
assurance program (Reference 16). 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 characteris-
tics of the method. If the method is to
be applied routinely to samples containing
high solids with very little moisture
(e.g., soils, filter cake, compost) or to
an alternate matrix, the high solids
reference matrix (Section 6.6.2) or the
alternate matrix (Section 6.6.4) is sub-
stituted for the reagent water matrix
(Section 6.6.1) in all performance tests.
8.1.1 The analyst shall make an initial demon-
stration of the ability to generate
acceptable accuracy and precision with
this method. This ability is established
as described in Section 8.2.
8.1.2 The analyst is permitted to modify this
method to improve separations or lower the
costs of measurements, provided that all
performance specifications are met. Each
time a modification is made to the method,
the analyst is required to repeat the pro-
cedures in Sections 7.2 through 7.4 and
Section 8.2 to demonstrate method
performance.
8.1.3 Analyses of blanks are required to demon-
strate freedom from contamination (Section
3.2). The procedures and criteria for
analysis of a blank are described in
Section 8.5.
8.1.4 The laboratory shall spike all samples
with labeled compounds to monitor method
performance. This test is described in
Section 8.3. When results of these spikes
indicate atypical method performance for
samples, the samples are diluted to bring
method performance within acceptable
limits. Procedures for dilutions are
given in Section 16.4.
8.1.5 The laboratory shall, on an ongoing basis,
demonstrate through calibration verifica-
tion and the analysis of the precision and
recovery standard that the analytical sys-
tem is in control. These procedures are
described in Sections 14.1 through 14.5.
8.1.6 The laboratory shall maintain records to
define the quality of data that is gener-
ated. Development of accuracy statements
is described in Section 8.4.
8.2 Initial precision and accuracy -- To
establish the ability to generate accept-
able precision and accuracy, the analyst
shall perform the following operations.
8.2.1 For low solids (aqueous samples), extract,
concentrate, and analyze four 1-liter
aliquots of reagent water spiked with the
diluted precision and recovery standard
(PARJ (Sections 6.14 and 10.3.4) according
to the procedures in Sections 10 through
13. For an alternate sample matrix, four
aliquots of the alternate matrix are used.
All sample processing steps, including
preparation (Section 10), extraction
(Section 11), and cleanup (Section 12)
that are to be used for processing samples
shall be included in this test.
8.2.2 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 PCDDs and PCDFs with a
labeled analog, and by internal standard
for labeled compounds. Calculate the
recovery of the labeled compounds.
8.2.3 For each unlabeled and labeled compound,
compare s and X with the corresponding
limits for initial precision and accuracy
in Table 7. 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
18
-------
performance is unacceptable for that
compound. Correct the problem and repeat
the test (Section 8.2). The concentration
limits in Table 7 for labeled compounds
are based on the requirement that the
recovery of each labeled compound be in
the range of 25-150%.
8.3 The laboratory shall spike all samples and
QC aliquots with the diluted labeled
compound spiking solution (Sections 6.10
and 10.3.2) to assess method performance
on the sample matrix.
8.3.1 Analyze each sample according to the
procedures in Sections 10 through 13.
8.3.2 Compute the percent recovery (R) of the
labeled compounds in the labeled compound
spiking standard and the cleanup standard
using the internal standard method
(Section 7.6).
8.3.3 The recovery of each labeled compound must
be within 25-150%. 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 17.
8.4 Method accuracy for samples shall be
assessed and records shall be maintained.
8.4.1 After the analysis of five samples of a
given matrix type (water, soil, sludge,
pulp, etc) for which the labeled compound
spiking standards pass the tests in
Section 8.3, compute the average percent
recovery (R) and the standard deviation of
the percent recovery (SR) for the labeled
compounds only. Express the accuracy
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 accuracy
interval is expressed as 70-110%.
8.4.2 Update the accuracy assessment for each
compound in each matrix on a regular basis
(e.g., after each 5-10 new accuracy
measurements).
8.5 Blanks -- Reference matrix blanks are
analyzed to demonstrate freedom from
contamination (Section 3.2).
8.5.1 Extract and concentrate a 1-1 iter reagent
water blank (Section 6.6.1), high solids
reference matrix blank (Section 6.6.2),
paper matrix blank (Section 6.6.3) or
alternate reference matrix blank (Section
6.6.4) with each sample set (samples
started through the extraction process on
the same 12-hour shift, to a maximum of 20
samples). Analyze the blank immediately
after analysis of the precision and
recovery standard (Section 14.5) to
demonstrate freedom from contamination.
8.5.2 If any of the PCDDs or PCDFs (Table 1) or
any potentially interfering compound is
found in blank at greater than the minimum
level (Table 2), assumina a response
factor of 1 relative to the C^-1,2,3,4-
TC0D internal standard for compounds not
listed in Table 1, analysis of samples is
halted until the source of contamination
is eliminated and a blank shows no
evidence of contamination at this level.
NOTE: All samples associated with a
contaminated method blank must be re-
extracted and reanalyzed before the
results may be reported for regulatory
compliance purposes.
8.6 The specifications contained in this
method can be met if the apparatus used is
calibrated properly and then maintained in
a calibrated state. The standards used
for calibration (Section 7), calibration
verification (Section 14.3), and for
initial (Section 8.2) and ongoing (Section
14.5) precision and recovery should be
identical, so that the most precise
results will be obtained. A GCMS instru-
ment will provide the most reproducible
results if dedicated to the settings and
conditions required for the analyses of
PCDDs and PCDFs by this method.
8.7 Depending on specific program require-
ments, 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.
9 SAMPLE COLLECTION, PRESERVATION, AND
HANDLING
9.1 Collect samples in amber glass containers
following conventional sampling practices
(Reference 17). Aqueous samples which
flow freely are collected in refrigerated
19
-------
bottles using automatic sampling equip*
ment. Solid samples are collected as grab
samples using wide mouth jars.
9.2 Maintain samples at 0-4 ®C m the dark
from the time of collection until
extraction. If residual chlorine is
present in aqueous samples, add 30 mg
sodium thiosulfate per liter of water.
EPA Methods 330.4 and 330.5 may be used to
measure residual chlorine (Reference 18).
9.3 Perform sample analysis uithin AO days of
extraction.
10 SAMPLE PREPARATION
The sample preparation process involves
modifying the physical form of the sample
so that the PCDDs and PCDFs 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
8 lists the phase(s) and quantity
extracted for various sample matrices.
Samples containing a solid phase and
samples containing particle sizes larger
than 1 mm require preparation prior to
extraction. Because PCDOs/PCOFs are
strongly associated with particulates, the
preparation of aqueous samples is depen-
dent on the solids content of the sample.
Aqueous samples containing less than one
percent solids are extracted in a
separatory funnel. A smaller sample
aliquot is used for aqueous samples
containing one percent solids or more.
For samples expected or known to contain
high levels of the PCDOs and/or PCDFs, the
smallest sample size representative of the
entire sample should be used, and the
sample extract should be diluted, if
necessary, per Section 16.4.
10.1 Determine percent solids
10.1.1 Weigh 5-10 g of sample (to three signifi-
cant figures) into a tared beaker. NOTE:
This aliquot is used only for determining
the sol ids. content of the sample, not for
analysis of PCDDs/PCDFs.
10.1.2 Dry overnight (12 hours minimum) at 110 ±5
°C, and cool in a dessicator.
10.1.3 Calculate percent solids as follows:
x 100
X sol ids =
weight of sample after drying
weight of sample before drying
10.2 Determine particle size
10.2.1 Spread the dried sample from Section
10.1.2 on a piece of filter paper or
aluminum foil in a fume hood or glove box.
10.2.2 Estimate the size of the particles in the
sample. If the size of the largest
particles is greater than 1 mm, the
particle size must be reduced to 1 mm or
less prior to extraction.
10.3 Preparation of aqueous samples containing
one percent solids or less -- The
extraction procedure for aqueous samples
containing less than or equal to one
percent solids involves filtering the
sample, extracting the particulate phase
and the filtrate separately, and combining
the extracts for analysis. The aqueous
portion is extracted by shaking with
methylene chloride in a separatory funnel.
The particulate material is extracted
using the SDS procedure.
10.3.1 Mark the original level of the sample on
the sample bottle for reference. Weigh
the sample in the bottle on a top loading
balance to ±1 g.
10.3.2 Dilute a sufficient volume of the labeled
compound stock solution by a factor of 50
with acetone to prepare the labeled
compound spiking solution. 1.0 mL of the
diluted solution is required for each
sample, but no more solution should be
prepared than can be used in one day.
Spike 1.0 mL of the diluted solution into
the sample bottle. Cap the bottle and mix
the sample by careful shaking. Allow the
sample to equilibrate for 1-2 hours, with
occasional shaking.
10.3.3 For each sample or sample set (to a
maximum of 20 samples) to be extracted
during the same 12-hour shift, place two
" " liter aliquots of reagent water in
1.0
clean 2 liter separatory flasks.
10.3.4 Spike 1.0 mL of the diluted labeled
compound spiking standard (Section 6.10)
into one reagent water aliquot. This
aliquot will serve as the blank. Dilute
10 uL of the precision and recovery
20
-------
standard (Section 6.14) to 2.0 mL with
acetone. Spike 1.0 mL of the diluted
precision and recovery standard into the
remaining reagent water aliquot. This
aliquot will serve as the PAR (Section
14.5).
