r/-o
METHODS FOR BENZIDINE. CHLORINATED ORGANIC COMPOUNDS.
PENTACHLORQPHENQL AND PESTICIDES
K ANU'WASTEWATER
INTERIM
Pending Issuance of
Methods for Organic Analysis
of Water and Wastes
U.S. ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
CINCINNATI, OHIO 42568
I
V
September 1978
-------
* v.
FOREWORD
This collection of methods for the determination of benzidine,
chlorinated organic compounds, pentachlorophenol and pesticides has been
assembled by the staff of the Environmental Monitoring and Support
Laboratory - Cincinnati (EMSL-Cinti.) for use by the NPOES Permits
Program.
These methods are as referenced in the Federal Register of
December 1, 1976 and are being provided only for the interim period until
the manual "Methods for Organic Analysis of Water and Wastes" becomes
available.
Dwight 6. Ballinger, Director
Environmental Monitoring and.Support Laboratory - Cincinnati
-------
DISCLAIMER
The mention of trade names or commercial products in ithis manual is for
illustration purposes, and does not constitute endorsement or recommendatior
by the U. S. Environmental Protection Agency.
111
-------
't.
TABLE OF CCMTEi'ITS
Method for Benzidine and Its Salts in Water and Wastewater
Method for Chlorinated Hydrocarbons in Water and Wastewater
Method for Organophosphorus Pesticides in Water and Waste-
water
Method for Polychlorinated Biphenyls (PC3s) in Water and Waste-
water
Method for Triazine Pesticides in Water and Wastewater
Method for 0-aryl Carbamate Pesticides in Water and Wastewater
Method for N-aryl Carbamate and Urea Pesticides in Water and'
Wastewater
Page
1
7
25
43
83
94
T04
Method for Chlorophenoxy Acid Pesticides in Water and Wastewater 115
Method for Volatile Chlorinated Organic Compounds in Water and
Wastewater
O
Method for Pentachlotfphenol in Water and Wastewater
Appendix I
Appendix II
Appendix III
Appendix IV
Bibliography '
130
140
141
146
149
151
154
iv
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PAGE REFERENCES
F.R.# Parameter
EPA 14th ed. ASTM US6S*
This Manual Std. Methods (1975)
9
14
94
95
Benzidine
Chlorinated orqanic
compounds:
Benzylchloride
Carbon tetrachloride
Chlorobenzene
Chloroform
Epichlorohydrin
Heptachloro epoxide
Methyl ene chloride
PCB-1016
PC8-1221
PCS- 1232
PCS- 1242
PCB-1248
PCS-1254
PCS-1260
1,1,2,2-Tetrachlorcethane
Tetr ach 1 oroethy 1 ene
1,2,4-Trichlorobenzene
1,1,2-Trichloroethane
Pentachlorophenol
Pesticides
Aldrin
Ametryn
Ami nocarb
Atraton
Atrazine
Azinphos methyl
Bar ban
BHC
Captan
Carbaryl
Carbophenothion
Chlordane
Chlorpropham
2,4-0
1
130
130
130
130
130'
_-
130
43
43
43
43
43
43
43
130
130
130
130
140
7
S3
94
S3
83
25
104
7
7
94
7
104
115
555
529
30
555
529 30
555
555
555
529
529
30
35
STORET
NUMBER
39120
32102
34301
32160
39420
34423
34671
39488
39492
39496
39500
39504.
39508
34475
39032
39330
39033
39640
39750
39350
-------
F.R.I Parameter
EPA 14th ed. ASTM USGS* STORFT
This Manual Std. Methods (1975) NUMBER
DOD
DDE
DOT
Demeton-0
Diazinon
Dicamba
Dichlorofenthion
Dichloran
Dicofol
Dieldrin
Dioxathion
Disulfoton
Diuron
Endosulfan
Edrin
Ethion
Fenuron
Fenuron - TCA
Heptachlor
" tsodrin
lindane
Linuron
Malathion
Methiocarfa
Methoxychlor
Mexacarfaate
Mirex
Monuron
Monuron-TCA
Neburon
Parathion methyl
Parathion ethyl
PCN8
Perthane
Prometon
Prometryn
Propazine
Propham
Proporur
Secfaumeton
Siduron
Si 1vex,
Simazine
Strobane
Swep
2,4,5-T
Terbuthylazine
7
7
7
25
25
115
;25
1P4
7
7
104
104
7
7
104
25
94
7
94
7
104
104
104
25
25
7
83
83
83
104
94
83
104
115
83
7
104
ITS
83
555
555
555
555
.529
529
529
555
555
555
555
555
555
555
555
555
555
529
529
529
529
529
529
529
30
30
30
30
30"
30
30
30
30
30
30
30
30
30
30
555
529
35
35
39360
39365
39370
39560
39570
39780
39010
39650
39388
39390
39398
39410
39430
39782
39530
39489
39755
39600
39540
39029
39034
39056
39057
39024
39052
39760
39055
vi
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F.R.# Parameter EPA 14th ed. ASTM US6S* STORET
This Manual Std. Methods (1975) NUMBER
Toxaphene ' 7 555 529 30 39400
Trlfluraline 7 ~ 39030
*Soerlitz, D. & Brown, E. "Methods for Analysis of Organic Substances in
Water," U.S. Geological Survey Techniques of Water-Resources Inv. Book 5, Ch.
A3 (1972).
-------
-------
METHQP FOR BENZIDINE AND ITS SALTS IN WASTEWATERS
1. Scope and Application
1.1 This method covers the determination for benzidine and its salts
in water and wastewaters. The method can be modified to apply
also to the determination of closely related materials as des-
cribed under Interferences (4.2)..
1.2 The salts of benzidine, such as benzidine sulfate, are measured
and reported as benzidine,'STORET NO. 39120.
1.3 The method detection limit is 0.2 Wl when analyzing 1 liter of
sample.
2. Summary
'2.1 The water sample is made basic and the benzidine is extracted
with ethyl acetate. Cleanup is accomplished by extracting the
benzidine from the ethyl acetate with hydrochloric acid.
Chloramine-T is added to the acid solution to oxidize the benzi-
dine. The yellow oxidation product is extracted with ethyl
acetate and measured with a scanning spectrophotometer. The
spectrum from 510 nm to 370 nm is used for qualitative identi-
fication.
3. Hazards
3.1 Benzidine is a known carcinogen. All manipulations of -this
method should be carried out in a hood with protection
1
-------
provided for the hands and arms of the analyst. Consult'OSHA
regulations (1) before working with benzidine.
4. Interferences
4.1 The multiple extractions effectively limit the interferences to
organic bases. The oxidation with Chloramine-T to form a yellow
product is very selective and has been described in detail
(2,3.). The use of the absorption spectrum for the identi-
fication of benzidine results in a highly specific procedure.
4.2 Some compounds having a structure very similar to benzidine will
interfere with the quantification, if present. Examples of
these interfering compounds are dichlorobenzidine, o-tolidine,
and dianisidine.
i . t> _ .
4.3 A general yellow background color in the extract will limit the
cell pathlength that can be employed and thus limit the sensi-
tivity of the method.
5. Apparatus and Materials
5.1 Spectrophotometer-visible, scanning (510-370 nm).
5.2 Separatory Funnels - 125 ml, 250 ml, 2000 ml.
5.3 Cells - 1 to 5 cm pathlength, 20 ml volume maximum.
6. Reagents, Solvents and Standards
6.1 Ethyl acetate
6.2 Hydrochloric acid (1 N) - Add 83 ml cone, hydrochloric acid to
water and-dilute to one liter.
6.3 Chloramine-T - 10% solution. Prepare fresh daily by dissolving
l.Og Chloramine-T in 10 ml distilled water.
-------
6.4 Stock standard (0.2 .ug/ul) - Dissolve 100.0 mg purified benzi-
dine in about 30 ml 1 N HC1. Dilute to 500 ml with water.
7. Preparation of Calibration Curve
7.1 To a series of 125-ml separatory funnels, add 45 ml of hydro-
chloric acid and 10 ml of ethyl acetate. Shake for one minute
to saturate the acid layers. Discard the solvent layers. Dose
the series with volumes from 1.0 to 20.0 jul-of stock standard,
using syringes.
7.2 Treat standards according to the Procedure beginning with 8.5.
8. Quality Control
8.1 Duplicate and spiked sample analyses are recommended as quality
control checks. Quality control charts should be developed
and used as a check on the analytical system. Quality control
check samples and performance evaluation samples should be
analyzed on a regular basis.
8.2 Each time a set of samples is extracted, a method blank is
determined on a volume of distilled water equivalent to that
used to dilute the sample.
9. Procedure
9.1 Adjust the sample pH to 8.5 to 9.0 with dilute NaOH or HC1.
9.2 Transfer 1 liter of sample to a 2000-ml separatory funnel. Add
150 ml ethyl acetate and shake for two minutes. Allow the
layers to separate,then drain the water layer into a
-------
second 2-liter separately funnel. Drain the solvent layer into
a 250-ml separatory funnel.
9.3 Repeat the extraction of the water layer twice more with
50-ml portions of ethyl acetate. Combine all solvent layers,
then discard the water layer.
9.4 Extract the solvent layer three times with 15-ml portions of
. --hydrochloric acid fay shaking 2 minutes and allowing the phases
to separate. Combine the acid layers in a glass stoppered
container for cold storage until time is available for analysis,
or transfer the layers directly into a 125-ml separatory funnel.
9.5 Prepare.the spectrophotometer so it is warmed and ready to use.
The remaining steps of the procedure must be performed rapidly
on one sample at a time. '
9.6 To the hydrochloric acid solution in a V25 ml separatory funnel,
add 1.0 ml chloramine-T solution and mix. Add
25.0-ml ethyl acetate with a pipet and snake for two rm'nuces.
Allow the layers to separate, then discard the. aqueous phase.
9.7 Filter the solvent layer through coarse filter paper and fill a
5-cm cell with the filtrate.
9.3 Scan the solvent from 510 nm to 370 nm, Ethyl acetate is used
for a blank with double beam instruments. Shorter pathlength
cells should be used in cases where absorbance exceeds 0.8.
10. Calculation of Results
10.1 Benzidine is identified by its absorbance maximum at
436 nm. Dichlorobenzidine gives similar response but has: its
absorfaance maximum at 445 nm..
-------
10.2 Construct a hasaline from the absorbance minimum at about 470 nm
to tha minimum at 350 nrn (or 420 nm minimum for samples with a
high background). Racord tha assorbanca ofthe peak maximum and
the absorbanca of tha constructed baseline at the 436 nm. Treat
samples snd standards in ths sane fashion.
10.3 Using the net absorbance values, prepare a calibration plot from
the standards. Determine the total micrograms in each sample
from this plot.
10.4 Divide the total micrograms by the sample volume, in liters, to
determine pg/1. Correct results for cell pathlength if
necessary.
11. Reporting Results
11.1 Report results in micrograms per liter as benzidine without
correction for recovery data. When duplicate and spike samples
are analyzed all data obtained should be reported.
12. Accuracy and Precision
12.1 When 1 liter samples of river water were dosed with 1.80 pg of
benzidine, an average of 1.24jug was recovered. The standard
deviation was 0.092 ,ug/l (n=S).
-------
REFERENCES:
1- Federal Register, Volume 39, Page 3779, Paragraph 1910.93; (January 29,
19747.
2. Classman, J. M., and Meigs, J. W., "Benzidine (4,4l-Diaminob-fphenyl) and
Substituted Benzidines", Arch. Industr. Hyg., 4, 519, (1951),;
3. Butt* L. T. and Strafford, N., "Papilloma of the Bladder in the Chemical
Industry. Anlaytical Methods for the Determination of Benzidine and
B-Naphtylamine, Recommended by A.B.C.M. Sub-Committee", J. Appl. Chem.,
£, 525 (1956). .-.
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METHOD FOR CHLORINATED HYDROCARBONS IN WATER AND WASTEWATER
1. Scope and Application
T.I This method covers the determination of various organo-
chlorine pesticides and heptachlor epoxide in water and
wastewater.
1.2 The following pesticides may be determined individually by this
method:
Parameter . Storet 'No.
Aldrin 39330
8HC
" Captan 39640
Chlordane 39350
ODD 39360
DDE - 39365
DOT 39370
Dichloran -
Oieldrin 39380
Endosulfan 39388
Endrin 39390
Heptachlor 39.410
Lindane 39782
Methoxychlor 39480
Mi rex 39755
PCNB 39029
Sfr-ohane
Toxaphene 39400
Trifluralin 39030
1.3 The following chlorinated organic compound may be determined
individually by this method:
Compound Storet No.
Heptachlor epoxide .
-------
2. Summary . . -. .
2.1 The method offers several analytical alternatives, dependent on
the analyst's assessment of the nature and extent of interfer-
ences and/or the complexity of the pesticide mixtures found,
Specifically, -the procedure describes the use of an effective
co-solvent for efficient sample extraction; provides, through
use of column chromatography and liquid-liquid partition,
methods for elimination of non-pesticide interferences and the
pre-separation of pesticide mixtures. Identification is made
by selective gas chromatographic separations and may be corro-
borated through the use of two or more unlike columns.
