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METHOD 513. DETERMINATION OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN
IN DRINKING WATER BY GAS CHROMATOGRAPHY WITH HIGH
RESOLUTION MASS SPECTROMETRY
July 1990
This method Is taken from the SW-846 Methods Manual,
Method 8280, and adapted to drinking water.
A. Alford-Stevens
James W. Eichelberger
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
HEADQUARTERS LIBRARY
ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
o
oo
33
HEADQu.., ' .
ENVIRONMLM*. ;
WASHINGTON, D.C. i
•LCTION AGENCY
+00
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METHOD 513
DETERMINATION OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN IN DRINKING WATER BY
GAS CHROMATOGRAPHY WITH HIGH-RESOLUTION MASS SPECTROMETRY
1. SCOPE AND APPLICATION § 1
1.1 This method provides procedures for identification and measurement
of 2,3,7,8-tetrachlorodibenzorp-dioxin (TCDD, CASRN 1746-01-6) at
concentrations of 20 pg/L to 2 ng/L in water sample extracts. The
minimum measurable concentration will vary among samples, depending
on the presence or absence of interfering compounds in a particular
sample.
1.2 A water sample may contain floating, suspended, and settled
particulate matter, which should not be removed by filtering before
extraction. The estimated solubility of 2,3,7,8-TCDD in water is
<50 ng/L (1), but larger measured concentrations can be caused by
TCDD associated with particulates.
1.3 Because 2,3,7,8-TCDD may be extremely toxic, safety procedures
described in Section 5 should be followed to prevent exposure of
laboratory personnel to materials containing this compound.
2. SUMMARY OF METHOD
2.1
^ iJM I I WJH» %f\r VVIJip WMIIV* ) s?Vy VLMV* «*•«* fe y W ) / y V/ llsLJU ^lllt«t^lll%Ai ^OUltNjlUlt*}
IS), are added to the water. The sample container is rinsed with
methylene chloride, which is then added to the water sample. The
water sample is extracted sequentially with three 60-mL portions of
methylene chloride. AN optional liquid-solid extraction procedure
using Empore disk technology is also described in this method. When
using this option, all surrogate compounds and internal standards
and other solutions are added just as in the liquid-liquid extraction
procedure.The combined extract is subjected to column chromatographic
procedures to remove sample components that may interfere with
detection and measurement of TCDD. A 10-juL aliquot of a solution
containing 13Ci;z-l,2,3,4-TCDD, which is used as a recovery standard
(RS), is addecf to the extract before concentration and analysis.
The sample extract is concentrated to 10 /iL, and a l-p,l or 2-/*L
aliquot is injected into a gas chromatograph (GC) equipped with a
fused silica capillary column and interfaced with a high resolution
mass spectrometer (MS). Selected characteristic ions are monitored
with high resolution MS (10,000 resolving power). Identification
of a sample component r'as TCDD is based on detection, of two
characteristic;'lions' (m/z 320 and 322) in the molecular ion cluster,
measurement of acceptable relative abundance of those two ions, and
34
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relative to the IS, "C^.S^.S-TCDD. Because the IS is a labeled
analog of the analyte, the procedure presumes that IS losses during
method procedures are equal to unlabeled TCDD losses. Therefore,
each calculated sample TCDD concentration has been compensated for
losses during sample preparation.
3. DEFINITIONS
3.1 Calibration limits — the minimum (20 pg/L) and maximum (2 ng/L)
concentration of 2,3,7,8-TCDD in solutions used to calibrate detector
response. In some samples, <20 pg/L of 2,3,7,8-TCDD may be detected
but measured concentrations will only be estimated concentrations.
In other samples, interferences may prevent identification and
measurement of 20 pg/L.
3.2 Concentration calibration solution — a solution containing known
amounts of the analyte (unlabeled 2,3,7,8-TCDD), the IS (13C "
2,3,7,8-TCDD), the SC (37Cl4-2,3,7,8-TCDD), and the RS (13C12-l,2,3,
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for the same Ion. The same number of scans must be integrated for
both areas. (The ratio of peak heights may be used instead of peak
areas.)
3.10 Surrogate compound (SC) — a compound (37Cl4-2,3,7,8-TCDD) that is
present in each calibration solution and is added at a low
concentration (20 pg/L) to each sample and blank before extraction.
Successful detection and measurement of the SC in each sample
provides some assurance that unlabeled 2,3,7,8-TCDD would be
detectable if present in the sample at >20 pg/L.
INTERFERENCES
4.1 An organic compound that has approximately the same GC retention
time 2,3,7,8-TCDD (within a few scans of the IS) and produces the
ions that are monitored to detect 2,3,7,8-TCDD is a potential
interference. Most frequently encountered interferences are other
sample components that are extracted along with TCDD; some potential
interferences are listed in Table 1. To minimize interference, high
purity reagents and solvents must be used and all equipment must be
meticulously cleaned. Laboratory reagent blanks must be analyzed
to demonstrate lack of contamination that would interfere with
2,3,7,8-TCDD measurement. Column chromatographic procedures are
used to remove some sample components; these procedures must be
performed carefully to minimize loss of 2,3,7,8-TCDD during attempts
to enrich its concentration relative to other sample components..
4.2 False positive identifications are produced only when an interfering
compound elutes from the GC column within 3 sec of the IS and
produces ions with exact masses and relative abundances very similar
to those for 2,3,7,8-TCDD. The specified GC column separates
2,3,7,8-TCDD from all 21 other TCDD isomers.
SAFETY
5.1 Because 2,3,7,8-TCDD has been identified as an animal carcinogen and
a possible human carcinogen, exposure to this compound and its
isotopically labeled analogs must be minimized (2,3). The
laboratory is responsible for maintaining a file of current OSHA
regulations regarding the safe handling of chemicals specified in
this method. A reference file of material data handling sheets
should also be made available to all personnel involved in analyses.
5.2 Each laboratory must develop a strict safety program for handling
2,3,7,8-TCDD. The following laboratory practices are recommended:
5.2.1 Minimize laboratory contamination by conducting all
manipulations in a hood.
5.2.2 Effluents of GC sample splitters and GC/MS vacuum pumps
should pass through either a column of activated carbon or
36
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be bubbled through a trap containing oil or high-boiling
alcohols.