10.3.5 Assemble a Buchner funnel on top of a
clean 1 L filtration flask. Apply a
vacuum to the flask, and pour the entire
contents of the sample bottle through a
glass fiber filter (Section 5.5.4) in the
Buchner funnel, swirling the sample
remaining in the bottle to suspend any
particulates.
10.3.6 Rinse the sample bottle twice with 5 mL of
reagent water to transfer any remaining
particulates onto the filter.
10.3.7 Rinse the any particulates off the sides
of the Buchner funnel with small quanti-
ties of reagent water.
10.3.8 Ueigh the empty sample bottle on a top-
loading balance to ±1 g. Determine the
weight of the sample by difference. Do
not discard the bottle at this point.
10.3.9 Extract the filtrates using the procedures
in Section 11.1.1.
10.3.10 Extract the particulates using the proce-
10.4
10.4.1
10.4.2
10.4.3
10.4.4
tAvi aw i imc i i i v*u i a it
dures in Section 11.1.2
Preparation of samples containing greater
than one percent solids
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 10.1.3) into a
clean beaker or glass jar.
Spike 1.0 mL of the diluted labeled
compound spiking solution (Section 10.3.2)
into the sample aliquot(s).
For each sample or sample set (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 6.6) into clean beakers or
glass jars.
Spike 1.0 mL of the diluted labeled
compound spiking solution into one
reference matrix aliquot. This aliquot
will serve as the blank. Spike 1.0 mL of
the diluted precision and recovery
10.4.5
10.4.6
standard (Section 10.3.4) into the
remaining reference matrix aliquot. This
aliquot will serve as the PAR (Section
14.5).
Stir or tumble and
aliquots for 1-2 hours.
equiIlbrate the
Extract the aliquots using the procedures
in Section 11.
10.5 Multiphase samples
10.5.1 Pressure filter the sample, blank, and PAR
aliquots through Whatman GF/D glass fiber
filter paper. If necessary, centrifuge
these aliquots for 30 minutes at greater
than 5000 rpm prior to filtration.
10.5.2 Discard any aqueous phase (if present).
Remove any non-aqueous liquid (if present)
and reserve for recombination with the
extract of the solid phase (Section
11.1.2.5). Prepare the filter papers of
the sample and OC aliquots for particle
size reduction and blending (Section
10.6).
10.6 Sample grinding, homogenization, or blend-
ing -- Samples with particle sizes greater
than 1 mm (as determined by Section
10.2.2) are subjected to grinding, homo-
genization, or blending. The method of
reducing particle size to less than 1 mm
is matrix dependent. In general, hard
particles can be reduced by grinding with
a mortar and pestle. Softer particles can
be reduced by grinding in a Wiley mill or
meat grinder, by homogenization, or by
blending.
10.6.1 Each size reducing preparation procedure
on each matrix shall be verified by run-
ning the tests in Section 8.2 before the
procedure is employed routinely.
10.6.2 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.
10.6.3 Grinding -- Tissue samples, certain papers
and pulps, slurries, and amorphous solids
can be ground in a Wiley mill or heavy
duty meat grinder. In some cases, reduc-
ing 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
Section 10.4.5 or 10.5.2 in a clean
21
-------
grinder. Do not allow the sample tempera-
ture to exceed 50 °C. Grind the blank and
reference matrix aliquots using a clean
grinder.
10.6.4 Homogemzation 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 sample, blank,
and PAR aliquots from Section 10.4.5,
10.5.2, or 10.6.3.
10.6.5 Extract the aliquots using the procedures
in Section 11.
11 EXTRACTION AND CONCENTRATION
11.1 Extraction of filtrates -- extract the
aqueous samples, blanks, and PAR aliquots
according to the following procedures.
11.1.1 Pour the filtered aqueous sample from the
filtration flask into a 2-L separatory
funnel. Rinse the flask twice with 5 mL
of reagent water and add these rinses to
the separatory funnel. Add 60 mL methy-
lene chloride to the sample bottle
(Section 10.3.8), seal,and shake 60
seconds to rinse the inner surface.
11.1.2 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 water phase for a
minimum of 10 minutes. If the emulsion
interface between layers is more than one-
third the volume of the solvent layer,
employ mechanical techniques to complete
the phase separation (e.g., a glass stir-
ring rod). Drain the methylene chloride
extract into a soI vent-rinsed glass funnel
approximately one-half full of clean
sodium sulfate. Set up the glass funnel
so that it will drain directly into a
solvent-rinsed 500-ml K-D concentrator
fitted with a 10 mL concentrator tube.
NOTE: Experience with aqueous samples
high in dissolved organic materials (e.g.,
paper mill effluents) has shown that acid-
ification 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 the Agency to date suggest that
the effect may be a result of the dilution
of the sample, and that the addition of
reagent water may serve the same function.
Mechanical techniques may still be neces-
sary to complete the phase separation. If
either of these techniques is utilized,
the laboratory must perform the startup
tests described in Section 8.2 using the
same techniques.
11.1.3 Extract the water sample two more times
using 60 mL of fresh methylene chloride
each time. Drain each extract through the
funnel containing the sodium sulfate into
the K-D concentrator. After the third
extraction, rinse the separatory funnel
with at least 20 mL of fresh methylene
chloride, and drain this rinse through the
sodium sulfate into the concentrator.
Repeat this rinse at least twice.
11.1.4 The extract of the filtrate must be
concentrated before it is combined with
the extract of the particulates for
further cleanup. Add one or two clean
boiling chips to the receiver and attach a
three-ball macro Snyder column. Pre-wet
the column by adding approximately 1 mL of
hexane 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.
11.1.5 Adjust the vertical position of the
apparatus and the water temperature as
required to complete the concentration in
15-20 minutes. At the proper rate of
distillation, the balls of the column will
actively chatter but the chambers will not
flood.
11.1.6 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 concen-
trator tube with 1-2 mL of hexane. A 5 mL
syringe is recommended for this operation.
11.1.7 The concentrated extracts of the filtrate
and the particulates are combined using
the procedures in Section 11.2.13.
11.2 Soxhlet/Dean-Stark extraction of solids --
Extract the solid samples, particulates,
blanks, and PAR aliquots using the follow-
ing procedure.
11.2.1 Charge a clean extraction thimble with 5.0
g of 100/200 mesh silica (Section 6.5.1.1)
22
-------
and 100 g of quartz sand (Section 6.3.2).
NOTE: Do not disturb the silica layer
throughout the extraction process.
11.2.2 Place the thimble in a clean extractor.
Place 30-40 mL of toluene in the receiver
and 200-250 mL of toluene in the flask.
11.2.3 Pre-extract the glassware by heating the
flask until the toluene is boiling. When
properly adjusted, 1-2 drops of toluene
per second will fall from the condensor
tip into the receiver. Extract the
apparatus for three hours minimum.
11.2.4 After pre-extraction, cool and disassemble
the apparatus. Rinse the thimble with
toluene and allow to air dry.
11.2.5 Load the wet sample from Sections 10.4.6,
10.5.2, 10.6.3, or 10.6.4, and any non-
aqueous liquid from Section 10.5.2 into
the thimble and manually mix into the sand
layer with a clean metal spatula carefully
breaking up any large lumps of sample. If
the material to be extracted is the
particulate matter from the filtration of
an aqueous sample, add the filter paper to
the thimble also.
11.2.6 Reassemble the pre-extracted SOS apparatus
and add a fresh charge of toluene to the
receiver and reflux flask.
11.2.7 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.
Check the apparatus for foaming frequently
during the first 2 hours of extraction.
If foaming occurs, reduce the reflux rate
until foaming subsides.
11.2.8 Drain the water from the receiver at 1-2
hours and 8-9 hours, or sooner if the
receiver fills with water. Reflux the
sample for a total of 16-24 hours. Cool
and disassemble the apparatus. Record the
total volume of water collected.
11.2.9 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.
11.2.10 For solid samples, the extract must be
concentrated to approximately 10 mL prior
to back extraction. For the particulates
filtered from an aqueous sample, the
extract must be concentrated prior to
combining with the extract of the
filtrate. Therefore, add one or two clean
boiling chips to the round bottom flask
and attach a three-ball macro Snyder
column. Pre-wet the column by adding
approximately 1 mL of toluene through the
top. Place the round bottom flask in a
heating mantle and apply heat as required
to complete the concentration in 15-20
minutes. At the proper rate of distilla-
tion, the balls of the column will
actively chatter but the chambers will not
flood.
11.2.11 When the liquid has reached an apparent
volume of 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.
11.2.12 If the extract is from a solid sample, not
the particulates from an aqueous sample,
transfer the concentrated extract to a 250
mL separatory funnel. Rinse the flask
with toluene and add the rinse to the
separatory funnel. Proceed with back
extraction per Section 11.3.
11.2.13 If the extract is from the particulates
from an aqueous sample, it must be com-
bined with the concentrated extract of the
filtrate (Section 11.1.7) prior to back
extraction. Assemble the glass funnel
filled approximately one-half full with
sodium sulfate from Section 11.1.2 such
that the funnel will drain into the K-D
concentrator from Section 11.1.7 contain-
ing the concentrated methylene chloride
extract of the filtrate. Pour the concen-
trated toluene extract of the particulates
through the sodium sulfate into the K-D
concentrator. Rinse the round-bottom
flask with three 15-20 mL volumes of
hexane, and pour each rinse through the
sodium sulfate into the K-D concentrator.