Detection and measurement is accomplished by electron capture,
microcoulometric or electrolytic conductivity gas chromato-
graphy. Results are reported in micrograms per liter.
2.2 . Confirmation of the identity of the compounds should be made by
GC-MS when a new or undefined sample type is being analyzed and
the concentration is adequate for such determination.
' 2.3 This method is recommended for use only by experienced pesti-
cide analysts or under the close supervision of such qualified
persons.
3. Interferences . . .
3.1 Solvents, reagents, glassware, and other sample processing
hardware may yield discrete artifacts and/or elevated
baselines, causing misinterpretation of gas chromatograms.
-------
All of these materials must be demonstrated to be free from
interferences under the conditions of the analysis. Specific
selection of reagents and purification of solvents by distill-
ation in all-glass systems may be required. Refer to Appendix
I.
3.2 The interferences in industrial effluents are high and varied
and often pose great difficulty in obtaining accurate and
precise measurement of organochlorine pesticides. Sample
clean-up procedures are generally required and may result in
the loss of certain organochlorine pesticides. Therefore,
great care should be exercised in the selection and use of
methods for eliminating or minimfzing interferences. It is not
possible to describe procedures for overcoming .all of the
interferences that may be encountered in industrial effluents.
3.3 Polychlorinated Biphenyls (PCSs) - Special attention is called
to industrial plasticizers and hydraulic fluids such as the
PCBs, which are a potential source of interference in pesticide
analysis. The presence of PCBs is indicated by a large number
of partially resolved or unresolved peaks which may occur
throughout the entire chromatogram. Particularly severe PCS
interference will require special separation procedures (1, 2).
3.4 Phthalate Esters - These compounds, widely used as
plasticizers, respond to the electron capture' detector and ara
a source of interference in the determination of organochlorine
pesticides using this detector. Water leaches these materials
from plastics, such as polyethylene bottles and tygon tubing.
-------
The presence of phthalate esters is implicated in samples that
respond to electron capture but not to the microcoulometric or
electrolytic-conductivity halogen detectors'or to the flame
photometric detector.
3.5 Organophosphorus Pesticides - A number of organophdsphorus
pesticides, such as those containing a nitrp group, e.g.,, para-
thion, also respond to the electron capture detector and may
interfere with the determination of the organochlorine pesti-
cides. Such compounds can. be identified by their response to.
the flame photometric-detector (3). . .
4. Apparatus and Materials
4.1 Gas Chromatograph - Equipped with glass lined injection port.
4.2 Detector Options: ,
4.2.1 Electron Capture - Radioactive (tritium or m'ckel-63)
4.2.2 Microcoulometric Titration
4.2.3 Electrolytic Conductivity-
4.3 Recorder - Potentiometric strip chart (10 in.) compatible with
the detector.
4.4 Gas Chromatographic Column Materials:
4.4.1 Tubing - Pyrex ()80 cm long X 4 mm ID)
4.4.2 Glass Wool - Silanized ' .
4.4.3 Solid Support - Gas-Chrom-Q (100-120 mesh)
4.4.4- Liquid Phases - Expressed as weight percent coated on
so.lid support* "
4.4.4.1 OV-1, 3%
.4.4.4.2 OV-2.10, '558: - '
10
-------
4.4.4,3 OV-17, 1.5% plus QF-1 or OV-210, 1.95%
4.4.4.4 QF-1, 6% plus SE-30, 4%
4.5 Kuderna-Danish (K-D) Glassware
4.5.1 Snyder Column - three-ball (macro) and two-ball
(micro)
4.5.2 Evaporative Flasks - 500 ml
4.5.3 Receiver Ampuls - 10 ml, graduated
4.5.4 AmpuT Stoppers
4.6 Chromatographic Column - Chromaflex (400 mm long x 19 mm ID)
with coarse fritted plate on bottom and Teflon stopcock; 250-ml
reservoir bulb at top of column with flared out funnel shape at
top of bulb - a special order (Kontes K-42054Q- 9011).
4.7 Chromatographic Column - pyrex (approximately 400 mm long x 20
mm ID) with coarse fritted plate on bottom.
4.8 Micro Syringes - 10, 25, 50 and 100 jul.
4.9- Separatory funnels - 125 ml, 1000 ml and 2000 ml with teflon
stopcock.
4.10 Blender - High speed, glass or stainless steel cup.
4.11 Graduated cylinders - 100 and 250 ml.
4.12 Florisil - PR Grade (60-100 mesh); purchase activated at
1250°F and store in the dark in glass containers with glass
stoppers or foil-lined screw caps. Before use, activate each
batch overnight at 130°C in foil-covered glass container.
Determine lauric-acid value (See Appendix II).
5. Reagents, Solvents, and Standards
5.1 Sodium Chloride - (ACS) Saturated solution in distilled water
11
-------
(pre-rinse Nad with hexane).
* ! .
5.2 Sodium Hydroxide - (ACS) 10 N in distilled water.
5.3 Sodium Sulfate - (ACS) Granular, anhydrous (conditioned at 400
C for 4 hrs.).
5.4 SuIfuric Acid - (ACS) Mix equal volumes of cone. HgSO^ with
distilled water. :
5.5 Diethyl Ether - Nanograde, redistilled in glass, if necessary.
5.5.1 Must be free of peroxides as indicated by EM Quant test
strips. (Test strips are available from EM Laboratories,
Inc., 500 Executive Blvd., Elmsford, M.Y. 10523.)
5.5.2 Procedures recommended for removal of peroxides are
provided with the test strips.
5.6 A'cetonitrile, Hexane, Methanol, Methylene Chloride, Petroleum
Ether (boiling range 30-60°C) - nanograde, redistill in glass
if necessary.
5.7 Pesticide Standards - Reference-grade.
6. Calibration
6.1 Gas chromatographic operating conditions are considered accept-
able if the response to dicapthon is at least 50% of full scale
when < 0-06 ng is injected for electron capture detection and
^100 ng is injected for microcoulometric or electrolytic.
conductivity detection. For all quantitative measurements, the
detector must be operated within its linear response range and
the detector noise level should be .less than 2% of full scale.
'6.2 Standards are injected frequently as a check on the stability
of operating conditions. Gas chromatograms of several standard
12
-------
pesticides are shown in Figures ls 2, 3 and 4 and provide
reference operating conditions for the four recommended columns.
6.3 The elution order and retention ratios of various organo-
chlorine pesticides are provided in Table 1, as a guide.
7. Quality Control
7.1 Duplicate and spiked sample analyses are recommended as quality
control checks. Quality control charts (4) should be developed
and used as a-check on the analytical system. Quality control
check samples and performance evaluation samples should be
analyzed on a regular basis.
7.2 Each time a set of samples is extracted, a method blank is
determined on a volume of distilled water equivalent to that
used to dilute the sample,
8. Sample Preparation
8.1 The sample size taken for analysis is dependent on the type of
sample and the sensitivity required for the purpose at hand.
Background information on the pesticide levels previously
detected at a given sampling site will assist in determining
the sample size required, as well as the final volume to which
the extract needs to be concentrated. A 1-liter sample is
usually taken for drinking water and ambient water analysis to
provide a detection limit of O.QSOto O.lQO^g/1. One-hundred
milliliters is usually adequate to provide a detection limit of
1 jjg/1 for industrial effluents.
13
-------
&
~e
S3
aa
re
«- 09
OS I
.
19 UJ trt «J
"~ t ra oa
- e.
*ei "5
14
-------
10 5
HETEXT10N TIME IH MINUTES
Figure 2. Column Picking: 5% OV-210, Carrier fias: Argon/Methane
at 70 ml/rain, Column Temperature: 180 C, Detector:
Electron Capture.
15
-------
XHHIW
16
-------
X3HIW
17
-------
Table 1
RETENTION RATIOS OF VARIOUS ORGANOCHLORINE PESTICIDES RELATIVE TO ALDRIN
Phase^ T.
Column Temp.
Argon/Methane
Carrier Flow
Pesticide
Trifluralin
«-BHC
PCNB
Lindane
Oichloran
Heptachlor .
Aldrin
Heptachlor Epoxide
Endosulfan I
p,p'-DDE
Dieldrin
Captan
Endrin
o,p'-DDT
p,p'-DOD
Endosulfan II
p,p'-ODT
Mirex
Methoxychlor
Aldrin
(Min. absolute)
1.5% OV-17
f
95% OF-12
200 C
60 ml/min
RR
0.39
0.54
0.68
0.69
0.77
o;s2 .
1.00
1.54
1.95
2.23
2.40
2.59
2.93
3.16
3.48
3.59
4.18
6.T
7.6
3.5
5%
OV-210
180 C
70 ml/min
RR
1.11
0.64
0.85
0.81
1.29
0.87
1.00
1.93
2.48 ..
2. 10.
3.00
4.09
3.56
2.70
3.75
4.59
4.07
3.78
6.5.
2.6
3%
OV-1
180 C
70 ml/min
RR
0.33
0.35
0.49
0.44
0.49
0.78
1.00
1.28 '
1.62
2.00
1.93
1.22
2.18
2.69
2.61
2.25
3.50
6.6
5.7
4.0.
5% QF-1
»K
4% SE-30
200 C
60 ml/min
RR
0.57
0.49
0.63
0.60
0.70
0.83
,1.00
1.43
1.79
1.82
2.12
1.94
:2.42
2.39
2.55
2.72
3.12
4.79
4,60
5.6
columns glass, 180 cm x 4 mm ID, solid support Gas-Chrom Q (100/120
mesh)
2OV-210 also may be used
18
-------
8.2 Quantitatively transfer the proper aliquot of sample from the
sample container into a two-liter separatory funnel. If less
than 800 ml is analyzed, dilute to one liter with interference
free distilled water.
9. Extraction
9.1 Add 60 ml of 15% methylene chloride in hexane (v:v) to the
sample in the separatory funnel and shake vigorously for two
minutes. .
9.2 Allow the mixed solvent to separate from the sample, then draw
the water into a one-liter Erlenmeyer flask. Pour the organic
layer into a 100 ml beaker and then pass it through a column
containing 3-4 inches of anhydrous sodium sulfate, and collect
it in a 500 ml K-0 flask equipped with'a 10 ml ampul. Return
the water phase to the separatory funnel. Rinse the Erlenmeyer
flask with a second 60-ml volume_of solvent; add the solvent to
the separatory funnel and complete the extraction procedure a
second time. Perform a third extraction in the same manner.
9.3 Concentrate the extract in the K-D evaporator on a hot water
bath.
9.4 Analyze by gas chromatography unless a need for cleanup is
indicated (See Section 10).
10. Clean-up and Separation Procedures
10.1 Interferences in the form of distinct peaks and/or high back-
ground in the initial gas chromatographic analysis, as well as
the physical characteristics of the extract (color, cloudiness,
viscosity) and background knowledge of the sample will indicate
19
-------
whether clean-up is required. When these interfere with
measurement of the pesticides, or affect column life or
detector sensitivity, proceed as directed below.
10,2 Acetonitrile Partition - This procedure is used to isolate fats
and oils from thel sample extracts. It should be noted that not
all pesticides are quantitatively recovered by this procedure.
The analyst must be aware of this and demonstrate the effi-
ciency of the partitioning for specific pesticides. All of the
pesticides listed in Scope (-1.2) with the exception of mi rex
are efficiently recovered.
10.2.1 Quantitatively:transfer the previously concentrated
extract to a 125-ml separatory funnel with enough
hexane to bring the final volume to 15 ml. Extract the
sample four times by shaking vigorously for one minute
with 30-ml portions of hexane-saturated acetonitrile.
10.2.2 Combine and transfer the acetonitrile phases to a
one-liter separatory funnel and add 650 ml of distilled
water and 40 ml of saturated sodium chloride solution.
Mix thoroughly for 30-45 seconds. Extract with two
100-ml portions of hexane by vigorously shaking about
15 seconds.
10.2.3 Combine the hexane extracts in a one-liter separatory
funnel and wash with two 100-ml portions of distilled
water. Discard the water layer and pour the hexane
layer through a 3-4 inch anhydrous sodium sulfate
column into a 500-ml K-0 flask equipped with a 10-ml
20
-------
ampul. Rinse the separatory funnel and column with
three 10-ml portions of hexane.
10.2.4 Concentrate the extracts to 6-10 ml in the K-0 evapor-
ator in a hot water bath.
10.2.5 Analyze by gas chromatography unless a need for further
cleanup is indicated.
10.3 Florisil Column Adsorption Chromatography
10.3.'1 Adjust the sample extract volume to 10 ml.
T0o3.2 Place a charge of activated Florisil (weight determined
by lauric-acid value, see Appendix II) in a Chromaflex
. column. After settling the Florisil by tapping the
column, add about one-half inch layer of anhydrous
granular-sodium sulfate to the top.
10.3.3 Pre-elute the column, after cooling, with 50-60 ml of
petroleum ether. Discard the eluate and just prior to
exposure of the sulfate layer to air, quantitatively
transfer the sample extract into the column by
decantation and subsequent petroleum ether wash- ings.
