EPA
United States Environmental Protection Agency
Office of Water Regulations and Standards
Industrial Technology Division
Office of Water July 1989
Method 1613: Tetra-through
Octa- Chlorinated Dioxins and
Furans by Isotope Dilution
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Introduction
Method 1613 was developed by the Industrial Technology
Division (ITD) within the United States Environmental
Protection Agency's (USEPA) Office of Water Regulations and
Standards (OURS) 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
standards setting limits on pollutant levels in industrial
discharges.
Method 1613 is a high resolution capillary column gas
chromatography (HRGO/high resolution mass spectrometry (HRMS)
method for analysis of tetra- through octa- chlorinated
dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) using
isotope dilution. Specificity is provided for determination
of the 2,3,7,8- substituted isomers of tetrachlorodibenzo-p-
dioxin (2,3,7,8-TCDD) and tetrachlorodibenzofuran (2,3,7.8-
TCOF).
I
Questions concerning the method or its application should be
addressed to:
U. A. Tel Hard
USEPA
Office of Water Regulations and Standards
401 M Street SW
Washington. DC 20460
202/382-7131
OR
USEPA OWRS
Sample Control Center
P.O. Box 1407
Alexandria, Virginia 22313
703/557-5040
Publication date: July 1989
Printed on Recycled Paper
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Method 1613 July 1989
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 (PCDDs) and dibenzofurans (PCOFs)
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 resolution
' capillary column gas chromatography
(HRGC)/high resolution mass spectrometry
(HRMS). Specificity is provided for
determination of the 2,3,7,8- substituted
isomers of tetrachlorodibenzo-p-dioxin
(2,3,7,8-TCDD) and tetrachlorodibenzofuran
(2,3,7,8-TCDF).
1.2 The method is based on EPA, industry,
commercial 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
interferences rather than instrumental
limitations. The levels in Table 2 typify
the minimum quantities that can be
detected with no interferences present.
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
laboratory that uses this method must
demonstrate the ability to generate
, acceptable results using the procedure in
1 Section 8.2.
2 SUMMARY OF METHOD
2.1 Stable isotopically labeled analogs of 16
of the PCDDs and PCDFs are added to each
sample. Samples containing coarse solids
are prepared for extraction by grinding or
homogenization. Water samples are
filtered and then extracted with methylene
chloride using separator/ funnel
procedures; the participates from the
water samples, soils, and other finely
divided solids are extracted using a
combined Soxhlet extraction/Dean-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.
Samples cleanup may include back
extraction with acid and/or base, and gel
permeation, alumina, silica gel, and
activated carbon chromatography. High
performance liquid chromatography (HPLC)
can be used for further isolation of the
2,3,7,8- isomers or other specific isomers
or congeners.
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
resolution (>10,000) mass spectrometer.
The labeled compounds serve to correct for
the variability of the analytical
technique.
2.4 Dioxins and furans are identified by
comparing GC retention time ranges 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 time ranges and m/z
abundance ratios agree within pre-defined
limits. By using a GC column or columns
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: 1) For the
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16 2.3,7,8-substituted isomers for which
labeled analogs are available (see Table
1), the GCMS system is calibrated and the
compound concentration is determined using
an isotope dilution technique; 2) For non-
2,3, 7,8-substituted isomers and the total
concentrations of all isomers within a
level of chlorination (i.e., total TCDO),
concentrations are determined assuming
response factors from the calibration of
labeled analogs at the same level of
chlorination. Although a labeled analog
of the octachlorinated dibenzofuran (OCDF)
is available, using high resolution mass
spectrometry, it produces an m/z that may
interfere with the identification and
quantisation of the native octachlorinated
dibenzo-p-dioxin (OCOD). Therefore, this
labeled analog has not been included in
the calibration standards, and the native
OCDF is quantitated against the labeled
OCDD.
2.6 The quality of the analysis is assured
through reproducible calibration and
testing of the extraction, cleanup, and
GCMS systems.
3 CONTAMINATION AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other
sample processing hardware may yield
artifacts and/or elevated , baselines
causing misinterpretation of chromatograms
(References 8-9). Specific selection of
reagents and purification of solvents by
distillation in all-glass systems may be
required. Where possible, reagents are
cleaned by extraction or solvent rinse.
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.
Sonication of glassware containing a
detergent solution for approximately 30 s
may aid in cleaning.
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, and then acetone, and methylene
chloride.
3.2.3 Do not bake reusable glassware in an oven.
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
extraction 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 for 2 minutes.
3.3 All materials used in the analysis shall
be demonstrated to be free from
interferences 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). 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 6.5.4) 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
encountered interferences are chlorinated-
biphenyls, methoxy biphenyls,
hydroxydiphenyl ethers, benzylphenyl
ethers, polynuclear aromatics, and
pesticides. Because very low levels of
PCDDs and PCDFs are measured by this
method, the elimination of interferences
is essential. The cleanup steps given in
Section 12 can be used to reduce or
eliminate these interferences and thereby
permit reliable determination of the PCDDs
and PCDFs the at levels shown in Table 2.
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 compounds should be
reduced to the lowest possible level.
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4.1.1 The 2,3,7,8-TCDD isomer has been found to
be acnegenic, carcinogenic, and
teratogenic in laboratory animal studies.
It is soluble in water to approximately
200 parts-per-trillion 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
purchase dilute standard solutions of the
analytes in this method. However, if
primary solutions are prepared, they shall
be prepared in a hood, and a NIOSH/MESA
approved toxic gas respirator shall be
worn when high concentrations are handled.
4.2 The laboratory is responsible for
maintaining a current awareness file of
OSHA regulations regarding the safe
handling of the chemicals specified in
this method. A reference file of 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 comprehensive 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 as
those employed in handling radioactive or
infectious materials. Well-ventilated,
controlled access laboratories are
required. Assistance in evaluating the
health hazards of particular laboratory
conditions may be obtained from certain
consulting laboratories and from State
Departments of Health or of Labor, many of
which have an industrial health service.
The PCDDs and PCDFs are extremely toxic to
laboratory animals. However, they have
been handled for years without injury in
analytical and biological laboratories.
Each laboratory must develop a strict
safety program for handling the PCDDs and
PCDFs. The following laboratory practices
are recommended (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 fume hood demonstrated to
have adequate air flow. Gross losses to
the laboratory ventilation system must not
be allowed. Handling of the dilute
solutions normally used in 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. 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.
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/MS should pass
through either a column of activated
charcoal or be bubbled through a trap
containing oil or high-boiling alcohols.
4.3.7 Waste
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.
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4.3.7.2 Disposal
4.3.7.2.1 The PCDDs and PCDFs decompose above 800
°C. Lou-level waste such as absorbent
paper, tissues, animal remains, and
plastic gloves may be burned in an
appropriate incinerator. Gross quantities
(milligrams) should be packaged securely
and disposed through commercial or
governmental channels which are capable of
handling extremely toxic wastes.
4.3.7.2.2 Liquid or soluble waste should be
dissolved in methanol or ethanol and
irradiated with ultraviolet light with a
wavelength greater than 290 nm 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 Personal decontamination -- Use any mild
soap with plenty of scrubbing action.
4.3.8.2 Glassware, tools, and surfaces
Chlorothene NU Solvent (Trademark of the
Dow Chemical Company) is the least toxic
solvent shown to be effective.
Satisfactory cleaning may be accomplished
by rinsing with Chlorothene, then washing
with any detergent and water. If
glassware is first rinsed with solvent,
then the dish water may be disposed of in
the sewer. Given the cost of disposal, it
is prudent to minimize solvent wastes.
4.3.9 Laundry -- Clothing known to be
contaminated should be collected in
plastic bags. Persons who convey the bags
and launder the clothing should be advised
of the hazard and trained in proper
handling. The clothing may be put into a
washer without contact if the launderer
knows of the potential problem. The
washer should be run through a cycle
before being used again for other
clothing.
4.3.10 Wipe tests -- A useful method of
determining cleanliness of work surfaces
and tools is to wipe the surface with a
piece of filter paper. Extraction and
analysis by GC 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 in the past.
4.3.11 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.
5 APPARATUS AND MATERIALS
5.1 Sampling equipment for discrete or
composite sampling.
5.1.1 Sample bottles and caps
5.1.1.1 Liquid samples (waters, sludges and
similar materials that contain less than
five percent solids) -- Sample bottle,
amber glass, 1.1 liters 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.
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 minimum
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 pump, a minimum length
of compressible silicone rubber tubing may
be used in the pump only. Before use, the
tubing shall be thoroughly rinsed with
methanol, followed by repeated rinsings
with reagent water to minimize sample
contamination. An integrating flow meter
is used to collect proportional composite
samples.
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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 Dessicator
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 vacuum 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 (SOS) extractor
(Figure 1)
i
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).
FIGURE 1 Soxhlet/Dean-Stark Extractor
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 or
baked at 450 °C for four hours minimum.
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5.5.2 Glass funnel -- 125 - 250 mL
5.5.3 Glass fiber filter paper (Whatman GF/D, or
equivalent)
5.5.A Drying column -- 15 to 20 mm 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 -- Hillipore
YT30 142 HW, 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 Cleanup apparatus
5.7.1 Automated gel permeation chromatograph
(Analytical Biochemical Labs, Inc,
Columbia, MO, Model GPC Autoprep 1002, or
. equivalent).
5.7.1.1 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).