Add one or two fresh boiling chips to the
receiver and attach the three-ball macro
Snyder column to the K-D concentrator.
Pre-wet the column by adding approximately
1 mL of hexane to the top of the column.
Concentrate the combined extract to
approximately 10 mL (the volume of the
toluene). 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. Transfer the contents
of the K-D concentrator to a pre-nnsed
250 mL separatory funnel. Rinse the flask
23
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and lower joint with three 5 mL volumes of
hexane, and add each rinse to the separa-
tory funnel. Proceed with back extraction
per Section 11.3.
11.3 Back extraction with base and acid
11.3.1 Spike 1.0 mL of the cleanup standard
(Section 6.11) into the separatory funnels
containing the sample and QC extracts
(Section 11.2.12 or 11.2.13).
11.3.2 Partition the extract against 50 mL of
potassium hydroxide solution (Section
6.1.1). Shake for 2 minutes with periodic
venting into a hood. Remove and discard
the aqueous layer. Repeat the base wash-
ing until no color is visible in the
aqueous layer, to a maximum of four wash-
ings. Minimize contact time between the
extract and the base to prevent degrada-
tion of the PCDDs and PCOFs. Stronger
potassium hydroxide solutions may be
employed for back extraction, provided
that the laboratory meets the specifica-
tions for labeled compound recovery and
demonstrates acceptable performance using
the procedures in Section 8.2.
11.3.3 Partition the extract against 50 mL of
sodium chloride solution (Section 6.1.3)
in the same way as with base. Discard the
aqueous layer.
11.3.4 Partition the extract against 50 mL of
sulfuric acid (Section 6.1.2) 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.
11.3 5 Repeat the partitioning against sodium
chloride solution and discard the aqueous
layer.
11.3 6 Pour each extract through a drying column
containing 7 to 10 cm of anhydrous sodium
sulfate. Rinse the separatory funnel wtth
30-50 mL of toluene and pour through the
drying column. Collect each extract in a
500 mL round bottom flask. Concentrate
and clean up the samples and QC aliquots
per Sections 11.4 and 12.
11.4 Macro-concentration -- Concentrate the
extracts in separate 100-mL round bottom
flasks on a rotary evaporator.
11.4.1 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 contamination check
if necessary. Between samples, three 2-3
mL aliquots of toluene should be rinsed
down the feed tube into a waste beaker.
11.4.2 Attach the round bottom flask containing
the sample extract to the rotary evapora-
tor. Slowly apply vacuum to the system,
and begin rotating the sample flask.
11.4.3 Lower the flask into the water bath and
adjust the speed of rotation and the
temperature as required to complete the
concentration in 15-20 minutes. At the
proper rate of concentration, the flow of
solvent into the receiving flask wilt be
steady, but no bumping or visible boiling
of the extract will occur. NOTE: If the
rate of concentration is too fast, analyte
loss may occur.
11.4.4 When the liquid in the concentration flask
has reached an apparent volume of 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 hexane.
11.4.5 Transfer the extract to a vial using three
2-3 mL rinses of hexane. Proceed with
micro-concentration and solvent exchange.
11.5 Micro-concentration and solvent exchange
11.5.1 Toluene extracts to be subjected to GPC or
HPLC cleanup are exchanged into methylene
chloride. Extracts that are to be cleaned
up using silica gel, alumina, and/or AX-
21/Celite are exchanged into hexane.
11.5.2 Transfer the vial containing the sample
extract to a nitrogen evaporation device.
Adj'ust 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.
11.5.3 Lower the vial into a 45 °C water bath and
continue concentrating.
11.5.4 When the volume of the liquid is approxi-
mately 100 uL, add 2-3 mL of the desired
solvent (methylene chloride or hexane) and
24
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11.5.5
11.5.6
11.5.7
11.5.8
12
12.1
12.1 1
12.1.2
12.1.3
continue concentration to approximately
100 uL. Repeat the addition of solvent
and concentrate once more.
If the extract is to be cleaned up by GPC
or HPLC, adjust the volume of the extract
to 5.0 mL uith methylene chloride.
Proceed with GPC cleanup (Section 12.2).
If the extract is to be cleaned up by
column chromatography (alumina, silica
gel, AX-21/Celite), bring the final volume
to 1.0 mL with hexane. Proceed with
column cleanups (Sections 12.3-12.5).
For extracts to be concentrated for
injection into the GCMS -- add 10 uL of
nonane to the vial. Evaporate the solvent
to the level of the nonane. Evaporate the
hexane in the vial to the level of the
nonane.
Seal the vial and label with the sample
number. Store in the dark at room temper-
ature until ready for GCMS analysis.
EXTRACT CLEANUP
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 require-
ments of Section 8.2 can be met using the
cleanup procedure.
Gel permeation chromatography (Section
12.2) removes many high molecular weight
interferences that cause GC column
performance to degrade. It may 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).
Acid, neutral, and basic silica gel, and
alumina (Sections 12.3 and 12.4) are used
to remove
interferences.
nonpolar
and
AX-21/Celite (Section 12.5) is
remove nonpolar interferences.
polar
used to
12.1.4 HPLC (Section 12.6) is used to provide
specificity for the 2,3,7,8-substituted
and other PCDD and PCDF isomers.
12.2 Gel permeation chromatography (GPC)
12.2.1 Column packing
12.2.1.1 Place 70-75 g of SX-3 Bio-beads in a 400-
500 mL beaker.
12.2.1.2 Cover the beads with methylene chloride
and allow to swell overnight (12 hours
minimum).
12.2.1.3 Transfer the swelled beads to the column
and pump solvent through the column, from
bottom to top, at 4.5-5.5 mL/min prior to
connecting the column to the detector.
12.2.1.4 After purging the column with solvent for
1-2 hours, adjust the column head pressure
to 7-10 psig and purge for 4-5 hours to
remove air. Maintain a head pressure of
7-10 psig. Connect the column to the
detector.
12.2.2 Column calibration
12.2.2.1 Load 5 mL of the calibration solution
(Section 6.4) into the sample loop.
12.2.2.2 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.
12.2.2.3 Set the "dump time" to allow >85 percent
removal of the corn oi I and >85 percent
collection of the phthalate.
12.2.2.4 Set the "collect time" to the peak minimum
between perylene and sulfur.
12.2.2.5 Verify the calibration with the calibra-
tion solution after every 20 extracts.
Calibration is verified if the recovery of
the pentachlorophenol is greater than 85
percent. 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 GPC system.
12.2.3 Extract cleanup -- GPC requires that the
column not be overloaded. The column
specified in this method is designed to
handle a maximum of 0.5 g of high
molecular weight material in a 5 mL
25
-------
extract. If the extract is known or
expected to contain more than 0.5 9, the
extract is split into aliquots for GPC and
the aliquots are combined after elution
from the colum. The residue content of
the extract may be obtained
gravimetrically by evaporating the solvent
from a 50 uL aliquot.
12.2.3.1 Filter the extract or load through the
filter holder to remove particulates.
Load the 5.0 mL extract onto the colum.
12.2.3.2 Elute the extract using the calibration
data determined in Section 12.2.2.
Collect the eluate in a clean 400-500 mL
beaker.
12.2.3.3 Rinse the sample loading tube thoroughly
with methylene chloride between extracts
to prepare for the next sample.
12.2.3.4 If a particularly dirty extract is
encountered, a 5.0 mL methylene chloride
blank shall be run through the system to
check for carry-over.
12.2.3.5 Concentrate the eluate per Section 11.2.1,
11.2.2, and 11.3.1 or 11.3.2 for further
cleanup or for injection into the GCMS.
12.3 Silica gel cleanup
12.3.1 Place a glass wool plug in a 15 mm i.d.
chromatography col ltd. Pack the column in
the following order (bottom to top): 1 g
silica gel (Section 6.5.1.1), four g basic
silica gel (Section 6.5.1.3), 1 g silica
gel, 8 g acid silica gel (Section
6.5.1.2), 2 g silica gel. Tap the colum
to settle the adsorbents.
12.3.2 Pre-rinse the colum with 50-100 mL of
hexane. Close the stopcock when the
hexane is within 1 mm of the sodium
sulfate. Discard the eluate. Check the
column for channeling. If channeling is
present, discard the column and prepare
another.
12.3.3 Apply the concentrated extract to the
column. Open the stopcock until the
extract is within 1 mm of the sodiun
sulfate.
12.3.4 Rinse the receiver twice with 1 mL
portions of hexane and apply separately to
the colum. Elute the PCDDs/PCDFs with
100 mL hexane and collect the eluate.
12.3.5 Concentrate the eluate per Section 11.4 or
11.5 for further cleanup or for injection
into the HPLC or GCMS.
12.3.6 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 colum. This
may be accomplished by increasing the
strengths of the acid and basic silica
gels. The acid silica gel (Section
6.5.1.2) may be increased in strength to
as much as 44X w/w (7.9 g sulfuric acid
added to 10 g silica gel). The basic
silica gel (Section 6.5.1.3) may be
increased in strength to as much as 33%
w/w (50 mL 1N NaOH added to 100 g silica
gel). 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 PCODs/PCDFs. Increasing the strengths
of the acid and basic silica gel may also
require different volunes 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
procedures in Section 8.2.
12.4 Alumna cleanup
12.4.1 Place a glass wool plug in a 15 mm i.d.
chromatography colum.