Adjust the elution rate to about 5 ml per minute and,
separately, collect up to three eluates in 500-ml K-0
flasks equipped with 10-ml ampuls (see Eluate
Composition 10.4.). Perform the first elution with
200 ml of 6% ethyl ether in petroleum ether, and the
second elution with 200 ml of 15% ethyl ether in
21
-------
petroleum ether. Perform the third elution with 200 ml
of 50% ethyl ether - petroleum ether and the fourth
elution with 200 ml of 100% ethyl ether.
10.3.4 Concentrate the eluates to 5-10 ml in the IC-D eva-
porator in a hot water bath.
10.3.5 Analyze by gas chromatography.
70.4 Eluate Composition - By using an equivalent quantity of any
batch of Florisil, as determined by its lauric acid value, the
pesticides will be separated into the eluates indicated below:
6% Eluate
Aldrin
BHC
Chlordane
ODD
ODE
15% Eluate
Endosulfan I
Endrin
Dieldrin
Dichloran
DDT Mi rex
Heptachlor PCNB
Heptachlor Epoxide Strobane
Lindane Toxaphene
Methoxychlor Trifluralin
: 50% Eluate
Endosulfan II
Captan
Certain thiophosphate pesticides will occur in each of the
above fractions as well as the 100% fraction. For additional
information regarding eluate composition, refer to the FDA
Pesticide Analytical Manual (5)».
'11. Calculation of Results
11.1 Determine the pesticide concentration by using the absolute
calibration procedure described below or the relative cali-
bration procedure described in-Appendix III.
22
-------
(1) Miprograms/liter = (A) (B) (vt)
(V-\) l»S'
A - nq standard
Standard area
B 3 Sample aliquot area
V-fs Volume of extract injected
V-t= Volume of total extract (jul)
Vss Volume of water extracted (ml)
12. Reporting Results
12.1 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are ana-
lyzed, all data obtained should be reported.
23
-------
REFERENCES:
1. Monsanto Methodology for Aroclors - Analysis of Environmental Materials
for Biphenyls, Analytical Chemistry Method 71-35, Monsanto Company,
St. Louis, Missouri, 63166, 1970.
2. "Method for Polychlorinated Biphenyls in Water and Wastewater", this
manual, p. 43.
3. "Method for Organophosphorus Pesticides in Water and Wastewater", this
manual, p. 25.
4. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories", Chapter 6, Section 6.4, U. S. Environmental Protection
Agency, National Environmental Research Center, Analytical Quality Con-
trol Laboratory, Cincinnati, Ohio, 45268, 1973.
5. "Pesticide Analytical Manual", U. S. Dept. of Health, Education and
Welfare, Food and Drug Administration, Washington, 0. C.
24
-------
METHOD FOR ORSANOPHOSPHORUS PESTICIDES IN WATER AND WASTEWATER
1. Scope and Application
1.1 This method covers the determination of various organophosphorus
pesticides in water and wastewater.
1.2 The following pesticides may be determined individually by this
method:
Parameter Storet No.
Azinphos methyl
Demeton-0 33560 .
Demeton-S
Diazinon " - 39570
Disulfoton 39010
Malathion 39530
Parathion methyl 39600
Parathion ethyl 39540
2, Summary
2.1 The method offers several analytical alternatives, dependent on
the analyst's assessment of the nature and extent of interferences
and the complexity of the pesticide mixtures found. Specifically,
the procedure describes the use of an effective co-solvent for
efficient sample extraction; provides, through use of the column
chromatography and liquid-liquid partition, methods for the
elimination of non-pesticide interferences and the preseparation
of pesticide mixtures. Identification is made by selective gas
chromatographic separation and may be corroborated through the use
of two or more unlike columns. Detection and measurement are best
accomplished by flame photometric gas chromatography using a
25
-------
phosphorus specific filter. The electron capture detector, though
non-specific, may also be used for those compounds to which it
responds. Results are reported in micrograms per liter,
2.2 Confirmation of the identity of the compounds should be made by
GC-MS when a new or undefined sample type is being analyzed and
the concentration is adequate for such determination.
2.3 This method is recommended for use only by experienced pesticide
analysts or under the close supervision of such qualified persons.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing hard-
ware may yield discrete artifacts and/or elevated baselines, caus-
ing misinterpretation of gas chromatograms. All of these .
materials must be demonstrated to be free from interferences under
the conditions of the analysis. Specific selection of reagents'
and purification of solvents by distillation in all-glass systems'"
may be required. Refer to Appendix I.
3.2 The- interferences in industrial effluents are high and varied and
often pose great difficulty in obtaining accurate and precise
measurement} of organophosphorus pesticides. Sample clean-up
procedures are generally;required and may result in the loss of
certain organophosphorus pesticides. Therefore, great care should
be exercised in the selection and use of methods for elfminating
or minimizing interferences. It is not possible to describe
procedures for overcoming all of the interferences that may be
encountered in industrial effluents.
26
-------
3.3 Compounds such as organochlorine pesticides, polychTor-mated
biphenyls and phthalate esters Interfere with the analysis of
organophosphorus pesticides by electron capture gas chro-
matography. When encountered, these interferences are overcome by
the use of the phosphorus specific flame photometric detector. If
such a detector is not available, these interferences may be
removed from the sample by using the clean-up procedures described
in the EPA methods for those compounds (1, 2).
3.4 Elemental sulfur will interfere with the determination of organo-
phosphorus 'pesticides by flame photometric and electron capture
gas chromatography. The elimination of elemental sulfur as an
interference is described in Section 10.5, Clean-up and Separation
Procedures.
4. Apparatus and Materials
4.1 Gas Crhomatograph - Equipped with glass lined injection port.
4.2 Detector options:
4.2.1 Flame Photometric - 526 mu phosphorus filter.
4.2.2 Electron Capture - Radioactive (tritium or nickel-63).
4.3 Recorder - Potentiometric strip chart (10 in.) compatible with the
detector.
4.4 Gas Chromatographic Column Materials:
4.4.1 Tubing - Pyrex (180 cm long x 4 mm ID)
4.4.2 Glass Wool - Silanized
4.4.3 Solid Support - Gas Chrom Q (100-120 mesh)
4.4.4 Liquid Phases - Expressed as weight percent coated on
solid support.
27
-------
4.4.4.1 OV-1, 3St .
4.4.4.2 OV-210, 5%
4.4.4.3. OV-17, 1.5% plus QF-1 or OV-210, 1
4.4.4.4 QF-1 or OV-210, 6% plus SE-30, 4%
4.5 Kuderna-Danish (K-0) Glassware
4.5.1 Snyder Column - three ball (macro) and two ball (micro)
4.5.2 Evaporative Flasks - 500 ml-.
4.5.3 Receiver Ampuls - 10 ml, graduated
4.5.4 Ampul Stoppers.
4.6 Chromatographic Column -. Chromaflex (400 mm x 19 mm ID) with
coarse fritted plate and Teflon stopcock on bottom; 250 ml
reservoir bulb at top of column with flared out funnel shape at
top of bulb - a special order (Kontes K-420540-9011).
4.7 Chromatographic Column - pyrex (approximately 400 mm long x 20 mm
ID) with coarse fritted plate on bottom.
4.8 Micro Syringes - 10, 25, 50 and TOOjjl.
4.9 Separatory funnels - 125 ml, 1000 ml and 2000 ml with Teflon
stopcock.
4.10 Micro-pipets - disposable (140 mm long x 5 mm. ID).
4.11 Blender - High speed, glass or stainless steel cup. ,
4.12 Graduated cylinders - 100 and 250 ml.
4.13 Florisil - PR Grade (50-100 mesh); purchase activated at 1250°F
and store in the dark in glass containers with glass stopper? or
foil-lined screw caps.. Before use, activate each batch overnight
at 130°C in foil-covered glass container. Determine lauric-acid
value (See Appendix II).
28
-------
4.14 Alumina - Woelm, neutral; deactivate by pipeting 1 ml of distilled
water into 125 ml ground glass-stoppered Erlenmeyer flask. Rotate
flask to distribute water over surface of glass. Immediately add
19.0 g fresh alumina through small powder funnel. Shake flask
containing mixture for two hours on a mechanical shaker (3).
5. Reagents, Solvents, and Standards
5.1 Sodium Chloride - (ACS) Saturated solution in distilled water
(pre-rinse NaCl with hexane).
5.2 Sodium Hydroxide - (ACS) 10.N in distilled water.
5.3 . Sodium Sulfate - (ACS) Granular, anhydrous (conditioned at 400*C
for 4 hrs.). .
.. 5.4 Sulfuric Acid -'(ACS) Mix equal volumes of cone. H2S04-with
- distilled water.
5.5 Oiethyl Ether -Nanograde, redistilled in glass, if necessary.
5.5.1 Must be free of peroxides as indicated by EM Quant test
strips. (Test strips are available from EM
Laboratories, Inc., 500 Executive Blvd., Emslford, N.Y-.
10523.)
5.5.2 Procedures recommended for removal of peroxides are
provided with the test strips.
5.6 Acetonitrile, Hexane, Methanol, Methylene Chloride, Petroleum
Ether (boiling range 30-60toC) - nanograde, redistill in glass if
necessary.
5.7 Pesticide Standards - Reference grade.
29
-------
6. Calibration
6.1 Gas chromatographic operating conditions are considered acceptable
If the response to dicapthon is at least 50% of full scale when <1.5
ng 1s Injected for flame photometric detection and <"0.06 ng is
Injected for electron capture detection. For all quantitative
measurements, the detector must be operated within its linear
response range and the-detector noise level should be less than 2%
of full scale.
6.2- Standards are injected frequently as a check on the stability of
operating conditions. Gas chromatograms of several standard
pesticides are shown in Figures 1, 2, 3 and 4 and provide reference
operating conditions for the four recommended columns.
6.3 The elution order and retention ratios of various organophosphorus
pesticides are provided in Table 1, as a guide.
7. Quality Control
7.1 Duplicate and spiked sample analyses are recommended as quality
control checks. Quality control charts (4) should be developed and
used as a check on the analytical system. Quality control check
samples and performance evaluation samples should be analyzed on a
regular basis.
7.2 Each time a set of samples is extracted, a method blank is
determined on a volume of distilled water equivalent to that used
to dilute the sample.
8. Sample Preparation
8.1 The sample size taken for analysis is dependent on the type of
sample and the sensitivity required for the purpose at hand.
30 :
-------
0 2 4 8 8 10
RETENTION TIME IN MINUTES
Figure 1. Column Packing: 1.5% OY-17 + 1.95 % QM?
Carrier Gas: Nitrogen at 70 ml/min, Column Temperature: 213 C,
Detector: Flame Photometric [Phospliorus).
31
-------
-CJJ
0
10
24 8 8
RETENTION TIME IN MINUTES
Figure 2. Column Packing: 5% OY-210, Carrier Gas: litrogen
at 60 ml/min, Column Temperature: 200 C, Detector:
Flame Photometric [Phosphorus].
32
-------
fl 2 4 8'8 10
RETENTION TIME IN MINUTES
Figure 3. Column Packing: 6% QF-1 +4% SE-30, Carrier Gas: Nitrogen
at 70 mi/min, Column Temperature: 215 C, Detector: FSame
Photometric (Phosphorus).
33
-------
2 4 6 8 10
RETENTION TIME IN MINUTES
Figure 4. Column Packing: 3% OY-1, Carrier Gas: Nitrogen at
60 ml/min, Column Temperature: 200 C, Detector: Flame
Photometric (Phosphorus).
34
-------
TABLE 1
RETENTION TIMES OF SOME ORGANOPHOSPHOROUS PESTICIDES
RELATIVE TO PARATHION
Liquid Phase1
Column Temp. ^
Nitrogen
Carrier Flow
Pesticide ,
Demeton^
Diazinon
Disulfoton
Ma lath ion
Parathion methyl
Parathion ethyl
Azinpnos methyl
Parathion
(min absolute)
1.5% OV-17
1.95£ QF-12
215 C
70 ml/min
RR
0.46
0.40
0.46
0.86
0.82
1.00
6.65
4.5
6% QF-1^
4% SE-30
215 C
70 ml/min
RR
0.25
0.43
0.38
0.45
0.78
0.80
1.00
4.15
6.6
5%
OV-210
200 C
60 ml/min
RR
0.20
.38
0.25
0.31
0.73
0.81
1.00
4.44
5.7
7%
OV-1
200 C
60 ml/min
RR
0.74
0.59
0.62
0.92-
0.79
1.00
4.68
wo I
n /ic>n
_ mesh.
2May substitute OV-210 for QF-1.
^Anomalous, multipeak response often encountered.
35
-------
Background information on the pesticide levels previously detected
at a given sampling site will assist in determining the sample
size required, as well as the final volume to which the extract
needs to be concen- trated. A 1-liter sample is usually taken for
drinking water and ambient water analysis to provide a-detection
limit of 0.050 to 0.100;jg/l. One-hundred milliliters is usually
adequate to provide a detection limit of 1 ^ig/1 for industrial
effluents.
8.2 Quantitatively transfer the proper aliquot of sample from the
sample container into a two-liter separatory funnel. If less than
a 800 ml is analyzed, dilute to one liter with interference free
distilled water.
