-------
GC/MS Conditions
Gas Chroraatography:
Capillary Column: a. Manufacturer - J&V Scientific
b. Liquid phase - DB-5
c. Length - 60 m
d. I. D - 0.25 ram
e. film thickness - 0.25 microns
Carrier gas : Helium
Head pressure : 28 psi
Flow thru column: 1 to 2 ral/min.
Injection type : Splitless for 30 sec.
Initial isothermal temperature :150 deg C for 30 sec.
Initial temperature program rate: to 190 deg C ballistically
Final temperature program rate : to 300 deg C
-------
SLUDGE SAMPLES
D100ML round bottom flask + boiling chip + sample <~2g) + 200ul
water + 200ul spiking solution + 50ml toluene.
2) Connect Dean/stark trap and heat apparatus to lOOdeg C until
volume of water collected in the resevolr is a constant value.
3) Disconnect apparatus , pipet toluene in side tube to sample
round bottom flask, discard water.
Rinse apparatus with 2 X 5ml toluene.
4) Filter sample though #54 Vhatman into a clean round bottom
flask. Rinse 1 st flask + filter with 2 X 5 ml toluene . Rotary
evaporate to ~lml.
5) go to step #9)
WATER SAMPLES
DMark the water meniscus on the side of the sample battle for later
determination of sample volume. Pour the entire sample into a 2-L
separatory funnel.
2) Add internal standard spiking solution to the sample in the
separatory funnel.
3) Add 60ml methylene chloride to the sample bottle, seal, and shake
for 30 s to rinse the inner surface. Transfer the solvent to the
separatory funnel and extract the sample by shaking the funnel for
two minutes with periodic venting to release e:ccess pressure. Allow
the organic layer to separate from the water phase for a minimum of
10 min. If the emulsion interface between layers is more than, one-
third the volume of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The optimum
technique depends on the sample, but may include stirring, filtering
at the emulsion though glass wool, cent ifugation, or ether physical
methods.
Collect the methylene chloride extract in a 250ml Erlenmeyer flask.
4) Added a second 60 ml volume of methylene chloride to the sample
bottle and repeat the extraction procedure, combining the extract in
the erlenmeycr flask. Perform a third extraction in the saire ranner
5) Assemble a Kuderna-Danish concentrator bv attaching a 10 mi
concentrator tube to a 500 ml evaporative flask.
6i Pour the combined extract into the KD concentrator. Rinse th^
•erlenraeyer flask with 3 X 10ml of methylene chloride to complete
the quantitative transfer.
7) Add one or more clean boiling chips to the evaporator and attach
a three-ball Snyder column. Prewet the Snyder column by adding about
1 ml of methylene chloride to the top. Place the KD apparatus an a
hot water bath 'x»50-65 deg C) .so that the ccncentator tube is
partially immersed in the hot water, and the entire lower rounded
surface of the r laslc is bathed with hot vapor. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 minutes. At the proper rate
of distillation the balls of the column will actively chatter but
the chambers will not flood with condensed solvent. When the
apparent volume of the liquid reaches 1 ml remove the KD apparatus
and allow to cool and drain for at least 10 rain.
8) Add 50 ml hexane and concentrate to 1 ml.
D-233
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PURIFICATION
9)Transfer to 250 ml separatory funnel with 5 X 5 ml hexane. Add 50
ml 5% tfaCl solution, shake for 2 min and. discard aqueous (bottom)
layer.
10) Add 40 ml 20% KOH (w/v) , shake for 2 min . discard aqueous
layer < bottom) repeat the base washing until no color is visible
the the bottom layer— a maximum of 4 times
11) Add 40 ml water shake for 2 min , discard aqueous layer
12) Add 40 ml concentrated sulfuric acid, shake for 2 min , discard
bottom layer. Repeat acid washing until no visible color is in the
bottom layer. A maximum or 4 times.
13) Add 40 ml water (care) shake for 2 min , discard bottom layer.
14 Filter upper layer though anhydrous sodium sulfate. Rinse with 2
X 5 ml hexane into 50 ml round bottom flask.
14) Rotary evaporate to near dryness a <35 deg C
15) Add 2 ml hexane to sample and have ready to load
25 ml pipet column with glasswool plug
+ 4g purified sodium sulfate (next page)
•f- 4g Voelm super neutral alumnia (desiccated)
+ 4g sodium sulfate
Vash with 10 ml hexane
When hexane layer reaches surface add sample the add 4 ml hexane
rinse ( 2 X 2> 'NOTE.. NEVER ALLOV SOLUTIONS TO GO BELOW SURFACE OF
SODIUM SULFATE.
Discard all the above elutants
Fr #1 = (into scintillation vial)
10 ml 8%(v/v) methylene chloride/ hexane —(hold.)
Fr #2 15ml 60%(v/v) methylene chloride/ hexane into a 50 ml rcur.d
bottom flask
Rotary evaporate to near dryness.
16> Prepare carbon column
9.5g 3iosil A silica Gel 24 hrs ® 225 deg C
+0.5g AX-21 carbon
mi:-: for 1 hour
2ml disposable pipet-broken at 1.3ml mark- glasswool plug at 0.0
mark
+ Biosil A silica gel to O.lml mark
+ carbon/Biosil A mixture to 0. 45ral mark
+ glasswool plug
Prewash column with :- 0.5ml 50% benzene/ methvlene chloride ,
9.5 ml 50% benzene/ methylene chloride,
10 ml toluene
add Iral hexane
17) add 1 ml hexane
load sample 0.2-0.4 ml in hexane
rinse with 2 X 0.2 ml hexane
add 5.0 ml hexane
add 10 ml 50 % benzene/ hexane
18) Turn column upside down
Elute with 10 ml toluene
transfer to sample tube and evaporate to near dryness with nitrogen
D-234
-------
Cgl A 50 gr A-540 basic alumina , activated 16-72hrs <2 130deg C
~ purified sodium sulfate on top
2gni Silica gel
1 era sodium sulfate on top
'_jjl 2 Directly under col 1
25ial pipet with glasswool plug
6gra A-948 Alumina with 10% water activated 16-72 hr-3
130deg C.
1cm sodiun sulfater on top
Load sample onto column 1
Rinse 2 X iral hexane
Wash with 162ml hexane —discard.
Remove column I .
Onto column 2 add 20ml 1% methylene chloride/hexane--save
Add 20ral 20% raethylene chloride/ hexane.
Evaporate Finished
D-235
-------
METHOD 8280
THE ANALYSIS OF POLYCHLORINATED DIBENZO-P-DIOXINS
AND POLYCHLORINATED DIBENZOFURANS
1.0 SCOPE AND APPLICATION
1.1 This method 1s appropriate for the determination of tetra-, penta-,
hexa-, hepta-, and octachlorinated dibenzo-p-diox1ns (PCDD's) and dibenzo-
furans (PCDF's) in chemical wastes including still bottoms, fuel oils,
sludges, fly ash, reactor residues, soil and water.
1.2 The sensitivity of this method is dependent upon the level of
interferents within a given matrix. Proposed quantification levels for target
analytes were 2 ppb in soil samples, up to 10 ppb in other solid wastes and
10 ppt in water. Actual values have been shown to vary by homologous series
and, to a lesser degree, by individual isomer. The total detection limit for
each CDD/CQF homologous series is determined by multiplying the detection
limit of a given isomer within that series by the number of peaks which can be
resolved under the gas chromatographic conditions.
1.3 Certain 2,3,7,8-substituted congeners are used to provide
calibration and method recovery information. Proper column selection and
access to reference Isomer standards, may in certain cases, provide isomer
specific data. Special Instructions are included which measure 2,3,7,8-
substituted congeners.
1.4 This method is recommended for use only by analysts experienced with
residue analysis and skilled in mass spectral analytical techniques.
1.5 Because of the extreme toxicity of these compounds, the analyst must
take necessary precautions to prevent exposure to himself, or to others, of
materials known or believed to contain PCDD's or PCDF's. Typical infectious
waste incinerators are probably not satisfactory
materials highly contaminated with PCOO's or PCDF's.
use these compounds should prepare a disposal plan to
by EPA's Dioxin Task Force (Contact Conrad Kleveno,
Street S.W., Washington, D.C. 20450). Additional
outlined in Appendix B.
devices for disposal of
A laboratory planning to
be reviewed and approved
WH-548A, U.S. EPA, 401 M
safety instructions are
2.0 SUMMARY OF THE METHOD
2.1 This procedure uses a matrix-specific extraction, analyte-specifie
cleanup, and high-resolution capillary column gas chromatography/low
resolution mass spectrometry (HRGC/LRMS) techniques.
2.2 If interferents are encountered,
cleanup procedures to aid the analyst in their
chart is shown in Figure 1.
the method
elimination.
provides selected
The analysis flow
8280 D-236
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Date September
1986
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Complex
Waste
Sample
(1) Add Internal Standards: 13C12-PCDO's
and I3C12-PCDF's.
(2) Perform matrix-specific extraction.
Sample
Extract
(1)
(2)
(3)
(4)
(5)
(6)
(7)
Wash with 20% KOH
Wash with 51 Nad
Wash with cone.