12.4.2 If using acid alumina, pack the colum by
adding 6 g acid alunina (Section 6.5.2.1).
If using basic alunina, substitute 6 g
basic alunina (Section 6.5.2.2). Tap the
colum to settle the adsorbents.
12.4.3 Pre-rinse the colum with 50-100 mL of
hexane. Close the stopcock when the
hexane is within 1 mm of the alumina.
12.4.4 Discard the eluate. Check the column for
channeling. If channeling is present,
discard the column and prepare another.
12.4.5 Apply the concentrated extract to the
colum. Open the stopcock until the
extract is within 1 mm of the alumina.
12.4.6 Rinse the receiver twice with 1 mL
portions of hexane and apply separately to
the colum. Elute the interfering
compounds with 100 mL hexane and discard
the eluate.
26
-------
12.4.7 The choice of eluting solvents will depend
on the choice of alunina (acid or basic)
made in Section 12.4.2.
12.4.7.1 If using acid alunina, elute the PCDDs and
PCDFs from the colunn with 20 ml methylene
chloride:hexane (20:80 v/v). Collect the
etuate.
12.4.7.2 If using basic alunina, elute the PCDDs
and PCDFs from the colunn with 20 mL
methylene chloride:hexane (50:50 v/v).
Collect the eluate.
12.4.8 Concentrate the eluate per Section 11.4 or
11.5 for further cleanup or for injection
into the HPLC or GOtS.
12.5 AX-21/Celite
12.5.1 Cut both ends from a 10 ml disposable
serological pipet to produce a 10 cm
colum. Fire polish both ends and flare
both ends if desired. Insert a glass wool
plug at one end, then pack the colum with
1 g of the activated AX-21/Celite to form
a 2 cm long adsorbent bed. Insert a glass
wool plug on top of the bed to hold the
adsorbent in place.
12.5.2 Pre-rinse the colunn with five mL of
toluene followed by 2 mL methylene
chloride:methanol:toluene (15:4:1 v/v), 1
mL methylene chloride:cyclohexane (1:1
v/v), and five mL hexane. If the flow
rate of eluate exceeds 0.5 mL per min,
discard the colunn.
12.5.3 When the solvent is within 1 mm of the
colunn packing, apply the sample extract
to the colunn. Rinse the sample container
twice with 1 mL portions of hexane and
apply separately to the colunn. Apply 2
mL of hexane to complete the transfer.
12.5.4 Elute the interfering compounds with 2 mL
of hexane, 2 mL of methylene
chloride:cyclohexane (1:1 v/v), and 2 mL
of methylene chloride: methanol:toluene
(15:4:1 v/v). Discard the eluate.
12.5.5 Invert the colunn and elute the PCDDs and
PCDFs with 20 mL of toluene. If carbon
particles are present in the eluate,
filter through glass fiber filter paper.
12.5.6 Concentrate the eluate per Section 11.4 or
11.5 for further cleanup or for injection
into the HPLC or GCMS.
12.6 HPLC (Reference 6)
12.6.1 Colum calibration
12.6.1.1 Prepare a calibration standard containing
the 2,3,7,8- isomers and/or other isomers
of interest at a concentration of approxi-
mately 500 pg/uL in methylene chloride.
12.6.1.2 Inject 30 uL of the calibration solution
into the HPLC and record the signal from
the detector. Collect the eluant for re-
use. The elution order will be the tetra-
through octa-isomers.
12.6.1.3 Establish the collect time for the tetra-
isoners and for the other isomers of
interest. Following calibration, flush
the injection system with copious
quantities of methylene chloride, includ-
ing a minimun of five 50-uL injections
while the detector is monitored, to ensure
that residual PCDDs and PCDFs are removed
from the system.
12.6.1.4 Verify the calibration with the calibra-
tion solution after every 20 extracts.
Calibration is verified if the recovery of
the PCDDs and PCDFs from the calibration
standard (Section 12.6.1.1) is 75-125
percent cotnpared to the calibration
(Section 12.6.1.2). 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.
12.6.2 Extract cleanup -- HPLC requires that the
colum not be overloaded. The colum
specified in this method is designed to
handle a maximun of 30 uL of extract. If
the extract cannot be concentrated to less
than 30 uL, it is split into fractions and
the fractions are combined after elution
from the colum.
12.6.2.1 Rinse the sides of the vial twice with 30
uL of methylene chloride and reduce to 30
uL with the blowdown apparatus.
12.6.2.2 Inject the 30 uL extract into the HPLC.
12.6.2.3 Elute the extract using the calibration
data determined in 12.6.1. Collect the
fraction(s) in a clean 20 mL concentrator
tube containing 5 mL of hexane:acetone
(1:1 v/v).
27
-------
12.6.2.4 If an extract containing greater than 100
ng/mL of total PCDD or PCDF is encoun-
tered, a 30 uL methylene chloride blank
shall be run through the system to check
for carry-over.
12.6.2.5 Concentrate the eluate per Section 11.5
for injection into the GCMS.
13 HRGC/HRMS ANALYSIS
13.1 Establish the operating conditions given
in Section 7.1.
13.2 Add 10 uL of the internal standard solu-
tion (Section 6.12) to the saciple extract
ioinediately 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 instrunent
internal standard solution. Rather, bring
the extract back to Its previous volume
(e.g., 19 uL) with pure nonane only.
13.3 Inject 1.0 uL of the concentrated extract
containing the internal standard solution,
using on-col urn or splitless injection.
Start the GC colusn Initial isothermal
hold tpon injection. Start MS data
collection after the solvent peak elutes.
Stop data collection after the octachloro-
dioxin and furan have eluted. Return the
colum to the initial tenperature for
analysis of the next extract or standard.
14 SYSTEM AND LABORATORY PERFORMANCE
14.1 At the beginning of each 12-hour shift
during which analyses are performed, GCMS
system performance and calibration are
verified for all unlabeled and labeled
compounds. For these tests, analysis of
the CS3 calibration verification (VER)
standard (Section 6.13 and Table 4) and
the isomer specificity test standards
(Sections 6.16 and Table 5) shall be used
to verify all performance criteria.
Adjustment and/or recalibration (per
Section 7) shall be performed until all
performance criteria are met. Only after
all performance criteria are met nay
samples, blanks, and precision and
recovery standards be analyzed.
14.2 MS resolution -- A static resolving power
of at least 10,000 (10 percent valley
definition) must be demonstrated at appro-
priate masses 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 7.1.2. Corrective
actions must be implemented whenever the
resolving power does not meet the
requirement.
14.3 Calibration verification
14.3.1 Inject the VER standard using the
procedure in Section 13.
14.3.2 The m/z abundance ratios for all PCDDs arid
PCDFs shall be within the limits In Table
3A; otherwise, the mass spectrometer shall
be adjusted until the m/z abundance ratios
fall within the limits specified, and the
verification test (Section 14.3.1)
repeated. If the adjustment alters the
resolution of the mass spectrometer, reso-
lution shall be verified (Section 7.1.2}
prior to repeat of the verification test.
14.3.3 The peaks representing each unlabeled and
labeled compound in the VER standard must
be present with a S/N of at least 10;
otherwise, the mass spectrometer shall be
adjusted and the verification test
(Section 14.3.1) repeated.
14.3.4 Confute the concentration of each
unlabeled compound by isotope dilution
(Section 7.5) for those compounds that
have labeled analogs (Table 1). Compute
the concentration of the labeled compounds
by the internal standard method. These
concentrations are computed based on the
averaged relative response and averaged
response factor from the calibration data
in Section 7.
14.3.5 For each compound, compare the concentra-
tion with the calibration verification
limit In Table 7. IT all compounds meet
the acceptance criteria, calibration has
been verified. If, however, any compound
fails, the measurement system Is not
performing properly for that compound. In
this event, prepare a fresh calibration
standard or correct the problem causing
the failure and repeat the resolution
(Section 14.2) and verification (Section
14.3.1) tests, or recalibrate (Section 7).
14.4 Retention times and GC resolution
14.4.1 Retention times
28
-------
14.4.1.1 Absolute -- The absolute retention times
of the 13C12-1,2,3,4-TCOO and C12*
1,2,3,7,8,9-HxCDD GCHS internal standards
shall be within ±15 seconds of the
retention times obtained during calibra-
tion (Section 7.2.4).
precision and recovery test (Section
14.5). The concentration limits in Table
7 for labeled compounds are based on the
requirement that the recovery of each
labeled compound be in the range of 25-
150%.
14.4.1.2 Relative -- The relative retention times
of unlabeled and labeled PCOOs and PCSFs
shall be within the limits given in Table
2.
14.4.2 GC resolution
14.4.2.1 Inject the isomer specificity standards
(Section 6.16) on their respective
colwris.
14.4.2.2 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 percent on their
respective colunns (Figure 3).
14.4.3 If the absolute retention time of any
compound is not within the limits
specified or 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
14.3.1) or recalibrate (Section 7).
14.5 Ongoing precision and accuracy
14.5.1 Analyze the extract of the diluted
precision and recovery standard (PAR)
(Section 10.3.4 or 10.4.4) prior to
analysis of samples from the same set.
14.5.2 Compute the concentration of each PCD0 and
PCDF by isotope dilution for those
compounds that have labeled analogs
(Section 7.5). Compute the concentration
of each labeled compound by the internal
standard method.