5.2.3 Liquid waste should be dissolved in methanol or ethanol and
irradiated with ultraviolet light at a wavelength <290 nm
for several days. Analyze the liquid wastes and dispose of
the solutions when 2,3,7,8-TCDD can no longer be detected.
5.3 The following precautions for safe handling of 2,3,7,8-TCDD in the
laboratory are presented as guidelines only. These precautions are
necessarily general in nature, because detailed specific
recommendations can be made only for the particular exposure and
circumstances of each individual use. Assistance in evaluating the
health hazards of particular conditions may be obtained from certain
consulting laboratories or from state Departments of Health or of
Labor, many of which have an industrial health service. Although
2,3,7,8-TCDD is extremely toxic to certain kinds of laboratory
animals, it has been handled for years without injury in analytical
and biological laboratories. Techniques used to handle radioactive
and infectious materials are applicable to 2,3,7,8-TCDD.
5.3.1 Protective equipment: Laboratory hood adequate for
radioactive work, safety glasses, and disposable plastic
gloves, apron or lab coat.
5.3.2 Training: Workers must be trained in the proper method of
removing contaminated gloves and clothing without contacting
the exterior surfaces.
5.3.3 Person hygiene: Thorough washing of hands and forearms
after each manipulation and before breaks (coffee, lunch,
and shift).
5.3.4 Confinement: Isolated work area, posted with signs,
segregated glassware and tools, plastic-backed absorbent
paper on benchtops.
5.3.5 Waste: Good technique includes minimizing contaminated
waste. Plastic liners should be used in waste cans.
5.3.6 Disposal of Wastes: Refer to the November 7, 1986, issue
of the Federal Register on Land Ban Rulings for details
concerning handling wastes containing dioxin.
5.3.7 Decontamination: Personnel - any mild soap with plenty of
scrubbing action. Glassware, tools, and surfaces - rinse
with 1,1,1-trichloroethane, then wash with any detergent and
water. Dish water may be disposed to the sewer after
percolation through a carbon filter. Solvent wastes should
be minimized, because they require special disposal through
commercial sources that are expensive.
37
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5.3.8 Laundry: Clothing known to be contaminated should be
disposed with the precautions described under "Disposal of
Hazardous Wastes". Laboratory coats or other clothing worn
in 2,3,7,8-TCDD work area may be laundered. Clothing 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 the problem.
The washer should be run through one full cycle before being
used again for other clothing.
5.3.9 Wipe tests: A useful method to determine cleanliness of
work surfaces and tools is to wipe a surface area of
2 in. X 1 ft. with an acetone-saturated laboratory wiper
held in a pair of clean stainless steel forceps. Combine
wipers to make one composite sample in an extraction jar
containing 200 mL acetone. Place an equal number of unused
wipers in 200 mL acetone and use as a control. Extract each
jar with a wrist-action shaker for 20 min. Transfer extract
to a Kuderna-Danish (K-D) apparatus fitted with a
concentrator tube and a three-ball Snyder column. Add two
boiling chips and concentrate the extract to an apparent
volume of 1.0 mL with the same techniques used for sample
extracts. Add 100 jitL of the sample fortification solution
that has not been diluted with acetone or 1.5 mL of the
acetone-diluted solution (Section 7.14), and continue all
extract preparation steps and analytical procedures
described for samples. If any 2,3,7,8-TCDD is detected,
report the result as a quantity (picograms) per wipe test.
A lower limit of calibration of 20 pg/composite wipe test
is expected. A positive response for the control sample is
8 pg/wipe test. When the sample contains >25 pg, steps must
be taken to correct the contamination. First vacuum the
working places (hoods, benches, sink) using a vacuum cleaner
equipped with a high-efficiency particulate absorbent filter
and then wash with a detergent. Analyze a new set of wipers
before personnel are allowed in work in the previously
contaminated area.
5.3.10 Inhalation: Any procedure that may produce airborne
contamination must be carried out with good ventilation.
Gross losses to a ventilation system must not be allowed.
Handling the dilute solutions normally used in analytical
and animal work presents no significant inhalation hazards
except in case of an accident.
5.3.11 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.
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,6. APPARATUS AND EQUIPMENT
6.1 Computerized GC/MS System
6.1.1 The GC must be capable of temperature programming and be
equipped with all required accessories, such as syringes,
gases, and capillary columns. The GC Injection port must
be designed for capillary columns. Splltless or on-column
Injection technique Is recommended. With this method, a
Z-pL Injection is used consistently. A 1-jttL injection
volume can be used, but the injection volume should be
constant throughout analyses of calibration solutions and
related blanks, sample extracts, and quality control
samples.
6.1.2 GC/MS interface components should withstand temperatures up
to 280°C. The interface should be designed so that
separation of 2,3,7,8-TCDD from all other TCDD isomers
achieved in the GC column is not appreciably degraded. Cold
spots or active surfaces (adsorption sites) in the interface
can cause peak tailing and broadening. The GC column should
be inserted directly into the MS ion source without being
exposed to the ionizing electron beam. Graphite ferrules
should be avoided in the injection port because they may
adsorb TCDD. Vespel or equivalent ferrules are recommended.
6.1.3 The static resolving power of the MS must be maintained at
>10,000 (10% valley). The MS must be operated in a selected
; ion monitoring (SIM) mode, and data must be acquired for the
ions listed in Table 2 during a total cycle time (including
instrument overhead time) of <1 s. Selection of the lock-
mass ion is left to the performing laboratory. Recommended
MS tuning conditions are provided in Section 9.1. The ADC
zero setting must allow peak-to-peak measurement of baseline
.noise for every monitored channel and allow good estimation
; of instrument resolving power.
6.1.4 A dedicated data system is used to control rapid SIM data
collection. Quantitation data (peak areas or peak heights)
must be acquired continuously and stored. The data system
must be capable of producing selected ion current profiles
(SICPs, which are displays of ion intensities as a function
of time) for each monitored ion, including the lock-mass
ion. Quantitation may be based on computer-generated peak
areas or on measured peak heights. The data system must be
capable of acquiring data for > five ions and generating
hard copies of SICPs for selected GC. retention time
intervals and permit measurement of baseline noise.