5.7.1.2 Syringe, 10 mL, with Luer fitting.
5.7.1.3 Syringe filter holder, stainless steel,
and glass fiber or Teflon filters (Gelman
4310, or equivalent).
5.7.1.4 UV detectors -- 254-mu, preparative or
semi-prep flow cell: (Isco, Inc., Type 6;
Schmadzu, 5 mm path length; Beckman-Altex
152W, 8 uL micro-prep flow cell, 2 mm
path; Pharmacia UV-1, 3 mm flow cell; LDC
Milton-Roy UV-3, monitor #1203; or
equivalent).
5.7.2 Reverse phase high performance liquid
chromatograph
5.7.2.1 Column oven and detector -- Perkin-Elmer
Model LC-65T (or equivalent) operated at
0.02 AUFS at 235 nm.
5.7.2.2 Injector -- Rheodyne 7120 (or equivalent)
with 50 uL sample loop.
5.7.2.3 Column -- Two 6.2 x 250 mm Zorbax-ODS
columns in series (DuPont Instruments
Division, Wilmington, DE, or equivalent),
operated at 50 °C with 2.0 mL/min methanol
isocratic effluent.
5.7.2.4 Pump -- Altex 110A (or equivalent).
5.7.3 Pipets
5.7.3.1 Disposable, Pasteur, 150 mm x 5 mm i.d.
(Fisher Sceintific 13-678-6A, or
equivalent).
5.7.3.2 Disposable, serological, 10 mL (6 mm
i.d.).
5.7.4 Chromatographic columns
5.7.4.1 150 mm x 8 mm i.d., (Kontes K-420155, or
equivalent) with coarse glass frit or
glass wool plug and 250 mL reservoir.
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 pump and chiller are
recommended, as use of tap water for
cooling the evaporator wastes large
volumes of water and can lead to
inconsistent performance as water
temperatures and pressures vary.
5.8.1.3 Round bottom flask -- 500 mL or larger,
with ground glass fitting compatible with
the rotary evaporator.
5.8.2 Nitrogen blowdown apparatus -- Equipped
with water bath controlled at 35 - 40 °C
(N-Evap, Organomation Associates, Inc.,
South Berlin, MA, or equivalent),
installed in a fume hood.
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5.8.3
5.9
Sample vials -- Amber glass, 2
Teflon-lined screw cap.
5.9.1
5.9.2
5.10
5.10.1
5.10.2
5.11
Gas chromatograph -- Shall have split less
or on-column injection port for capillary
column, temperature program with
isothermal hold, and shall meet all of the
performance specifications in Section 14.
GC Column for PCDDs and PCDFs and for
isomer specificity for 2,3,7,8-TCDO -- 60
±5 m x 0.32 ±0.02 mm i.d.; 0.25 urn 5X
phenyl, 94X methyl, 1X vinyl silicone
bonded phase fused silica capillary column
(J & W OB-5, or equivalent).
GC Column for isomer specificity for
2,3,7,8-TCDF -- 30 ±5 m x 0.32 tO.02 mm
i.d.; 0.25 urn bonded phase fused silica
capillary column (J & W DB-225, or
equivalent).
Mass spectrometer -- 28 - 40 eV electron
impact ionization, shall repetitively
selectively monitor 11 exact m/z's minimum
at high resolution (>10,000) during a
period of approximately 1 second.
The groups of m/z's to be monitored are
shown in Table 3. Each group or
descriptor shall be monitored in
succession as a function of GC retention
time to ensure that all PCODs and PCOFs
are detected. The theoretical abundance
ratios for the m/z's are given in Table
3A, along with the control limits of each
ratio.
The mass spectrometer shall be operated in
a mass drift correction mode, using
perfluorokerosene (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.
Variations 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.
GC/MS interface -- The mass spectrometer
shall be interfaced to the GC such that
the end of the capillary column terminates
within 1 cm of the ion source but does not
5 ml with intercept the electron or ion beams. All
portions of the column which connect the
GC to the ion source shall remain at or
above the column temperature during
analysis to preclude condensation of less
volatile compounds.
5.12 Data system -- Shall collect and record
and store MS data.
5.12.1 Data acquisition -- The signal at each
exact m/z shall be collected repetitively
throughout the monitoring period and
stored on a mass storage device.
5.12.2 Response factors and multipoint
calibrations -- The data system shall be
used to record and maintain lists of
response factors (response ratios for
isotope dilution) and multi-point
calibration curves. Computations of
relative standard deviation (coefficient
of variation) are used to test calibration
linearity. Statistics on initial (Section
8.2) and ongoing (Section U.5)
performance ' shall be computed and
maintained.
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 SuIfuric acid -- Reagent grade (specific
gravity 1.84).
6.1.3 Sodium chloride -- Reagent grade, prepare
a five percent (w/v) solution in reagent
water.
6.2 Solution drying and evaporation
6.2.1 Solution drying -- Sodium sulfate, reagent
grade, granular anhydrous (Baker 3375, or
equivalent), rinsed with methylene
chloride (20 ml/g), baked at 400 °C for
one hour minimum, cooled in a dessicator,
and stored in a pre-cleaned glass bottle
with screw cap that prevents moisture from
entering.
6.2.2 Prepurified nitrogen
6.3 Solvents -- Acetone, toluene, cyclohexane,
hexane, nonane, methanol, methylene
chloride, and nonane: distiI led-in-glass,
pesticide quality, lot certified to be
free of interferences.
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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 -
6.5.3.1 Activated carbon --
Development Company,
equivalent). Prewash
dry in vacuo at 110 °C.
AX-21 (Anderson
Adrian, MI, or
with met Hanoi and
131-1340, or
with methylene
°C for one hour
dessicator, and
200 mesh (Bio-Rad
equivalent), rinsed
chloride, baked at 250
minimum, cooled in a
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 4.4 g of concentrated
sulfuric acid with 10.0 g activated silica
gel. 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. 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
6.5.2.1 Neutral alumina -- Bio-Rad Laboratories
132-1140 Neutral Alumina Ag 7 (or
equivalent). Heat to 600 °C for 24 hours
minimum. Store at 130 °C in a covered
flask. Use within five days of baking at
600 "C.
6.5.2.2 Acid alumina -- Bio-Rad Laboratories 132-
1340 Acid Alumina AG 4 (or equivalent).
Activate by heating to 130 °C for 12 hours
minimum.
6.5.2.3 Basic alumina -- Bio-Rad Laboratories 132-
1240 Basic Alumina AG 10 (or equivalent).
Activate by heating to 600 °C for 24 hours
minimum. Alternatively, activate by
heating alumina in a tube furnace at 650 -
700 °C under an air flow of approximately
400 cc/min. To avoid melting the alumina,
do not heat over 700 °C. Store at 130 °C
in a covered flask. Use within five days
of baking.
6.5.3 AX-21/Celite
6.5.3.2
6.5.3.3
Celite 545
equivalent).
(Supelco 2-0199, or
Thoroughly mix 5.35 g AX-21 and 62.0 g
Celite 545 to produce a 7.9% w/w mixture.
Activate the mixture at 130 °C for six
hours minimum. Store in a dessicator.
6.5.4 White quartz sand, 60/70 mesh -- For
Soxhlet/Dean-Stark extraction, (Aldrich
Chemical Co, Milwaukee UI Cat No.
27,437-9, or equivalent). Bake at 450 "C
for four hours minimum.
6.6 Reference matrices
6.6.1 Reagent water -- Water in which the PCDDs
and PCDFs and interfering compounds are
not detected by this method.
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
equivalent) glass fiber filter paper in
which the PCDDs and PCDFs and interfering
compounds 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
solutions or mixtures with certification
to their purity, concentration, and
authenticity, or prepared from materials
of known purity and composition. If
8
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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
solutions (Cambridge Isotope Laboratories,
Cambridge, MA, or equivalent). Observe
the safety precautions in Section 4, and
the recommendation in Section 4.1.2.
6.8.2
6.8.3
6.9
6.10
6.11
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 TCDO is completely dissolved, transfer
the solution to a clean 15 ml vial with
Teflon-lined cap.
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.
Secondary standard -- Using stock
solutions (Section 6.8), prepare secondary
standard solutions containing the
compounds and concentrations shown in
Table 4 in nonane.
Labeled compound spiking standard -- From
stock standard solutions prepared as
above, or from purchased mixtures, prepare
this standard to contain the labeled
compounds at the concentrations shown in
Table 4 in nonane. This solution is
diluted with acetone prior to use (Section
10.3.2).
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
concentration shown in Table 4 in nonane.
6.13 Calibration standards (CS1 through CSS) --
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
relative response (labeled to unlabeled)
and response factor to be measured as a
function 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 recovery. This solution contains the
analytes and labeled compounds at the
concentrations 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
solutions -- Used to define the beginning
and ending retention times for the dioxin
and furan isomers.
6.15.1 DB-5 column window defining standard --
Cambridge Isotope Laboratories ED-1732-A,
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 DB-5 column -- 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
7.1.2 Obtain a selected ion current profile of
each analyte in Table 4 at the 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.
7.2 The ion abundance ratios, minimum levels,
and absolute retention times -- Inject the
CS1 calibration solution (Table 4) per the
procedure in Section 13 and the conditions
in Table 2.
7.2.1 Measure the selected ion current profile
(SICP) areas for each analyte and compute
the ion abundance ratios specified in
Table 3. Compare the computed ratio to
the theoretical ratio given in Table 3.