14.5.3 For each unlabeled and labeled compound,
conpare the concentration with the limits
for ongoing accuracy in Table 7. 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-extract the sample set
(Section 10) and repeat the ongoing
14.5.4
Add results which pass the specifications
in Section 14.5.3 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 state-
ment of laboratory accuracy for each PCOD
and PCDF in each matrix type by calculat-
ing the average percent recovery (R) and
the standard deviation of percent recovery
Express the accuracy as a recovery
- 2S„ to R ~ 2S„. For
(S„).
interval from R
example, if R
accuracy is 85-105X.
= 95 XR
and S_
Si.
the
15 QUALITATIVE DETERMINATION
For a gas chromatographic peak to be
identified as a PCOD or PCDF (either a
in labeled or a labeled compound), it must
meet all of the criteria in Sections 15.1-
15.4.
15.1 The signals for the two exact m/z's being
monitored (Table 3) must be present, and
must maximize within ~ 2 seconds of one
another.
15.2 The signal-to-noise ratio (S/N) of each of
the two exact m/z's must be greater than
or equal to 2.5 for a sample extract, and
greater than or equal to 10 for a calibra-
tion standard (see Sections 7.2.3 and
14.3.3).
15.3 The ratio of the integrated ion currents
of both the exact m/z's monitored must be
within the limits in Table 3A.
15.4 The relative retention time of the peaks
representing a unlabeled 2,3,7,8-
substituted PCOD or PCDF must be within
the limits given in Table 2. The
retention time of peaks representing non-
2,3, 7, 8- substituted PCDDs or PCDFs must be
within the retention time windows
established in Section 7.3.
15.5 Confirmatory analysis -- Isomer
specificity for all of the 2,3,7,8-substi-
tuted analytes cannot be attained by
analysis on the DB-5 (or equivalent) GC
colunn alone. The lack of specificity is
29
-------
of greatest concern for the unlabeled
2,3,7,8-TCDF. Therefore, any sample In
which 2,3,7,8-TCDF fs identified by
analysis on a D8-5 (or equivalent) GC
colum must have a confirmatory analysis
performed on a D8-225, SP-2330, or equiva-
lent GC colum. The operating conditions
in Section 7.1.1 may be adjusted for
analyses on the second GC colum, but the
GCMS must meet the mass resolution and
calibration specifications in Section 7.
15.6 If any gas chromatographic peak meets the
identification criteria in Sections 15.1,
15.2, and 15.4, but does qo£ meet the ion
abundance ratio criterion (Section 15.3),
and is not a labeled analog, that sample
must be analyzed on a second GC colum, as
in Section 15.5 above. Interferences co-
eluting in either of the two m/z's may
cause the ion abmdance ratio to fall out-
side of the limits in Table 3A. If the
ion abundance ratio of the peak fails to
meet the criteria on the second GC colum,
then the peak does not represent a PCDD or
PCDF. If the peak does meet all of the
criteria in Sections 15.1-15.4 on the
second GC colum, then calculate the
concentration of that peak from the
analysis on the second GC colum, accord-
ing to the procedures in Section 16.
15.7 If any gas chromatographic peak that
represents a labeled analog does not meet
all of the identification criteria in
Sections 15.1-15.4 on the second GC
colum, then the results may not be
reported for regulatory compliance
purposes and a new aliquot of the sample
must be extracted and analyzed.
16 QUANTITATIVE DETERMINATION
16.1 Isotope dilution -- By adding a known
amount of a labeled compound to every
sample prior to extraction, correction for
recovery of the unlabeled compound can be
made because the unlabeled compound and
its labeled analog exhibit similar effects
upon extraction, concentration, and gas
chromatography. Relative response (RR)
values are used in conjunction with cali-
bration data described in Section 7.5 to
determine concentrations directly, so long
as labeled compound spiking levels are
constant, using the following equation:
(A 1 ~ A 2) C,
C (ng/mL) » " " la
ex i j
RF
where, C is the concentration of the
ex
compound in the extract and the other
terms are as defined in Section 7.6.1.
(NOTE: There is only one m/z for the
^Cl-labeled standard.)
30
-------
16.3 The concentration of the in labeled
compound 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 10), as follows:
IP * V 1
Concentration ¦ ex ex
in solid (ng/Kg) y
s
where,
V is the extract volune in mL.
ex
U& is the saople weight in Kg.
16.4 The concentration of the unlabeled
compound 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 10.3),
as follows:
Concentration (C x V_ )
3 ex ex
in aqueous phase ^——
(pg/L) V8
where,
V is the extract volune in mL.
ex
V. is the sample volune in liters,
s
16.5 If the SICP areas at the quantitation
m/z's for any compound exceed the calibra-
tion range of the system, a smaller sampte
aliquot is extracted.
16.5.1 For aqueous samples containing one percent
solids or less, dilute 100 mL, 10 mL,
etc., of sample to 1 liter with reagent
water and extract per Section 11.
16.5.2 For samples containing greater than one
percent solids, extract an amount of
sample equal to 1/10, 1/100, etc., of the
amount determined in Section 10.1.3.
Extract per Section 10.4.
16.5.3 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
instrunent internal standard to 100 pg/ul
in the extract, and analyze an aliquot of
this diluted extract by the internal
standard method.
16.6 Results are reported to three significant
figures for the ui labeled and labeled
isomers fotnd in all standards, blanks,
and samples. For aqueous samples, the
units are pg/L; for samples containing
greater than one percent solids (soils,
sediments, filter cake, compost), the
inits are ng/Kg based on the dry weight of
the sample.
16.6.1 Results for samples which have been
diluted are reported at the least dilute
level at which the areas at the quantita-
tion m/z's are within the calibration
range (Section 16.5).
16.6.2 For unlabeled compounds having a labeled
analog, results are reported at the least
dilute level at tdiich the area at the
quantitation m/z is within the calibration
range (Section 16.5) and the labeled
compound recovery is within the normal
range for the method (Section 17.4).
16.6.3 Additionally, the total concentrations of
all isomers in an individual level of
chlorination (i.e., total TCDD, total
PeCDD, etc.) are reported to three signi-
ficant figures in units of pg/L, for both
dioxins and furans. The total or ng/Kg
concentration in each level of chlorina-
tion is the sun of the concentrations of
all .isomers identified in that level,
including any non-2,3,7,8-subst i tuted
isomers.
17 ANALYSIS OF COMPLEX SAMPLES
17.1 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 11);
others may overload the GC column and/or
mass spectrometer.
17.2 Analyze a smaller aliquot of the sample
(Section 16.4) when the extract will not
concentrate to 20 uL after all cleanup
procedures have been exhausted.
17.3 Recovery of labeled compound spiking
standards -- In most samples, recoveries
of the labeled compound spiking standards
will be similar to those from reagent
water or from the alternate matrix
(Section 6.6). If recovery is outside of
the 25-150* range, a diluted sample
(Section 16.4) shall be analyzed. If the
recoveries of the labeled compound spiking
standards in the diluted sample are
outside of the limits (per the criteria
above), then the verification standard
(Section 14.3) shall be analyzed and
calibration verified (Section 14.3.4). if
the calibration cannot be verified, a new
31
-------
calibration must be performed and the
original sample extract reanalyzed. If
the calibration is verified and the
diluted sample does not meet the limits
for labeled compound recovery, then the
method does not apply to the sample being
analyzed and the result may not be
reported for regulatory compliance
purposes.
18 METHGO PERFORMANCE
The performance specifications in this
method are based on the analyses of more
than 400 samples, representing matrices
from at least five industrial categories.
These specifications will be updated
periodically as more data are received,
and each time the procedures in the method
are revised.
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Procedures and Quality Assurance for
Multimedia Analysis of Polychlorinated
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Las Vegas, Nevada, June 1987.
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Special Analytical Services Samples",
Triangle Laboratories, Inc., 801-10
Capitola Dr, Research Triangle Park, NC
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6 Lamparski, L.L., and Nestrick, T.J.,
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Levels", Analytical Chemistry. 52: 2045-
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7 Lamparski, L.L., and Nestrick, T.J.,
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in Matrices Containing Water",
Chemosnhere. 19:27-31, 1989.
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chlorodibenzo-p-dloxin". Environmental
Toxicoloqical 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,
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CDC, NIOSH, Publication 77-206, August
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13 "Standard Methods for the Examination of
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later revisions, American Public Health
Association, 1015 15th St, N.U.,
Washington, DC 20005, 46: Section 108
(Safety), 1985.
14 "Method 613 -- 2,3,7,8-Tetrachlorodibenzo-
p-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 "Handbook of Analytical Quality Control in
Water and Wastewater Laboratories", USEPA
EMSL, Cincinnati, OH 45268, EPA-600/4-79-
019, March 1979.
32
-------
17 "Standard Practice for Sampling Water",
ASTM Annual Book of Standards, ASTN, 1916
Race Street, Philadelphia, PA 19103-1187,
1980.
18 "Methods 330.4 and 330.5 for Total
Residual Chlorine", USEPA, EMSL, Cincin-
nati, OH 45268, EPA 600/4-70-020, March
1979.
19 Barnes, Donald G., Kutz, Frederick U., and
Bottimore, David P., "Update of Toxicity
Equivalency Factors (TEFs) for Estimating
Risks Associated with Exposures to
Mixtures of Chlorinated Dibenzo-p-Dtoxins
and Dibenzofurans (CODs/CDFs)", Risk
Assessment Forua, USEPA, Washington, DC
20460, February 1989.