Wash with 51 NaCl
Dry extract
Solvent exchange
Alumina column
601 CH2C12/nexane
Fraction
(1) Concentrate eluate
(2) Perform carbon column cleanup
(3) Add recovery standard(s)-13C12-l,2,3,4-TCDO
Analyze by GC/MS
Flyure 1. Method 8280 flow chart for sample extraction and cleanup as
used for the analysis of PCOO's and PCDF's In complex waste samples.
8280 D-237
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Date September 1986
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3.0 INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines which may cause
misinterpretation of chromatographic data. All of these materials must be
demonstrated to be free from Interferents under the conditions of analysis by
running laboratory method blanks.
3.2 The use of high purity reagents and solvents helps to minimize
interference problems. Purification of solvents by distillation in all glass
systems may be required.
3.3 Interferents co-extracted from the sample will vary considerably
from source to source, depending upon the industrial process being sampled.
PCDD's and PCDF's are often associated with other interfering chlorinated
compounds such as PCB's and polychlorinated diphenyl ethers which may be found
at concentrations several orders of magnitude higher than that of the analytes
of interest. Retention times of target analytes must be verified using
reference standards. These values must correspond to the retention time
windows established 1n Section 6-3. While certain cleanup techniques are
provided as part of this method, unique samples may require additional cleanup
techniques to achieve the method detection limit (Section 11.6) stated in
Table 8.
3.4 High resolution capillary columns are used to resolve as many PCOD
and PCDF isomers as possible; however, no single column is known to resolve
all of the isomers.
3.5 Aqueous samples cannot be aliquoted from sample containers. The
entire sample must be used and the sample container washed/rinsed out with the
extracting solvent.
4.0 APPARATUS AND MATERIALS
4.1 Sampling equipment for discrete or composite sampling;
4.1.1 Grab sample bottle—amber glass, 1-liter or 1-quart volume.
French or Boston Round design 1s recommended. The container must be acid
washed and solvent rinsed before use to minimize interferences.
4.1.2 Bottle caps—threaded to screw onto the sample bottles. Caps
must be lined with Teflon. Solvent washed foil, used with the shiny side
toward the sample, may be substituted for Teflon if the sample is not
corrosive. Apply tape around cap to completely seal cap to bottom.
4.1.3 Compositing equipment—automatic or manual compositing
system. No tygon or rubber tubing may be used, and the system must
incorporate glass sample containers for the collection of a minimum of
250 ml. Sample containers must be kept refrigerated after sampling.
4.2 Water bath—heated, with concentric ring cover, capable of
temperature control (+2*C). The bath should be used in a hood.
8280 D-238
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4.3 Gas chromatograph/mass spectrometer data system;
4.3.1 Gas chromatograph: An analytical system with a temperature-
programmable gas chromatograph and all required accessories including
syringes, analytical columns, and gases.
4.3.2 Fused silica capillary columns are required. As shown in
Table 1, three columns were evaluated using a column performance check
mixture containing 1,2,3,4-TCDD, 2,3,7,8-TCDD, 1,2,3,4,7 PeCDD,
1,2,3,4,7,8-HxCDO, 1,2,3,4,6,7,8-HpCDD, OCDD, and 2,3,7,8-TCDF.
The columns include the following: (a) 50-m CP-Sil-88 programmed 60*-
190* at 20Vminute, then 19CT-240* at SVminute; (b) DB-5 (30-m x 0.25-mm
I.D.; 0.25-um film thickness) programmed 170* for 10 minutes, then 17CT-
320* at SVminute, hold at 3204C for 20 minutes; (c) 30-m SP-2250
programmed 70*-320* at 10'/minute. Column/conditions (a) provide good
separation of 2,3,7,8-TCDD from the other TCDD's at the expense of longer
retention times for higher homologs. Column/conditions (b) and (c) can
also provide acceptable separation of 2,3,7,8-TCDD. Resolution of
2,3,7,8-TCDD from the other TCDD's is better on column (c), but column
(b) is more rugged, and may provide better separation from certain
classes of interferents. Data presented 1n Figure 2 and Tables 1 to 8 of
this Method were obtained using a DB-5 column with temperature
programming described in (b) above. However, any capillary column which
provides separation of 2,3,7,8-TCDD from all other TCDD isomers
equivalent to that specified in Section 6.3 may be used; this separation
must be demonstrated and documented using the performance test mixture
described in Paragraph §.3.
4.3.3 Mass spectrometer: A low resolution Instrument is specified,
utilizing 70 volts (nominal) electron energy in the electron impact
ionization mode. The system must be capable of selected ion monitoring
(SIM) for at least 11 ions simultaneously, with a cycle time of 1 sec or
less. Minimum integration time for SIM is 50 ms per m/z. The use of
systems not capable of monitoring 11 ions simultaneously will require the
analyst to make multiple injections.
4.3.4 GC/MS Interface: Any GC-to-MS interface that gives an
acceptable calibration response for each analyte of interest at the
concentration required and achieves the required tuning performance
criteria (see Paragraphs 6.1.-6.3) may be used. GC-to-MS interfaces
constructed of all glass or glass-lined materials are required. Glass
can be deactivated by silanlzing with dichlorodimethylsilane. Inserting
a fused silica column directly into the MS source is recommended; care
must be taken not to expose the end of the column to the electron beam.
4.3.5 Data system: A computer system must be interfaced to the
mass spectrometer. The system must allow for the continuous acquisition
and storage on machine-readable media of all data obtained throughout the
duration of the chromatographic program. The computer must have software
that can search any GC/MS data file for ions of a specific mass and can
plot such ion abundances versus time or scan number. This type of plot
8280 D-239
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Date September 1986
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1s defined as an Selected Ion Current Profile (SICP). Software must also
be able to Integrate the abundance, 1n any SICP, between specified time
or scan number limits.
4.4 P1pets-01sposable, Pasteur, 150-mm long x 5-mm I.D. (Fisher
Scientific Company, No. 13-678-6A, or equivalent).
4.4.1 P1pet, disposable, serologlcal 10-mL (American Scientific
Products No. P4644-10, or equivalent) for preparation of the carbon
column specified 1n Paragraph 4.19.
4.5 Amber glass bottle (500-mL, Teflon-lined screw-cap).
4.6 Reacti-vial 2-mL, amber glass (Pierce Chemical Company). These
should be sllanized prior to use.
4.7 500-mL Erlenmeyer flask (American Scientific Products Cat. No. f4295
SOOfO) fitted with Teflon stoppers (ASP No. S9058-8, or equivalent).
4.8 Wrist Action Shaker (VWR No. 57040-049, or equivalent).
4.9 125-mL and 2-L Separatory Funnels (Fisher Scientific Company,
No. 10-437-5b, or equivalent).
4.10 500-mL Kuderna-Oanlsh fitted with a 10-mL concentrator tube and
3-ball Snyder column (Ace Glass No. 6707-02, 6707-12, 6575-02, or equivalent).
4.11 Teflon boiling chips (Berghof/American Inc., Main St., Raymond, New
Hampshire 03077, No, 15021-450, or equivalent). Wash with hexane prior to
use.
4.12 300-mHi x 10.5-mm glass chromatographic column fitted with Teflon
stopcock.
4.13 15-mL conical concentrator tubes (Kontes No. K-288250, or
equivalent).
4.14 Adaptors for concentrator tubes (14/20 to 19/22) (Ace Glass No.
9092-20, or equivalent).
4.15 Nitrogen blowdown apparatus (N-Evap (reg. trademark) Analytical
Evaporator Model 111, Organomatlon Associates Inc., Northborough,
Massachusetts or equivalent). Teflon tubing connection to trap and gas
regulator is required.
4.16 Microflex conical vials 2.0-mL (Kontes K-749000, or equivalent).
4.17 Filter paper (Whatman No. 54, or equivalent). Glass fiber filters
or glass wool plugs are also recommended.
4.18 Solvent reservoir (125-mL) Kontes; (special order item) 12.5-cm
diameter, compatible with gravity carbon column.
8280 D-240
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Date September 1986
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4.19 Carbon column (gravity flow); Prepare carbon/silica gel packing
material bymixing5 percent (byweight) active carbon AX-21 (Anderson
Development Co., Adrain, Michigan), pre-washed with methanol and dried i_n
vacuo at 110*C and 95 percent (by weight) Silica gel (Type 60, EM reagent 70
to 230 mesh, CMS No. 393-066) followed by activation of the mixture at 130*
for 6 hr. Prepare a 10-mL disposable serological pi pet by cutting off each
end to achieve a 4-1n. column. F1re polish both ends; flare 1f desired.
Insert a glass-wool plug at one end and pack with 1 g of the carbon/silica gel
mixture. Cap the packing with a glass-wool plug. (Attach reservoir to column
for addition of solvents).
Option: Carbon column (HPLC): A silanized glass HPLC column (10 mm x 7
cm), or equivalent, which contains 1 g of a packing prepared by mixing 5
percent (by weight) active carbon AX-21, (Anderson Development Co., Adrian,
Michigan), washed with methanol and dried 1_n vacuo at 110*C, and 95 percent
(by weight) 10 urn silica (Spherisorb S10W from Phase Separations, Inc.,
Norwalk, Connecticut). The mixture must then be stirred and sieved through a
38-um screen (U.S. Sieve Designation 400-mesh, American Scientific Products,
No. S1212-400, or equivalent) to remove any clumps.1
4.20 HPLC pump with loop valve (1.0 ml) injector to be used in the
optional carbon column cleanup procedure.