9. Extraction
9.1 Add 60 ml of 15% methylene chloride in hexane (v:v) to the sample
in the separatory funnel and shake vigorously for two minutes.
9.2 Allow the mixed solvent to separate from the sample, then draw the
water into a one-liter Erlenmeyer flask. Pour the organic layer
into a 100 ml beaker and then pass it through a column containing
3-4 inches of anhydrous sodium sulfate, and collect it in a 500 ml
K-D flask equipped with a 10 ml ampul. Return the water phase to
the separatory funnel. Rinse the Erlenmeyer flask with a second
60 ml volume of solvent; add the solvent to the separatory funnel
and complete the extraction procedure a second time. Perform a
third extraction in the same manner.
9.3 Concentrate the extract in the K-D evaporator on a hot water bath.
36
-------
9.4 Analyze by gas chromatography unless a need for cleanup is indi-
cated. (See Section 10).
10. Clean-up and Separation Procedures
10.1 Interferences in the form of distinct peaks and/or high background
in the iaitial gas chromatographic analysis, as well as the
physical characteristics of the extract (color, cloudiness,
viscosity) and background knowledge of the sample source will
indicate whether clean-up is required. When these interfere with
measurement of the pesticides, or affect column life or detector
sensitivity, proceed as directed below. The use of these
procedures is not required for samples free of interferences.
They are provided as options to the analyst to be used when'needed.
10.2' Acetonitrile Partition - This procedure is used to separate fats
and oils from the sample extracts. It should be noted that not
all pesticides are quantitatively recovered by this procedure.
The analyst must be aware of this and demonstrate the efficiency
of the partitioning for specific pesticides.
10.2.1 Quantitatively transfer the previously concentrated
extract to a 125-ml separatory funnel with enough hexane
to bring the final volume to 15 ml. Extract the sample
four times by shaking vigorously for one minute with 30
ml portions of hexane-saturated ace-tonitrile.
10.2.2 Combine and transfer the acetonitrile phases to a
one-liter separatory funnel and add 650 ml of distilled
water and 40 ml of saturated sodium chloride solution.
Mix thoroughly for 30-45 seconds. Extract with two
37
-------
100 ml portions of hexane by vigorously shaking about 15
seconds.
10.2.3 Combine the hexane extracts in a one-liter separatory
funnel and wash with two 100 ml portions of distilled
water. Discard the water layer and pour the hexane
layer through a 3-4 inch anhydrous sodium sulfate column
into a 500-ml K-D flask equipped with a 10-ml ampul.
Rinse the separatory funnel and column with three 10 ml
portions of hexane.
10.2.4 Concentrate the extracts to 6-10 ml in the K-D
evaporator in a hot water bath.
10.2.5 Analyze by gas chromatography unless a need for further
clean-up is indicated.
10.3 Florisil Column Adsorption Chromatography
10.3.1 Adjust the sample extract volume to 10 ml.
10.3.2 Place a charge of activated Florisil (weight determined
fay lauric-acid value, see Appendix II) in a Chromaflex .
column. After settling the Florisil by tapping the
column, add about one-half inch layer of anhydrous
granular sodium sulfate to the top.
10.3.3 Pre-elute the column, after cooling, with 50-SO ml of
petroleum ether. Discard the eluate and just prior to
exposure of the sulfate layer to air, quantitatively
transfer the sample extract into the column by
decantation and subsequent petro- leum ether washings.
Adjust the elution rate to about 5 ml per; minute and,
38 -
-------
separately, collect up to four eluates In 500-ml K-0
flasks equipped with 10-inl ampuls. (See Eluate Compos-
ition, 10.4.) Perform the first elution with 200 ml of
6% ethyl ether in petroleum ether, and.the second
elution with 200 ml of 15% ethyl ether in petroleum
ether. Perform the third elution with 200 ml of 50%
ethyl ether - petroleum ether and the fourth elution
with 200 ml of 100% ethyl ether.
10.3.4 Concentrate the eluates to 6-10 ml in the K-0 evaporator
in a hot water bath.
10.3.5 Analyze by gas chromatography.
10.4 Eluate Composition --By iTsing an equivalent quantity of any batch
of Florisil as determined by its lauric-acid value, the pesticides
will be separated into the eluates indicated below:
6% Eluate 15% Eluate
Demeton Diazinon
Oisulfoton Malathion (trace)
Parathion Methyl
50% Eluate ' 100% Eluate
Malathion Azinphos methyl (30%)
Azinphos methyl (20%)
For additional information regarding eluate composition, refer
to the FDA Pesticide Analytical Manual (5).
10.5 Removal of Sulfur - If elemental sulfur interferes with the gas
chromatographic analysis, it can be removed by the use of an
alumina microcolumn.
10.5.1 Adjust the sample extract volume to 0.5 ml in a K-0
39
-------
apparatus, using a two-ball Snyder microcolumn.
10.5.2 Plug a disposable pipet with a small quantity of glass
wool. Add enough alumina to produce a 3-cm column after
settling. Top the alumina with a 0.5-cm layer of
anhydrous sodium sulfate.
10.5.3 Quantitatively transfer the concentrated extract to the
alumina microcolumn using a 100 jjl syringe. Rinse the
ampul with 200 pi of hexane and add to the microcolumn.
10.5.4 Elute the microcolumn with 3 ml of hexane and discard the
first eluate which contains the elemental sulfur*
10.5.5 Next elute the column with 5 ml of 10% hexane in
methylene chloride. Collect the eluate in a 10 ml
graduated ampul.
10.5.6 Analyze by gas chromatography. -
NOTE: If the electron capture detector is to be used methylene
. chloride must be removed... To do this, attach the ampul
to a K-D apparatus (500-ml flask and 3-ball Snyder
column) and concentrate to about 0.5 ml. Adjust volume
as required prior to analysis. -
11. Calculation of Results
11.1 Determine the pesticide concentration by using the absolute
calibration procedure described below or the relative cali-
bration procedure described in Appendix III.
40
-------
(1) Micrograms/liter = iA)Jll_(ltl
V (ViJ ivs) '
A = no standard
Standard area
B = Sample aliquot area
V-j = Volume of extract Injected (.ul) a
Vt = Volume of total extract Ul)
Vs s Volume of water extracted (ml)
12. Reporting Results
12.1 Report results in micrograms per liter without correction for
recovery data. When 'duplicate and spiked samples are analyzed
all data obtained should be reported.
41
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REFERENCES:
1. "Method for Chlorinated Hydrocarbons in Water and Wastewater", this
manual, p. 7.
2. "Method for Polychlorinated EJiphenyls (PCBs) in Water and Wastewater",
this manual, p. 43.
3. Law, L. M. and Georlitz, D. F., "Microcolumn Chromatographic Clean-up
for the Analysis of Pesticides in Water", Journal of the Association
for Analytical Chemists, 53_, 1276 (1970). ~~
4. "Handbook for Analytical Quality Control in Water and .Wastewater
Laboratories", Chapter 6, Section 6.4, U. S! Environmental Protection
Agency, National Environmental Research Center, Analytical Quality Con-
trol Laboratory, Cincinnati, Ohio, 45268, 1973.
5. "Pesticide Analytical Manual", U. S. Dept. of Health, Education and
Welfare, Food and Drug Administration, Washington, D. C.
42
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METHOD FOR POLYCHLORINATEO BIPHENYLS (PCBs) IN WATER AND WASTEWATER
1. Scope and Application
1.1 This method covers the determination of various polych-lorinated
biphenyl (PCS) mixtures in water and wastewater.
1.2 The following mixtures of chlorinated biphenyls (Aroclors) may be
determined by this method:
Parameter Storet No.
PCS-1016 34671
PCS-1221 39488
PC8-1232 39492
PCS-1242 39496
PCB-1248 39500
PCS-1254 39504
PCB-1260 39508
1.3 The method is an extension of the Method for Chlorinated
Hydrocarbons in Water and Wastewater (1). It is designed so
that determination of both the PCBs and the organochlorine
pesticides may be made on the same sample.
2. Summary
2.1 The PCBs and the organochlorine pesticides are co-extracted by
liquid-liquid extraction and, insofar as possible, the two
classes of compounds separated from one another prior to gas
chromatographic determination. A combination of the standard
Florisil column cleanup procedure and a silica gel microcolumn
separation procedure (2)(3) are employed. Identification is
43
-------
made from gas chromatographic patterns obtained through the use
of two or more unlike columns. Detection and measurement is
accomplished using an electron capture, microcoulometric, or
electrolytic conductivity detector. Techniques for confirming
qualitative identification are suggested.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing
* A
hardware may yield discrete artifacts and/or elevated baselines
causing misinterpretation of gas chromatograms. All of these
materials must be demonstrated to be free from interferences
under the conditions of the analysis. Specific selection of
reagents and the purification of solvents by distillation in
all-glass systems may be required.' Refer to Appendix I.
3.2 The interferences in industrial effluents are high and varied
and pose great difficulty in obtaining accurate and precise
measurement of PCBs and organochlorine pesticides. Separation
and clean-up procedures are generally required and may result
in the loss of certain organochlorine compounds. Therefore,
'great care should be exercised in the selection and use of
methods for eliminating or minimizing interferences. It is not
possible to describe procedures for overcoming all of the
interferences that may be encountered in industrial effluents.
3.3 Phthalate esters, certain organophosphorus pesticides, and
elemental sulfur will interfere when using electron capture for
detection. These materials do not interfere when the
44
-------
nricrocoulometric or electrolytic conductivity detectors are
used in the halogen mode.
3.4 Organochlorine pesticides and other halogenated compounds
constitute interferences in the determination of PCBs. Most of
these are separated by the method described below. However,
certain compounds, if present in the sample, will occur with
the PCSs. Included are: Sulfur, Heptachlor, aldrin, DDE,
technical chlordane, mirex, and to some extent, o,p'-DDT and
p,p'-ODT.
4. Apparatus and Materials
4.1 Gas Chromatograph - Equipped with glass lined injection port.
4.2 Detector Options:
4.2.1 Electron Capture - Radioactive (tritium or nickel-63)
4.2.2 Microcoulometric Titration
4.2.3 Electrolytic Conductivity
4.3 Recorder - Potentiometric strip chart (10 in.) compatible with
the detector.
4.4 Gas Chromatographic Column Materials:
4.4.1 Tubing - Pyrex (180 cm long X 4 mm ID)
4.4.2 Glass Wool - Silanized
4.4.3 Solid Support - Gas-Chrom Q (100-120 mesh)
4.4.4 Liquid Phases - Expressed as weight percent coated on
solid support.
4.4.4.1 SE-30 or.OV-1, 3%
4.4.4.2 OV-17, 1.5% + QF-1 or OV-210, 1.95%
45
-------
4.5 Kuderna-Danish (K-D) Glassware
4.5.1. Snyder Column - three-ball (macro) and two-ball (micro)
4.5.2 Evaporative Flasks - 500 ml
4.5.3 Receiver Ampuls - 10 ml, graduated
4.5.4 Ampul Stoppers
4.6 Chromatographic Column - Chromaflex (400 mm long x 19 mm ID)
'"with coarse fritted plate on bottom and Teflon stopcock; 250-ml
reservoir bulb at top of column with flared out funnel shape at
top of bulb - a special order (Kontes K-420540-9011)..
4.7 Chromatographic Co-lumn - pyrex (approximately 400 mm 'long x 20
mm ID) with coarse fritted plate on bottom.
4.8 Micro Co.lumn Pyrex - constructed according to Figure T.
4.9 Capillary pipets disposable (5-3/4 in.) with rubber bulb
(Scientific Products P5205-1). ,
4.10 Low pressure regulator - 0 to 5 PSIG - with low-flow needle
valve (see Figure 1, Matheson Model 70).
4.11 Beaker - 100 ml
4.12 Micro Syringes - 10, 25, 50 and 100 ul.
4.13 Separatory funnels - 125 ml, 1000 ml and 2000 ml with Teflon
stopcock.
4.14 Blender - High speed, glass or stainless steel cup.
4.15 Graduated cylinders - 100 and 250 ml.
4.16 Florisil - PR Grade (60-100 mesh); purchase activated1 at
1250°F and store in the dark in glass containers with glass
stoppers or foil-lined screw caps. Before use, activate each
46
-------
PRESSURE
COMPRESSED
AIR
SUPPLY
I
SHUT-OFF
VALVE
0-5
PSIG
REGULATOR
NEEDLE
VALVE
FLEXIBLE
TUBING
SILICA GEL
5 cm
i cm
g
$ 10/30
!5mi
RESERVOIR
§ 10/30
23cm x 4.2mm i,D.
2 cm x 2 mm l.D.
FIGURE I. MICROCOLUMN SYSTEM
47
-------
batch overnight at 130°C in foil-covered glass container.
Determine lauric-acid value (See Appendix II).
4.17 Silica gel - Davison code 950-08008-226 (60/200 mesh).
4.18 Glass Wool - Hexane extracted.
4.19 Centrifuge Tubes - Pyrex calibrated (15 ml).
5. Reagents, Solvents, and Standards
5.1 Sodium Chloride - (ACS) Saturated solution in distilled water
(pre-rinse NaCl with hexane).