7.2.2 All PCDDs 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; otherwise,
the mass spectrometer shall be adjusted
and this test repeated until the minimum
levels in Table 2 are met.
7.2.4 The retention times of C12-1,2,3,4-TCDD
and C12-1,2,3,7,8,9-HxCDF (the internal
standards, Section 6.12) shall exceed 27
and 38 minutes, respectively, on the DB-5
column, and the retention time of C._-
1,2,3,4-TCDD shall exceed 17 minutes on
the DB-225 column; otherwise, the GC
temperature program shall be adjusted and
this test repeated until the 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 - 20).
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
recalibrate (Section 7.2 through 7.4).
7.5 Calibration with isotope dilution
Isotope dilution is used when 1) labeled
compounds are available, 2) interferences
do not preclude its use, and 3) the SICP
area for the analyte at the exact m/z
(Table 3) is in the calibration range for
the analyte. The reference compound for
each native and labeled compound is shown
in Table 6. Alternate labeled compounds
and quant i tat ion m/z's may be used based
on availability. If any of the above
conditions preclude isotope dilution, the
internal standard method (Section 7.6) is
used.
7.5.1 A calibration curve encompassing the
concentration range is prepared for each
compound to be determined. The relative
response (native to labeled) vs
concentration in standard solutions is
plotted or computed using a linear
regression. Relative response (RR) is
determined according to the procedures
described below. A minimum of five data
points are employed for calibration.
10
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6-MAY-88 Sir: Voltage 705 Sys: DB5US
Sample 1 Injection 1 Group 2 Mass 303.9016
100
80
60
40
20
1,3,6,8-TCDF
Norm: 3044
1,2,8,9-TCDF
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
100
80
60
40
20
1,3,6,8-TCDD
Norm: 481
1,2,8.9-TCDD
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
11
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6-MAY-88 Sir: Voltage 705 Sys: DB5US
Sample 1 Injection 1 Group 2 Mass 339.8597
100
80
60-
40
20-
Norm: 652
1,3,4,6,8-PeCDF
1,2,3,8,9-PeCDF
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
100
80
60
40
20
1,2,4,7,9-PeCDD
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
12
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6-MAY-88 Sir: Voltage 705 Sys: DB5US
Sample 1 Injection 1 Group 3 Mass 373.8208
100
80-
60'
40-
20-
Norm: 560
1,2,3,4,6,8-HxCDF
1,2,3,4,8,9-HxCDF
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
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
13
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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
\s
Norm: 336
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-HpCDD
lOOn
80
60
40
20
0
Norm: 282
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-I
0
OCDF
Norm: 13
45:20 46:40 48:00 49:20 50:40 52:00 53:20 54:40 56:00 57:20
Sample 1
100
80
60
40
20
0
6-MAY-88 Sir: Voltage 705 Sys: DB5US
Injection 1 Group 4 Mass 457.7377
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
14
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3A DB225 Column
21-APR-88 Sir: Voltage 705 Sys: DB225
Sample 1 Injection 1 Group 1 Mass 305.8987
Text: COLUMN PERFORMANCE
100
80
60
40
20
2.3,7,8-TCDF
2,3,4,7-TCDF
Norm: 3466
1,2,3,9-TCDF
16:1016:20^6:30 16:40 16:50 17:00 17:10 17:20 17:30 17:40 17:50 18:00
3B DBS Column
FIGURE 3 Valley between 2,3,7,8- Tetra Dioxin and Furan Isomers and Other Closely Eluted Isomers
15
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7.5.2 The relative response of a PCDO or PCDF to
its labeled analog is determined from
isotope ratio ' values computed from
acquired data. Three isotope ratios are
used in this process:
Rx = the isotope ratio measured for the
pure pollutant.
Ry = the isotope ratio measured for the
labeled compound.
Rm = the isotope ratio, of an analytical
mixture of pollutant and labeled
compounds .
The m/z's are selected such that Rx > Ry.
If Rm is not between 2Ry and O.SRx, the
method does not apply and the sample is
analyzed by the internal standard method.
7.5.4
7.5.3
When there is no overlap between the GC
peaks or the quant i tat ion m/z's, as occurs
with nearly all of the PCODs and PCDFs and
their respective labeled analogs, the RR
is calculated per the following:
Rx = [area m1/z]
i
at the retention time of
native compound.
the
Ry = 1
[area m2/z]
at the retention time of the
labeled compound (RT2).
Rm = [area at m1/z (at RT2)]
[area at m2/z (at RT1)]
as measured in the mixture of the
native and labeled compounds
(Figure 4) (RT1).
AREA AT
M,(Z
FIGURE 4 Selected Ion Current Profiles for
Chromatographically Resolved Labeled (m.2/z)
and Unlabeled (m-j/z) Pairs.
7.5.5
7.6
7.6.1
7.6.1.1
7.6.1.2
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 RR at each
concentration.
Linearity -- If the ratio of relative
response to concentration for any compound
is constant (less than 20 percent
coefficient of variation) over the 5-point
calibration range, an averaged relative
response/concentration ratio may be used
for that compound; otherwise, the complete
calibration curve for that compound shall
be used over the 5-point calibration
range.
Calibration by internal standard -- The
internal standard method is applied to
determination of compounds having no
labeled analog, and to measurement of
labeled compounds for intra- laboratory
statistics (Sections 8.4 and 14.5.4).
Response factors -- Calibration requires
the determination of response factors (RF)
defined by the following equation:
RF
(A
Cis>
(Ais x V
where,
A is the area of the exact m/z for the
compound in the calibration standard.
A. is the area of the exact m/z for the
internal standard.
Cis
is the concentration of the GCHS
internal standard (Section 6.12 and Table
4) in pg/uL.
C is the concentration of the compound in
the calibration standard in pg/uL.
The response factor is determined for at
least five concentrations appropriate to
the response of each compound (Section
6.13); nominally, 0.5, 2, 10, 40, and 200
ng/mL. The amount of internal standard
added to each calibration solution and
extract is the same (100 ng/mL) so that
Cis remains constant. The RF is plotted
vs concentration for each compound in the
standard (Cs) to produce a calibration
curve.
Linearity -- If the response factor (RF)
for any compound is constant (less than 35
16
-------
percent coefficient of variation) over the
5-point calibration range, an averaged
response factor may be used for that
compound; otherwise, the complete
calibration curve for that compound shall
be used over the 5-point range.
7.7 Combined calibration -- By using
calibration 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 U.3) by analyzing the
calibration verification standard (VER,
Table 4). Recalibration is required if
calibration verification criteria (Section
14.3.4) cannot be met.
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 charac
1 teristics 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 substituted for the reagent
water matrix (Section 6.6.1) in all
performance tests.
8.1.1 The analyst shall make an initial
demonstration of the ability to generate
acceptable accuracy and precision with
this method. This ability is established
as described in Section 8.2.
8.1.2 The analyst is permitted to modify this
method to improve separations or lower the
costs of measurements, provided all
performance specifications are met. Each
time a modification is made to the method,
the analyst is required to repeat the
procedures in Sections 7.2 through 7.4 and
Section 8.2
performance.
to demonstrate method
8.1.3 Analyses of blanks are required to
demonstrate 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
verification and the analysis of the
precision and recovery standard that the
analytical system 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
generated. Development of accuracy
statements is described in Section 8.4.
8.2 Initial precision and accuracy -- To
establish the ability to generate
acceptable 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
(PAR) (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 recovery (X) in ng/mL
and the standard deviation of the recovery
(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 and PCDDs and PCDFs
with no labeled analog.
17
-------
8.2.3 For each 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 performance is
unacceptable for that compound. Correct
the problem and repeat the test (Section
8.2).
8.3 The laboratory shall spike all samples and
QC aliquots with the diluted labeled
compound spiking standard (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 (P) 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 Compare the labeled compound recovery for
each compound with the corresponding
limits in Table 7. If the recovery of any
compound falls outside its limit, 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 (P) and the standard deviation of
the percent recovery (sp) for the labeled
compounds only. Express the accuracy
assessment as a percent recovery interval
from P - 2sp to P + 2sp for each matrix.
For example, if P = 90% and sp = 10X 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-liter 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 PCODs or PCOFs (Table 1) or
any potentially interfering compound is
found in blank at greater than the minimum
level (Table 2), assuming a response
factor of 1 relative to the c.2-1,2,3,4-
TCOD 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.
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 GCHS
instrument will provide the most
reproducible results if dedicated to the
settings and conditions required for the
analyses of PCODs and PCOFs by this
method.
8.7 Depending on specific program
requirements, field replicates may be
collected to determine the precision of
the sampling technique, and spiked samples
may be required to determine the accuracy
of the analysis when the internal standard
method is used.
9 SAMPLE COLLECTION, PRESERVATION, ANO
HANDLING
• 9.1 Collect samples in glass containers
following conventional sampling practices
(Reference 17). Aqueous samples which
18
-------
flow freely are collected in refrigerated
bottles using automatic sampling
equipment. Solid samples are collected as
grab samples using wide mouth jars.
9.2 Maintain samples at 0 - 4 °C from the time
of collection until extraction. If
residual chlorine is present in aqueous
samples, add 80 mg sodium thiosulfate per
liter of water. EPA Methods 330.4 and
330.5 may be used to measure residual
chlorine (Reference 18).