4.21 Dean-Stark trap, 5- or 10-mL with T joints, (Fisher Scientific
Company, No. 09-146-5, or equivalent) condenser and 125-mL flask.
4.22 Continuous liquid-liquid extractor (Hershberg-Wolfe type, Lab Glass
No. LG-6915; or equivalent.).
4.23 Roto-evaporator, R-110. Buchi/Brinkman - American Scientific No.
£5045-10; or equivalent.
5.0 REAGENTS
5.1 Potassium hydroxide (ASC): 20 percent (w/v) in distilled water.
5.2 Sulfuric acid (ACS), concentrated.
5.3 Methylene chloride, hexane, benzene, petroleum ether, methanol,
tridecane, Isooctane, toluene, cyclohexane. Distilled in glass or highest
available purity.
5.4 Prepare stock standards in a glovebox from concentrates or neat
materials. The stock solutions (50 ppm) are stored in the dark at 4*C, and
checked frequently for signs of degradation or evaporation, especially just
prior to the preparation of working standards.
1 The carbon column preparation and use is adapted from W. A. Korfmacher,
L. G. Rushing, D. M. Nestorick, H. C. Thompson, Jr., R. K. Mitchum, and J. R.
Kominsky, Journal of High Resolution Chromatography and Chromatography
Communications, 8, 12-19 (1985).
8280 D-241
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Date September 1986
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5.5 Alumina, neutral, Super 1, Woelm, 80/200 mesh. Store in a sealed
container at room temperature 1n a desiccator over self-Indicating silica gel.
5.6 Prepurifled nitrogen gas.
5.7 Anhydrous sodium sulfate (reagent grade): Extracted by manual
shaking with several portions of hexane and dried at 100*C.
water.
5.8 Sodium chloride - (analytical reagent), 5 percent (w/v) in distilled
6.0 CALIBRATION
6.1 Two types of calibration procedures are required. One type, initial
calibration, is required before any samples are analyzed and is required
intermittently throughout sample analyses as dictated by results of routine
calibration procedures described below. The other type, routine calibration,
consists of analyzing the column performance check solution and a
concentration calibration solution of 500 ng/ml (Paragraph 6.2). No samples
are to be analyzed until acceptable calibration as described in Paragraphs 6.3
and 6.6 1s demonstrated and documented.
6.2 Initial calibration:
6.2.1 Prepare multi-level calibration standards^ keeping one of
the recovery standards and the internal standard at fixed concentrations (500
ng/mL). Additional Internal standards (^Ci2-OCDD 1,000 ng/ml) are
recommended when quantification of the hepta- and octa-isomers is required.
The use of separate internal standards for the PCDF's is also recommended.
Each calibration standard should contain the following compounds:
2,3,7,8-TCDQ,
1,2,3,7,8-PeCDD
1,2,3,4,7,8-HxCDO
1,2,3,4,6,7,8-HpCDD
2,3,7,8-TCOF
l,2,3,7,8,PeCOF
1,2,3,4,7,8-HxCOF
1,2,3,4,6,7,8-HpCDF
or any available
or any available
or any available
or any available
or any available
or any available
OCDO, OCOF, 13Ci2-2,3,7,8-TCOO,
2,3,7,8,X-PeCDD isomer,
2,3,7,8,X,Y-HxCOD isomer,
2,3,7,8,X,Y,Z-HpCDD isomer,
2f3,7,8,X-PeCDF Isomer,
2,3,7,8,X,Y,HxCDF isomer,
2f3,7,8,X,Y,Z-HpCDF Isomer,
and 13C12-OCOO.
2 13Ci2~labeled analytes are available from Cambridge Isotope Laboratory,
Woburn, Massachusetts. Proper quantification requires the use of a specific
labeled isomer for each congener to be determined. When labeled PCOO's and
PCDF's of each homolog are available, their use will be required consistent
with the technique of isotopic dilution.
8280 D-242
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Recommended concentration levels for standard analytes are 200, 500, 1,000,
2,000, and 5,000 ng/mL. These values may be adjusted 1n order to Insure that
the analyte concentration falls within the calibration range. Two uL
Injections of calibration standards should be made. However, some GC/MS
instruments may require the use of a 1-uL Injection volume; if this Injection
volume is used then all Injections of standards, sample extracts and blank
extracts must also be made at this Injection volume. Calculation of relative
response factors is described 1n Paragraph 11.1.2. Standards must be analyzed
used 1n the final sample extract. A wider
for higher level samples provided it can be
range of the method, and the Identification
criteria defined in Paragraph 10.4 are met. All standards must be stored in
an isolated refrigerator at 4*C and protected from light. Calibration
standard solutions must be replaced routinely after six months.
using the same solvent as
calibration range is useful
described within the linear
6.3 Establish operating parameters for the GC/MS system; the instrument
should be tuned to meet the Isotopic ratio criteria listed in Table 3 for
PCOD's and PCDF's. Once tuning and mass calibration procedures have been
completed, a column performance check mixture^ containing the isomers listed
below should be injected into the GC/MS system:
TCDD 1,3,6,8; 1,2,8,9; 2,3,7,8; 1,2,3,4; 1,2,3,7; 1,2,3,9
PeCDD 1,2,4,6,8; 1,2,3,8,9
HxCDO 1,2,3,4,6,9; 1,2,3,4,6,7
HpCDD 1,2,3,4,6,7,8; 1,2,3,4,6,7,9
OCDD 1,2,3,4,6,7,8,9
TCOF 1,3,6,8; 1,2,8,9
PeCDF 1,3,4,6,8; 1,2,3,8,9
HxCOF 1,2,3,4,6,8; 1,2,3,4,8,9
HpCDF 1,2,3,4,6,7,8; 1,2,3,4,7,8,9
OCDF 1,2,3,4,6,7,8,9
Because of the known overlap between the late-eluting tetra-isomers and
the early-eluting penta-isomers under certain column conditions, it may be
necessary to perform two injections to define the TCOD/TCOF and PeCDO/PeCOF
elution windows, respectively. Use of this performance check mixture will
enable the following parameters to be checked: (a) the retention windows for
each of the homologues, (b) the GC resolution of 2,3,7,8-TCDO and 1,2,3,4-
TCOO, and (c) the relative 1on abundance criteria listed for PCDD's and PCDF's
in Table 3. GC column performance should be checked daily for resolution and
peak shape using this check mixture.
The chromatographic peak separation between 2,3,7,8-TCDD and 1,2,3,4-TCDD
must be resolved with a valley of £25 percent, where
Valley Percent * (x/y) (100)
x = measured as in Figure 2
y = the peak height of 2,3,7,8-TCOD
3 Performance check mixtures are available from Brehm Laboratory, Wright
State University, Dayton, Ohio.
8280 D-243
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It 1s the responsibility of the laboratory to verify the conditions
suitable for maximum resolution of 2,3,7,8-TCOO from all other TCOD isomers.
The peak representing 2,3,7,8-TCDD should be labeled and Identified as such on
all chromatograms.
6.4 Acceptable SIM sensitivity is verified by achieving a minimum
signal-to-nolse ratio of 50:1 for the m/z 320 ion of 2,3,7,8-TCDD obtained
from injection of the 200 ng/mL calibration standard.
6.5 From injections of the 5 calibration standards, calculate the
relative response factors (RRF's) of analytes vs. the appropriate internal
standards, as described in Paragraph 11.1.2. Relative response factors for
the hepta- and octa-chlorinated CDD's and CDF's are to be calculated using the
corresponding ^c^-octachlorinated standards.
6.6 For each analyte calculate the mean relative response factor (RRF),
the standard deviation, and the percent relative standard deviation from
triplicate determinations of relative response factors for each calibration
standard solution.
6.7 The percent relative standard deviations (based on triplicate
analysis) of the relative response factors for each calibration standard
solution should not exceed 15 percent. If this condition is not satisfied,
remedial action should be taken.
6.8 The Laboratory must not proceed with analysis of samples before
determining and documenting acceptable calibration with the criteria specified
in Paragraphs 6.3 and 6.7.
6.9 Routine calibration;
6.9.1 Inject a 2-uL aliquot of the column performance check
mixture. Acquire at least five data points for each GC peak and use the
same data acquisition time for each of the ions being monitored.
NOTE: The same data acquisition parameters previously used to
analyze concentration calibration solutions during initial
calibration must be used for the performance check solution.
The column performance check solution must be run at the
beginning and end of a 12 hr period. If the contractor
laboratory operates during consecutive 12-hr periods
(shifts), analysis of the performance check solution at the
beginning of each 12-hr period and at the end of the final
12-hr period is sufficient.
Determine and document acceptable column performance as described in
Paragraph 6.3.
6.9.2 Inject a 2-uL aliquot of the calibration standard solution at
500 ng/mL at the beginning of a 2-hr period. Determine and document
acceptable calibration as specified 1n Paragraph 6.3, i.e., SIM
sensitivity and relative ion abundance criteria. The measured RRF's of
8280 D-244
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all analytes must be within +30 percent of the mean values established by
initial analyses of the calibration standard solutions.
7.0 QUALITY CONTROL
7.1 Before processing any samples, the analyst must demonstrate through
the analysis of a method blank that all glassware and reagents are
interferent-free at the method detection limit of the matrix of interest.