6.2 GC Column. ' Two narrow bore, fused silica capillary columns coated
with phenyl cyanppropyl si 11 cone are recommended; one is a 60-m SP-
39
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2330 and the other is a 50-m CP-SIL 88. Any capillary column that
separates 2,3,7,8-T€DD from all other TCDO isomers may be used, but
this separation must be demonstrated. At the beginning of each 12-h
period during which analyses are to be performed, column operating
conditions must be demonstrated to achieve the required separation
on the column to be used for samples. Operating conditions known
to produce acceptable results with the recommended columns are shown
in Table 3.
6.3 Miscellaneous Equipment.
6.3.1 Nitrogen evaporation apparatus with variable flow rate.
6.3.2 Balances capable of accurately weighing to 0.01 g and
0.0001 g.
6.3.3 Centrifuge.
6.3.4 Water bath equipped with concentric ring covers and capable
of being temperature controlled within ±2°C.
6.3.5 Glove box.
6.3.6 Drying oven.
6.3.7 Minivials — 1-mL amber borosilicate glass with
conical-shaped reservoir and screw caps lined with
Teflon-faced silicone disks.
6.3.8 Pipets, disposable, Pasteur, 150 mm X 5 mm i.d.
6.3.9 Separatory funnels, 2 L with Teflon stopcock.
6.3.10 Kuderna-Danish concentrator, 500 ml, fitted with 10-mL
concentrator tube and three-ball Snyder column.
6.3.11 Teflon boiling chips washed with hexane before use.
6.3.12 Chromatography column, glass, 300 m X 10.5 mm i.d., fitted
with Teflon stopcock.
6.3.13 Adapters for concentrator tubes.
^
6.3.14 Continuous liquid-liquid extractor (optional).
6.3.15 Glass funnels, appropriate size to accommodate filter paper
used to filter extract (volume of approximately 170 mL).
6.3.16 Desiccator.
6.4 CAUTION: To avoid the risk of using contaminated glassware, all
glassware that is reused must be meticulously cleaned as soon as
40
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7.
•possible after use. Rinse glassware with the last solvent used In
It. Wash with hot Water containing detergent. Rinse with copious
amounts of tap water and several portions of distilled. Drain dry
and heat in a muffle furnace at 400°C for 15-30 win. Volumetric
glassware must not be heated in a muffle furnace. Some thermally
.stable materials (such as PCBs) may not be removed by heating in a
muffle furnace. In these cases, rinsing with high-purity acetone
and hexane may be substituted for muffle-furnace heating. After the
glassware is dry and cool, rinse it with hexane and store it
inverted or capped with solvent-rinsed aluminum foil in a clean
environment.
6.5 TCDD concentrations of concern in water are much lower than those
of concern in many other sample types. Extreme care must be taken
to prevent cross-contamination between water and other samples. The
use of separate glassware for water samples is recommended.
6.6 Empore extraction disks, C-18, 47mm.
6.7 Mi 111 pore Standard Filter Apparatus (or equivalent) to hold disks,
all glass
REAGENTS AND CONSUMABLE MATERIALS
7.1 Alumina, acidic (BioRad Lab. #132-1240 or equivalent). Extract in
, ., a Soxhlet apparatus with methylene chloride for 6 h (> 3 cycles/h)
and activate it by heating in a foil-covered glass container for
24 h at 190°C.
7.2 Carbon, (Amoco PX-21 or equivalent).
7.3 Glass wool. Extract with methylene chloride and hexane and air-dry
before use. Store in a clean glass jar.
1 '''•
7.4 Potassium hydroxide, ACS grade.
7;5 Potassium silicate. Slowly dissolve 56 g of reagent grade potassium
hydroxide in 300 mL of anhydrous methanol in a 1-L round bottom
: flask. Add slowly with swirling 100 g silica gel (prewashed and
activated). With a rotary evaporation apparatus with no vacuum
applied, rotate the flask and heat to 55°C for 90 min. After the
mixture cools to room temperature, pour it into a large glass column
containing a plug of glass wool at the end. Wash the mixture into
the column with methanol, and then add 200 mL of methanol. When the
methanol level reaches the bed of sorbent, add 200 mL of methylene
chloride to the column. Push the methylene chloride through the
column of sorbent by applying nitrogen pressure to dry or partially
dry the sorbent, which is then activated at 130°C overnight.
7.6 Silica gel, high purity grade, type 60, 70-230 mesh. Extract in a
.Soxhlet apparatus with methylene chloride for 6 h (>3 cycles/h) and
41
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activate by heating in a foil-covered glass container for 24 h at
130°C.
7.7 Silica gel impregnated with 40% (w/w) sulfuric acid. Add two parts
(by weight) concentrated sulfuric acid to three parts (by weight)
silica gel (extracted and activated), mix with a glass rod until
free of lumps, and store in a screw-capped glass bottle.
7.8 Silica gel/Carbon. To a 20-g portion of silica gel add 500 mg
carbon, and blend until the mixture is a uniform color.
7.9 Sodium sulfate, granular, anhydrous.
7.10 Solvents, high purity, distilled-in-glass, or highest available
purity: methylene chloride, hexane, benzene, methanol, tridecane,
isooctane, toluene, cyclohexane, and acetone.
7.11 Sulfuric acid, concentrated, ACS grade, specific gravity 1.84.
7.12 Concentration Calibration Solutions (Table 4) — Five (or more)
tridecane solutions (CAL 1-5) containing unlabeled 2,3,7,8-TCDD and
isotopically labeled TCDDs. All five solutions contain unlabeled
2,3,7,8-TCDD at varying concentrations and the IS (13C1?-2,3,7,8-
TCDD, CASRN 80494-19-5) and the RS (13C12-1,2,3,4-TCDD) each at a
constant concentration. Three of these solutions also contain the
surrogate compound (SC, 37Cl4-2,3,7,8-TCDD, CASRN 85508-50-5) at
varying concentrations. All standards required for preparing CALs
are commercially available but must be verified by comparison with
the National Bureau of Standards certified solution SRM-1614, which
contains 67.8 ng/mL of unlabeled 2,3,7,8-TCDD and 65.9 ng/mL of 13C-
labeled 2,3,7,8-TCDD at 23°C. Note: CALs can be prepared by
diluting calibration solutions used in Contract Laboratory Program
procedures for 2,3,7,8-TCDD determinations with low resolution MS;
to obtain appropriate IS concentrations for CAL 4, however, solvent
containing the IS must be used for dilution. Calibration solutions
used for USEPA Method.8290 can also be used to determine RFs for
2,3,7,8-TCDD; with those solutions the lower calibration
concentration may be higher (25 pg/L rather than 20 pg/L) or lower,
depending on injected volume of calibration solution. Because
Method 8290 solutions do not contain the SC, however, one or three
additional solutions containing that compound will be necessary to
measure its RF relative to the IS. Assuming adequate
reproducibility of RF measurements, triplicate analyses of one
solution (recommended SC concentration of 1.2 pg/A*L) or single
analysis of three solutions (0..6 to 1.8 pg/MU Table 4) are
acceptable.