9.3 Begin sample extraction within one year of
collection, and analyze all extracts
within 40 days of extraction.
10 SAMPLE PREPARATION
The sample preparation process involves
modifying the physical form of the sample
so that the PCDDs and PCOFs 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 PCDDs/PCOFs are
strongly associated with particulates, the
preparation of aqueous samples is
dependent 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 PCDDs and/or PCOFs, 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
significant figures) into a tared beaker.
NOTE: This aliquot is used only for
determining the solids 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:
10.2
% solids =
weight of sample after drying
weight of sample before drying
Determine particle size
x 100
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
less than one percent solids -- The
extraction procedure for aqueous samples
containing less than one percent solids
involves filtering the sample, extracting
the paniculate 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 SOS 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 spiking standard by a, factor of
50 with acetone. 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) to be extracted during the
same 12-hour shift, place two 1.0 liter
aliquots of reagent water in 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
20 uL of the precision and recovery
standard (Section 6.14) to 1.0 ml with
acetone. Spike 1.0 mL of the diluted
precision and recovery standard into the
19
-------
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
quantities of reagent water.
10.3.8 Weigh 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.
10.3.10 Extract the particulates using the
procedures in Section 11.
10.4 Preparation of samples containing greater
than one percent solids
10.4.1 Weigh a well-mixed aliquot of each sample
(of the same matrix type) sufficient to
provide 10 g of dry solids (based on the
solids determination in 10.1.3) into a
clean beaker or glass jar.
10.4.2 Spike 1.0 ml of the diluted labeled
compound spiking solution (Section 10.3.2)
into the sample aliquot(s).
10.4.3 For each sample or sample set (to a
maximum of 20) 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.
10.4.4 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
standard (Section 10.3.4) into the
remaining reference matrix aliquot. This
aliquot will serve as the PAR (Section
14.5).
10.4.5 Stir or tumble and equilibrate the
aliquots for 1-2 hours.
10.4.6 Extract the aliquots using the procedures
in Section 11.
10.5 Multi-phase 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 QC aliquots for particle
size reduction and blending (Section
10.6).
10.6 Sample grinding, homogenization, or
blending -- Samples with particle sizes
greater than 1 mm (as determined by
Section 10.2.2) are subjected to grinding,
homogenization, or blending. The method
of reducing particle size to less than 1
mm is matrix dependent. In general, hard
particles can be reduced by grinding with
a mortar and pestle. Softer particles can
be reduced by grinding in a Wiley mill or
meat grinder, by homogenization, or by
blending.
10.6.1 Each size reducing preparation procedure
on each matrix shall be verified by
running 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,
reducing the temperature of the sample to
freezing or to dry ice or liquid nitrogen
temperatures can aid in the grinding
process. Grind the sample aliquots from
Section 10.4.5 or 10.5.2 in a clean
grinder. Do not allow the sample
temperature to exceed 50 °C. Grind the
blank and reference matrix aliquots using
a clean grinder.
20
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10.6.4 Homogem'zation 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
11.1.1 Extraction of filtrates -- extract the
aqueous samples, blanks, and PAR aliquots
according to the following procedures.
11.1.1.1 j Pour filtered aqueous sample into a 2-L
separatory funnel. Add 60 mL methylene
chloride to the sample bottle, seal,and
shake 60 seconds to rinse the inner
surface.
11.1.1.2 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
stirring rod). Drain the methylene
chloride extract into a 500-mL KD
concentrator.
11.1.1.3 Extract the water sample two more times
using 60 mL of fresh methylene chloride
each time. Drain each extract into the KD
concentrator. After the third extraction,
rinse the separatory funnel with at least
30 mL of fresh methylene chloride.
11.1.2 Soxhlet/Dean-Stark extraction of solids --
Extract the solid samples, participates,
blanks, and PAR aliquots using the
following procedure.
11.1.2.1 Charge a clean extraction thimble with 5.0
g of 100/200 mesh silica (Section 6.5.1.1)
and 100 g of quartz sand (Section 6.5.4).
NOTE: Do not disturb the silica layer
throughout the extraction process.
11.1.2.2 Place the thimble in a clean extractor.
Place 30 - 40 ml of toluene in the
receiver and 200 - 250 mL in the flask.
11.1.2.3 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 condenser
tip into the receiver. Extract the
apparatus for 3 hours minimum.
11.1.2.4 After pre-extraction, cool and disassemble
the apparatus. Rinse the thimble with
toluene and allow to air dry.
11.1.2.5 Load the wet sample from Section 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.
11.1.2.6 Reassemble the pre-extracted SOS apparatus
and add a fresh charge of toluene to the
receiver and reflux flask.
11.1.2.7 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.
11.1.2.8 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.
11.1.2.9 Remove the distilling flask, estimate and
record the volume of extract (to the
nearest 100 mL), and pour the extract from
the receiver and flask into a 500 mL
separatory funnel. Rinse the receiver and
flask with toluene and add to the
separatory funnel. Proceed with back
extraction per Section 11.1.3.
11.1.3 Back extraction with base and acid
11.1.3.1 Spike 1.0 mL of the cleanup standard
(Section 6.11) into the separatory funnels
containing the sample and QC extracts
(Section 11.1.1.3 or 11.1.2.9).
21
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11.1.3.2 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
washing until no color is visible in the
aqueous layer, to a maximum of four
washings. Minimize contact time between
the extract and the base to prevent
degradation of the PCDDs and PCDFs.
11.1.3.3 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.
11.1.3.4 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.
11.1.3.5 Repeat the partitioning against sodium
chloride solution and discard the aqueous
layer.
11.1.3.6 Pour each extract through a drying column
containing 7 to 10 cm of anhydrous sodium
sulfate. Rinse the separatory funnel with
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 11.2 and 12.
11.2 Concentration
11.2.1 Macro-concentration -- Concentrate the
extracts in separate 500 ml round bottom
flasks on a rotary evaporator.
11.2.1.1 Assemble the rotary evaporator according
to manufacturer's instructions, and warm
the water bath to 45 °C. On a daily
basis, preclean the rotary evaporator by
concentrating 100 ml of clean extraction
solvent through the system. Archive both
the concentrated solvent and the solvent
in the catch flask for contamination check
if necessary. Between samples, three 2 -
3 ml aliquots of toluene should be rinsed
down the feed tube into a waste beaker.
11.2.1.2 Attach the round bottom flask containing
the sample extract to the rotary
evaporator. Slowly apply vacuum to the
system, and begin rotating the sample
flask.
11.2.1.3 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 will be
steady, but no bumping or visible boiling
of the extract will occur. NOTE: If the
rate of concentration is too fast, analyte
loss may occur.
11.2.1.4 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.
11.2.1.5 Transfer the extract to a vial using three
2 - 3 mL rinses of hexane. Proceed with
micro-concentrat ion and solvent exchange.
11.2.1.6 The extracts of the filtered aqueous
sample and its particulates must be
combined prior to cleanup and analysis.
Transfer the concentrated extract of the
aqueous sample to the flask containing the
concentrated particulate extract. Rinse
the flask twice with 5 ml toluene, and add
these rinses to the flask with the
combined extracts. Reattach the flask to
the rotary evaporator and continue to
concentrate the combined extract until the
volume is approximately 2 ml. Proceed
with micro-concentration and solvent
exchange.
11.2.2 Micro-concentration and solvent exchange
11.2.2.1 Toluene extracts to be subjected to GPC
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.
Extracts to be subjected to HPLC are
exchanged into nonane.
11.2.2.2 Transfer the vial containing the sample
extract to a nitrogen evaporation device.
Adjust the flow of nitrogen so that the
surface of the solvent is just visibly
disturbed. NOTE: A large vortex in the
solvent may cause analyte loss.
11.2.2.3 Lower the vial into a 45 °C water bath and
continue concentrating.
22
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11.2.2.4 When the volume of the liquid is
approximately 100 uL, add 2 - 3 mL of the
desired solvent (methylene chloride or
hexane) and continue concentration to
approximately 100 uL. Repeat the addition
of solvent and concentrate once more.
11.2.2.5 If the extract is to be cleaned up by GPC.
adjust the volume of the extract to 5.0 ml
with methylene chloride. Proceed with GPC
cleanup (Section 12.2).
11.2.2.6 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).
11.2.2.7 For extracts to be concentrated for
injection into the HPLC or 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.
11.2.2.8 Seal the vial and label with the sample
number. Store in the dark at room
temperature until ready for HPLC or GCMS.
12 EXTRACT CLEANUP
12.1 | Cleanup may not be necessary for
relatively clean samples (e.g., treated
effluents, groundwater, drinking water).
If particular circumstances require the
use of a cleanup procedure, the analyst
may use any or all of the procedures below
or any other appropriate procedure.
Before using a cleanup procedure, the
analyst must demonstrate that the
requirements of Section 8.2 can be met
using the cleanup procedure.
12.1.1
12.1.2
12.1.31
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 nonpolar and polar
interferences.
AX-21/Celite (Section 12.5) is
remove nonpolar interferences.
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 PCOF 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 oil 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
calibration 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
23
-------
extract. If the extract is known or
expected to contain more than 0.5 g, the
extract is split into aliquots for GPC and
the aliquots are combined after elation
from the column. The solids 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 column.
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 column. 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, 1 g sodium
sulfate (Section 6.2.1). Tap the column
to settle the adsorbents.
12.3.2 Pre-rinse the column 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 sodium
sulfate.