5.2 Sodium Hydroxide - (ACS) 10 N in distilled water.
5.3 Sodium Sulfate - (ACS) Granular, anhydrous .(conditioned at 400°
C for 4 hrs.K
5.4 Sulfuric Acid - (ACS) Mix equal volumes of cone. H2S04 w1t}l
distilled water. . -
. 5.5 Diethyl Ether - Nanograde, redistilled in glass, if necessary.
5.5.1 Must be free of peroxides as indicated by EM Quant test
strips. (Test strips are-available from EM Labora-
tories, Inc., 500 Executive Blvd., Elmsford, N.Y.
10523).
5.5.2 Procedures recommended for removal of peroxides are
provided with the test strips.
5.6 n-Hexane - Pesticide quality (NOT MIXED HEXANES).
5.7 Acetonitrile, Hexane, Methanol, Methylene Chloride, Petroleum
Ether (boiling range 30-60°C) - pesticide quality, redistill in
glass if necessary.
'5.8 Standards - Aroclors 1221, 1232, 1242, 1248, 1254, 1260,, and
.1016.
48
-------
5,9 Anti-static Solution - STATNUL, Daystrom, Inc., Weston Instru-
ment Division, Newark, N.J., 95212.
6. Calibration
^^^^"^^^^
6.1 Gas chromatographic operating conditions are considered accept-
i - '
'' able if the response to dicapthon is at least 50% of full scale
: when < 0.06 ng is injected for electron capture detection and <
100 ng is injected for microcoulometric or electrolytic con-
ductivity detection. For all quantitative measurements, the
detector must be operated within its linear response range and
the detector noise level should be less than 2% of full scale.
6.2 Standards are injected frequently as a check on the stability
of operating conditions, detector and column. Example chro-
matograms are shown In Figures 3 through 8 and provide
reference operating conditions.
7. Quality Control
7.1 Duplicate and spiked sample analyses are recommended as quality
control checks. Quality control charts (4) should be developed
and used as a check on the analytical system. Quality control
check samples and performance evaluation samples should be
analyzed on a regular basis.
7.2 Each time a set of samples is extracted, a method blank is
determined on a volume of distilled water equivalent to that
used to dilute the sample.
8. Sample Preparation
8.1 Blend the sample if suspended matter is present and adjust pH
49
-------
to near neutral (pH 6.5-7.5) with 50% sulfuric acid or 10 N
sodium hydroxide.
8.2 For sensitivity requirement of 1 ug/1, when using micro-
coulometric or electrolytic conductivity methods for detection
take 1000 ml of sample for analysis. If interferences pose no
problem, the sensitivity of the electron capture detector
should permit as little as 100 ml of sample to be used. Back-
ground information on the extent and nature of interferences
will assist the analyst in choosing the required sample size
and preferred detector.
8.3 Quantitatively transfer the proper aliquot into a two-liter
separatory funnel and dilute to. one liter.
9. Extraction ...
9.1 Add 60 ml of 15^ methylene chloride in hexane (v:v) to the
sample in the separatory funnel and shake vigorously for two
minutes.
9.2 Allow the mixed solvent to separate from the sample, then draw
the water into a one-liter Erlenmeyer flask. Pour the organic
layer into a 100-ml beaker and then pass it through a column
containing 3-4 inches of anhydrous sodium sulfate, and collect
it in a 500-ml K-0 flask equipped with a 10 mi-ampul. Return
the water phase to the separatory funnel. Rinse the Erlenmeyer
flask with a second 60-ml volume of solvent; add the solvent to
the separatory funnel and complete the extraction procedure a
second time. Perform a third extraction in the same manner.
50
-------
9.3 Concentrate the extract in the K-D evaporator on a hot water
bath.
9.4 Qualitatively analyze the sample by gas chromatography with an
electron capture detector. From the response obtained decide:
a. If there are any organochlorine pesticides present.
b. If there are any PCBs present.
d If there is a combination of a and b.
d. If elemental sulfur is present.
e. If the response is too complex to determine a, b or c.
f. If no response, concentrate to 1.0 ml or less, as required,
and repeat the analysis looking for a, b, c, d, and e.
Samples containing Aroclors with a low percentage of
chlorine, e.g., 1221 and 1232, may require this concentra-
tion in order to achieve the detection limit of 1 ug/1.
Trace quantities of PCBs are often masked by background
which usually occur in samples.
9.5 If condition _a exists, quantitatively determine the organo-
chlorine pesticides according to (1).
N
9.6 If condition b_ exists, PCBs only are present; no further
separation or cleanup is necessary. Quantitatively determine
the PCBs according to step 11.
9.7 If condition £ exists, compare peaks obtained from the sample
to those of standard Aroclors and make a judgment as to wh'ich
Aroclors may be present. To separate the PCBs from the organo-
chlorine pesticides, continue as outlined in 10.4.
51
-------
9.8 If condition d_ exists, separate the sulfur from the sample
using the method outlined in 10.3 followed by the method in
10.5.
9.9 If condition e, exists, the following macro cleanup and separa-
i tion procedures (10.2 and 10.3) should be employed and, if
necessary, followed by the micro separation procedures (10.4
and 10.5).
10. Cleanup and Separation Procedures
10.1 Interferences in the form of distinct peaks and/or high back-
ground in the initial gas chromatographic analysis, as well as
the physical characteristics of the extract (color, cloudiness,
viscosity) and background knowledge of the sample will Indicate
whether clean-up is required. When these interfere with
measurement of the PCBs, or affect column life or detector
sensitivity, proceed as directed below.
10.2 Acetonitrile Partition - This procedure is used to'remove fats
and oils from the sample extracts. It should be noted that not
all pesticides are quantitatively recovered by this procedure.
The analyst must be aware of this and demonstrate the effi-
ciency of the partitioning for the compounds of interest.
10.2.1 Quantitatively transfer the previously concentrated
extract to a 125-ml separatory funnel with enough hexane
to bring the final volume to 15 ml. Extract the sample
four times fay shaking vigorously for one minute with
30-ml portions of hexane-saturate'd acetonitrile.
52
-------
10.2.2 Combine and transfer the acetonitrile phases to a
one-liter separatory funnel and add 650 ml of distilled
water and 40 ml of saturated sodium chloride solution.
Mix thoroughly for 30-45 seconds. Extract with two
100-ml portions of hexane by vigorously shaking about 15
seconds.
10.2.3 Combine the hexane extracts in a one-liter separatory
funnel and wash with two 100-ml portions of distilled
water. Discard the water layer and pour the hexane
layer through a 3-4 inch anhydrous sodium sulfate column
into a 500-ml K-0 flask equipped with a 10-ml ampul.
Rinse the separatory funnel and column with three 10-ml
portions of hexane.
10.2.4 Concentrate the extracts to 6-10 ml in the K-0 eva-
porator in a hot water bath.,
10.2.5 Analyze by gas chromatography unless a need for further
cleanup is indicated.
10.3 Florisil Column Adsorption Chromatography
10;3.1 Adjust the sample extract volume to 10 ml.
10..-3.2 Place a charge of activated Florisil (weight determined
by lauric-acid value, see Appendix II) in a Chromaflex
column. After settling the Florisil by tapping the
column, add about one-half inch layer of anhydrous
granular sodium sulfate to the top.
53
-------
10.3.3 Pre-elute the column, after cooling, with 50-60 ml of
petroleum ether. Discard the eluate and just prior to
exposure of the sulfate layer to air, quantitatively
transfer the sample extract into the column by
decantation and-subsequent petroleum ether washings.
Adjust the elution rate to about 5 ml per minute and,
separately, collect up to three eluates in 500-ml K-D
,
flasks equipped with 10-ml ampuls (see Eluate Composi-
tion 10.4.). Perform the first elution,with 200 ml of
6% ethyl ether in petroleum ether, and the second
elution with 200 ml of 15% ethyl ether in petroleum
ether. Perform the third elution with 200 ml of 50%
ethyl"ether - petroleum ether and the fourth elution
with 200 ml of 100% ethyl ether.
10.3.3.1 Eluate Composition - By using an equivalent
quantity of any batch of Florisil as deter-
mined by its lauric acid value, the pesti-
cides will be separated into the eluates
indicated as follows.
6% Eluate
Aldrin
8HC
Chlordane
ODD
ODE
DDT
Heptach 1 or
Heptachlor Epoxide
Lindane *
Methoxych 1 or
Mi rex
Pentachloro-
nitrobenzene
Strobane
Toxaphene
TrifTuralin
PCBs
15% Eluate
Endosulfan I
Endrin
Dieldrin
Dichloran
Phthalate esters
54
50% Eluate
Endosulfan I!
Captan
-------
Certain thiophosphate pesticides will occur in
each of the above fractions as well as the 100%
fraction. For additional information regarding
eluate composition, refer to the FDA Pesticide
Analytical Manual (5).
10.3.4 Concentrate the eluates to 6-10 ml in the K-0 evaporator
in a hot water bath.
10.3.5 Analyze by gas chromatography.
10.4 Silica Gel Micro-Column Separation Procedure (6)
10.4.1 Activation for Silica Gel
10.4.1.1 Place about 20 gm of silica gel in a 100-ml
beaker. Activate at 180°C for. approximately
16 hours. Transfer the silica gel to a 100-ml
. . glass-stoppered bottle. When cool, cover with
about 35 ml of 0.50% diethyl ether in benzene
(volume:volume). -Keep bottle well sealed. If
silica gel collects on the ground glass
surfaces, wash off with the above solvent
before resealing. Always maintain an excess of
the mixed solvent in bottle (aproximately 1/2
in. above silica gel). Silica gel can be
effectively stored in this manner for several
days.
10.4.2 Preparation of the Chromatographic Column
10.4.2.1 Pack the lower 2 mm ID section of the micro-
column with glass wool. Permanently mark
55
-------
the column 120 mm above the glass wool. Using
a clean rubber bulb from a disposable pipet
seal the lower end of the microcolumn. Fill
the microcolumn with 0.50% ether in benzene
(v:v) to the bottom of the- 10/30 joint (Figure
1). Using a disposable capillary pipet,
transfer several aliquots of the silica gel
slurry into the microcolumn. After approxi-
mately 1 cm of silica gel collects in the
bottom of the microcolumn, remove the rubber
bulb seal, tap the column to insure that the
silica gel reaches the 120 ± 2 mm mark. Be
sure that there are no air bubbles in the
column. Add about 10 mm of sodium sulfate to
the top of the silica gel. Under low humidity
conditions, the s'ilica gel may coat the sides
of the column and not settle properly. This
can be minimized by wiping the outside of the
column with an anti-static solution.
10.4.2.2 Deactivation of the Silica Gel
a. Fill the microcolumn to the base of the
10/30 joint with the 0.50% ether-benzene
mixture, assemble reservoir (using spring
clamps) and fill with approximately 15 ml
of the 0.50% ether-benzene mixture.: Attach
the air pressure device (using spring
56
-------
clamps) and adjust the elution rate to
approximately 1 ml/min. with the air
pressure control. Release the air pressure
and detach reservoir just as the last of
the solvent enters the sodium sulfate.
Fill the column with n-hexane (not mixed
hexanes) to the base of the 10/30 fitting.
Evaporate all residual benzene from the
reservoir, assemble the reservoir section
and fill with 5 ml of n-hexane. Apply air
pressure and remove the reservoir just as
the n-hexane enters the sodium sulfate.
The column is now ready for' use.
b. Pipet a 1.0 ml aliquot of the concentrated
sample extract (previously reduced to a
total volume of 2.0 ml) on to the column.
As the last of the sample passes into the
sodium sulfate layer, rinse down the
internal wall of the column twice with 0.25
ml of n-hexane. Then assemble the upper
section of the column. As the last of the
n-hexane rinse reaches the surface of the
sodium sulfate, add enough n-hexane (volume
predetermined, see 10.4.3) to just elute
all of the PCBs present in the sample.
Apply air pressure and adjust until the
57
-------
flow is 1 ml/min. Collect the desired
volume of eluate (predetermined, see
10.4.3) in an accurately calibrated ampul.
As the last of the n-hexane reaches the
surface of the sodium sulfate, release the
air pressure and change the collection
ampul. " ' .
c.. Fill the column with 0.50% diethyl ether in
benzene, again apply air pressure and
adjust flow to 1 ml/min. Collect the
eluate until all of the organochlorine
pesticides of interest have been eluted
(volume predetermined, see 10.4.3).
d. Analyze the eluates by gas chromatography.
10.4.3 Determination of Elution Volumes
10.4.3.1 The elution volumes for the PCBs and the
pesticides depend upon a number of factors
which are difficult to control. These include
variation in:
a. Mesh size of the silica gel
b. Adsorption properties of the silica gel
c. Polar contaminants present in the eluting
solvent
d. Polar materials present in the sample and
sample solvent
58
-------
e. The dimensions of the microcolumns
Therefore, the optimum elution volume must
be experimentally determined each time a
factor is changed. To determine the
elution volumes, add standard mixtures of
Aroclors and pesticides to the column and
serially collect 1-ml elution volumes.
Analyze the individual eluates by gas
chromatography and determine the cut-off
volume for n-hexane and for ether-benzene.