Each time a set of samples is extracted, or there is a change in reagents, a
method blank must be processed as a safeguard against laboratory
contamination.
7.2 A laboratory "method blank" must be run along with each analytical
batch (20 or fewer samples). A method blank is performed by executing all of
the specified extraction and cleanup steps, except for the introduction of a
sample. The method blank is also dosed with the internal standards. For
water samples, one liter of deionized and/or distilled water should be used as
the method blank. Mineral oil may be used as the method blank for other
matrices.
7.3 The laboratory will be expected to analyze performance evaluation
samples as provided by the EPA on a periodic basis throughout the course of a
given project. Additional sample analyses will not be permitted if the
performance criteria are not achieved. Corrective action must be taken and
acceptable performance must be demonstrated before sample analyses can resume.
7.4 Samples may be split with other participating labs on a periodic
basis to ensure interlaboratory consistency. At least one sample per set of
24 must be run in duplicate to determine intralaboratory precision.
7.5 Field duplicates (individual samples taken from the same location at
the same time) should be analyzed periodically to determine the total
precision (field and lab).
7.6 Where appropriate, "field blanks" will be provided to monitor for
possible cross-contamination of samples in the field. The typical "field
blank" will consist of uncontaminated soil (background soil taken off-site).
7.7 GC column performance must be demonstrated initially and verified
prior to analyzing any sample in a 12-hr period. The GC column performance
check solution must be analyzed under the same chromatographic and mass
spectrometric conditions used for other samples and standards.
7.8 Before using any cleanup procedure, the analyst must process a
series of calibration standards (Paragraph 6.2) through the procedure to
validate elution patterns and the absence of Interferents from reagents. Both
alumina column and carbon column performance must be checked. Routinely check
the 8 percent CH2Cl2/hexane eluate of environmental extracts from the alumina
column for presence of target analytes.
NOTE: This fraction is intended to contain a high level of interferents
and analysis near the method detection limit may not be possible.
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8.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
8.1 Grab and composite samples must be collected in glass containers.
Conventional sampling practices must be followed. The bottle must not be
prewashed with sample before collection. Composite samples should be
collected in glass containers. Sampling equipment must be free of tygon,
rubber tubing, other potential sources of contamination which may absorb the
target analytes.
8.2 All samples must be stored at 4*C, extracted within 30 days and
completely analyzed within 45 days of collection.
9.0 EXTRACTION AND. CLEANUP PROCEDURES
9.1 Internal standard addition. Use a sample aliquot of 1 g to 1,000 mL
(typical sample size requirements for each type of matrix are provided in
Paragraph 9.2) of the chemical waste or soil to be analyzed. Transfer the
sample to a tared flask and determine the weight of the sample. Add an
appropriate quantity of 13Ci2-2,3,7,8-TCDD, and any other material which is to
be used as an internal standard, (Paragraph 6.2). All samples should be
spiked with at least one internal standard, for example, 13Ci2-2,3,7,8-TCDD,
to give a concentration of 500 ng/mL 1n the final concentrated extract. As an
example, a 10 g sample concentrated to a final volume of 100 uL requires the
addition of 50 ng of 13Ci2-2,3,7,8-TCDD, assuming 100% recovery. Adoption of
different calibration so>ution sets (as needed to achieve different
quantification limits for different congeners) will require a change in the
fortification level. Individual concentration levels for each homologous
series must be specified.
9.2 Extracti on
9.2.1 Sludge/fuel oil. Extract aqueous sludge samples by refluxing
a sample (e.g. 2 g) with 50 mL of toluene (benzene) in a 125-mL flask
fitted with a Dean-Stark water separator. Continue refluxing the sample
until all the water has been removed. Cool the sample, filter the
toluene extract through a fiber filter, or equivalent, into a 100-mL
round bottom flask. Rinse the filter with 10 mL of toluene, combine the
extract and rinsate. Concentrate the combined solution to near dryness
using a rotary evaporator at 50*C. Use of an inert gas to concentrate
the extract is also permitted. Proceed with Step 9.2.4.
9.2.2 Still bottom. Extract still bottom samples by mixing a
sample (e.g., 1.0 g) with 10 mL of toluene (benzene) in a small beaker
and filtering the solution through a glass fiber filter (or equivalent)
into a 50-mL round bottom flask. Rinse the beaker and filter with 10 mL
of toluene. Concentrate the combined toluene solution to near dryness
using a rotary evaporator at 50'C while connected to a water aspirator.
Proceed with Step 9.2.4.
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9.2.3 Fly ash. Extract fly ash samples by placing a sample (e.g.
10 g) and an equivalent amount of anhydrous sodium sulfate 1n a Soxhlet
extraction apparatus charged with 100 mL of toluene (benzene) and extract
for 16 hr using a three cycle/hour schedule. Cool and filter the toluene
extract through a glass fiber filter paper Into a 500-mL round bottom
flask. Rinse the filter with 5 mL of toluene. Concentrate the combined
toluene solution to near dryness using a rotary evaporator at 50*C.
Proceed with Step 9.2.4.
9.2.4 Transfer the residue to a 125-mL separatory funnel using
15 mL of hexane. Rinse the flask with two 5-mL aliquots of hexane and
add the rinses to the funnel. Shake 2 min with .50 mi of 5% NaCI
solution, discard the aqueous layer and proceed with Step 9.3.
9.2.5 Soil. Extract soil samples by placing the sample (e.g. 10 g)
and an equivalent amount of anhydrous sodium sulfate in a 500-mL
Erlenmeyer flask fitted with a Teflon stopper. Add 20 mL of methanol and
80 mL of petroleum ether, in that order, to the flask. Shake on a wrist-
action shaker for two hr. The solid portion of sample should mix freely.
If a smaller soil aliquot is used, scale down the amount of methanol
proportionally.
9.2.5.1 Filter the extract from Paragraph 9.2.5 through a
glass funnel fitted with a glass fiber filter and filled with
anhydrous sodium sulfate into a 500-mL Kuderna-Oanish (KO)
concentrator fitted with a 10-mL concentrator tube. Add 50 mL of
petroleum ether to the Erlenmeyer flask, restopper the flask and
swirl the sample gently, remove the stopper carefully and decant the
solvent through the funnel as above. Repeat this procedure with two
additional 50-mL aliquots of petroleum ether. Wash the sodium
sulfate 1n the funnel with two additional 5-mL portions of petroleum
ether.
9.2.5.2 Add a Teflon or PFTE boiling chip and a three-ball
Snyder column to the KQ flask. Concentrate in a 70*C water bath to
an apparent volume of 10 ml. Remove the apparatus from the water
bath and allow 1t to cool for 5 min.
9.2.5.3 Add 50 mL of hexane and a new boiling chip to the KD
flask. Concentrate in a water bath to an apparent volume of 10 ml.
Remove the apparatus from the water bath and allow to cool for 5
min.
9.2.5.4 Remove and invert the Snyder column and rinse it down
into the KO with two 1-mL portions of hexane. Decant the contents
of the KD and concentrator tube into a 125-mL separatory funnel.
Rinse the KD with two additional 5-mL portions of hexane, combine.
Proceed with Step 9.3.
9.2.6 Aqueous samples: Mark the water meniscus on the side of the
1-L sample bottle for later determination of the exact sample volume.
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Pour the entire sample (approximately 1-L) Into a 2-L separatory funnel.
Proceed with Step 9.2.6.1.
NOTE: A continuous liquid-liquid extractor may be used 1n place of
a separatory funnel when experience with a sample from a
given source Indicates that a serious emulsion problem will
result or an emulsion 1s encountered using a separatory
funnel. Add 60 ml of methylene chloride to the sample
bottle, seal, and shake for 30 sec to rinse the Inner
surface. Transfer the solvent to the extractor. Repeat the
sample bottle rinse with an additional 50- to 100-mL portion
of methylene chloride and add the rinse to the extractor.
Add 200 to 500 mL of methylene chloride to the distilling
flask; add sufficient reagent water to ensure proper
operation, and extract for 24 hr. Allow to cool, then detach
the distilling flask. Dry and concentrate the extract as
described in Paragraphs 9.2.6.1 and 9.2.6.2. Proceed with
Paragraph 9.2.6.3.
9.2.6.1 Add 60 ml methylene chloride to the sample bottle,
seal and shake 30 sec to rinse the inner surface. Transfer the
solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 m1n with periodic venting. Allow the organic layer
to separate from the water phase for a minimum of 10 min. If the
emulsion interface between layers is more than one-third the volume
of the solvent layer, the analyst must employ mechanical techniques
to complete the phase separation. Collect the methylene chloride
(3 x 60 mL) directly Into a 500-mL Kuderna-Oanish concentrator
(mounted with a 10-mL concentrator tube) by passing the sample
extracts through a filter funnel packed with a glass wool plug and
5 g of anhydrous sodium sulfate. After the third extraction, rinse
the sodium sulfate with an additional 30 ml of methylene chloride to
ensure quantitative transfer.
9.2.6.2 Attach a Snyder column and concentrate the extract on
a water bath until the apparent volume of the liquid reaches 5 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 hexane, re-attach the
Snyder column and concentrate to approximately 5 mL. Add a new
boiling chip to the K-D apparatus before proceeding with the second
concentration step.