12.3.4 Rinse the receiver twice with 1 mL
portions of hexane and apply separately to
the column. Elute the PCDDs/PCDFs with
100 mL hexane and collect the eluate.
12.3.5
12.4
12.4.1
12.4.2
Concentrate the eluate per Section 11.2.1
or 11.2.2 for further cleanup or for
injection into the HPLC or GCMS.
12.4.3
12.4.4
12.4.5
12.4.6
12.4.7
12.4.8
12.5
12.5.1
Alumina cleanup
Place a glass wool plug in a 15 mm
chromatography column.
i.d.
12.5.2
Pack the column in the following order
(bottom to top): 1 g neutral alumina
(Section 6.5.2.1), 3 g basic alumina
(Section 6.5.2.2), 1 g neutral alumina, 6
g acid alumina (Section 6.5.2.3), 2 g
neutral alumina, 1 g sodium sulfate
(Section 6.2.1). Tap the column to settle
the adsorbents.
Pre-rinse the column 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.
Apply the concentrated extract to the
column. Open the stopcock until the
extract is within 1 mm of the sodium
sulfate.
Rinse the receiver twice with 1 mL
portions of hexane and apply separately to
the column. Elute the interfering
compounds with 100 mL hexane and discard
the eluate.
Elute the PCDDs and PCDFs with 20 mL of
methylene chloride:hexane (1:1 v/v).
Concentrate the eluate per Section 11.2.1
or 11.2.2 for further cleanup or for
injection into the HPLC or GCMS.
AX-21/Celite
Cut both ends from a 10 mL disposable
serological pi pet to produce a 10 cm
column. Fire polish both ends and flare
both ends if desired. Insert a glass wool
plug at. one end, then pack the column 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.
Pre-rinse the column 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
24
-------
v/v), and five ml hexane. If the flow
rate of eluate exceeds 0.5 mL per min,
discard the column.
12.5.3 When the solvent is within 1 mm of the
column packing, apply the sample extract
to the column. Rinse the sample container
twice with 1 ml portions of hexane and
apply separately to the column. Apply 2
ml of hexane to complete the transfer.
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 column 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 11.2.1
or 11.2.2 for further cleanup or for
injection into the HPLC or GCMS.
12.6 HPLC (Reference 6)
12.6.1 Column 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
approximately 500 pg/uL in chloroform.
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-
isomers and for the other isomers of
interest. Following calibration, flush
the injection system with copious
quantities of chloroform, including a
minimum 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
calibration 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 compared 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
column not be overloaded. The column
specified in this method is designed to
handle a maximum of 30 uL of extract. If
the extract cannot be concentrated to less
than 30 uL, -it is split into fractions and
the fractions are combined after elution
from the column.
12.6.2.1 Rinse the sides of the vial twice with 30
uL of chloroform and reduce to the level
of the nonane with the blowdown apparatus.
Rinse the sides of the vial with 20 uL of
chloroform to bring the extract volume to
30 uL.
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).
12.6.2.4 If an extract containing greater than 100
ng/mL of total PCDD or PCDF is
encountered, a 30 uL chloroform blank
shall be run through the system to check
for carry-over.
12.6.2.5 Concentrate the eluate per Section 11.2.2
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
solution (Section 6.12) to the sample
extract immediately prior to injection to
minimize the possibility of loss by
evaporation, adsorption, or reaction. If
an extract is to be reanalyzed, do not add
more instrument 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 standatd solution,
using on-column or split less injection.
Start the GC column initial isothermal
hold upon injection. Start MS data
collection after the solvent peak elutes.
Stop data collection after the octachloro-
25
-------
dioxin and furan have eluted. Return the
column to the initial temperature 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 native 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 recall* brat ion (per
Section 7} shall be performed until all
performance criteria are met. Only after
all performance criteria are met may
samples, blanks, and precision and
recovery standards be analyzed.
14.2 Mass spectrometer resolution -- A static
resolving power of at least 10,000 (10
percent valley definition) must be
demonstrated at appropriate 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. Corrective actions must be
implemented whenever the resolving power
does not meet the requirement.
14.2.1 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
instrument 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.
14.2.2 By using a PFK molecular leak, tune the
instrument 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.
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 PCODs and
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,
resolution shall be verified (Section
14.2) prior to repeat of the verification
test.
14.3.3 Compute the concentration of each native
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.
14.3.4 For each compound, compare the
concentration with the calibration
verification limit in Table 7. If 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
26
-------
14.4.1.1 Absolute "The absolute retention times
of the 13C12-1,2.3,4-TCDD and C^-
1,2,3,7,8,9-HxCDF GCMS internal standards
shall be within ±15 seconds of the
retention times obtained during
calibration (Section 7.2.4).
14.4.1.2 Relative -- The relative retention times
of native and labeled PCDDs and PCDFs
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
columns.
14.4.2.2 The valley height between 2,3,7,8-TCDO 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 columns (Figure 3).
14.4.3 If the absolute or relative 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 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 PCOO or
PCDF by isotope dilution (Section 7.5) for
those compounds that have labeled analogs.
Compute the concentration of the labeled
compounds by the internal standard method.
14.5.3 For each compound, compare 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
precision and recovery test (Section
14.5).
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
statement of laboratory accuracy for each
PCDD and PCDF in each matrix type by
calculating the average percent recovery
(R) and the standard deviation of percent
recovery (sr). Express the accuracy as a
recovery interval from R - 2sr to R + 2sr.
For example, if R = 95X and sr = 5X, the
accuracy is 85 - 105%.
15 QUALITATIVE DETERMINATION
Identification is accomplished by
comparison of data from analysis of a
sample or blank with data for authentic
standards. For compounds for which the
relative retention times are known,
identification is confirmed per Sections
15.1 and 15.2.
15.1 Labeled compounds and native PCDDs and
PCDFs having no labeled analog
15.1.1 The signals for the exact m/z's being
monitored (Table 3A) shall be present and
shall maximize within the same two
consecutive scans.
15.1.2 Either (1) the ratio of the background
corrected exact SICP areas, or (2) the
corrected relative intensities of the
exact m/z's at the GC peak maximum shall
be within the limits in Table 3A.
15.1.3 For the individual labeled compounds and
individual PCDDs and PCDFs, the relative
retention time shall be within the limits
specified in Table 2.
15.2 PCDDs and PCDFs having a labeled analog
15.2.1 The signals for the exact m/z's being
monitored (Table 3) shall be present and
shall maximize within the same two
consecutive scans.
15.2.2 The ratio of the ion abundances of the
exact m/z's at the GC peak maximum shall
agree within the limits in Table 3.
27
-------
15.2.3 The relative retention time between the
native compound and its labeled analog
shall be within the windows specified in
Table 2.
15.3 If identification is ambiguous, an
experienced spectrometrist (Section 1.5)
is to determine the presence or absence of
the compound.
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 native compound can be
made because the native compound and its
labeled analog exhibit the same effects
upon extraction, concentration, and gas
chromatography. Relative response (RR)
values for sample mixtures are used in
conjunction with calibration data
described in Section 7.5 to determine
concentrations directly, so long as
labeled compound spiking levels are
constant.
16.1.1 Because of a potential interference, the
labeled analog of OCOF is not added to the
sample. Therefore, this native analyte is
quant itated against the labeled OCDD.
16.1.2 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 native compound. Therefore, the
native 1,2,3,7,8,9-HxCDD is quantitated
using the avtrage 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.
16.1.3 Any peaks representing non-2,3,7,8-
substituted dioxins or furans are
quantitated using an average of the
response factors from all the labeled
2,3,7,8- isomers in the same level of
chlorination.
16.2 Internal standard -- Compute the
concentrations of the labeled analogs and
the cleanup standard in the extract using
the response factors determined from
calibration data (Section 7.6) and the
following equation:
where C i s
compound in
Cex (ng/ml) =
(A
Cis>
16.3
16.4
16.4.1
16.4.2
16.4.3
16.5
16.5.1
the concentration of the
the extract and the other
terms are as defined in Section 7.6.1.
The concentration of the native 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:
Concentration
in solid (ng/kg)
(Cex x Vex>
where,
* is
U is the sample weight in Kg.
V is the extract volume in ml.
cx
If the SICP area at the quant i tat ion m/z
for any compound exceeds the calibration
range of the system, a smaller sample
aliquot is extracted.
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.
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 10.1.3. Extract per
Section 10.4.
If a smaller sample size will not be
representative of the entire sample,
dilute the sample extract by a factor of
10, adjust the concentration of the
instrument internal standard to 100 pg/uL
in the extract, and analyze an aliquot of
this diluted extract by the internal
standard method.
Results are reported to three significant
figures for the native and labeled isomers
found in all standards, blanks, and
samples. For aqueous samples, the units
are ng/L; for samples containing one
percent or greater solids (soils,
sediments, filter, cake, compost), the
-units are ng/kg, based on the dry weight
of the sample.
Results for samples which have been
diluted are reported at the least dilute
level at which the area at the
quantitat ion m/z is within the calibration
range (Section 16.4).
(A-s x RF)
28
-------
16.5.2 For native compounds having a labeled
analog, results are reported at the least
dilute level at which the area at the
quant itat ion m/z is within the calibration
range (Section 16.4) and the labeled
compound recovery is within the normal
range for the method (Section 17.4).