7.12.1 Each of CALs 1-5 contains the IS at a concentration of
50 pg/ML; if 100% of the IS is extracted, 10 ML of this
solution is equivalent to a 10-jxL extract of a 1-L sample
to which 500 pg of IS was added before extraction. If 100%
42
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of the analyte is extracted, CALs 1-5 contain unlabeled
2,3,7,8-TCDD at concentrations that are equivalent to 10-nl
extracts of 1-L samples containing 20 pg to 2 ng.
7.12.2 CALs 1-3 contain the SC (37C1-2,3,7,8-TCDD) at a
concentration of 0.6 pg/^L, 1.2 pg/*iL, and 1.8 pg//iL,
respectively; 10 jxL of those solutions are equivalent to
10 juL extracts containing 30%, 60%, and 90%, respectively,
of the amount of SC added to each 1-L sample before
extraction.
7.12.3 Store CALS in 1-mL amber minivials at room temperature in
, the dark.
7.13 Column Performance Check Solution -- contains a mixture of TCDDs
including the IS, the SC, unlabeled 2,3,7,8-TCDD, 1,2,3,4-TCDD
(CASRN 30746-58-8), 1,4,7,8-TCDD(CASRN 40581-94-0), 1,2,3,7,-TCDD
(CASRN 67028-18-6), 1,2,3,8-TCDD (CASRN 53555-02-5), 1,2,7,8-TCDD
(CASRN 34816-53-0), and 1,2,6,7-TCDD (CASRN 40581-90-6). Other
TCDDs can be present. Except for the IS and SC, solution component
concentrations are not critical. The IS concentration should be
• 10 ± 1 pg/ML and the SC concentration should be 0.6 ±0.1 pg/L,
because ions produced by these compounds will be used to check
signal-tornoise ratios.
7.14 Sample Fortification Solution. A solution containing the IS at a
concentration of 5 to 25 pg/^L and the SC at a concentration of 0.2
. , ' to 1 pg/^L, but with the ratio of IS to SC always 25:1. The
, , solution solvent is not critical; mix 20 to 100 pi, as appropriate
to produce needed IS and SC concentrations (50 pg/L and 2 pg/L,
respectively) of this solution with 1.5 mL of acetone. Add the
resulting solution to each sample and blank before extraction.
7J5 Recovery Standard Solution. A tridecane solution containing the
recovery standard, 13C12-1,2,3,4-TCDD at a concentration of 50 pg/ML.
A lQ-fj.1 aliquot of this solution is added to each sample and blank
extract before concentrating the extract to its final volume for
analysis (Section 11.4.1).
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 Samples must be collected in glass containers. The container should
not be rinsed with sample before collection.
8.2 Samples may be stored under ambient conditions as long as
temperature extremes (below freezing or >90°F) are avoided. To
prevent photo-decomposition, samples must be protected from light
from the time of collection until extraction.
8.3 All samples must be extracted within 90 days after collection and
completely analyzed within 40 days after extraction.
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9. GC/MS SYSTEM CALIBRATION.
9.1 MS Performance.
9.1.1 The MS must be operated In the electron ionization mode, and
a static resolving power of >10,000 (10% valley definition)
at >m/z 334 must be demonstrated before any analysis is
performed. The resolving power must be documented by
recording the mass profile of the reference peak. The
format of the peak profile representation must allow manual
determination of resolution (i.e., the horizontal axis must
be a calibrated mass scale (amu or ppm per division). The
peak width at 5% peak height must appear on the hard copy
and cannot exceed 100 ppm. Static resolving power must be
checked at the beginning and end of each 12-h period of
operation. A visual check of static resolution is
recommended before and after each analysis.
9.1.2 Chromatography time may exceed the long-term mass stability
of the high resolution MS, and mass drift of a few ppm can
affect the accuracy of measured masses. Therefore, a mass
drift correction is required. A lock mass ion from the
reference compound (high boiling perfluorokerosene, PFK, is
recommended) is used to calibrate the MS. An acceptable
lock mass is an ion with mass larger than the lightest mass
monitored but less than the heaviest ion monitored. The
amount of. PFK introduced into the ion chamber during
analysis should be adjusted so that the amplitude of the
lock mass ion is <10% full scale. Excessive PFK may cause
noise problems and ion source contamination.
9.1.3 Using a PFK molecular leak, tune the MS to obtain resolving
power of >10,000 (10% valley) at m/z 334. Using a
reference peak near m/z 320, verify that the exact mass of
the reference peak is within 5 ppm of the known mass. The
low- and high-mass reference ions must be selected to
provide the voltage jump required to detect ions from m/z
320 through m/z 334. (Note: With a qualitative
confirmation option in Section 11.5.5, detected ion range
will be m/z 257 to m/z 334.)
9.1.4 MS resolving power must be demonstrated by recording the
mass peak profile of the high-mass reference signal obtained
using the low-mass ion as a reference. The minimum
resolving power of 10,000 must be demonstrated on the high-
mass ion while it is transmitted at a lower accelerating
voltage than the low-mass reference ion, which is
transmitted at full sensitivity. The peak profile
representation must allow manual determination of the.
resolution (i.e., the horizontal axis must be a calibrated
mass scale in amu or ppm per division. The measured peak
44
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width at 5% of the peak maximum must appear on the hard copy
and cannot exceed 100 ppm at the high mass.