Rinse the flask and the lower joint with 2 x 5 mL hexane and combine
rinses with extract to give a final volume of about 15 mL.
9.2.6.3 Determine the original sample volume by refilling the
sample bottle to the mark and transferring the liquid to a 1,000-mL
graduated cylinder. Record the sample volume to the nearest 5 mL.
Proceed with Paragraph 9.3.
9.3 In a 250-mL Separatory funnel, partition the solvent (15 mL hexane)
against 40 mL of 20 percent (w/v) potassium hydroxide. Shake for 2 min.
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Remove and discard the aqueous layer (bottom). Repeat the base washing until
no color is visible in the bottom layer (perform base washings a maximum of
four times). Strong base (KOH) is 'known to degrade certain PCOD/PCDF's,
contact time must be minimized.
9.4 Partition the solvent (15 ml hexane) against 40 ml of 5 percent
(w/v) sodium chloride. Shake for 2 m1n. Remove and discard aqueous layer
(bottom).
NOTE: Care should be taken due to the heat of neutralization and
hydration.
9.5 Partition the solvent (15 ml hexane) against 40 ml of concentrated
sulfuric acid. Shake for 2 min. Remove and discard the aqueous layer
(bottom). Repeat the acid washings until no color is visible in the acid
layer. (Perform acid washings a maximum of four times.)
9.6 Partition the extract against 40 ml of 5 percent (w/v) sodium
chloride. Shake for 2 min. Remove and discard the aqueous layer (bottom).
Dry the organic layer by pouring through a funnel containing anhydrous sodium
sulfate into a 50-mL round bottom flask, wash the separatory funnel with two
15-mL portions of hexane, pour through the funnel, and combine the hexane
extracts. Concentrate the hexane solution to near dryness with a rotary
evaporator (35'C water bath), making sure all traces of toluene are removed.
(Use of blowdown with an inert gas to concentrate the extract is also
permitted).
9.7 Pack a gravity column (glass 300-mm x 10.5-mm), fitted with a Teflon
stopcock, in the following manner:
Insert a glass-wool plug into the bottom of the column. Add a 4-g layer
of sodium sulfate. Add a 4-g layer of Woelm super 1 neutral alumina. Tap the
top of the column gently. Woelm super 1 neutral alumina need not be activated
or cleaned prior to use but should be stored 1n a sealed desiccator. Add a 4-
g layer of sodium sulfate to cover the alumina. Elute with 10 ml of hexane
and close the stopcock just prior to the exposure of the sodium sulfate layer
to air. Discard the eluant. Check the column for channeling. If channeling
is present discard the column. Do not tap a wetted column.
9.8 Dissolve the residue from Step 9.6 in 2 ml of hexane and apply the
hexane solution to the top of the column. Elute with enough hexane (3-4 mL)
to complete the transfer of the sample cleanly to the surface of the alumina.
Discard the eluant.
9.8.1 Elute with 10 ml of 8 percent (v/v) methylene chloride in
hexane. Check by GC/MS analysis that no PCOD's or PCDF's are eluted in
this fraction. See Paragraph 9.9.1.
9.8.2 Elute the PCDD's and PCDF's from the column with 15 mL of 60
percent (v/v) methylene chloride 1n hexane and collect this fraction in a
conical shaped (15-mL) concentrator tube.
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9.9 Carbon column cleanup;
Prepare a carbon column as described In Paragraph 4.18.
9.9.1 Using a carefully regulated stream of nitrogen (Paragraph
4.15), concentrate the 8 percent fraction from the alumina column
(Paragraph 9.8.1) to about 1 ml. Wash the sides of the tube with a small
volume of hexane (1 to 2 ml) and reconcentrate to about 1 ml. Save this
8 percent concentrate for GC/MS analysis to check for breakthrough of
PCDO's and PCDF's. Concentrate the 60 percent fraction (Paragraph 9.8.2)
to about 2 to 3 ml. Rinse the carbon with 5 ml cyclohexane/methylene
chloride (50:50 v/v) 1n the forward direction of flow and then in the
reverse direction of flow. While still in the reverse direction of flow,
transfer the sample concentrate to the column and elute with 10 ml of
cyclohexane/methylene chloride (50:50 v/v) and 5 ml of methylene
chloride/methanol/benzene (75:20:5, v/v). Save all above eluates and
combine (this fraction may be used as a check on column efficiency). Now
turn the column over and in the direction of forward flow elute the
PCDD/PCDF fraction with 20 ml toluene.
NOTE: Be sure no carbon fines are present in the eluant.
9.9.2 Alternate carbon column cleanup. Proceed as in Section 9.9.1
to obtain the 60 percent fraction re-concentrated to 400 ul which is
transferred to an HPLC Injector loop (1 ml). The Injector loop is
connected to the optional column described 1n Paragraph 4.18. Rinse the
centrifuge tube with 500 ul of hexane and add this rinsate to the
injector loop. Load the combined concentrate and rinsate onto the
column. Elute the column at 2 ml/min, ambient temperature, with 30 ml of
cyclohexane/methylene chloride 1:1 (v/v). Discard the eluant. Backflush
the column with 40 ml toluene to elute and collect PCDO's and PCDF's
(entire fraction). The column is then discarded and 30 ml of
cyclohexane/methylene chloride 1:1 (v/v) is pumped through a new column
to prepare 1t for the next sample.
9.9.3 Evaporate the toluene fraction to about 1 ml on a rotary
evaporator using a water bath at 50*C. Transfer to a 2.0-ml Reacti-vial
using a toluene rinse and concentrate to the desired volume using a
stream of N£. The final volume should be 100 ul for soil samples and
500 ul for sludge, still bottom, and fly ash samples; this is provided
for guidance, the correct volume will depend on the relative concentra-
tion of target analytes. Extracts which are determined to be outside the
calibration range for Individual analytes must be diluted or a smaller
portion of the sample must be re-extracted. Gently swirl the solvent on
the lower portion of the vessel to ensure complete dissolution of the
PCDO's and PCDF's.
9.10 Approximately 1 hr before HRGC/LRMS analysis, transfer an aliquot
of the extract to a micro-vial (Paragraph 4.16). Add to this sufficient
recovery standard (13Ci2l,2,3,4-TCDO) to give a concentration of 500 ng/ml.
(Example: 36 ul aliquot of extract and 4 ul of recovery standard solution.
Remember to adjust the final result to correct for this dilution. Inject an
appropriate aliquot (1 or 2 ul) of the sample into the GC/MS instrument.
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10.0 6C/MS ANALYSIS
10.1 When toluene 1s employed as the final solvent use of a bonded phase
column from Paragraph 4.3.2 1s recommended. Solvent exchange into tridecane
1s required for other liquid phases or nonbonded columns (CP-S11-88).
NOTE: Chromatographic conditions must be adjusted to account for solvent
boiling points.
10.2 Calculate response factors for standards relative to the Internal
standards, 13Ci2-2,3,7,8-TCOO and 13c12-OCOD (see Section 11). Add the
recovery standard (13Ci2-l»2,3,4-TCDD) to the samples prior to injection. The
concentration of the recovery standard 1n the sample extract must be the same
as that 1n the calibration standards used to measure the response factors.
10.3 Analyze samples with selected ion monitoring, using all of the ions
listed in Table 2. It 1s recommended that the GC/MS run be divided into five
selected 1on monitoring sections, namely: (1) 243, 257,, 304, 306, 320, 322,
332, 334, 340, 356, 376 (TCOO's, TCDF's, "C^-labeled internal and recovery
standards, PeCDO's, PeCDF's, HxCOE); (2) 277, 293, 306, 332, 338, 340, 342,
354, 356, 358, 410 (peCDD's, PeCDF's, HpCOE); (3) 311, 327, 340, 356, 372,
374, 376, 388, 390, 392, 446, (HxCOD's, HxCDF's, OCDE); (4) 345, 361, 374,
390, 406, 408, 410, 422, 424, 426, 480 (HpCOD's, HpCOF's, NCOE) and (5) 379,
395, 408, 424, 442, 444, 458, 460, 470, 472, 514 (OCDO, OCDF, 13Ci2-OCOD,
DCDE). Cycle time not to exceed 1 sec/descriptor. It 1s recommended that
selected 1on monitoring section 1 should be applied during the GC run to
encompass the retention window (determined 1n Paragraph 6.3) of the first- and
Iast-elut1ng tetra-chlorlnated Isomers. If a response 1s observed at m/z 340
or 356, then the SC/MS analysis must be repeated; selected ion monitoring
section 2 should then be applied to encompass the retention window of the
first- and Iast-elut1ng penta-chlorinated isomers. HxCDE, HpCDE, OCDE, NCOE,
DCDE, are abbreviations for hexa-, hepta-, octa-, nona-, and decachlorinated
diphenyl ether, respectively.
10.4 Identfffcation criteria for PCDD's and PCDF's;
10.4.1 All of the characteristic ions, i.e. quantisation ion,
confirmation Ions, listed 1n Table 2 for each class of PCDD and PCDF,
must be present in the reconstructed 1on chromatogram. It is desirable
that the M - COC1 1on be monitored as an additional requirement.
Detection limits will be based on quantitation ions within the molecules
in cluster.
10.4.2 The maximum intensity of each of the specified charac-
teristic Ions must coincide within 2 scans or 2 sec.