16.5.3 Additionally, the total concentrations of
all isomers in an individual level of
chlorination (i.e. total TCDD, total
PeCOD, etc.) are reported to three
significant figures in units of ng/L, for
both dioxins and furans. The total or
ng/kg concentration in each level of
chlorination is the sum of the
concentrations of all isomers identified
in that level, including any non-2,3,7,8-
substituted 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 col urn and/or
mass spectrometer.
17.2 i 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 Interferences at the primary m/z -- If an
interference occurs at the primary
quantitat ion m/z (Table 3) for any native
or labeled compound, the alternate m/z is
used for quantitat ion.
17.4 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
1 the limits given in Table 7, a diluted
sample (Section 16.4) is 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
. 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 METHOD PERFORMANCE
EPA is in the process of developing
performance data for this draft method.
When these tests are complete, the
specifications in this method will be
modified based on these data, and the
supporting documents will be referenced in
this section.
REFERENCES
1 Tondeur, Yves, "Method 8290: Analytical
Procedures and Quality Assurance for
Multimedia Analysis of Pol/chlorinated
Oibenzo-p-dioxins and Dibenzofurans by
High-Resolution Gas Chromatography/High-
Resolution Mass Spectrometry," USEPA,
EMSL-Las Vegas, Nevada, June 1987.
2 "Measurement of 2,3,7,8-Tetrachlorinated
Dibenzo-p-dioxin (TCDD) and 2,3,7,8-
Tetrachlorinated Dibenzofuran (TCDF) in
Pulp, Sludges, Process Samples and
Uastewaters from Pulp and Paper Mills",
Wright State University, Dayton OH
45435, June 1988.
3 "NCASI Procedures for the Preparation and
Isomer Specific Analysis of Pulp and Paper
Industry Samples for 2,3,7,8-TCDD and
2,3,7,8- TCDF", National Council of the
Paper Industry for Air and Stream
Improvement, 260 Madison Av, New York NY
10016, Technical Bulletin No. 551, Pre-
release Copy, July 1988.
4 "Analytical Procedures and Quality
Assurance Plan for the Determination of
PCDD/PCDF in Fish", U.S. Environmental
Protection Agency, Environmental Research
Laboratory, 6201 Congdon Blvd., Duluth MN
55804, April 1988.
5 Yves Tondeur, "Proposed GC/MS Methodology
for the Analysis of PCDDs and PCDFs in
Special Analytical Services Samples",
Triangle Laboratories, Inc., 801-10
Capitola Dr, Research Triangle Park NC
27713, January 1988; updated by personal
communication September 1988.
29
-------
6 Lamparski, L.L., and Nestrick, T.J.,
"Determination of Tetra-, Hexa-, Hepta-,
and Octachlorodibenzo-p-dioxin Isomers in
Particulate Samples at Parts per Trillion
Levels", "Anal. Chem." 52, 2045-2054
(1980).
7 Lamparski, L.L., and Nestrick, T.J.,
"Novel Extraction Device for the
Determination of Chlorinated Dibenzo-p-
dioxins (PCOOs) and Dibenzofurans (PCDFs)
in Matrices Containing Water", Personal
Communication, July 1988.
8 Patterson, D.G., et. al. "Control of
Interferences in the Analysis of Human
Adipose Tissue for 2,3,7,8-Tetra-
chlorodibenzo-p-dioxin", "Environ.
Toxicol. Chem.," 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", U.S. EPA,
Environmental Monitoring Systems
Laboratory, Las Vegas NV 89114, EPA
600/4-86-004. January 1986.
10 "Working with Carcinogens," DHEW, PHS,
CDC, NIOSH, Publication 77-206, (Aug
1977).
11 "OSHA Safety and Health Standards, General
Industry" OSHA 2206,' 29 CFR 1910 (Jan
1976).
12 "Safety in Academic Chemistry
Laboratories," ACS Committee on Chemical
Safety (1979).
13 "Standard Methods for the Examination of
Water and Wastewater", 16th Ed. and Later
Revisions, American Public Health
Association, 1015 15th St, N.W.,
Washington DC 20005, Section 108
"Safety", 46 (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).
17 "Standard Practice for Sampling Water,"
ASTM Annual Book of Standards, ASTM,
Philadelphia. PA, 76 (1980).
18 "Methods 330.4 and 330.5 for Total
Residual Chlorine," USEPA, EMSL,
Cincinnati, OH 45268, EPA 600/4-70-020
(March 1979).
30
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Table 1
POLYCHLORIMATED DIBENZODIOXINS AND FURANS DETERMINED BY ISOTOPE DILUTION AND INTERNAL STANDARD
HIGH RESOLUTION GAS CHROMATOGRAPHY (HRGO/HIGH RESOLUTION MASS SPECTROMETRY (HRMS)
PCDDs/PCOFs (1)
I somer/Congener
2,3,7.8-TCDD
Total -TCDD
2,3.7,8-TCDF
Total -TCOF
1,2,3,7,8-PeCDD
Total-PeCDD
1,2,3,7,8-PeCDF
2,3,4.7.8-PeCDF
Total-PeCDF
1,2,3,4,7,8-HxCDD
1.2,3,6,7,8-HxCDD
1,2,3, 7,8, 9-HXCDD
Total-HxCDD
1,2, 3,4. 7,8- HxCDF
1,2.3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7,8-HxCDF
Total -HxCDF
1,2,3,4,6,7,8-HpCDD
Total -HpCOD
1, 2,3,4,6, 7,8-HpCOF
1,2,3,4,7,8,9-HpCDF
Total-HpCDF
OCDD
OCDF
(1) Polychlorinated dioxins
CAS Registry
1746-01-6
41903-57-5
51207-31-9
55722-27-5
40321-76-4
36088-22-9
57117-41-6
57117-31-4
30402-15-4
39227-28-6
57653-85-7
19408-74-3
34465-4608
70648-26-9
57117-44-9
72918-21-9
60851-34-5
35822-46-9
37871-00-4
67562-39-4
55673-89-7
38998-75-3
3268-87-9
39001-02-0
and furans
TCDD = Tetrachlorodibenzo-p-dioxin
PeCDD - Pentachlorodibenzo-p-dioxin
HxCDD - Hexachlorodibenzo-p-dioxin
HpCDD = Heptachlorodibenzo-p-dioxin
OCDD = Octachlorodibenzo-p-dioxin
Labeled Analog CAS Registry
"c12-2,3,7,8-TCDD
37Cl4-2,3',7,8-TCDD
13C12-2,3,7,8-TCDF
13C12-1,2,3,7,8-PeCDD
13C12-1,2,3,7,8-PeCDF
13C12-2,3,4,7.8-PeCDF
13C12-1,2,3,4,7,8-HxCDD
13C12-1,2,3,6,7,8-HxCDD
13C12-1,2,3,7,8,9-HxCDD(2)
13C12-1,2,3,4,7,8-HxCDF
13C12-1,2,3,6,7,8-HxCDF
13C12-1,2,3,7,8,9-HxCDF
13C,,-2,3,4,6,7,8-HxCDF
13C12-1,2,3,4,6,7,8-HpCDD
13C12-1,2,3.4,6,7,8-HpCDF
13C12-1,2,3,4,7.8,9-HpCDF
13C12-OCDD
TCDF » Tetrachlorodibenzofuran
PeCDF = Pentachlorodibenzofuran
HxCDF = Hexachlorodibenzofuran
MpCDF = Heptachlorodibenzofuran
OCDF - Octachlorodibenzofuran
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
(2) Labeled analog is used as an internal standard and therefore cannot be used for quant itat ion by isotope
dilution.