9.2 Initial Calibration
9.2.1 GC column performance. The laboratory must verify GC
conditions necessary for required separation of 2,3,7,8-TCDD
from other TCDD isomers. Inject 2 ML of the performance
check solution and acquire SIM data for the five ions in
Table 2 (nominal m/z 320, 322, 328, 332, and 334) within a
total cycle time of <1 s. Acquire at least five scans for
; each ion across each GC peak and use the same data
acquisition time for each ion monitored. The peak
representing 2,3,7,8-TCDD and peaks representing any other
TCDD isomers must be resolved with a valley of <25% (Figure
1), Valley % - 100 x/y, where y is peak height of 2,3,7,8-
TCDD and x is measured as shown in Figure 1 between 2,3,7,8-
TCDD and its closest eluting isomer. CAUTION: The same
data acquisition parameters must be used to analyze all
calibration and performance check solutions.
9.2.2 MS calibration and sensitivity check. Ratio of integrated
ion current for m/z 320 to m/z 322 produced by unlabeled
2.3,7,8-TCDD and for m/z 332 and 334 produced by the IS
("C-labeled 2,3,7,8-TCDD) must be >0.67 and <0.87. The S/N
ratio for m/z 328 produced by the SC (13C-labeled 1,2,3,4-
TCDD) must be >2.5 and the S/N ratio for m/z 332 produced
by the IS must be >10.
.,, 9.2.3 Using the same GC and MS conditions, analyze a 2-/iL aliquot
of the medium concentration CAL (CAL 3). Check ion ratios
specified in Section 9.2.2. If criteria are met, analyze
a 2-/iL,aliquot of each of the four (or more), remaining CALS.
9.2.4 For each CAL, ensure that ion ratios (Section 9.2.2) are
acceptable. For CAL 1 (the lowest concentration CAL) data,
ensure that each ion produces a signal-to-noise (S/N) ratio
of >2.5. Display a SICP for a region of the chromatogram
near the elution time of 2,3,7,8-TCDD but where no analyte
or interference peak is present. The preferred width of the
display is about 10 X full width at half height of the IS
peak. The "noise" is the height (measured from the lowest
point in the display window) of the largest signal not
attributable to any eluting substance.
•>. 9.2.5 RF Measurements. Using data acquired for each CAL,
calculate the RF for unlabeled 2,3,7,8-TCDD, the SC (37CL-
2,3,7,8-TCDD), and the RS (13C12-2,3,7,8-TCDD) relative to
the IS (13C12-2,3,7,8-TCDD) with the following equation:
RF- VQi./"AI.QX
45
-------
where Ax= the sum of integrated ion abundances of m/z
320 and 322 for unlabeled 2,3,7,8-TCDD, the
abundance of m/z 328 for the SC, or the
abundances of m/z 332 and 334 for the RS.
A, « the sum of integrated ion abundances of m/z
332 and 334 for the IS,
Qis - injected quantity of IS, and
Qx = injected quantity of unlabeled 2,3,7,8-TCDD,
the SC, or the RS.
RF is a unitless number; units used to express
quantities must be equivalent.
9.2.6 For each compound (unlabeled 2,3,7,8-TCDD, the SC, and the
RS), calculate a mean RF and the relative standard deviation
(RSD) of the five measured RFs. When RSD exceeds 20%,
analyze additional aliquots of appropriate CALs to obtain
an acceptable RSD of RFs over the entire concentration
range, or take action to improve GC/MS performance.
9.3 Routine Calibration. If a laboratory operates during only one <12-h
period (shift) each day, routine calibration procedures must be
performed at the beginning (after mass calibration and successful
analysis of the performance check solution to ensure adequate
sensitivity and acceptable ion ratios) of that shift, and the
performance check solution must be analyzed again at the end of that
shift to validate data acquired during the shift. If the laboratory
operates during consecutive shifts, routine calibration procedures
must be performed at the beginning of each shift, but analysis of
the performance check solution at the beginning of each shift and
at the end of the final 12-h period is sufficient.
9.3.1 Inject a 2-/iL aliquot of CAL 3, and analyze with the same
conditions used during Initial Calibration.
9.3.2 Demonstrate acceptable performance for ions abundance
ratios, and demonstrate that each measured RF for unlabeled
2,3,7,8-TCDD, the SC, and the RS is within 20% of the
appropriate mean RF measured during initial calibration.
If one or more of these criteria are not met, up to two
additional attempts can be made before remedial action is
necessary and the entire initial calibration process is
repeated. Corrective action may include increasing the
detector sensitivity, baking the GC column, clipping a short
length (about 0.3-0.5 m) of the injector side of the GC
column, washing or replacing the GC column, and cleaning the
46
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ion source. If degradation of the standards in CALs Is
.-.. suspected, a fresh set of CALs should be. used for repeating
••: initial calibration procedures.
10. QUALITY CONTROL
10.1 Laboratory Reagent Blank. Perform all steps in the analytical
procedure using all reagents, standards, equipment, apparatus,
glassware, and solvents that would be used for a sample analysis,
but omit a water sample, and substitute 1 L of reagent water.
10.1.1 Analyze two laboratory reagent blanks (LRBs) before sample
analyses begin and when a new batch of solvents or reagents
is used for sample extraction. Do not add any IS, SC or RS
to one blank; this will allow demonstration that reagents
contain no impurities producing any ion current above the
level of background noise for monitored ions.
10.1.2 Criteria for acceptable LRBs.
10.1.2.1 When no IS, SC, or RS is present, no ion current
.:-... above the level- of background S/N is detected for
!, . any monitored ion within 20 s of the retention
times previously measured for labeled 1,2,3,4-
TCDD or for unlabeled and labeled 2,3,7,8-TCDD.
10.1.2.2 When the IS is present, no ion current'for m/z
259, 320, or 322 is observed that is >2% of the
abundance of m/z 332 within 5 scans of the IS
peak maximum.
" 10.1.3 Corrective action for unacceptable LRB. Check solvents,
.:',,.'. reagents, apparatus, and glassware to locate and eliminate
\ the source of contamination before any samples are extracted
and analyzed. Purify or discard contaminated reagents and
solvents. .