10.4.3 The relative intensity of the selected, isotopic ions within
the molecular ion cluster of a homologous series of PCDD's of PCDF's must
lie within the range specified in Table 3.
10.4.4 The GC peaks assigned to a given homologous series must have
retention times within the window established for that series by the
column performance solution.
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10.5 Quantltate the PCDO and PCDF peaks from the response relative to
the appropriate Internal standard. Recovery of each Internal standard) vs.
the recovery standard must be greater than 40 percent. It Is recommended that
samples with recoveries of less than 40 percent or greater than 120 percent be
re-extracted and re-analyzed.
NOTE: These criteria are used to assess method performance; when
properly applied, Isotope dilution techniques are Independent of
internal standard recovery.
In those circumstances where these procedures do not yield a definitive
conclusion, the use of high resolution mass spectrometry or HRGC/MS/MS is
suggested.
11.0 CALCULATIONS
NOTE: The relative response factors of a given congener within any
homologous series are known to be different. However, for
purposes of these calculations, 1t will be assumed that every
congener within a given series has the same relative response
factor. In order to minimize the effect of this assumption on
risk assessment, a 2,3,7,8-substltuted Isomer that 1s
commercially available was chosen as representative of each
series. All relative response factor calculations for a given
homologous series are based on that compound.
11.1 Determine the concentration of Individual Isomers of tetra-, penta,
and hexa-CDD/CDF according to the equation:
Qis x A
Concentration, ng/g = G x A^ x RRF
where:
Qis = ng of internal standard HCi2-2,3,7,8-TCDO, added to the sample
before extraction.
G = g of sample extracted.
As = area of quantltation ion of the compound of interest.
Ajs = area of quantltation ion (m/z 334) of the Internal standard,
!3Ci2-2,3,7,8-TCDD.
RRF = response factor of the quantltation ion of the compound of
interest relative to m/z 334 of 13Ci2-2,3,7,8-TCOO.
NOTE: Any dilution factor Introduced by following the procedure in
Paragraph 9.10 should be applied to this calculation.
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11.1.1 Determine the concentration of individual isomers of hepta-
CDD/CDF and the concentration of OCDD and OCDF according to the equation:
Q1s x As
Concentration, ng/g = G x A1$ x RRF
where:
Qis * "9 of Internal standard 13Ci2-°cDDf added to the sample before
extraction.
G » g of sample extracted.
AS = area of quantisation ion of the compound of interest.
Ais = area of quantitation ion (m/z 472) of the internal standard,
13c12-OCDO.
RRF = response factor of the quantitation ion of the compound of
interest relative to m/z 472 of 13Ci2-OCDD.
NOTE: Any dilution factor introduced by following the procedure in
Paragraph 9.10 should be applied to this calculation.
11.1.2 Relative response factors are calculated using data obtained
from the analysis of multi-level calibration standards according to the
equation:
RRF s A x C
Ais x Ls
where:
AS = area of quantitation ion of the compound of interest.
Ais * area °f quantitation ion of the appropriate internal standard
(m/z 334 for 13c12-2,3,7,8-TCDD: m/z 472 for 13c12-OCDD).
Cfs = concentration of the appropriate internal standard,
13Ci2-2,3,7,8-TCDD or "c^-OCDO)
Cs = concentration of the compound of interest.
11.1.3 The concentrations of unknown isomers of TCDD shall be
calculated using the mean RRF determined for 2,3,7,8-TCDD.
The concentrations of unknown Isomers of PeCDD shall be calculated
using the mean RRF determined for 1,2,3,7,8-PeCDO or any available
2,3,7,8,X-PeCDD isomer.
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The concentrations of unknown Isomers of HxCDD shall be calculated
using the mean RRF determined for 1,2,3,4,7,8-HxCDO or any available
2,3,7,8,-X,Y-HXCDD Isomer.
The concentrations of unknown Isomers of HpCDO shall be calculated
using the mean RRF determined for 1,2,3,4,6,7,8-HpCOO or any available
2,3,7,8,X,YlZ-HpCDD isomer.
The concentrations of unknown Isomers of TCDF shall be calculated
using the mean RRF determined for 2,3,7,8-TCOF.
The concentrations of unknown Isomers of PeCDF shall be calculated
using the mean RRF determined for 1,2,3,7,8-PeCDF or any available
2,3,7,8,X-PeCDF isomer.
The concentrations of unknown Isomers of HxCDF shall be calculated
using the mean RRF determined for 1,2,4,7,8-HxCDF or any available
2,3,7,8-X.Y-HxCDF isomer.
The concentrations of unknown Isomers of HpCDF shall be calculated
using the mean RRF determined for 1,2,3,4,6,7,8-HpCQF or any available
2,3,7f8,X,Y,Z-HpCDF Isomer.
The concentration of the octa-CDO and octa-COF shall be calculated
using the mean RRF determined for each.
Mean relative response factors for selected PCDD's and PCDF's are
given in Table 4.
11.1.4 Calculate the percent recovery, Rjs, for each internal
standard in the sample extract, using the equation:
A. Q
" - —!s_._x__rs— _
"is Ars x RFp x Qis
where:
Ars = Area of quantitation 1on (m/z 334) of the recovery standard,
l3Ci2-l,2,3,4-TCDD.
Qrs = ng of recovery standard, ^2-1,2, 3, 4-TCDD, added to
extract.
The response factor for determination of recovery is calculated using
data obtained from the analysis of the multi -level calibration standards
according to the equation:
's
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where:
Crs = Concentration of the recovery standard,
11.1.5 Calculation of total concentration of all Isomers within
each homologous series of PCDD's and PCDF's.
Total concentration , Sum of the concentrations of the Individual
of PCOO's or PCOF's PCDO or PCDF Isomers
11.4 Report results 1n nanograms per gram; when duplicate and spiked
samples are reanalyzed, all data obtained should be reported.
11.5 Accuracy and Precision. Table 5 gives the precision data for
revised Method 8280 for selected analytes In the matrices shown. Table 6
lists recovery data for the same analyses. Table 2 shows the linear range and
variation of response factors for selected analyte standards. Table 8
provides the method detection limits as measured in specific sample matrices.
11.6 Method Detection Limit. The Method Detection Limit (MDL) is
defined as the minimum concentration of a substance that can be measured and
reported with 99 percent confidence that the value is above zero. The
procedure used to determine the MDL values reported 1n Table 8 was obtained
from Appendix A of EPA Test Methods manual, EPA-600/4-82-057 July 1982,
"Methods for Organic Chemical Analysis of Municipal and Industrial
Wastewater."
11.7 Maximum Holding Time (MHT). Is that time at which a 10 percent
change in the analyte concentration (C^io) occurs and the precision of the
method of measurement allows the 10 percent change to be statistically
different from the 0 percent change (Cto) at the 90 percent confidence level.
When the precision of the method is not sufficient to statistically
discriminate a 10 percent change in the concentration from 0 percent change,
then the maximum holding time 1s that time where the percent change in the
analyte concentration (Ctn) is statistically different than the concentration
at 0 percent change (Cto) and greater than 10 percent change at the 90 percent
confidence level.
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TABLE 1. REPRESENTATIVE GAS CHROMATOGRAPH RETENTION TIMES* OF ANALYTES
Analyte
2,3,7,8-TCOF
2,3,7,8-TCDO
1,2,3,4-TCOO
1,2,3,4.7-PeCDD
1,2,3, 4,7, 8-HxCDD
1,2,3,4,6,7,8-HpCOD
OCDO
50-ra
CP-Sn-88
25.2
23.6
24.1
30.0
39.5
57.0
NM
30-m
OB-5
17.8
17.4
17.3
20.1
22.1
24.1
25.6
3— m
SP-2250
26.7
26.7
26.5
28.1
30.6
33.7
NM
*Retention time in min, using temperature programs shown below.
NM = not measured.
Temperature Programs;
CP-S11-88 60'C-190'C at 20'/m1n; 190*-240* at SVmln.
D8-5 170*, 10 min; then at 8*/min to 320*C, hold
30 m x 0.25 mm at 320*C 20 min (until OCDD elutes).
Thin film (0.25 um)
SP-2250 70*-320* at lOVmtnute.
Column Manufacturers
CP-S11-88 Chrompack, Incorporated, Brldgewater, New Jersey
OB-5, J and W Scientific, Incorporated, Rancho Cordova,
California
SP-2250 Supelco, Incorporated, Bellefonte, Pennsylvania
8280 D-256
Revision
Date September 1986
-------
TABLE 2. IONS SPECIFIED3 FOR SELECTED ION MONITORING
FOR PCDD'S AND PCDF'S
Quantltation
1on
Confirmation
ions
M-COC1
PCDD'S
13c12-Tetra
Tetra
Penta
Hexa
Hepta
Octa
PCDF's
334
322
356
390
424
460
472
332
320
354;358
388,-392
422;426
458
470
257
293
327
361
395
Tetra
Penta
Hexa
Hepta
Octa
306
340
374
408
444
304
338; 342
372;376
406; 410
442
243
277
311
345
379
alons at m/z 376 (HxCDE), 410 (HpCDE), 446 (OCOE), 480 (NCDE) and 514 (DCDE)
are also Included in the scan monitoring sections (1) to (5), respectively.
See Paragraph 10.3.