31
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Table 2
RETENTION TIMES AND MINIMUM LEVELS FOR PCDDs AND PCDFs
Compound
Compounds using C.2-1,2,3
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
Labeled Compounds
13C12-2,3,7,8-TCDF
13C12-1,2,3,4-TCDD
13C12-2,3,7,8-TCDD
37Cl4-2,3,7,8-TCDD
13C12-1,2,3,7,8-PeCDF
13C12-2.3,4,7,8-PeCDF
13C12-1,2,3,7,8-PeCDD
Compounds using C.p-1,2,3
Native Compounds
1,2,3,4,7,8-HxCDF
1,2,3,6,7,8-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
1,2,3,7,8,9-HxCDF
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
Labeled Compounds
13C,, -1,2, 3. 4. 7,8- HxCDF
17
C12-1,2,3,6,7,8-HxCDF
13C,,-1,2,3,4,7,8-HxCDD
13
IJC12-1,2,3,6,7,8-HxCDD
13C,.,-1,2,3f7,8t9-HxCDD
17
"c,,-!, 2, 3, 7,8,9- HxCDF
13
C,,-1,2,3,4,6.7,8-HpCDF
12 ,,,,., HV
IJC12-1,2,3,4,6.7.8-HpCDD
13C.,-1,2f3,4,7,8,9-HpCDF
«C -OCDD
13cJ2-OCDF
Absolute
Retention
Time
(Minutes)
Minimum Level (2)
Retention
Time
Reference
Relative
Retention
Time (1)
Water
P9/U
ppq
Solid
ng/kg
PPt
Extract
pg/uL
ppb
,4-TCDD as internal standard
26.35
27.24
31.16
32.16
32.45
26.35
27.03
27.22
27.23
31.16
32.15
32.44
,7,8,9-HxCDD
36.19
36.29
37.19
37.30
37.36
38.07
38.23
40.55
42.27
43.01
46.56
47.05
36.18
36.27
37.29
37.38
38.06
38.23
40.54
42.27
43.01
46.55
47.04
3C12-2,3,7,8-TCDF
,C12-2,3,7,8-TCDD
"c12-1,2,3,7.8-PeCDF
"c 12-2,3,4,7,8-PeCDF
'3C12-1,2,3,7,8-PeCDD
13C12-1,2,3,4-TCDD
13C12-1,2,3,4-TCDD
13C12-1,2,3,4-TCDD
13C12-1.2,3,4-TCDD
13C12-1,2,3,4-TCDF
13C12-1,2,3.4-TCDD
13C12-1,2,3,4-TCDD
as internal standard
]3C -1,2,3,4,7,8-HxCDF
^C12-1,2.3,6,7.8-HxCDF
C12-2, 3,4, 6,7,8- HxCDF
]3C12- ,2,3,4,7,8-HxCDD
3C.2- ,2,3,6,7,8-HxCDD
"C12- ,2,3,6,7.8-HxCDD
"C.2- ,2,3,7,8,9-HxCDF
C12- ,2,3,4,6,7,8-HpCDF
3C.2- ,2,3,4,6,7,8-HpCDD
3C -1,2,3,4,7,8,9-HpCDF
]3C12-OCDD
C12-OCDD
13C,,-1,2.3,7,8,9-HxCDD
17
aC12-1,2,3,7,8.9-HxCDD
13C..,-1,2,3,7,8.9-HxCOD
13
°C12-1,2,3,7,8,9-HxCDD
"c,--!, 2,3, 7,8,9- HxCOD
17
JC...-1,2,3.7,8,9-HxCDD
13
0C,,-1,2,3,7,8,9-HxCDD
13
C12-1,2,3,7,8,9-HxCDD
13C,,-1,2,3,7,8,9-HxCDD
IT F • * f 1
13C,,-1,2,3,7,8,9-HxCDD
., 12 ' '
C12-1,2,3,7.8,9-HxCDD
0.999 -
0.999 -
0.999 -
0.999 -
0.999 -
0.970 -
1.000 -
1.002 -
1.002 -
1.147 -
1.183 -
1.194 -
0.999 -
0.999 -
0.999 -
0.999 -
0.999 -
0.999 -
0.999 -
0.999 -
0.999 -
0.999 -
0.999 -
1.007 -
0.946 -
0.948 -
0.975 -
0.977 -
1.000 -
0.999 -
1.060 -
1.105 -
1.124 -
1.217 -
1.229 -
1.001
1.001
1.001
1.001
1.001
0.980
1.000
1.012
1.013
1.159
1.196
1.206
1.001
1.001
1.001
1.001
1.001
1.001
1.001
1.001
1.001
1.001
1.001
1.013
0.956
0.958
0.985
0.987
1.000
1.010
1.071
1.116
1.136
1.230
1.242
10
10
50
50
50
50
50
50
50
50
50
50
50
50
50
100
100
1
1
5
5
5
5
5
5
5
5
5
5
5
5
5
10
10
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
(1) Initial specifications are estimated based on isotope dilution and internal standard data from Method
These specifications may be revised when further data have been collected by EPA using Method 1613.
(2) Level at which the analytical system will give acceptable SICP and calibration.
1625.
32
-------
Table 3
DESCRIPTORS, MASSES. M/Z TYPES, AND ELEMENTAL COMPOSITIONS OF THE CDDs AND CDFs (1)
Descriptor
Number
1
1
2
3
Accurate
m/z (2)
292.9825
303.9016
305.8987
315.9419
317.9389
319.8965
321.8936
327.8847
330.9792
331.9368
333.9339
375.8364
339.8597
341.8567
351.9000
353.8970
354.9792
355.8546
357.8516
367.8949
369.8919
409.7974
373.8208
375.8178
383.8639
385.8610
389.8157
391.8127
392.9760
401.8559
403.8529
430.9729
445.7555
m/z
Type
Lock
M
M+2
M
M+2
M
M+2
M
QC
M
M+2
M+2
M+2
M+4
M+2
M+4
Lock
M+2
M+4
M+2
M+4
M+2
M+2
M+4
M
M+2
M+2
M+4
Lock
M+2
H+4
QC
M+4
Elemental Composition
C7F11
C12 H4 ^U °
C,, H. 37Cl. 0
12 4 4
13C,, H. 35Cl, 0
12 4 4
13 35 . 37 .
C12 H4 Cl, Cl 0
C,_ H. 35Cl. 0,
12 4 42
C,- H. 35Cl, 37Cl 0-
12 4 3 2
C12 H4 ^U °2
C7F13
13C12 H4 35C14 02
13. , 35 37
C12 H4 Cl, Cl 02
C,, H. 35CU 37Cl 0
12 4 5
C., H, 35Cl, 37Cl 0
12 3 4
C,, H, 35C13 37C12 0
13 35.. 37
C12 H3 C14 Cl 0
13C H 35cl 37cl Q
C9F13
C12 H3 35cl4 37cl °2
C12 H3 35cl3 37cl2 °2
13 35 . 37..
C12 H3 C14 Cl °2
13. H 35 37
C12 H3 C13 C12 °2
C12 H3 35cl6 3/Cl °
C12 H2 35C15 37Cl 0
C12 H2 ^U 37cl2 °
13C12 H2 35C16 0
13C12 H, 35C15 37Cl 0
C12 H2 35cl5 3?Cl °2
C H 35Cl 37Cl 0
C12 H2 C14 C12 °2
C9F15
13C H 35cl 37cl 0
13C H 35cl 37cl
C9F13
Compound
(3)
PFK
TCDF
TCDF
TCDF(4)
TCDFC4)
TCDD
TCDD
TCDOC4)
PFK
TCDD<4)
TCDD(4)
HxCDPE
PeCDF
PeCDF
PeCDF(4)
PeCDF(4)
PFK
PeCDD
PeCDD
PeCDD(4)
PeCDD(4)
HpCDPE
HxCDF
HxCDF
HxCDF(4)
HxCDF(4)
HxCDD
HxCDD
PFK
HxCDD(4)
HxCDD(4)
PFK
OCDPE
Primary
m/z?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
33
-------
DESCRIPTORS, MASSES, M/Z
Table 3 (continued)
TYPES, AND ELEMENTAL COMPOSITIONS OF
THE CDDs AND CDFs (1)
Descriptor Accurate m/z
Number m/z (2) Type
4 407.7818 M+2
409.7789 M+4
417.8253 M
419.8220 M+2
423.7766 M+2
425.7737 M+4
430.9729 Lock
435.8169 M+2
437.8140 M+4
479.7165 M+4
5 441.7428 M+2
442.9728 Lock
443.7399 M+4
457.7377 M+2
459.7348 M+4
469.7779 M+2
471.7750 M+4
513.6775 M+4
(1) From Reference 5
(2) Nuclidic masses used:
H = 1.007825 C = 12.00000
0 = 15.994915 35Cl = 34.968853
(3) Compound abbreviations:
Chlorinated dibenzo-p-dioxins
TCDD = Tetrachlorodibenzo-p-dioxin
PeCDD = Pentachlorodibenzo-p-dioxin
HxCDD = Hexachlorodibenzo-p-dioxin
HpCDD = Heptachlorodibenzo-p-dioxin
OCDD = Octachlorodibenzo-p-dioxin
Elemental Composition
C,, H 35Cl., 37Cl 0
l£ O
35 37
C12 H 03C15 3 C12 0
13C H 35Cl 0
C12 H C17 °
13C,, H 35Cl, 37Cl 0
l£ O
C12 H 35C16 37Cl O,
C12 H 35C15 37C12 02
C9F17
13 35 37
C12 H C16 Ct °2
13 35 . 37
C12 H C15 C12 °2
C12 H 35C17 37C12 0
c12 35ci7 37ci o
C10 F17
C12 35cl6 3/Cl2 °
c,2 35ci7 37ci o.
c,, 35ci., 37ci, o,
12 6 22
13C12 35C17 37Cl O,
C12 35cl8 37cl2 °
13C = 13.003355
37Cl = 36.965903
Compound
(3)
HpCDF
HpCDF
HpCDF(4)
HpCDF(4)
HpCDD
HpCDD
PFK
HpCDD (4)
HpCDD(4)
NCDPE
OCDF
PFK
OCDF
OCDD
OCDD
OCDD (4)
OCDD (4)
DCDPE
F = 18.9984
Primary
m/z?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Chlorinated diphenyl ethers
HxCDPE =
HpCDPE =
OCDPE
NCDPE =
DCDPE =
Hexachlorodiphenyl ether
Heptachlorodiphenyl ether
Octachlorodiphenyl ether
Nonachlorodiphenyl ether
Decachlorodiphenyl ether
Chlorinated dibenzofurans
TCDF = Tetrachlorodibenzofuran
PeCDF = Pentachlorodibenzofuran
HxCDF = Hexachlorodibenzofuran
HpCDF = Heptachlorodibenzofuran
(4) Labeled compound
Lock mass and QC compound
PFK = Perfluorokerosene
34
-------
Table 3A
THEORETICAL M/Z RATIOS AND CONTROL LIMITS
No. of
Chlorine
Atoms
4
5
6
6 (2)
7
7 (3)
8
m/z's
Forming
Ratio
M/M+2
M+2/M+4
H+2/M+4
M/M+2
M+2/M+4
M/H+2
M+2/M+4
Theoretical
Ratio
0.77
1.55
1.24
0.51
1.05
0.44
0.89
Control
Lower
0.65
1.32
1.05
0.43
0.88
0.37
0.76
Limitsd)
Upper
0.89
1.78
1.43
0.59
1.20
0.51
1.02
(1) Represent ± 15X windows around the theoretical ion
abundance ratios.