Figure 2 shows the retention order of the
various PCS components and of the pesti-
cides. Using this information, prepare the
mixtures required for calibraton of the
microcolumn.
10.4.3.2 In determining the*volume of hexane required to
elute the PCBs the sample volume (1 ml) and the
volume of n-hexane used to rinse the column
wall must be considered. Thus, if it is
determined that a 10.0-ml elution volume is
required to elute the PCBs, the volume of
hexane to be added in addition to the sample
volume but including the rinse volume should be
9.5 ml.
59
-------
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60
-------
10.4.3.3 Figure 2 shows that as the average chlorine
content of a PCB mixture decreases the solvent
.volume for complete elution increases. Quali-
tative determination (9.4) indicates which
Aroclors are present and provides the basis for
selection of the ideal elution volume. This
helps to minimize the quantity of organo-
chlorine pesticides which will elute along with
the low percent chlorine PCBs and insures the
most efficient separations possible for
accurate analysis.
10.4.3.4 For critical analysis where the PCBs'and pesti-
cides are not separated completely, the column
should be accurately calibrated according to
(10.4.3.1) to determine the percent of material
of interest that eTutes in each fraction. Then
flush the column with an additional 15 ml of
0.50% ether in benzene followed by 5 ml of
n-hexane and use this reconditioned column for
the sample separation. Using this technique
one can accurately predict the amount (%) of
materials in each micro column fraction.
10.5 Micro Column Separation of Sulfur, PCBs, and Pesticides
10.5.1 See procedure for preparation and packing micro column
in PCB analysis section (10.4.1 and 10.4.2).
61
-------
10.5.2 Microcolumn Calibration
10.5.2.1 Calibrate the microcolumn for sulfur and PCB
separation by collecting 1.0-ml fractions and
analyzing them by gas chromatography to
determine the following:
1} The fraction with the first eluting PCBs
(those present in 1260),
2) The fraction with the last eluting PCBs
(those present in 1221),
3) The elution volume for sulfur,
4) The elution volume for the pesticides of
interest in the 0.50% ether-benzene
fraction.
From these data determine the following:
1) The eluting volume containing only sulfur
(Fraction I)r ,
2) The eluting volume containing the last of
the sulfur and the early eluting PCBs
(Fraction II),
3) The eluting volume containing the remaining
PCBs (Fraction III),
4) The ether-benzene eluting volume containing
the pesticides of interest (Fraction IV).
10.5.3 Separation Procedure
10.5.3.1 Carefully concentrate the 6% eluate from the
62
-------
florisil column to 2.0 ml in the graduated
ampul on a warm water bath.
10.5.3.2-Place 1.0 ml (50%) of the concentrate into the
microcolumn with a 1-ml pipet. Be careful not
to get any sulfur crystals into the pipet.
10.5.3.3 Collect Fractions I and II in calibrated
centrifuge tube's. Collect Fractions III and IV
in calibrated ground glass stoppered ampuls.
10.5.3.4 Sulfur Removal (7) - Add 1 to 2 drops of
mercury to Fraction II stopper and place on a
wrist-action shaker. A black precipitate
indicates the presence of sulfur. After
approximately 20 minutes the-mercury may become
entirely reacted or deactivated by the
precipitate. The sample should be quanti-
tatively transferred to a clean centrifuge .tube
and additional mercury added. When crystals
are present in the sample, three treatments may
be necessary to remove all the sulfur. After
all the sulfur has been removedfrom Fraction II
(check using gas chromatography) combine
Fractions II and III. Adjust the volume to 10
ml and analyze by gas chromatography. Be sure
no mercury is transferred to the combined
Fractions II and III, since it can react with
certain pesticides.
63
-------
By combining Fractions II and III, if PCBs are
present, it is possible to identify the
Aroclor(s) present and a quantitative analysis
can be performed accordingly. Fraction I can
be discarded since it only contains the bulk of
the sulfur. Analyze Fractions III and IV for
the PCBs and pesticides. If DDT and its
homologs, aldrin, heptachlor, or technical
chlordane are present along with the PCBs, an
additional microcolumn separation can be
performed which may help to further separate
the PCBs from the pesticides (See 10.4),,.
11. Quantitative Determination
U.I Measure the volume of n-hexane eluate containing the PCBs and
inject 1 to 5/
-------
Microgram/ liter =
A = ng of Standard Injected
mm
2
B = of Sample Peak Areas - (mm )
V. = Volume of sample injected
vt = Vo1ume of Extract (vl) from which sample
is injected into gas chromatograph
V = Volume of water sample extracted (ml)
N = 2 when micro column used
1 when micro column not used
Peak Area = Peak height (mm x Peak Width at 1/2
height
11.2.2 For complex situatons, use the calibration method
described below (8). Small variations in components
between different Aroclor batches make it necessary to
obtain samples of several specific Aroclors. These
reference Aroclors can be obtained from the Southeast
Environmental Research Laboratory, EPA, Athens, Georgia,
30601. The procedure is as follows:
11.2.2.1 Using the OV-1 column, chromatograph a known
quantity of each Aroclor reference standard.
Also chromatograph a sample of p,p'-DOE.
Suggested concentration of each standard is 0.1
ng/ul for the Aroclors and 0.02 ng/yl for the
p9p'-ODE.
65
-------
11.2.2.2 Determine the relative retention time (RRT) of
each PCB peak in the resulting chromatograms
using p,p'-DDE as 100. i
x 10°
RRT =
DDE
RRT = Relative Retention Time
RT = Retention time of peak of interest
RTODE = Retention time of p,p'-DDE
Retention time is measured as that distance in
mm between the first appearance of the solvent
peak and the maximum for the compound.
11.2.2.3 To calibrate the instrument for each PCB
measure the area of each peak.
Area = Peak height -(mm) x Peak width at 1/2
height. Using Tables 1 through 6 obtain the
proper mean weight factor, then determine the
response factor .ng/mm .
V
ng/rnn^
(mean weight percent)
100 '
(Area)
rig.,- = ng of Aroclor Standard Injected
Mean weight percent - obtained from Tables 1
through 6.
11.2.2.4 Calculate the RRT value and the area for each
PCB peak in the sample chromatogram. Compare
the sample chromatogram to those obtained for
each reference Aroclor standard. If it; is
66
-------
Table 1
Composition of Aroclor 1221 (8)
RRTa
n
14
16
19
21
28
32
37"
40
Mean
Weight
Percervt
31.8
19.3
10.1
2.8
20.8
5.4
1.4
1.7
Relative
Std. Dev.b
15.8
9.1
9.7
9.7
9.3
13.9
30.1
48.8
Number of
Chlorines0
1
1
2
2
2
2 85%
3 15%
2 10%
3 90%
3
aRetention time relative to p,p'-DDE=100. Measured from first appearance
of solvent. Overlapping peaks that are quantitated as one peak are
bracketed.
bStandard deviation of seventeen results as a percentage of the mean of
the results.
cFrom GC-MS data. Peaks containing mixtures of isomers of different
chlorine numbers are bracketed.
67
-------
Table 2
Composition of Aroclor 1232 (8)
RRTa
n
14
16
20
21
28
32
37
40
47
54
58
70
78
Mean
Weight
Percent '
16.2
9.9
7.1
17.8
9.6
3.9
6.8
6.4
4.2
3.4
2.6
4.6
1.7
Relative .
Std. Dev.
3.4
2.5
6.8
.2.4
3.4
4.7
2.5
2.7
4. 1
3.4
3.7
3.1
7.5
Number of
Chlorines
1
1
2
2
2
3
3
3
3
4
3
4
4
4
5
4
40%
60%
33%
67%
90%
10%,
Total
94.2
Detention time relative to p,p'-QDE=100. Measured from first appearance
of solvent. Overlapping peaks that are quantitated as one peak are
bracketed.
bStandard deviation of four results as a mean of the results.
cFrom SC-MS data. Peaks containing mixtures of isomers of different
chlorine numbers are bracketed.
68
-------
Table 3
Composition of Aroclor 1242 (8)
RRTa
n
16
21
28
32
37
40
47
54
58
70
78
84
98
104
125
146
Mean
Weight
Percent
1.1
2.9
11.3
n.o
6.1
11.5
n.i
8.8
6.8
5.6
10.3
3.6
2o7
1.5
2.3
1.6
1.0
Relative ,
Std. Dev.
35.7
4.2
3.0
5.0
4.7
5.7
6.2
4.3
2.9
3.3
2.8
4.2
9.7
9:4
16.4
20.4
19.9
Number of
Chlorines
1
2
2
2
3
3
3
3
4
3.
4
4
4
5
4
5
5
5
5
6
5
6
25%
75%
33%
67%
90%
10%
85%
15%
75%
25%
Detention time relative to p,p'-ODE=100. Measured from first appearance
of solvent.
bStandard deviation of six results as a percentage of the mean of the
results.
cFrom GC-MS data. Peaks containing mixtures of isomers of different
chlorine numbers are bracketed.
69
-------
Table 4
Composition of Aroclor 1248 (8)
RRTa
21
28
32
47
40
47 '
54
58
70
78
84
98
104
.112
125
146
Mean
Wei ght
Percent
1.2
5.2
3.2
8.3
8.3
15.6
9.7
. 9.3 -
19.0
6.6
4.9
3.2
3.3
1.2
2.6
1.5
Relative .
Std. Dev.
23.9
3.3
3.8
3.6
3.9
1.1
6.0
5.8
1.4
2.7
2.6
3.2
3.6
"6.6
5.9
10.0
Number of
Chlorines
2
3
3
3
3
4
4
3
4
4
4
. 5
4
5
5
4
5
5
5
6
5
6
85%
15%
10%
90%
80%
20%
10%
90%
90%
10%
85%
15%
Total
103.1
Detention time relative to p,p'-DDE-100. Measured from first appearance
of solvent.
^Standard deviation of six results as a percentage of the mean of the.
results.
cFrom GC-MS data. Peaks containing mixtures of isomers of different
chlorine numbers are bracketed.
,70
-------
Table 5
Composition of Aroclor 1254 (8)
RRTa
47
54
58
70
84
98
104
125
146
160
174
203
232
Mean
Weight
Percent
6.2
2.9
1.4
13.2
17.3
7.5
13.6
15.0
-
10.4
1.3
8.4
1.8
1.0
Relative .
Std. Dev.
3.7
2.6
2.8
2.7
1.9
5.3
3.8
2.4
2.7
' 8 ..4
5.5
18.6
26.1
Number of
Chlorines0
4
4
4
4
5
5
5
5
5
6
5
6
6
6
6
7
25%
75%
70%
80%
30%
70%
Total 100.0
Detention time relative to p,p'-DDE=100. Measured from first appearance
of solvent.
bStandard deviation of six results as a percentage of the mean of the
results.
cFrom GC-MS data. Peaks containing mixtures of isomers are bracketed.
71
-------
Table 6
Composition of Aroclor 1260 (8)
RRTa
70
84
98
104
117
125
146
160
174
203
232
244
280
332
372
448
528
Mean
Wei ght
Percent
2.7
4.7
3.8
3.3
12.3
14.1
4.9
12.4
9.3
9.8
11.0
4.2
4.0
.6
1.5
Relative , Number of
Std. Dev. Chlorines
6.3
1.6
3.5
6.7
3.3
3.6
, 2.2 .
-
2.7
4.0 ,-
: " 3.4
-2.4
5.0
8.6
25.3
10.2
5
5
ci
5
6
6
5
6
6
- 6
7
6
6
7
a
6
7
7;
7
8
8
8
60%
40%
15%
85%
50%
50%
10%
90%
10%
90%
Total
98.6
Detention time relative to p,p'-DDE=100. Measured from first appearance
of solvent. Overlapping peaks that are quantitated as one peak are
bracketed.
^Standard deviation of six results as a mean of the results.
cFrom GC-MS data. Peaks containing mixtures of isomers of different
chlorine numbers are bracketed.
dComposition determined at the center of peak 104.
eComposition determined at the center of peak 232.
72
-------
apparent that the PCB peaks present are due to
only one Aroclor, then calculate the concen-
tration of each PCB using the following formula:
?
ng PCB = ng/mm x Area
2
Where Area = Area (mm ) of sample peak
ng/mm = Response factor for that peak :
measured.
Then add the nanograms of PCBs present in the
injection to get the total number of nanograms
of PCBs present. Use the following formula to
calculate the concentration of PCBs in the
sample:
Micrograms/Liter =
- V = volume of water extracted (ml)
Vt = volume of extract (yl)
V. = volume of samp-le injected (uD
ng = sum of all the PCBs in nanograms for that
Aroclor identified
N = 2 when microcolumn used
N = 1 when microcolumn not used
The value can then be reported as micro-
grams/liter PCBs or as the Aroclor. For
samples containing more than one Aroclor, use
Figure 9 chromatogram divisional flow chart to
assign a proper response factor to each peak
and also identify the "most likely" Aroclors
73
-------
present. Calculate the ng of each PCJ3 isomer
present and sum them according to the
divisional flow chart. Using the formula
above, calculate the concentration of the
various Ar.oclors present in the sample.
12. Reporting Results
12.1 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
all data obtained should be reported.