TABLE 3. CRITERIA FOR ISOTOPIC RATIO MEASUREMENTS FOR PCDO'S AND PCDF'S
Selected ions (m/z)
Relative intensity
PCDD'S
Tetra
Penta
Hexa
Hepta
Octa
PCDF's
Tetra
Penta
Hexa
Hepta
Octa
320/322
358/356
392/390
426/424
458/460
304/306
342/340
376/374
410/408
442/444
0.65-0.89
0.55-0.75
0.69-0.93
0.83-1.12
0.75-1.01
0.65-0.89
0.55-0.75
0.69-0.93
0.83-1.12
0.75-1.01
8280 D-257
Revision 0
Date September 1986
-------
TABLE 4. MEAN RELATIVE RESPONSE FACTORS OF CALIBRATION STANDARDS
Analyte
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
1,2,3,4,7,8-HxCDD
l,2,3,4,6,7,8-HpCDOb
OCDDb
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
1,2,3,4,7,8-HxCDF
1, 2,3.4,6, 7,8-HpCDFb
OCDFb
13C12-2,3,7,8-TCDD
13Ci2-l,2,3,4-TCDD
13C12-OCDD
RRFa
1.13
0.70
0.51
1.08
1.30
1.70
1.25
0.84
1.19
1.57
1.00
0.75
1.00
RSDX
(n - 5)
3.9
10.1
6.6
6.6
7.2
8.0
8.7
9.4
3.8
8.6
-
4.6
-
Quantisation ion
(m/z)
322
356
390
424
460
306
340
374
444
408
334
334
472
aThe RRF value is the mean of the five determinations made. Nominal weights
injected were 0.2, 0.5, 1.0, 2.0 and 5.0 ng.
bRRF values for these analytes were determined relative to 13Ci2-OCDD. All
other RRF's were determined relative to 13Ci2-2,3,7,8-TCDD.
Instrument Conditions/Tune - GC/MS system was tuned as specified in
Paragraph 6.3. RRF data was acquired under
SIM control, as specified in Paragraph 10.3.
GC Program - The GC column temperature was programmed as specified in
Paragraph 4.3.2(b).
8280 D-258
Revision
Date September 1986
-------
TABLE 5. PRECISION DATA FOR REVISED METHOD 8280
Compound
2,3,7,8-TCDD
1,2,3,4-TCOD
1,3,6,8-TCDD
1,3,7,9-TCDD
1,3,7,8-TCDD
1,2,7,8-TCDD
1,2,8,9-TCDD
-
Analyte
Matrix3
clay
son
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
level (ng/g)
Native
N0b
378
NO
NO
487
NO
NO
NO
38.5
NO
NO
NO
NO
19.1
227
NO
NO
NO
58.4
NO
ND
NO
ND
16.0
422
NO
NO
ND
2.6
NO
ND
NO
NO
NO
ND
Native
+ spike
5.0
378
125
46
487
5.0
25.0
125
38.5
2500
2.5
25.0
125
19.1
2727
2.5
25.0
125.0
58.4
2500
5.0
25.0
125
16.0
2920
5.0
25.0
125
2.6
2500
5.0
25.0
125
46
2500
N
4
4
4
2
4
3
4
4
4
4
4
4
4
2
2
4
4
4
2
2
4
4
4
4
2
4
4
4
3
2
4
4
4
2
2
Percent
RSO
4.4
2.8
4.8
-
24
1.7
1.1
9.0
7.9
-
7.0
5.1
3.1
-
-
19
2.3
6.5
-
-
7.3
1.3
5.8
3.5
-
7.7
9.0
7.7
23
-
10
0.6
1.9
-
-
8280 D-259
Revision
Date September 1986
-------
TABLE 5 (Continued)
Compound
1, 2,3,4, 7-PeCDD
1,2,3, 7,8-PeCDD
1,2,3,4,7,8-HxCDD
1,2,3,4,6,7,8-HpCDO
1,2,7,8-TCDF
1,2,3,7,8-PeCDF
1,2,3,4,7,8-HxCDF
*
Analyte
Matrix3
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge0
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom3
clay
v >»j
soil
sludge
fly ash
still bottom
level (ng/g)
Native
NO
NO
NO
25.8
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
8760
NO
NO
NO
NO
NO
7.4
NO
NO
NO
NO
NO
25600
NO
NO
13.6
24.2
NO
Native
+ spike
5.0
25.0
125
25.8
2500
5.0
25.0
125
46
2500
5.0
25.0
125
46
2500
5.0
25.0
8780
_
-
5.0
25.0
125
7.4
2500
5.0
25.0
125
46
28100
5.0
25.0
139
24.2
2500
N
4
4
4
2
2
4
4
4
2
2
4
4
4
2
2
4
4
4
_
-
4
4
4
3
2
4
4
4
2
2
4
4
4
4
2
Percent
RSO
10
2.8
4.6
6.9
-
25
20
4.7
_
-
38
8.8
3.4
_
-
.
_
_
_
-
3.9
1.0
7.2
7.6
-
6.1
5.0
4.8
_
-
26
6.8
5.6
13.5
-
8280 D-260
Revision 0
Date September 1986
-------
TABLE 5. (Continued)
Compound
OCDF
Analyte
Matrix3
clay
soil
sludge
fly ash
still bottom
level (ng/g)
Native Percent
Native + spike N RSD
NO -
NO -
192 317 4 3.3
NO -
NO ...
amatrix types:
clay: pottery clay.
soil: Times Beach, Missouri, soil blended to form a homogeneous sample.
This sample was analyzed as a performance evaluation sample for the Contract
Laboratory Program (CLP) in April 1983. The results from EMSL-LV and 8
contract laboratories using the CLP protocol were 305.8 ng/g 2,3,7,8-TCDD
with a standard deviation of 81.0.
fly ash: ash from a municipal incinerator; resource recovery ash No. 1.
still bottom: distillation bottoms (tar) from 2,4-dichlorophenol production.
sludge: sludge from cooling tower which received both creosote and
pentachlorophenolic wastewaters.
Cleanup of clay, soil and fly ash samples was through alumina column only.
(Carbon column not used.)
- not detected at concentration injected (final volume 0.1 mL or greater).
cEstimated concentration out of calibration range of standards.
8280 D-261
Revision
Date September 1986
-------
TABLE 6. RECOVERY DATA FOR REVISED METHOD 8280
Compound
2,3,7,8-TCDD
1,2,3,4-TCDO
1,3,6,8-TCDD
1,3,7,9-TCDD
1,3,7,8-TCDD
1,2,7,8-TCDD
1,2,8,9-TCDD
Matrix3
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
Nativeb
(ng/g)
NO
378
NO
NO
487
NO
NO
NO
38.5
NO
NO
NO
NO
19.1
227
NO
NO
NO
58.4
NO
NO
NO
NO
16.0
615
NO
NO
NO
2.6
NO
NO
NO
NO
NO
NO
Sp1kedc
level
(ng/g)
5.0
-
125
46
-
5.0
25.0
125
46
2500
2.5
25.0
125
46
2500
2.5
25.0
125
46
2500
5.0
25.0
125
46
2500
5.0
25.0
125
46
2500
5.0
25.0
125
46
2500
Mean
percent
recovery
61.7
-
90.0
90.0
-
67.0
60.3
73.1
105.6
93.8
39.4
64.0
64.5
127.5
80.2
68.5
61.3
78.4
85.0
91.7
68.0
79.3
78.9
80.2
90.5
68.0
75.3
80.4
90.4
88.4
59.7
60.3
72.8
114.3
81.2
8280 D-262
Revision 0
Date September 1986
-------
TABLE 6. (Continued)
Compound
1,2,3,4,7-PeCDD
1,2,3,7,8-PeCDD
1,2,3,4,7,8-HxCDD
1,2,3,4,6,7,8-HpCDD
2,3,7,8-TCDD
(C-13)
1,2,7,8-TCOF
1,2,3,7,8-PeCDF
Matrix2
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludged
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash — .-
still bottom
Native13
(ng/g)
NO
NO
NO
25.8
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO •
ND
NO
8780
ND
NO
ND
ND
ND
NO
NO
NO
ND
ND
7.4
ND
ND
ND
ND
ND
25600
Spikedc
level
(ng/g)
5.0
25.0
125
46
2500
5.0
25.0
125
46
2500
5.0
25.0
125
46
2500
5.0
25.0
125
-
-
5.0
25.0
125
46
2500
5.0
25.0
125
46
2500
5.0
25.0
125
46
2500
Mean
percent
recovery
58.4
62.2
79.2
102.4
81.8
61.7
68.4
81.5
104.9
84.0
46.8
65.0
81.9
125.4
89.1
NO
ND
-
-
64.9
78.8
78.6
88.6
69.7
65.4
71.1
80.4
90.4
104.5
57.4
64.4
84.8
105.8
-
8280 D-263
Revision Q
Date September 1986
-------
TABLE 6. (Continued)
Compound
1,2,3,4,7,8-HxCDF
OCDF
Matrix3
clay
soil
sludge
fly ash
still bottom
clay
soil
sludge
fly ash
still bottom
Nat1veb
(ng/g)
NO
NO
13.6
24.2
NO
NO
NO
192
NO
NO
Sp1kedc
level
(ng/g)
5.0
25.0
125
46
2500
.