33
-------
Table 1
POLYCHLORINATED DIBENZOOIOXINS AND FURAMS DETERMINED BY ISOTOPE DILUTION AND INTERNAL STANDARD
HIGH RESOLUTION GAS CHROMATOGRAPHY (HRGC)/H1GH RESOLUTION MASS SPECTROMETRY (HRMS)
PCDDs/PCDFs (1)
Isomer/Congener
CAS Registry
Labeled Analog
CAS Registry
2,3,7,8-TCDD
TCDD
8-TCDF
TCDF
7,8-PeCDD
PeCDD
7,8-PeCDF
7,8-PeCDF
PeCDF
4,7,8-HxCDD
6.7.8-HxCDD
7.8.9-HxCOD
HxCDD
4,7,8-HxCDF
6.7.8-HxCDF
7.8.9-HxCDF
6,7,8-HxCDF
HxCDF
4,6,7,8-HpCOD
HpCDD
4.6.7.8-HpCDF
4.7.8.9-HpCDF
HpCDF
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
34465-4608
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
"c12-2,3,7,8-TCDD
C 1^-2,3,7,8-TCDD
13
C12-2,3,7,8-TCDF
13,
13
13
C12-1,2,3,7,8-PeCDD
C^-1,2,3,7,8-PeCDF
C12-2,3,4,7,8-PeCDF
13C12-1,2,3,4,7,8-HxCDD
J|c12-1,2,3,6,7,8-HxCDD
C^2*1,2,3,7,8,9-HxCDD(2)
C12-1,2,3,4,7,8-HxCDF
C12-1,2,3,6,7,8-HxCDF
C12-1,2,3,7,8,9-HxCDF
C12-2,3,4,6,7,8-HxCDF
C12-1,2,3.4,6,7,8-HpCDD
13
U
13
13,
"c12-1, 2,3,4,6,7,8-HpCDF
C12-1,2,3,4,7,8,9-HpCDF
13
c12-ocdd
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
none
(1) Polychlorinated dioxins and furans
TCDD
= Tetrachlorodibenzo-p-dioxin
TCDF
=
TetrachIorodibenzofuran
PeCDD
= Pentachlorodibenzo-p-dioxin
PeCDF
3
Pentachlorodi benzofuran
HxCDD
= Hexachlorodibenzo-p-dioxin
HxCDF
3
HexachIorodi benzofuran
HpCDD
= Heptachlorodibenzo-p-dioxin
HpCDF
a
HeptachIorod i benzofuran
OCDD
= Octachlorodibenzo-p-dioxin
OCDF
3
Octachlorodibenzofuran
Labeled
analog is used as an internal standard and
therefore
is
not used for quantitation of the native
compound.
34
-------
Table 2
RETENTION TIMES AND MINIMUM LEVELS FOR PCODs AND PCDFs
Minimum Level (1)
Compound
Retent ion
Time
Reference
Relative
Retention
Time
Water
P9/L
ppq
Sol id
ng/kg
PP«
Extract
pg/uL
PPb
Compounds using 13C12*1,2,3,4-TCDD as internal standard
Native Compounds
2,3,7,8-TCDF
2,3,7,8-TCDD
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,7,8-PeCDD
"c12-2,3,7,8-TCDF
C^2*2,3,7,8-TCDD
"C12-1,2,3,7,8-PeCDF
C12-2,3,4,7,8-PeCDF
C^2*1,2,3,7,8-PeCDD
0.993 - 1.009
0.993 - 1.009
0.918 - 1.076
0.999 - 1.001
0.987 - 1.016
Labeled Compounds
"c12-2,3,7,8-TCDF
C12-1,2,3,4-TCDD
"c12-2,3,7,8-TCDD
Cl^-2,3,7,8-TCDD
13C12-1,2,3,7,8-PeCDF
"c12-2,3,4,7,8-PeCDF
13C12-1,2,3,7,8-PeCDD
13c
13^2
1312
13c12
1312
«c"
12
13C12-1,2,3,4-TC0D
1,2,3,4-TCDD
1,2,3,4-TCDD
1,2,3,4-TCDD
1,2,3,4-TCDD
1,2,3,4-TCDD
1,2,3,4-TCDD
0.931
1.000
0.993
1.002
1.091
1.123
1.134
0.994
1.000
1.036
1.013
1.371
1.408
1.428
13,
Compounds using
Native Compounds
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
1,2,3,4,7,8-HxCDD
1.2.3.6.7.8-HxCDD
1.2.3.7.8.9-HxCDD
1,2,3,4,6,7,8-HpCDF
1.2.3.4.6.7.8-HpCDD
1.2.3.4.7.8.9-HpCDF
OCDD
OCDF
C12-1,2,3,7,8,9.
Labeled Compounds
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
1,2,3,4,7,8-HxCDD
1.2.3.6.7.8-HxCDD
1.2.3.7.8.9-HxCDD
2#3,4,6,7,8-HpCDF
2,3,4,6,7,8-HpCDD
13C
1312
I3".
13 12
13c12'
I312-
1312-
13C
13 12 '
C«p-1
13 I2
C -1
12 1
2,3,4,7,8,9-HpCDF
c12-ocdd
HxCDD as internal standard
«C
13(
13(
"(
13<
13(
13C
13«
"«
13C
"<
13<
«(
13(
13(
13(
13(
13<
13(
13,
c
2
1
2,3,7,8,9-HxCOD
0.947
0.992
c
2
1
2,3,7,8,9-HxCDD
0.940
1.006
c
2
1
2,3,7,8,9-HxCDD
0.993
1.017
c
2
1
2,3,7,8,9-HxCDD
0.971
1.000
c
2
1
2,3,7,8,9-HxCDD
0.974
1.002
c
2
1
2,3,7,8,9-HxCDD
0.975
1.006
c
2
1
2,3,7,8,9-HxCDD
1.000
1.000
c
2
1
2,3,7,8,9-HxCDD
0.953
1.172
c
2
1
2,3,7,8,9-HxCDD
1.023
1.125
c
2
1
2,3,7,8,9-HxCDD
1.024
1.148
c
2'
1
2,3,7,8,9-HxCDD
1.050
1.275
10
10
50
50
50
0.5
0.5
2.5
2.5
2.5
C
2
1,2,3,4,7,8-HxCDF
0.986
1.015
50
5
2.5
c
2
1,2,3,6,7,8-HxCDF
0.973
1.025
50
5
2.5
c
2
1,2,3,7,8,9-HxCDF
0.937
1.068
50
5
2.5
c
2
2,3,4,6,7,8-HxCDF
0.999
1.001
50
5
2.5
c
2
1,2,3,4,7,8-HxCDD
0.999
1.001
50
5
2.5
c
2
1,2,3,6,7,8-HxCDD
0.992
1.009
50
5
2.5
c
2
1,2,3,6,7,8-HxCDD
0.986
1.016
50
5
2.5
c
2
1,2,3,4,6,7,8-HpCDF
0.930
1.022
50
5
2.5
c
2
1,2,3,4,6,7,8-HpCDD
0.986
1.016
50
5
2.5
(c
2
1,2,3,4,7,8,9-HpCDF
0.896
1.079
50
5
2.5
lc
2
OCDD
0.996
1.005
100
10
5.0
c
2
OCDD
0.995
1.013
100
10
5.0
(1) Level at which the analytical system will give acceptable SICP and calibration.
35
-------
Table 3
DESCRIPTORS, MASSES, M/Z TYPES, AND ELEMENTAL COMPOSITIONS OF THE CDDs AND COFs (1)
Descriptor Accurate m/z Compound Primary
Number m/z (2) Type Elemental Composition (3) m/z?
292.9825
Lock
C7 F11
PFK
303.9016
M
C12 H4 35c14 0
TCOF
Yes
305.8987
M+2
c12 h4 35ci337ci 0
TCDF
315.9419
M
13c12 H4 35c14 0
TCDF(4)
Yes
317.9389
M+2
13c12 h4 35ci3 37Cl O
TCDF(4)
319.8965
M
C12 H4 35c14 °2
TC0D
Yes
321.8936
M+2
C12 H4 35c13 37cl °2
TCDD
327.8847
M
C12 H4 37c14 °2
TCDD(5)
330.9792
QC
C7 F13
PFK
331.9368
M
"C12 H4 35c14 °2
TCDD(4)
Yes
333.9339
M+2
TCDD(4)
375.8364
M+2
C12 35Clj 37ci 0
HxCDPE
339.8597
M+2
C12 h 35cl4 37cl 0
PeCDF
Yes
341.8567
M+4
C12 Hj »Cl, 37Cl2 0
PeCDF
351.9000
M+2
13C12 h3 »Cl4 37ci 0
PeCDF(4)
Yes
353.8970
M+4
13c12 h3 35ci3 37ci2 0
PeCDF(4)
354.9792
Lock
C9 F13
PFK
355.8546
M+2
C12 h3 35CIa 37Cl 02
PeCDD
Yes
357.8516
M+4
C12 «3 ""a 37cI2 °2
PeCDD
367.8949
M+2
% K, 35Cl4 *e, 02
PeC0D(4)
Yes
369.8919
M+4
13 35 t 37 .