74
-------
\
37
AROCLOR 1242
Figure 3. Column: 3% OV-1, Carrier Gas: Nitrogen at 60 ml/min,
Column Temperature: 170 C, Detector: Electron Capture
75
-------
I
70
84
AROCLOR 1254
104
125
146
Figure 4. Column: 3% OY-1, Carrier Gas: Nitrogen at 61) mi/min,
Column Temperature: 170 C, Detector: Electron Capture.
76
-------
146
125 (I 174
AROCLOR 1260
280
5. Column: 3% OV-1, Carrier Gas: Nitrogen at 60 ml/mm,
Column Temperature: 170 G, Detector: Electron Capture.
77
-------
AROCLOR 1242
I
I
I
I
I
I
1
3
21
24
6 9 12 15 18
RETENTION TIME IN MINUTES
Figure 6. Column: 1.5% OV-17 + 1.95% QF-1, Carrier fias: Mitrogen
at 60 ml/min, Column Temperature: 200 C, Detector: Electron Capture.
78
-------
131FNI
S
eu
C/» «J
u-a
S oi
CM uj M
ea
i
kM
= .S
-------
o
,S3
O
03
a
cs
ISM
a
S3
to
03
0.
E
as
&
=1
taa
CM K S5
ts
£
3-
euo
133fH|-
80
-------
RRT of first peak < 47?
YES
NO
Is there a distinct
peak with RRT 78?
YES
/ V
RRT 47-58?
YES
Use 1242 for
peaks 1 RRT 84
Use 1242 for
peaks - RRT 70
Use 1254
for peaks
1 RRT 104
NO
RRT- 70?
Is there a distinct
peak with RRT 117?
YES
NO
Use 1254 for all
peaks- RRT 174
Use 1260 for
all other peaks
Use 1260 for
all peaks
Figure 9. Chromatogram Division Flowchart (8).
81
-------
REFERENCES:
1. "Method for Chlorinated Hydrocarbons in Water and Wastewater", this
manual, p. 7.
2. Leoni, V., "The Separation of Fifty Pesticides and Related Compounds and
Polychlorinated Biphenyls into Four Groups by Silica Gel Microcolumn
Chromatography", Journal of Chromatographv, 62, 63 (1971).
3. McClure, V. E., "Precisely Deactivated Adsorbents Applied to the Separa-
tion of Chlorinated Hydrocarbons", Journal of Chromatographv, 70, 168
(1972). a__*_j. _
4. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories", Chapter 6, Section 6.4, U. S. Environmental Protection
Agency, National Environmental Research Center, Analytical Quality
Control Laboratory, Cincinnati, Ohio, 45268, 1972.
5. "Pesticide Analytical Manual", U. S.'Dept. of Health, Education and
Welfare, Food and Drug Administration, Washington, D. C.
6. Bellar, T. A. and Lichtenberg, J.. J., "Method for the Determination of
. Polychlorinated Biphenyls 'in Water and Sediment", U. S. Environmental
Protection Agency', National Environmental Research Center, Analytical
Quality Control Laboratory, Cincinnati, Ohio, 45268, 1973.
7. Goerlitz, D. F. and Law, L. M., "Note on Removal of Sulfur Interferences
from Sediment Extracts for Pesticide Analysis", Bulletin of Environmental
Contamination and Toxicology, 6_, 9 (1971). . !
8. Webb, R. G. and McCall, A. C,, "Quantitative PCB Standards for Electron
Capture Gas Chromatography", Journal of Chromatographic Science, 11, 366
(1973).
82
-------
METHOD FOR TRIAZINE PESTICIDES IN WATER AND WASTEWATER
V. Scope and Application
1.1 This method covers the determination of various symmetrical triazine
pesticides in water and wastewaters.
1.2 The following pesticides may be determined individually by this
method:
Parameter Storet No.
Ametryn
Altraton
Atrazine 39033
Prometon . 39056
Prometryn 39057
Propazine 39024
Secbumeton
Simazine 39055
Terbuthylazine
2. Summary
2.1 The method describes an efficient sample extraction procedure
and provides, through use of column chromatography, a method
for the elimination of non-pesticide interferences and the
pre-separation of pesticide mixtures. Identification is made
by selective gas chromatographic separation, and measurement
is accomplished by the use of an electroytic conductivity
detector (CCD) in the nitrogen mode or a nitrogen specific
thermionic detector. Results are reported in micrograms per
liter.
83
-------
2.2 This method is recommended for use only by experienced
pesticide analysts or under the close supervision of such
qualified persons.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing
hardware may yield discrete artifacts and/or elevated
baselines causing misinterpretation of gas chromatograms.
All of these materials must be demonstrated to be free from
interferences under the conditions of the analysis. Specific
selection of reagents and purification of solvents by
distillation in all-glass systems may be required. Refer to
Appendix I.
3.2 The interferences in industrial effluents" are high and varied
and often pose great difficulty in obtaining accurate and
precise measurement of triazine pesticides. The use of a
specific detector supported by an .optional column cleanup
procedure will eliminate many of these interferences.'
3.3 Nitrogen containing compounds other than the triazines may
interfere.
4. Apparatus and Materials
- 4.1 Gas Chromatograph - Equipped with glass lined injection port.
4.2 Detector Options
4.2.1 Electrolytic Conductivity.
4.2.2 Nitrogen specific thermionic
4.3 Recorder - Potentiometric strip chart (10 in.) compatible
with the detector.
84
-------
4.4 Gas Chromatographic Column Materials:
4.4,1 Tubing - Pyrex (180 cm long x 4 mm ID)
4.4.2 Glass Wool - Silam'zed
4.4.3 Solid Support - Gas Chrom Q (100-120 mesh)
4.4.4 Liquid Phases - Expressed as weight percent coated on
solid support.
4.4.4.1 - Garbowax 20M, 1%
4.5 Kuderna-Danish (K-0) Glassware
4.5.1 Snyder Column - three ball (macro) and two ball (micro)
4.5.2 Evaporative Flasks - 500 ml
4.5.3 Receiver Ampuls - 10 ml, graduated
4.5.4 Ampul Stoppers
4.6 Chromatographic Column - Chromaflex (400 mm x 19-mm ID) with
coarse fritted plate and Teflon stopcock on bottom; 250 ml
reservoir bulb at top of column with flared out funnel shape
at top of bulb - a special order (Kontes K-420540-9011).
4.7 Chromatographic Column - Pyrex (approximately 400 mm long x
20 mm ID) with coarse fritted plate on bottom,
4.8 Micro Syringes - 10, 25, 50 and 100 jul.
4.9 Separatory funnels - 2000 ml with Teflon stopcock.
4.10 Blender - High speed, glass or stainless steel cup.
4.11 Graduated Cylinders - 1000 ml.
4.12 Florisil - PR Grade (60-100 mesh); purchase activated at
1250°F and store in the dark in glass containers with glass
stoppers or foil-lined screw caps. Before use, activate each
85
-------
batch overnight at 130°C in foil-covered glass container.
Determine 1 auric acid value (See Appendix II).
5. Reagents, Solvents, and Standards
5.1 Sodium Hydroxide - (ACS) 10 N in distilled water.
5.2 Sodium Sulfate (ACS) Granu-lar, anhydrous (conditioned at
400 C for 4 hrs.).
5.3 Sulfuric Acid - (ACS) Mix equal volumes of cone. H2S04
with distilled water. '
5.4' Diethyl Ether - Pesticide Quality, redistilled in glass, if
necessary
5.4.1 Must be free of peroxides as indicated fay EM Quant:
Test strips. (Test strips are available from EM
Laboratories, Inc., 500 Executive Blvd., Elmsford.,
N.Y. 10523.)
5.4.2 Procedures recommended for removal of peroxides are
provided with the test strips.
5.5 Hexane, Methanol, Methylene Chloride, Petroleum Ether
(boiling range 30-60°C) - pesticide quality, redistill in
glass if necessary. .
5.6 Pesticide Standards - Reference grade.
6. Calibration .
6.1 Gas chromatographic operating conditions are considered
optimum when an injection of^ 20 ng of each triazine will "
yield a peak at least 50% of full scale deflection with the
modified Coulson detector (1). For all quantitative
86
-------
measurements, the detector must be operated within its linear
response range and the detector noise level should be less
than 2% of ful.l scale.
6.2 Inject standards frequently as a check on the stability of
operating conditions. A chromatogram of a mixture of several
pesticides is shown in Figure 1 and provides reference
operating conditions for the-recommended column.
6.3 The elution order and retention ratios of various
organophosphorus pesticides are provided in Table 1, as a
guide.
7. Quality Control
7.1 Duplicate and spiked sample analyses are recommended as
quality control checks. Quality control charts (2) should be
developed and used as a check on the analytical system.
Quality control check samples and performance evaluation
samples should be analyzed on a regular basis.
7.2 Each time a set of samples is extracted, a method blank is
determined on a volume of distilled water equivalent to that
used to dilute the sample.
8. Sample Preparation
8.1 Blend the sample if suspended matter is present and adjust pH
to near neutral (pH 6.5-7.5) with 50% sulfuric acid or ION
sodium hydroxide.
8.2 Quantitatively transfer a 1000 ml aliquot into a two-liter
separatory funnel.
87
-------
ce
4 6 8 10
RETENTION TIME IN MINUTES
14
Figure 1. Column Packing: 1% Carbowax 20M on Gas-Ghrom Q (100/120 mesh],
Column Temperature: 155 C, Carrier Gas: Helium at 80 ml/min,
Detector: Electrolytic Conductivity.
88
-------
TABLE 1
RETENTION RATIOS OF VARIOUS TRIAZINE
PESTICIDES RELATIVE TO ATRAZINE
Pesticide Retention Ratio
Prometon 0»52
Atraton 0-67
Propazine 0«71
Terbuthylazine 0.78
Secbumeton 0-88
Atrazine 1.00
Prometryne 1-^0
Simazine T-35
Ametryne ' 1-48
Absolute retention time of atrazine = 10.1 minutes
89
-------
9. Extraction
9.1 Add 60 ml methylene chloride to the sample in the separatory
funnel and shake vigorously for two minutes.
9.2 Allow the solvent to separate from the sample, draw the ;
organic layer into a 100-ml beaker, then pass the organic
layer through a chromatographic column containing 3-4 inches
anhydrous sodium sulfate, and collect it in a 500-ml K-D
flask equipped with a 10 ml ampul. Add a second 60-ml volume
of solvent to the separatory funnel and complete the
extraction procedure a second time. Perform a third
extraction in the same manner.
9.3 ' Concentrate the extract to 10 ml. in a K-D evaporator on a hot
water bath. Disconnect the Snyder column just long enough to
add 10 ml hexane to the K-D flask and then continue the
concentration to about 5-6 ml. This operation is to displace
methylene chloride and give a final hexane solution. If the
need for cleanup is indicated, continue to Florisil Column
Cleanup (10 below).
9.4 If further cleanup is not required, replace the Snyder column
and flask with a micro-Snyder column and continue the
concentration to 0.5-1.0 ml. Analyze this final concentrate
by gas chromatography.
10. Florisil Column Adsorption Chromatography
10.1 Adjust the sample extract volume to 10 ml.
90
-------
10.2 Place a charge of activated Florisil (weight determined by
lauric acid value, see Appendix II) in a Chromaflex column*
After settling the Florisil by tapping the column, add about
oner-half inch layer of anhydrous granular sodium sulfate to
the top.
10.3 Pre-elute the column, after cooling, with 50-60 ml of
petroleum ether. Discard the eluate and just prior to
exposure of the sulfate layer to air, quantitatively
transfer the sample extract into the column by decantation
and subsequent petroleum ether washings. Adjust the elution
rate to about 5 ml per minute and, separately, collect up to
four eluates in 500-ml K-D flasks equipped with 10-ml
ampuls. (See Eluate Composition, 10.4.) Perform the first
elution with 200 ml of 6% ethyl ether in petroleum ether,
and the second elution with 200 ml of 15% ethyl ether in
petroleum ether. Perform the third elution with 200 ml of
50% ethyl ether - petroleum ether and the fourth elution
with 200 ml of 100% ethyl ether.
10.4 Eluate Composition - By using an equivalent quantity of any
batch of Florisil as determined by its lauric acid value,
the pesticides will be separated into the eluates indicated
as follows:
91
-------
Eluate
50% Eluate
100% Eluate
Propazine (90%) Propazine (10%) Atraton
Terbuthylazine (30%) Terbuthylazine(70%) Secbumeton
Atrazine (20%) Atrazine (80%) Prometon
Ametryne
Prometryne
Simazine
10.5 Concentrate the eluates to 6-10 ml in the K-D evaporator in a
hot water bath. Change to the micro-Snyder column and continue
concentration to 0.5-1.0 ml.
10.6 Analyze by gas chromatography.
11. Calculation of Results
11.1 Determine the pesticide concentration by using the absolute
calibration procedure .described below or the relative
calibration procedure described in Appendix- III.