-
125
-
—
Mean
percent
recovery
54.2
68.5
82.2
91.0
92.9
_
-
86.8
-
-
amatrix types:
clay: pottery clay,
soil: Times Beach, Missouri soil blended to form a homogeneous sample. This
sample was analyzed as a performance evaluation sample for the Contract
Laboratory Program (CLP) in April 1983. The results from EMSL-LV and 8
contract laboratories using the CLP protocol were 305.8 ng/g 2,3,7,8-TCOO
with a standard deviation of 81.0.
fly ash: ash from a municipal incinerator: resource recovery ash No. 1.
still bottom: distillation bottoms (tar) from 2,4-dichlorophenol production.
sludge: sludge from cooling tower which received both creosote and
pentachlorophenol wastewaters.
The clay, soil and fly ash samples were subjected to alumina column cleanup,
no carbon column was used.
volume of concentrate 0.1 mL or greater, NO means below quantification
limit, 2 or more samples analyzed.
cAmount of analyte added to sample, 2 or more samples analyzed.
^Estimated concentration out of calibration range of standards.
8280 D-264
Revision 0
Date September 1986
-------
TABLE 7. LINEAR RANGE AND VARIATIOIN OF RESPONSE FACTORS
Analyte Linear range tested (pg) nb
l,2,7,8-TCOFa
2,3,7,8-TCDOa
2,3,7,8-TCDF
50-6000
50-7000
300-4000
8
7
5
Mean RF
1.634
0.721
2.208
XRSO
12.0
11.9
7.9
aResponse factors for these analytes were calculated using 2,3,7,8-TCDF as the
internal standard. The response factors for 2,3,7,8-TCDF were calculated vs.
13C12-1,2,3,4-TCDD.
bEach value of n represents a different concentration level.
8280 D-265
Revision
Date September 1986
-------
TABLE 8. METHOD DETECTION LIMITS OF I3C12 - LABELED PCDD'S and PCDF'S
IN REAGENT WATER (PPT) AND ENVIRONMENTAL SAMPLES (PPB)
13C .-Labeled
Analyte
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
1,2,3,6,7,8-HxCDD
1,2,3,4,6, 7,8-HpCDD
OCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
1,2,3,4,7,8-HxCDF
Reagent
Water
0.44
1.27
2.21
2.77
3.93
0.63
1.64
2.53
Missouri
Soil5
0.17
0.70
1.25
1.87
2.35
0.11
0.33
0.83
??
Ash
0.07
0.25
0.55
1.41
2.27
0.06
0.16
0.30
Industrial
Sludge0
0.82
1.34
2.30
4.65
6.44
0.46
0.92
2.17
Stilly
Bottom
1.81
2.46
6.21
4.59
10. 1
0.26
1.61
2.27
Fuel
Oil*
0.75
2.09
5.02
8.14
23.2
0.46
0.83
2.09
Fuel Oil/
Sawdust
0.13
0.18
0.36
0.51
1.48
0.4'J
0.43
2.22
.Sample size I ,000 mL.
Sample size 10 g.
.Sample size 2 g.
Sample size 1 g.
Note: The final sample-extract volume was 100 uL for all samples.
Matrix types used in MDL Study:
- Reagent water: distilled, deionized laboratory water.
- Missouri soil: soil blended to form a homogeneous sample.
- Fly-ash: alkaline ash recovered from the electrostatic precipitator of
a coal-burning power plant.
- Industrial sludge: sludge from cooling tower which received creosotic
and pentachlorophenolic wastewaters. Sample was ca. 70 percent water,
mixed with oil and sludge.
- Still-bottom: distillation bottoms (tar) from 2,4-dichlorophenol
production.
- Fuel oil: wood-preservative solution from the modified Thermal Process
tanks. Sample was an oily liquid (>90 percent oil) containing no
water.
- Fuel oil/Sawdust: sawdust was obtained as a very fine powder from the
local lumber yard. Fuel oil (described above) was mixed ac che 4
percent (w/w) level.
Procedure used for the Determination of Method Detection Limits was obtained
from "Methods for Organic Chemical Analysis of Municipal and Industrial
Uastewater" Appendix A, EPA-600/4-82-057, July 1982. Using this procedure,
the method detection limit is defined as the minimum concentration of a
substance that can be measured and reported with 99 percent confidence tnat
the value is above zero.
8280 D_266
Revision 0
Date September 1986
-------
100.0-1
a
a>
O 73
(vi n>
rt <
O
n> =>
50-
0)
*-•
c
0
15:00
18:00 21:00 24:00
Retention Time
27:00
Figure 2. Mass Chromatogram of Selected PCOD and PCOF Congeners,
-------
METHOO 8290
oi8ENzo-p-oroxiiNs ANO POLYCHI_OPIN*TEO
6. 1
o
Perform Initial
callBratlon on
CC/MS «»«t*«"
6.9
10.2
Calculate
resoonse
factor* for
stanaaras
Oo rout in*
calioratlon
to. 3
Analyze
•a«ol*s wttn
•elected ion
monitor ma
». a
Extract
•amole uitng
aooroorlate
xetnoa for tn*
••••te matrix
9.9
Preoare
caroan column:
do caroon
column cleanuc
10.3
Quantltate PCOO
and PCQP ocakc
O
O«termln«
concentrations
and reoort
reault*
f Stoo J
8280 D-268
Revision o
Date September 1986
-------
APPENDIX A
SIGNAL-TO-NOISE DETERMINATION METHODS
MANUAL DETERMINATION
This method corresponds to a manual determination of the S/N from a GC/MS
signal, based on the measurement of Its peak height relative to the baseline
noise. The procedure 1s composed of four steps as outlined below. (Refer to
Figure 1 for the following discussion).
1.
2.
3.
Estimate the peak-to-peak noise (N) by tracing the two lines (EI and
£2) defining the noise envelope. The lines should pass through the
estimated statistical mean of the positive and the negative peak
excursions as shown 1n Figure 1. In addition, the signal offset (0)
should be set high enough such that negative-going noise (except for
spurious negative spikes) is recorded.
Draw the line (C) corresponding to the mean
segments defining the noise envelope.
noise between the
Measure the height of the GC/MS signal (S) at the apex of the peak
relative to the mean noise C. For noisy GC/MS signals, the average
peak height should be measured from the estimated mean apex signal D
between £3 and £4.
4. Compute the S/N.
This method of S/N measurement 1s a
noise measurement in analytical chemistry.
conventional, accepted method of
INTERACTIVE COMPUTER GRAPHICAL METHOD
This method calls for the measurement of the GC/MS peak area using the
computer data system and Eq. 1:
A/t
S/N » A:/2t f Ar/2t
where t is the elution time window (time interval, t2~t2, at the base of the
peak used to measure the peak area A). (Refer to Figure 2, for the following
discussion) .
left
and Ar correspond to the areas of the noise level in a region to the
and to the right (Ar) of the GC peak of interest.
8280 D-269
Revision 0
Date September 1986
-------
The procedure to determine the S/N 1s as follows:
1. Estimate the average negative peak excursions of the noise (I.e.,
the low segment-E£-of the noise envelope). Line £3 should pass
through the estimated statistical mean of the negative-going noise
excursions. As stated earlier, It 1s Important to have the signal
offset (0) set high enough such that negative-going noise 1s
recorded.
2. Using the cross-hairs of the video display terminal, measure the
peak area (A) above a baseline corresponding to the mean negative
noise value (£2) and between the time tj and t2 where the GC/MS peak
Intersects the baseline, £3. Make note of the time width t^tg-ti.
3. Following a similar procedure as described above, measure the area
of the noise 1n a region to the left (Aj) and to the right (Ar) of
the GC/MS signal using a time window twice the size of t, that is,
2 x t.
The analyst must sound judgement in regard to the proper selection of
interference-free regions in the measurement of Aj and Ar. It is not
recommended to perform these noise measurements (Aj and Ar) in remote regions
exceeding ten time widths (lOt).
4. Compute the S/N using Eq. 1.
NOTE: If the noise does not occupy at least 10 percent of the vertical
axis (I.e., the noise envelope cannot be defined accurately), then
it is necessary to amplify the vertical axis so that the noise
occupies 20 percent of the terminal display (see Figure 3).
8280 D-270
Revision
Date September 1986
-------
FIGURE CAPTIONS
Figure l. Manual determination of S/N.
The peak height (S) 1s measured between the mean noise (lines C and
D). These mean signal values are obtained by tracing the line
between the baseline average noise extremes, EI and £2. and between
the apex average noise extremes, £3 and £4, at the apex of the
signal. Note, 1t is Imperative that the Instrument's Interface
amplifier electronic's zero offset be set high enough such that
negative-going baseline noise is recorded.
Figure 2. Interactive determination of S/N.
The peak area (A) is measured above the baseline average negative
noise £2 and between times tj and t£. The noise is obtained from
the areas AI and Ar measured to the left and to the right of the
peak of interest using time windows Tj and Tr (TjsTr=2t).
Figure 3. Interactive determination of S/N.
A) Area measurements without amplification of the vertical axis.
Note that the noise cannot be determined accurately by visual
means. 8) Area measurements after amplification (10X) of the
vertical axis so that the noise level occupies approximately 20
percent of the display, thus enabling a better visual estimation of
the baseline noise, EI, £2, and C.
8280 D-271
Revision Q
Date September 1986