10.2 Field Blanks. An acceptable field blank must meet criteria in
Section 10.1.2.2. When results for a field blank are acceptable,
analysis of an LRB is not needed with that sample batch. When field
blank results are not acceptable, analysis^of an LRB is needed; if
LRB results are acceptable, data for samples associated with the
field blank must be accompanied by pertinent information about the
nature and amount of contamination observed in the field blank.
10.3 Corrective action for unacceptable performance check solution data.
When the MS sensitivity requirement (Section 9.2.2) is not met at
the end of a 12-h period in which sample extracts were analyzed, all
related sample extracts must be reanalyzed after criteria have been
met. When other criteria (ion ratios or GC resolution) are not met,
; ,. all sample extracts that produced positive results or potential
47
-------
positive results must be reanalyzed after calibration criteria have
been met.
11. PROCEDURE
11.1 Sample Extraction ~ Liquid-Liquid Extraction
11.1.1 Mark the water meniscus on the side of the 1-L sample bottle
for later determination of the exact sample volume. Pour
the entire sample (approximately 1 L) into a 2-L separatory
funnel. A continuous liquid-liquid extractor may be used
instead of a separatory funnel.
11.1.2 Add 1.5 mL of the sample fortification solution (Section
7.14) to the sample in the separatory funnel.
11.1.3 Add 60 mL of methylene chloride to the sample bottle, seal
and shake 30 s to rinse the inner surface. Transfer the
solvent to the separatory funnel and extract the sample by
shaking the funnel for 2 min with periodic venting. Allow
the organic layer to separate from the water phase for a
minimum of 10 min. If an emulsion interface between layers
exists, the analyst may use mechanical techniques to
complete the phase separation. Collect the methylene
chloride layer directly into a 500-mL Kuderna Danish (K-D)
concentrator (mounted with a 10-mL concentrator tube) by
passing the sample extract through a filter funnel packed
with a glass wool plug and 5-g of anhydrous sodium sulfate.
Repeat the extraction with two additional 60-mL portions of
methylene chloride, filtering each extract before adding it
to the K-D concentrator. After the third extraction, rinse
the sodium sulfate with an additional 30 mL of methylene
chloride to ensure quantitative transfer, and add rinse to
composite extract.
11.1.4 Add one or two clean boiling chips to the evaporative flask
and attach a Snyder column. Prewet the Snyder column by
adding about 1 mL of methylene chloride to the top. Place
the K-D apparatus on a hot water bath (60-65°C) so that the
concentrator tube is partially immersed in the hot water,
and the entire lower rounded surface of the flask is bathed
with hot vapor. Concentrate the extract until the apparent
volume of the liquid reaches 1 mL. Remove the K-D apparatus
and allow it to drain and cool for at least 10 min. Remove
the Snyder column, add 50 mL of hexane and a new boiling
chip and reattach the Snyder column. Increase the water
bath temperature to 85-90°C and concentrate the extract to
approximately 1 mL. Rinse the flask and the lower joint
with 1-2 mL hexane. Concentrate the extract to 1 mL under
a gentle stream of nitrogen. If further extract processing
is to be delayed, the extract should be quantitatively
48
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transferred to a Teflon-sealed, amber, screw-cap vial and
stored refrigerated and protected from light.
11.1.5 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the liquid to a 1000 ml
graduated cylinder. Record the sample volume to the nearest
5 ml.
11.2 Sample Extraction -- Liquid-Solid Extraction
11.2.1 Preparation of disks
11.2.1.1 Insert the disk into the 47mm filter apparatus.
Wash the disk with about .10 mL of benzene by
adding the solvent to the disk, pulling about
half through the disk and allowing it to soak the
disk for about a minute, then pulling the
remaining rinse solvent through the disk. With
the vacuum on, pull air through the disk for
about one minute.
11.2.1.2 Pre-wet the disk with 10 ml methanol {MeOH) by
adding the MeOH to the disk, pulling about half
through the disk and allowing it to soak for
about a minute, then pulling MOST of the MeOH
through. A layer of MeOH should be left on the
surface of the disk, which should not be allowed
to go dry from this point until the end of the
sample extraction. This is an important step to
ensure uniform flow and good analyte recoveries.
11.2.1.3 Rinse the disk with 10 ml reagent water by adding
the water to the disk and pulling most through,
again leaving a layer on the surface of the disk.
11.2.2 Mark the water miniscus on the side of the 1-L sample bottle
for later determination of the exact sample volume.
11.2.3 Add the water sample, to which all necessary surrogate
compounds and internal standards have been added according
to Section 11.1.2, to the reservoir and turn on the vacuum
to begin the extraction. Aspirator vacuum should be adjusted
to allow the sample to pass through the disk in
approximately 20 minutes. Extract the entire sample,
draining as much water as possible from the sample
container. After all the sample has passed through, draw
air through the disk for about 10 minutes to remove some of
the residual water.
11.2.4 Remove the filtration top from the apparatus, but do not
disassemble the reservoir and fritted base. Empty the
49
-------
water from the flask and insert a suitable sample tube to
contain the eluate. The only constraint on the sample tube
is that it fit around the drip tip of the fritted base.
; Reassemble the apparatus.
11.2.5 Add 5 ml benzene to the sample bottle and rinse the inside
of the container. Transfer this benzene to the disk with a
dispo- pipet or other suitable vessel, rinsing the sides of
the filtration reservoir in the process. Pull about half of
the benzene through the disk, release the vacuum, and allow
the disk to soak for about a minute. Pull the remaining
benzene through the disk.
11.2.6 Repeat the above step twice. Pour the combined eluates
through a small funnel containing about 3 grams of anhydrous
\ ' sodium sulfate. The sodium sulfate may be contained in a
prerinsed filter paper, or by a plug of prerinsed glass wool
- in the stem of the funnel. Rinse the sodium sulfate with a
5 ml aliquot of benzene.
11.2.7 Quantitatively transfer the combined eluate to a suitable
graduated concentrator tube, and rinse the test tube with
^ ' benzene. Using micro-Kuderna-Danish or nitrogen blowdown,
concentrate the eluate almost to dryness, then add hexane
to bring the volume to 1 ml for sample extract cleanup.
11.2.8 Determine the original sample volume by refilling the sample
bottle to the mark, and transferring the liquid to a 1000
ml graduated cylinder. Record the sample volume to the
• " nearest 5 ml.