12 3 3 2 2
PeCDD(4)
409.7974
M+2
C12 H3 35c16 37(:l 0
HpCOPE
373.8208
M+2
C12 H2 35Clg 37Cl O
HxCDF
Yes
375.8178
M+4
C12 H2 35c14 37c12 0
HxCDF
383.8639
M
13c12 H2 35c16 0
HxCDF(4)
Yes
385.8610
M+2
13c12 h2 35ci5 37Cl O
HxCDF(4)
389.8157
M+2
C12 H2 35c15 37cl °2
HxCDD
Yes
391.8127
M+4
C12 H2 35Cl4 37Cl2 02
HxCDD
392.9760
Lock
C9 F15
PFK
401.8559
M+2
H2 35Cl5 37Cl 02
HxCDD(4)
Yes
403.8529
M+4
13c12 H2 35C14 37ci2 02
HxCDD(4)
430.9729
QC
C9 F13
PFK
445.7555
M+4
C12 H2 35c16 37c12 0
OCDPE
36
-------
DESCRIPTORS,
MASSES, M/Z TYPES,
Table 3 (continued)
AND ELEMENTAL COMPOSITIONS OF
THE CODs AND CDFs (1)
Descriptor
Number
Accurate
m/z (2)
m/z
Type
Elemental Conpositlon
Compound
(3)
Primary
m/z?
4
407.7818
H+2
C12 H 35cl6 37cl 0
HpCOF
Yes
409.7789
M+4
C12 H 35cl5 37c12 0
HpCOF
417.8253
M
13c12 h 35Cl7 0
HpC0F(4)
Yes
419.8220
H+2
13C12 h 35ci6 37Cl 0
HpCDF(4)
423.7766
H+2
C12 H 35cl6 37cl °2
HpCOD
Yes
425.7737
M+4
C12 H 35cl5 37c12 °2
HpCOD
430.9729
Lock
C9 F17
PFK
435.8169
H+2
13c12 H 35Cl6 37Cl 02
HpC0D(4)
Yes
437.8140
M+4
13C12 H 35CIj 37Cl2 02
HpCDD(4)
479.7165
M+4
C12 H 35Cl7 37Cl2 O
NCDPE •
5
441.7428
H+2
C12 35Cl7 37ci 0
OCDF
Yes
442.9728
Lock
C10 F17
PFK
443.7399
M+4
C12 35cl6 37c12 0
OCDF
457.7377
H+2
C12 35Cl7 37Cl 02
OCDD
Yes
459.7348
M+4
C 35Cl 37Cl 0
12 6 2 2
OCDD
469.7779
M+2
13C12 35Cl7 37Cl 02
OCOD(4)
Yes
471.7750
H+4
13C 35cl 37Cl 0
12 6 2 2
OCDD(4)
513.6775
M+4
C 35Cl 37Cl 0
12 8 2
DCDPE
(1) From Reference 5
(2) Nuclidic masses used:
H s 1.007825 C ° 12.00000
0 = 15.994915 35Cl = 34.968853
(3) Compound abbreviations:
Chlorinated dibenzo-p-dioxins
TC0D = Tetrachlorodibenzo-p-dioxln
PeCOO = Pentachlorodibenzo-p-dioxin
HxCOO " Hexachlorodlbenzo-p-dioxin
HpCOD = Heptachlorodibenzo-p-dioxin
OCDD = Octachlorodibenzo-p-dioxin
Chlorinated dibenzofurans
TCDF = Tetrachlorodibenzofuran
PeCDF ° Pentachlorodibenzofuran
HxCOF ° Hexachlorodibenzofuran
HpCOF o Heptachlorodibenzofuran
(4) Labeled compound
(5) There is only one m/z for 37Cl^-2,3,7,8-TCDD (cleanup standard).
13C = 13.003355 F = 18.9984
37Cl = 36.965903
Chlorinated diphenyl ethers
HxCDPE = Hexachlorodiphenyl ether
HpCOPE = Heptachlorodiphenyl ether
OCOPE = Octachlorodiphenyl ether
NCDPE = Nonachlorodiphenyl ether
OCOPE = Decachlorodiphenyl ether
Lock mass and QC compound
PFK = Perfluorokerosene
37
-------
Table 3A
THEORETICAL ION ABUNDANCE RATIOS AND CONTROL LIMITS
No. of
m/z's
Chlorine
Forming
Theoretical
Control
LimitsO)
Atoms
Ratio
Ratio
Lower
Upper
4 (2)
M/M+2
0.77
0.65
0.89
5
M+2/M+4
1.55
1.32
1.78
6
H+2/H+4
1.24
1.05
1.43
6 (3)
H/M+2
0.51
0.43
0.59
7
M+2/M+4
1.05
0.88
1.20
7 (4)
M/M+2
0.44
0.37
0.51
8
M+2/M+4
0.89
0.76
1.02
(1) Represent + 15X windows around the theoretical ion
abundance ratios.
(2) Does not apply to ^Cl^-2,3,7,8-TCDD (cleanup
standard).
(3) Used for ^C-HxCDF only.
(4) Used for ^C-HpCDF only.
38
-------
Table 4
CONCENTRATIONS OF SOLUTIONS CONTAINING LABELED AND UNLABELED PCDDS AND PCDFS --
STOCK AND SPIKING SOLUTIONS
Compound
Labeled
Compound
Stock
Solution (1)
(ng/mL)
Labeled
Compound
Spiking
Solution (2)
(ng/mL)
PAR
Stock
Solution (3)
(ng/mL)
Cleanup
Standard
Spiking
Solution (4)
(ng/mL)
Internal
Standard
Spiking
Solution (5)
(ng/mL)
Native CDDs and CDFs
2,3,7,8-TCDD
2,3,7,8-TCOF
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-HxCOD
1.2.3.7.8.9-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
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
Labeled CDDs and CDFs
13C12-2,3,7,8-TCDD
13
13i
13,
13-
13C12-2,3,7,8-TCDF
C,2"1»2,3,7,8-PeCDD
C12-1,2,3,7,8-PeCDF
C12-2,3,4,7,8-PeCDF
C12-1,2,3,4,7,8-HxCDD
13C12-1,2,3,6,7,8-HxCDD
13C12-1,2,3,4,7,8-HxCDF
13C12*1,2,3,6,7,8-HxCDF
13C^2*1,2,3,7,8,9-HxCDF
13C12-2,3,4,6,7,8-HxCDF
13C12-1,2,3,4,6f7,8-HpCDD
13C12-l,2,3,4,6,7,8-HpCDF
13C12-1,2,3,4,7 8,9-HpCDF
13c12-ocdd
Cleanup Standard
37Cl4-2,3,7,8-TCDD
Internal Standards
13C12-1,2,3,4-TCDD
13
C12-1,2,3,7,8,9-HxCDD
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
40
40
200
200
200
200
200
200
200
200
200
200
200
200
200
400
400
0.8
200
200
(1) Section 6.10 - prepared in nonane and diluted to prepare spiking solution.
(2) Section 10.3.2 - prepared from stock solution daily.
(3) Precision and Recovery (PAR) standard, Section 6.14 - prepared in nonane and diluted to prepare spiking
solution in Section 10.3.4.
(4) Section 6.11 - prepared in nonane.
(5) Section 6.12 - prepared in nonane.
39
-------
Table 4 (continued)
CONCENTRATIONS OF SOLUTIONS CONTAINING LABELED AND UNLABELED PCDDS AND PCDFS
CALIBRATION AND VERIFICATION SOLUTIONS
Compound
CS1
(ng/mL)
CS2
(ng/mL)
VER(6)
CS3
(ng/mL)
CS4
(ng/mL)
CS5
(ng/mL)
Native CDDs and CDFs
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-HxCOD
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
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
Labeled CDDs and CDFs
13C12-2,3,7,8-TCDD
13C12-2,3,7,8-TCDF
13C12-1,2,3,7,8-PeCDD
]3C12-1,2,3,7,8-PeCDF
C12-2,3,4,7,8-PeC0F
C12-1,2,3,4,7,8-HxCDD
13C12-1,2,3,6,7,8-HxCDD
13C12-1,2,3,4,7,8-HxCDF
13C12-1,2,3,6,7,8-HxCDF
13C12-1,2,3,7,8,9-HxCDF
13C12-2,3,4,6,7,8-HxCDF
]3C12-1,2,3,4,6,7,8-HpCDD
3C12-1,2,3,4,6,7,8-HpCDF
13C12-1,2,3,4,7 8,9-HpCDF
c12-ocdd
Cleanup Standard
37Cl4-2,3,7,8-TCDD
Internal Standards
13C12-1,2,3,4-TCDD
13,
C12-1,2,3,7,8,9-HxCDD
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
100
200
0.5
100
100
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
100
200
100
100
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
100
100
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
100
200
40
100
100
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
100
200
200
100
100
(6) Section 14.3 - calibration verification (VER) solution.