(2) Used for 13C-HxCDF only.
(3) Used for 13C-HpCDF only.
35
-------
Table 4
CONCENTRATIONS OF SOLUTIONS CONTAINING LABELED AND UNLABELED CDDS AND CDFS
Stock
Solution
(1)
Compound ng/mL
Native CDDs and COFs
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-PeCOF
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-HpCOD
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7.8,9-HpCDF
OCDD
OCOF
Labeled Compound Spiking Standards
13C12-2,3,7,8-TCDD
13C12-2.3.7.8-TCOF
13C12-1.2.3,7,8-PeCDD
13C12-1,2,3.7,8-PeCOF
13C12-2,3,4.7.8-PeCDF
13C,,-1,2.3.4.7,8-HxCOD
„ 12 ' ' ' '
13C12-1,2.3,6,7,8-HxCDD
13C,,-1,2,3,4.7,8-HxCDF
13 12
'•>C,,-1,2,3,6.7,8-HxCOF
"c12-1,2,3,7.8.9-HxCOF
13C,--2,3.4.6,7,8-HxCDF
13C12-1, 2.3,4,6, 7,8-HpCOO
13C,,-1.2,3,4,6.7.8-HpCDF
l3C12-1,2.3,4,7,8,9-HpCOF
13
C12-OCDD
-
-
-
-
-
.
-
-
.
-
-
.
-
.
-
-
-
100
100
100
100
100
100
100
100
100
100
100
100
100
100
200
Spike
Solutions
(2)
ng/mL
-
-
-
-
-
.
-
-
.
-
-
.
-
.
-
-
-
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
Calibration and Verification Solutions
CS1
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
CS2
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
ng/mL
VER(3)
CS3 CS4
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
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
CSS
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
PAR(4)
ng/mL
40
40
200
200
200
200
200
200
200
200
200
200
200
200
200
400
400
-
-
-
-
-
.
-
-
-
-
-
-
-
-
-
Cleanup Standard
37,
Cl4-2,3,7,8-TCOD
Internal Standards
13,
13,
C12-1.2.3.4-TCDD
JC12-1.2.3.7.8,9-HxCDD
0.8
200
200
0.5
100
100
100
100
10
100
100
40
100
100
200
100
100
(1) Stock solution (Section 6.10} - Prepared in nonane, and diluted daily with acetone to prepare the spiking
solution (Section 10.3.2).
(2) Spiking solutions (Sections 6.11, 6.12, 8.3, 10.3.2, and 10.4.2).
Calibration verification solution (Section 14.3).
Precision and recovery standard (Section 6.14) - Prepared in nonane, and diluted daily with acetone to
prepare the spiking solution (Section 10.3.4).
(3)
(4)
36
-------
Table 5
GC RETENTION TIME WINDOW DEFINING MIXTURES AND ISOMER
SPECIFICITY TEST MIXTURES
DB-5 Column GC Retention Time Window Defining Standard
(Section 6.15)
Congener First Eluted Last Eluted
TCDF
TCOD
PeCDF
PeCDD
HxXCDF
HxCDD
HpCOF
HpCDD
1,3,6,8-
1,3.6,8-
1.3.4,6.8-
1,2,4,7,9-
1,2,3,4,6,8-
1,2,4,6,7,9-
1.2,3.4,6,7,8-
1.2,3,4,6,7,9-
1,2,8,9-
1,2,8,9-
1,2,3,8,9-
1,2,3,8.9-
1,2,3,4,8,9-
1,2,3,4,6,7-
1.2,3,4,7.8,9-
1,2,3,4.6.7.8-
DB-5 TCDD Isomer Specificity Test Standard
(Section 6.16.1)
1.2,3.4-TCDD 1,2.3.7-TCOD
1,2,7,8-TCDD 1,2,3,8-TCDD
1,4,7,8-TCDD 2,3,7,8-TCDD
DB-225 Column TCDF Isomer Specificity Test Standard
(Section 6.16.2)
2,3,4,7-TCDF
2,3,7.8-TCDF
1,2,3,9-TCDF
37
-------
Table 6
REFERENCE COMPOUNDS FOR NATIVE AND LABELED PCDDS AND PCDFS
Labeled PCDDs and PCDFs
Native PCDDs and PCDFs
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
Reference Compound
13C12-2,3.7,8-TCDD
13C12-2,3,7,8-TCDF
13C12-1,2,3,7,8-PeCDD
13C12-1,2,3,7,8-PeCDF
13C12-2,3,4,7.8-PeCDF
13C12-1,2,3,4,7,8-HxCDD
13C12-1,2,3,6,7,8-HxCDD
13C12-1.2,3,7,8,9-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
13C12-1,2,3,4,6,7,8-HpCDD
13C12-1,2,3,4.6,7,8-HpCDF
13C..,-1,2,3,4,7,8,9-HpCDF
12 ,,,,,, H*.
"C , -OCDD
13
13C12-OCDD
Reference Compound
C,,-2, 3, 7,8-TCDD
,, 12
™C12-2.3,7,8-TCDF
13C1,-1,2,3,7,8-PeCDD
13 '
1:SC12-1,2,3,7,8-PeCDF
13
'°C12-2,3,4,7,8-PeCDF
13C..,-1,2,3,4,7,8-HxCDD
13 12
C-.-I^.S^^.S-HxCDD
15 12 ' ' '
C12-1,2,3.7,8,9-HxCDD
13C,,-1,2,3,4,7,8-HxCDF
13 ''
C,--1,2,3,6,7,8-HxCDF
„ 12
'°C12-1,2,3,7,8,9-HxCDF
13C12"2,3,4,6,7,8-HxCDF
13C12-1,2,3,4,6,7,8-HPCDD
13 '*
C.,-1,2,3,4,6,7,8-HpCDF
13 12 ^
°C,--1f2,3,4,7,8,9-HpCDF
13C,,-OCDO
37
Cl^-2, 3. 7,8-TCDD
13C,,-1,2,3,4-TCDD
13
°C12-1,2,3,4-TCDD
13C 2-1,2,3,4-TCDD
17
IJC12-1,2,3,4-TCDD
13C12-1,2,3.4-TCDD
13C., -1,2, 3, 7, 8,9-HxCDD
•IT * * * * *
13C., -1,2, 3, 7, 8,9-HxCDD
•5C12-1,2,3,7,8,9-HxCDD
13C1,-1,2,3,7f8,9-HxCDD
13 l<:
°C12-1,2,3,7,8,9-HxCDD
13C12-1,2,3,7,8,9-HxCDD
13C12-1,2,3,7,8,9-HxCDD
13C,,-1, 2,3, 7,8,9- HxCDD
13
C12-1,2,3,7,8,9-HxCDD
"c-.-I^.S^.B^-HxCDD
13
C,,-1, 2,3,7,8, 9-HxCDD
13
'°C12-1.2,3,4-TCDD
Table 7
ACCEPTANCE CRITERIA FOR PERFORMANCE TESTS (1)
PCDDs/PCDFs
Compound
by internal standard
13C-tetra-hepta CDD and CDF
PCDDs/PCDFs
pent a
37Cl-tetra CDD
13C-octa CDD
by isotope dilution
tetra CDD and CDF
- hepta CDD and CDF
octa CDD and CDF
Test
Concen-
t rat ion
(ng/mL)
100
40
200
40
200
400
Initial
Precision
and Accuracy
Sec
s
32
13
64
9
45
90
8.2.3
X
60 -
24 -
120 -
30 -
150 -
300 -
145
58
290
52
260
520
Labeled
Compound
Recovery
Sec 8.3
and 1
P (5
25 -
25 -
25 -
25 -
25 -
25 -
Calibration
Verification
Sec
14.5
'.•) (ug/mL)
150
150
150
150
150
150
65
26
130
30
150
300
- 140
- 56
- 280
- 52
- 260
- 520
Ongoing
Accuracy
Sec
R
55
22
110
28
140
280
14.6
(%)
- 150
- 60
- 300
- 56
- 280
- 560
(1) Based on data from Method 1625.
38
-------
Table 8
SAMPLE PHASE AND QUANTITY EXTRACTED FOR VARIOUS MATRICES
Sample Matrix (1)
SINGLE PHASE
Aqueous
Solid
Organic
Example
Drinking water
Groundwater
Treated wastewater
Dry soil
Compost
Ash
Waste solvent
Waste oil
Organic polymer
Percent Quantity
Solids Phase Extracted
<1 (2) 1000 mL
>20 Solid 10 g
<1 Organic 10 g
MULTIPHASE
Liquid/Solid
Aqueous/solid
Organic/solid
Liquid/Liquid
Aqueous/organic
Aqueous/organic/
sol id
Wet soil
Untreated effluent
Digested municipal sludge
Filter cake
Paper pulp
Tissue
Industrial sludge
Oily waste
In-process effluent
Untreated effluent
Drum waste
Untreated effluent
Drum waste
1-30
1-100
Solid
Both
Organic
Organic
& solid
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 analysis.
39
------- |