11.3 Sample Extract Cleanup
11.3.1 Chromatography columns 1 and 2, described below, are
recommended for every sample extract. A third column
containing silica gel and carbon may be useful for removal
"•""' - of interferences from some sample extracts and may be used
at the analyst's discretion. Because each cleanup procedure
• ' increases the chances of analyte loss, such procedures
should be minimized. Criteria for predicting when the
carbon column will be needed are not available, but that
column is probably not needed for finished drinking water
samples that have been filtered through granular activated
carbon.
^ 11.3.2 Column Preparation
11.3.2.1 Column 1. Place 1.0 g of silica gel (See NOTE)
into a 1.0 cm X 20 cm column and tap the column
gently to settle the silica gel. Add 2 g
potassium hydroxide impregnated silica gel, 1 g
silica gel, 4.0 g of sulfuric acid impregnated
50
-------
silica gel, and 2 g silica gel. Tap column
gently after each addition. NOTE: The silica
gel for this application is partially deactivated
with 1% water immediately before packing the
column.
11.3.2.2 Column 2. Place 6.0 g of alumina into a 1.0 cm
X 30 cm column and tap the column gently to
settle the alumina. Add a 1-cm layer of purified
sodium sulfate to the top of the alumina.
11.3.2.3 Add hexane to each column until the packing is
free of channels and air bubbles. A small
positive pressure (5 psi) of clean nitrogen can
be used if needed.
11.3.3 Quantitatively transfer the sample extract to the top of the
silica gel in column 1. Rinse the concentrator tube with
two 0.5 ml portions of hexane; transfer rinses to Column 1.
With 90 ml of hexane, elute the extract from Column 1
directly into Column 2.
11.3.4 Add an additional 20 ml of hexane to Column 2 and elute
until the hexane level is just below the top of the sodium
sulfate; discard the eluted hexane.
11.3.5 Add 20 ml of 20% methylene chloride/80% hexane (v/v) to
Column 2 and collect the eluate.
11.3.6 If carbon column cleanup is selected, proceed with Section
11.3.7. If not, proceed with Section 11.3.8.
11.3.7 Optional cleanup with Column 3. Reduce the volume of eluate
from Column 2 to about 1 ml in a K-D apparatus. Transfer
the concentrated eluate from Column 2 to a 4 mm X 200 mm
column (2 ml disposable pipette) containing 200 mg silica
gel/carbon. Elute with 15 ml methylene chloride and 15 mL
80% methylene chloride/20% benzene (v/v). in forward
direction of flow. Discard these fractions. .Elute TCDD
with 15 ml toluene in a reverse direction flow. Collect
this eluate.
11.3.8 Concentrate the eluate (either the toluene fraction from
'Section 11.3.7 or the methylene chloride/hexane fraction
from Section 11.3.5) to a small volume {<0.5 ml) and
transfer to a 1-mL minivial. Store the extract in the dark
at 4°C until just before analysis. Note: The final volume
is adjusted to 10 /uL immediately before 6C/MS analysis.
li.4 GC/MS Analysis of Extracts
51
-------
11.4.1 Remove the sample or blank extract from storage and allow
it to warm to ambient laboratory temperature. Add a 10-jiL
aliquot of the RS solution (Section 7.15) to the extract and
reduce the extract volume to 10 ML with a stream of dry,
purified nitrogen.
11.4.2 Inject a 2-/iL aliquot of the extract into the GC, operated
under conditions previously used to produce acceptable
results with the performance check solution.
11.4.3 Acquire SIM data using the same analytical conditions
previously used to determine RFs.
11.5 Identification Criteria
11.5.1 Obtain SICPs for each ion monitored.
11.5.2 The abundance of m/z 332 relative to m/z 334 produced by the
IS must be >0.67 and <0.87, and these ions must maximize
within 1 scan of each other. Retention time should be
within ±5 scans of that observed during the most recent
acceptable calibration. For good performance, the retention
time of the IS must be reproducible to ±5 scans from one
injection to the next. Over the course of a 12-h work
period, the IS retention time should be reproducible within
+10 scans. Less reproducible IS retention times indicate
serious chromatography problems that should be corrected
before further sample analyses.
11.5.3 The sample component must produce a signal for both ions
monitored to detect and measure unlabeled 2,3,7,8-TCDO, and
the abundance of m/z 320 relative to m/z 322 must be >0.67
and <0.87. All ions must maximize within 1 scan of each
other and within 3 sec of the IS.
11.5.4 The S/N ratio for each unlabeled TCDD and SC ion must be
>2.5 and must not have saturated the detector; the S/N ratio
for each IS and RS ion must be >10 and must not have
saturated the detector.
11.5.5 Additional qualitative confirmation can be obtained by
monitoring m/z 257 and 259 (fragment ions produced by loss
of COC1 from the analyte) along with ions previously
specified or by reanalysis of an aliquot of the extract to
monitor m/z 257 and 259 along with m/z 268 and 270, fragment
ions produced by loss of COC1 from the IS. The relative
abundance of m/z 257 to 259 and m/z 268 to 270 should be 0.9
to 1.1, and the abundance of 259 to 270 should be the same
as the ratio of 322 to 334 measured in the previous
injection. Although variable with instrumental conditions,
the abundance of fragment ions relative to molecular ions
52
-------
is approximately 30-45% for each compound; therefore, the
detection limit for these ions will be greater than for
molecular ions.
12. CALCULATIONS
12.1 From appropriate SICPs of nominal m/z 259, 320 and 322, obtain and
record the spectrum number of the apex of the chromatographic peak
produced by unlabeled TCDD and the area of the entire chroma-
tographic peak.
12.2 Calculate the concentration using the formular
Cx «
-------
Ar. .= sum of areas for m/z 332 and m/z 334 produced by the
RS,
Qrs = quantity (picograms) of RS added to the sample,
RF = mean RF measured for the RS relative to the IS during
initial calibration, and
V = Volume (liters) of water extracted.
12.5 Report calculated concentrations with three significant figures when
measured concentration is >100 pg/L and with two significant figure
when value is <100 pg/L. The recovery of the IS should be >40% and
IS
12.6 Estimated Maximum Possible Concentration (EMPC) -- For samples in
which no unlabeled 2,3,7,8-TCDD is detected, calculate the EMPC,
which is the concentration required to produce a signal with S/N
ratio of 2.5. The background signal level (area or height) within
±5 scans of the IS peak is determined as previously described and
is multiplied by 2.5. With the following formula, the product is
related to the estimated unlabeled TCDD concentration required to
produce a signal equivalent of 2.5 S/N.