40
-------
Table 5
GC RETENTION TIME WINDOW DEFINING STANDARD MIXTURES AND
ISOMER SPECIFICITY TEST STANDARD MIXTURES
DB-5 Colimn GC Retention Time Window Defining Standard
(Section 6.15)
Congener First Eluted last Eluted
TCDF 1,3,6,8- 1,2.8,9-
TCDD 1,3,6,8- 1.2,8,9-
PeCDF 1,3,4,6,8- 1,2,3,8,9-
PeCDD 1,2,4,7,9- 1,2,3,8,9-
HxCDF 1,2,3,4,6,8- 1,2,3,4,8,9-
HxCDD 1,2,4,6,7,9- 1,2,3,4,6,7-
HpCDF 1,2,3,4,6,7,8- 1,2,3,4,7,8,9-
HpCDD 1,2,3,4.6.7,9- 1,2,3,4,6,7,8-
DB-5 TCDD Isomer Specificity Test Standard
(Section 6.16.1)
D8-225 Column TCDF Isomer Specificity Test Standard
(Section 6.16.2)
1,2,3,4-TCDD
1,2,7,8-TCDD
1,4,7,8-TCDD
1.2.3.7-TCOD
1.2.3.8-TCDD
2,3,7,8-TCDD
2.3.4.7-TCDF
2.3.7.8-TCDF
1.2.3.9-TCDF
-------
Table 6
REFERENCE COMPOUNDS FOR QUANTITATION OF NATIVE AND LABELED PCDDS AND PCDFS
Native PCODs and PCDFs
Reference Compound
Labeled PCODs and PCDFs
Reference Compound
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
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
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
13
13
13
13
13
13
13
C12-2,3,7,8-TCDD
C12-2,3,7,8-TCDF
C12-1,2,3,7,8-PeCDD
|c12-1,2,3,7,8-PeCDF
C12-2,3,4,7,8-PeCDF
13
13
13
13
C12-1,2,3,6,7,8-HxC0D
<1)
C^2"1,2,3,4,7,8-HxCDF
C12-1,2,3,6,7,8-HxCDF
'c^2" 1,2,3,7,8,9-HxCDF
!C12-2,3,4,6,7,8-HxCDF
13
13
13
13
13
C12-2,3,7,8-TCDD
^C^2-2,3,7,8-TCDF
C12-1,2,3,7,8-PeCDD
^12"1,2,3,7,8-PeCDF
lC12-2,3,4,7,8-PeCDF
'c12-1,2,3,4,7,8-HxCDD 13C-2-1,2,3,4,7,8-HxCDD
13
13
13
13
12
'c12"1,2,3,6,7,8-HxCDD
'c12-1,2,3,7,8,9-HxCDD
C^2*1,2,3,4,7,8-HxCDF
C12-1,2,3,6,7,8-HxCDF
13C12-1,2,3f4,6,7,8-HpCDD
13
13
1 f 2,3,7,8,9-HxCOF
13C12"2,3,4,6,7,8-HxCDF
^2-1,2,3,4,6,7.8-HpCDD
13
Ci2*1'2,3.4,7,8,9-HpCDF
13
C---OCDD
13
ijc12-ocdd
13
12
C1?-1,2,3,4,7,8,9-HpCDF
c12-ocdd
Ct4-2,3,7,8-TCDD
13
37
13
13,
13,
13,
13
13r
13(
13(
13,
13,
13,
13,
13,
13C12*1,2,3,4,6,7,8-HpCDF 13C12*1,2,3,4,6,7,8-HpCDF 13C
13(
13,
'12
'12
'12
'12
'12
=12
'12
'12
'12
'12
C12-1,2,3,4-TCDD
C12-1,2,3,4-TCOD
C12-1,2,3,4-TCOD
C12-1,2,3,4-TCDD
C12-1,2,3,4-TCDD
1,2,3,7,8,9-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,7,8,9-HxCDD
C^2*1,2,3,7,8,9-HxCDD
13C12-1,2.3,4-TCDD
(1) 1,2,3,7,8,9-HxCDD is quantified using the average responses for the 13C12-1,2,3,4,7,8-HxCDD and 1JC
1,2,3,6,7,8-HxCDD.
12
42
-------
Table 7
ACCEPTANCE CRITERIA FOR PERFORMANCE TESTS
Test
I PR (2)
Compound
Cone. (1)
(ng/mL)
s
(ng/mL)
X
(ng/mL)
0PR(2)
(ng/mL)
VER
(ng/mL)
2,3,7,8-TCDD
10
1.5
3.9 -
20.6
5.9
14.2
8.6
11.6
2,3,7,8-TCDF
10
2.0
3.2 -
26.8
6.6
12.7
8.8
- 11.3
1,2,3,7,8-PeCDD
50
4.2
47.5 -
50.5
35.6
58.1
44.2
- 56.6
1,2,3,7,8-PeCDF
50
4.6
44.2 -
54.0
36.7
57.3
46.7
- 53.5
2,3,4,7,8-PeCDF
50
4.2
45.3 -
50.3
37.8
56.9
47.2
53.0
1,2,3,4,7,8-HxCOD
50
5.5
30.9 -
70.2
35.1
60.4
37.6
- 66.5
1,2,3,6,7,8-HxCDD
50
5.5
33.2 -
65.9
33.3
64.4
39.7
- 63.0
1,2,3,7,8,9-HxCDD
50
9.5
22.7 -
90.9
31.8
61.2
42.6
58.7
1,2,3,4,7,8-HxCDF
50
6.3
25.2 -
92.0
36.9
58.8
41.5
- 60.2
1,2,3,6,7,8-HxCDF
50
4.0
39.1 -
54.4
34.8
58.8
40.5
- 61.7
1,2,3,7,8,9-HxCDF
50
4.0
37.9 -
62.9
37.1
55.7
45.7
54.5
2,3,4,6,7,8-HxCDF
50
5.0
27.4 -
85.5
35.7
60.0
44.1
• 56.7
1,2,3,4,6,7,8-HpCDD
50
6.4
27.4 -
76.5
37.5
56.8
41.6
- 60.2
1,2.3,4,6,7,8-HpCDF
50
3.6
39.5 -
62.1
37.4
60.5
43.1
58.0
1,2,3,4,7,8,9-HpCDF
50
4.2
36.6 -
64.9
36.9
60.6
43.6
- 57.3
OCDD
100
13.0
69.4 -
154.6
75.6
118.7
87.5
- 114.4
OCDF
100
45.0
46.1 -
139.8
69.5
127.0
83.9
- 119.2
13C12-2,3,7,8-TC0D
13Cl2-2,3,7,8-TCDF
100
100
-
25.0 -
25.0 -
150.0
150.0
25.0
25.0
150.0
150.0
90.0
87.7
- 111.2
- 114.0
3C12-1,2,3,7,8-PeCDD
100
-
25.0 -
150.0
25.0
150.0
80.6
- 124.0
3C12-1,2,3,7,8-PeC0F
100
-
25.0 -
150.0
25.0
150.0
81.8
- 122.3
3C12-2.3,4,7,8-PeCDF
100
-
25.0 -
150.0
25.0
150.0
83.0
- 120.5
12*1,2,3,4,7,8-HxCDO
100
-
25.0 -
150.0
25.0
150.0
76.1
- 131.3
12-1,2,3,6,7,8-HxCDD
100
-
25.0 -
150.0
25.0
150.0
84.0
- 119.1
12-1,2,3,4,7,8-HxCOF
100
-
25.0 -
150.0
25.0
150.0
85.2
- 117.4
12*1,2,3,6,7,8-HxCDF
100
-
25.0 -
150.0
25.0
150.0
85.0
- 117.7
12-1,2,3,7,8,9-HxCDF
100
-
25.0 -
150.0
25.0
150.0
89.5
- 111.7
¦j2* 2,3,4,6,7,8-HxCDF
100
-
25.0 -
150.0
25.0
150.0
85.7
- 116.7
' 1,2,3,4,6,7,8-HpCDD
100
-
25.0 -
150.0
25.0
150.0
82.2
- 121.6
-1.2.3,4,6,7,8-HpCOF
100
-
25.0 -
150.0
25.0
150.0
88.5
- 113.1
-1,2,3,4,7,8,9-HpCDF
100
-
25.0 -
150.0
25.0
150.0
89.0
- 112.4
13c12-ocdd
37Cl4-2,3,7,8-TCDD
200
10
-
50.0 -
2.5 -
300.0
15.0
50.0
2.5
300.0
15.0
164.2
6.1
- 243.6
11.6
13,
13,
'12
"12
"12
(1) All specifications are given as concentrations in the final extract or standard solution.
(2) s = standard deviation of the concentration; X = average concentration. Concentration limits for labeled
compounds in IPR and OPR aliquots are based on requirements for labeled compound recovery of 25-150X (Sections
8.2.3 and 14.5.3).
43
-------
Table 8
SAMPLE PHASE AND QUANTITY EXTRACTED FOR VARIOUS MATRICES
Sample Matrix (1)
Example
Percent
Solids
Phase
Quantity
Extracted
SINGLE PHASE
Aqueous
Solid
Organic
MULTIPHASE
Liquid/Solid
Aqueous/solid
Organic/solid
Liquid/Liquid
Aqueous/organi c
Aqueous/organ ic/
solid
Drinking water
Groundwater
Treated wastewater
Dry soil
Compost
Ash
Waste solvent
Waste oiI
Organic polymer
Wet soiI
Untreated effluent
Digested municipal sludge
Filter cake
Paper pulp
Tissue
Industrial sludge
Oily waste
In-process effluent
Untreated effluent
Drun waste
Untreated effluent
Drun waste
>20
1-30
1-100
<1
>1
(2)
Solid
Organic
Solid
Both
Organic
Organi c
& solid
1000 mL
10 g
10 g
10 g
10 g
10 g
10 g
(1) The exact matrix may be vague for some samples. In general, when the CDDs and 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.
(2) Aqueous samples are filtered after spiking with labeled analogs. The filtrate and the material trapped on
the filter are extracted separately, and then the extracts are combined for cleanup arid analysis.
44
------- |