EMPC = 2.5 ' Bx • Qis / Ais • RF ' V
Bx = background (height or area) for either nominal
m/z 320 or 322 within ±5 scans of the IS peak,
peak height or area (depending on selection for
Bx) for nominal m/z 332 when m/z 320 is used to
determine Bx or nominal m/z 334 when m/z 322 is
used to determine Bx> and
Qis, RF, and V retain previous definitions.
12.7 An interference results when sample a component elutes in the
retention time window for 2,3,7,8-TCDD and produces both monitored
TCDD ions but measured relative abundances do not meet
identification criteria. Any ion with S/N of <2.5 should be
ignored. Ions producing S/N of >2.5 but with unacceptable relative
abundance should be treated as an interference, and a quantitative
estimate of that interference should be calculated using the
equation in Section 12.2. Interferences observed in blanks and also
present in samples should not be reported as a sample interference
but should be reported as a blank interference.
12.8 Table 5 lists results of analyses of fortified reagent water samples
carried out using the Empore disk extraction method according to the
procedure detailed in Section 11.2. Even though this method was
developed for only 2,3,7,8-TCDD, since the other dioxins and furans
54
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had been studied, the results were included. The fortifying levels
were 0.16 ng/L for the tetra isomers, 0.8 ng/L for the penta, hexa,
and hepta isomers, and 1.6 ng/L for the octa isomers. The average
recovery for all isomers in all replicate analyses is 80% with an
11% relative standard deviation. No clean up was done on these
samples.
13. REFERENCES
1. "Water Solubility of 2,3,7,8-Tetrachlorodibenzo-p-dioxin," L. Marple,
R. Brunck, and L. Throop, Environ; Sci. and Techno!. 1986, 20(2), 180-
182.
2. "Carcinogens -- Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Centers for Disease
Control, NIOSH, Pub. #77-206, August 1977.
3. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Ed., 1979.
4. Statement of Work, Dixoin Analysis, Soil/Sediment and Water Matrices,
IFB WA86-K357, September 1986.
55
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TABLE 1. POTENTIAL INTERFERENCES
Compound
Heptachlorobiphenyl
Nonachlorobiphenyl
Tetrachl oromethoxy-
biphenyl
Tetrachl orobenzyl -
phenyl ether
DDT
DDE
Pentachl orobenzyl -
phenyl ether
Tetrachl oroxanthene
Hydroxytetrachl oro-
dibenzofuran
Tetrachl orophenyl-
benzoqulnone
Interfering
Formul a
M+ - 2 35C1
M* - 4 35C1
M+ - 3 35C137C1
M*
M+
M*
M*
M+ - H35C1
M* - H35C1
M*
M*
M+ - H35C1
M+ - H35C1
M+
M+
M*
M+
M*
M+
Ion
m/z
321.867
319.8521
321.8491
319.9329
321.9299
319.9329
321.9300
319.9321
321.9292
319.9321
321.9292
319.9143
321.9ll4
319.9143
321.9114
319.8966
321.8936
319.8966
321.8936
Required
Resolution
12,476
7,189
7,233
8,805
8,848
8,813
8,843
9,006
9,050
9,011
9,050
18,043
18, 104
18,043
18,104
—
-
56
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TABLE 2. IONS TO BE MONITORED
Accurate
Mass
Elemental
Composition
Compound
258.9298
319.8965
321.8936
327.8847
331.9368
and
333.9339
and
C12H435C1402
C12H435C1337C102
C12H437C1402
"C12H435C1337C102
Unlabeled 2,3,7,8-TCDD
Unlabeled 2,3,7,8-TCDD
Unlabeled 2,3,7,8-TCDD
37
Cl4-2,3,7,8-TCDD(SC)
13
C12-2,3,7,8-TCDD (IS)
13C12-1,2,3,4-TCDD (RS)
13
Cl2-2,3,7,8-TCDD (IS)
13,
C12-1,2,3,4-TCDD (RS)
57
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TABLE 3. GC OPERATING CONDITION GUIDELINES
Column coating
Film thickness
i
Column dimensions
Helium* linear
velocity
Initial temperature
Initial time
Temperature program
Retention time of
2,3,7,8-TCDD
SP-2330
0.2 urn
60 m X 0.24 mm
28-29 cm/sec
at 240°C
70°C
4 min
Rapid increase to 200°C;
200°C to 240°C at
4°C/min
24 min
CP-SIL 88
0.22 urn
50 m X 0.22 mm
28-29 cm/sec
at 240°C
45°C
3 min
Rapid increase to 190°C;
190°C to 240°C at
5°C/min
26 min
*Hydrogen is an acceptable carrier gas.
58
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TABLE 4. COMPOSITION OF CONCENTRATION CALIBRATION SOLUTIONS
Analyte
Surrogate Cmpd. Internal Std. Recovery Std.
Unlabeled
CAL f 2,3,7,8-TCDD
37C1 - 13C
2,3,47,8-TCDD 2,3,^8-TCDD
13
c,,-
2 pg//iL
0.6 pg//iL
50
30
2
3
4
5
10
50
100
200
1.2
1.8
0
0
50
50
50
50
30
30
30
30
59
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TABLE 5. RECOVERY OF CHLORINATED DIOXINS AND FURANS FROM
FORTIFIED* REAGENT WATER USING EMPORE DISK EXTRACTION
Compound
TCDF
TCDD
PCDF
PCDD
HxCDF
HxCDD
HpCDF
HpCDD
OCDF
OCDD
No. Samples
2
2
4
2
8
6
4
2
2
2
% Recovery
72
75
78
86
83
80
77
80
91
82
% RSD
6
.0
11
5
16
11
23
10
15
11
* Fortifying levels were 0.16 ng/L for the tetra isomers, 0.8 ng/L
for the penta and hexa Isomers, and 1.6 ng/L for the octa isomers.
Analyses were carried out using the procedure described in Section 11.2
of this method.
60
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I
i
61
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