(1) Micrograms/liter = (A) (B) (Vtl
A = ng standard
Standard area
B = Sample aliquot area
Vj = Volume of extract injected (yl)
Vt = Volume of total extract G-il)
v<
12. Reporting Results
12.1 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed
all data obtained should be reported. "
s Volume of water extracted (ml)
92
-------
REFERENCES:
Patchett, G. 6., "Evaluation of the Electrolytic Conductivity Detector
for Residue Analyses of Nitrogen-Containing Pesticides", Journal of
Chroma tographic Science, 8., 155 (1970).
"Handbook for Analytical Quality Control in Water and Wastewater
Laboratories", Chapter 6, Section 6.4, U. S. Environmental Protection
Agency, National Environmental Research Center, Analytical Quality
Control Laboratory, Cincinnati, Ohio, 45268, 1972. (Revised edition
to be available soon.)
93
-------
METHOD FOR 0-ARYL CARBAMATE PESTICIDES IN WATER AND WASTEWATER
Scope and Application
1.1 This method covers the determination of various 0-aryl. carbamate
pesticides in water and wastewater. <
1.2 The following pesticides may be determined individually by this
method:
Parameter Storet No.
Aminocarfa
Carbaryl
Methiocarb
Mexacarbate
Propoxur
39750
Summary
2.1 A measured volume of water is extracted with methylene
chloride. The concentrated extract is cleaned up with a
Florisil column. Appropriate fractions from the column are
concentrated and portions are separated by'thin-layer
chromatography. The carbamates are hydrolyzed on the layer and
the hydrolysis products are reacted with 2,6-dibromoquinone
chlorimide to yield specific colored products. Quantitative
measurement is achieved by visually comparing the responses of
sample extracts to the responses of standards on the same
thin-layer. Identifications are confirmed by changing the pH
of the layer and observing color changes of the reaction
products. Results are reported in micrograms per liter.
94
-------
2.2 This method is recommended for use only by experienced
pesticide analysts or under the close supervision of such
qualified persons.
3. Interferences
3.1 Direct interferences may be encountered from phenols that may
be present in the sample. These materials react with the
chromogenic reagent and yield reaction products .sim-ilar to
those of the carbamates. In cases where phenols are suspected
of interfering with a determination, a different solvent system
should be used to attempt to isolate the carbamates.
3.2 Indirect interferences may be encountered from naturally
colored materials whose presence masks the chromogenic reaction.
4. Apparatus and Materials
.4.1 Thin-layer plates - Glass plates ('200 x 200 mm) coated with
0.25 mm layer of Silica Gel G (gypsum binder),
4.2 Spotting Template
4.3 Developing Chamber
4.4 Sprayer - 20 ml capacity
4.5 Kuderna-Danish (K-D) Glassware (Kontes)
4.5.1 Snyder Column - three ball (K-503000)
. 4.5.2 Micro-Snyder Column -two ball (K-569001)
4.5.3 Evaporative Flasks - 500 ml (K-570001)
4.5.4 Receiver Ampuls - 10 ml graduated (K-570050)
4.5.5 Ampul Stoppers
95
-------
4.6 Chromatographic Column - Chromaflex (400 mm long x 19 mm ID) with
coarse fritted plate on bottom and Teflon stopcock; 250 ml
reservoir bulb at top of column with flared out funnel shape at
top of bulb - a special order (Kontes K-420540-9011).
4.7 Chromatographic Column - Pyrex (approximately 400 mm long x 20 mm
ID) with coarse fritted plate on bottom.
4.8 Micro Syringes - 10, 25, 50 and 100 jul.
*
4.9 Separator/ Funnel - 2000 ml, with Teflon stopcock.
4.10 Blender - High speed, glass or stainless steel cup.
4.11 Florisil - PR Grade (60-80 mesh); purchase activated at 1250°F
and store in the dark in glass containers with glass stoppers or
foil-lined screw caps. Before use activate each batch overnight
at 130°C in foil-covered glass container. Determine lauric
acid value (see Appendix II). !
5. Reagents, Solvents, and Standards
5.1 Sodium Hydroxide - (ACS) 10 N in distilled water.
5.2 Sodium Sulfate - (ACS) Granular, anhydrous.
5.3 Sulfuric Acid - (ACS) Mix equal volumes of cone. H2S04 with
distilled water. .
5.4 Diethyl Ether - Nanograde, redistilled in glass, if necessary.
5.4.1 Must be free of peroxides as indicated by EM Quant test
strips. (Test strips are available from EM Laboratories,
Inc., 500 Executive Blvd., Elmsford, N.Y. 10523.)
5.4.2 Procedures recommended for removal of peroxides are
provided with the test strips.
96
-------
5.5 Hexane, Methanol, Methylene Chloride, Petroleum Ether -
nanograde, redistill in glass if necessary.
5.6 .Pesticide Standards - Reference grade.
5.8.1 TLC Standards - 0.100 ^ig/ul in chloroform.
5.7 Chromogenic agent - Dissolve 0.2 g 2,6-dibromoquinone chlorimide
in 20 ml chloroform.
5.8 Buffer solution - 0.1 N sodium borate in water.
6. Calibration
6.1 To insure even solvent travel up the layer, the tank used for-
layer development must be thoroughly saturated with developing
solvent before it is used. This may be achieved by lining the
inner walls of the tank with chromatography paper and introducing
the solvent 1-2 hours before use.
6.2 Samples and standards should be introduced to the layer using a
syringe, micropipet or other suitable device that permits all the
spots to be about the same size and as small as possible.. An air
stream directed on the layer during spotting will speed solvent
evaporation and help to maintain small spots.
6.3 For qualitative and quantitative work, spot a series representing
0.1-1.0 jjg of a pesticide. Tables 1 and 2 present color
responses and Rf values for several solvent systems.
7. Quality Control
7.1 Duplicate and spiked sample analyses are recommended as quality
control checks. Quality control charts should be developed
and used as a check on the analytical system. Quality control
97
-------
Table 1
Values of 0-Aryl Carbamate Pesticides in Several Solvent Systems
Carbaryl
Aminocarb
Mexacarbate
Methiocarb
Propoxur
A
0.26
0.26
0.34
0.31
0.27
B
0.22
0.02
0.22
0.31
0.10
.C
0.48
0.46
0.54
0.55
0.53
D
0.41
0.52
0.53
0.55
0.59
E
0.58
0.54
0.60
0.59
0.60
F
0.24
0.04
0.24
0.28
0.13
Solvent Systems:
A. Hexane/acetone (3:1)
B. Methylene chloride
C. Benzene/acetone (4:1)
D. Benzene/cyclohexane/diethylamine (5:2:2)
E. Ethyl acetate
F. Chloroform
98
-------
Table 2
Color Responses and Detection Limit for 0-Aryl Carbamates
~ 'Colors; "" Detection
Before After ' Limit
Buffer Buffer (ug)
Carbaryl Brown Red-Purple 0.1
Aminocarb Gray Green 0.1
Mexacarbate Gray Sreen 0.1
Methiocarb Brown Tan O.Z
Propoxur Blue Blue 0,1
99
-------
check samples and performance evaluation samples should be
analyzed on a regular basis.
7.2 Each time a set of samples is extracted, a method blank is
determined on a volume of distilled water equivalent to that used
to dilute the sample. , i
8. Sample Preparation . '
8.1 Blend the sample if suspended matter is present and adjust pH to
near neutral (pH 6.5-7.5) with 50% sulfuric acid or 10 N sodium
hydroxide.
8.2 quantitatively transfer a one-liter aliquot into, a two-liter
separatory funnel.
9. Extraction
9.1 Add 60 ml of methylene chloride to the sample in the separatory
funnel and shake vigorously for two minutes.
9.2 Allow the solvent to separate from the sample, draw the organic
layer .into a 100-ml beaker, then pass the organic layer through a
chromatographic column containing 3-4 inches anhydrous sodium
sulfate, and collect it in a 500-ml K-0 flask equipped with a
10-ml ampul. Add a second 60-ml volume of solvent to the
separatory funnel and complete the extraction procedure a second
time. Perform a third extraction in the same manner.
?
9.3 Concentrate the extract to 10 ml in a K-D evaporator on a hot
water bath. Disconnect the Snyder column just long enough to add
10 ml of hexane to the K-D flask and then continue the
concentration to about 5-6 ml. If the need for cleanup is
indicated, continue to Florisil Column Cleanup (10 below).
TOO
-------
9.4 If further cleanup Is not required, replace the Snyder column and
r
flask with a micro-Snyder column and continue the concentration
to 0.5-1.0 ml. Analyze this final concentrate by thin-layer
chromatography (Section 11).
10. Florisil Column Cleanup
10.1 Adjust the sample extract to 10. ml with hexane.
10.2 Place a charge of activated Florisil (weight determined by
lauric-acid value, see Appendix II) in a Chromaflex column.
After settling the Florisil by'tapping the column, add about
one-half inch layer of anhydrous granular sodium sulfate to the
top. .
10.3 Pre-elute the column, after cooling, with 50-60 ml of petroleum
ether. Discard the eluate and just prior to exposure of the
sulfate layer to air, quantitatively transfer the sample extract
into the column by decantation. and .subsequent petroleum ether
washings. Adjust the elution rate to about 5 ml per minute and,
separately collect the fo'ur eluates in 500-ml K-D flasks equipped
with 10-ml ampuls-. Perform the first elution with 200 ml of 6%
ethyl ether in petroleum ether, and the second elution with 200
ml of 15% ethyl ether in petroleum ether. Perform the third
elution with 200 ml of 50% ethyl ether - petroleum ether and the
fourth elution with 200 ml of 100% ethyl ether.
10.3.1 Eluate Composition - By using an equivalent quantity of
any batch of Florisil as determined by its 1 auric acid
value, the pesticides will be separated into the eluates
indicated as follows:
101
-------
50% E1uate 100% Eluate
Carbaryl (70%) Carbaryl (30%)
Mexacarbate Aminocarb
Propoxur
10.4 Concentrate the eluates to 6 - 10 ml in the K-D evaporator in a
hot water bath. Change to the micro-Snyder column and continue
concentration to 0.5-1.0 ml.
10.5 Analyze according to 11. below.
11. Separation and Detection
11.1 Carefully spot 10% of the extract on a thin layer. On the same
plate spot several pesticides or mixtures for screening
purposes, or a series of 1, 2, 4, 6, 8 and 10 jjl of specific
standards for quantitative analysis.
11.2 Develop the'layers 10 cm in a tank saturated with solvent
vapors. Remove the plate and allow it to dry.
11.3 Spray the layer rapidly and evenly with about 10-15 ml
chromogenic reagent. Heat the layer-in an oven at 110° C for 15
minutes. The pesticides will appear with colors as indicated in
Table 2. Make quantitative estimates by visually comparing the
intensity and size of the spots with those of the series of
standards.
11.4 Spray the layer with sodium borate reagent and observe the color
shift of the reaction products. The color shift'must be the
same for sample and standard for identification to be confirmed.
102
-------
12. Calculation of Results
12.1. Determine the concentration of pesticide in a sample by
comparing the response in a sample to that of a quantity of
standard treated on the same layer. Divide the result, in
micrograms, by the fraction of extract spotted to convert to
micrograms per liter.
13. Reporting Results
'13.1 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed
all data obtained should be reported.
103
-------
METHOD FOR N-ARYL CARBAMATE AND UREA PESTICIDES IN WATER AND WASTEWATER
1. Scope and Application
1.1 This method covers the determination of various N-aryl carbamate
and urea pesticides in water and wastewater.
The following pesticides may be determined individually by this
method:
1.2
Parameter
Barban
Chlorpropham
Diuron
Fenuron
Fenuron-TCA
Linuron
Monuron
Monuron-TCA
Neburon
Propham
Siduron
Swep
Storet No.
39650
39052
2. Summary
2.1 A measured volume of water is extracted with methylene chloride
and the concentrated extract is cleaned up with a Florisil
column. Appropriate fractions from the column are concentrated
and portions are separated by thin-layer chromatography. The
pesticides are hydrolyzed to primary amines, which in turn are
chemically converted to diazonium salts. The layer is sprayed
with 1-naphthol and the products appear as colored spots.
Quantitative measurement is achieved by visually comparing the
104
-------
responses of sample extracts to the responses of standards on the
same thin layer. Results are reported in micrograms per liter.
2.2 This method is recommended for use, only by experienced pesticide
analysts or under the close supervision of such qualified persons.
3. Interferences .
3.1 Direct interferences may be encountered from aromatic amines that
may be present in the sample. These materials react with the
chromogenic reagent and yield reaction products similar to those
of the pesticides. In cases where amines are suspected of
' interfering with a determination, a different solvent system
should be used to attempt to isolate the pesticides on the layer.
3,2 Indirect interferences may be encountered from naturally colored
materials whose presence masks the chromogenic reaction.
4. Apparatus and Materials
4.1 Thin-layer plates - Glass plates (200 x 200 mm) coated with 0.25
mm layer of Silica Gel 6 (gypsum binder).
4.2 Spotting Template
4.3 Developing Chamber
4.4 Sprayer - 20 ml capacity
4.5 Kuderna-Danish (K-D) Glassware (Kontes)
4.5.1 Snyder Column - three ball (K-503000)
4,5.2 Micro-Snyder Column - two ball (K-569001)
4.5.3 Evaporative Flasks T 500 ml (K-570001)
4.5.4 Receiver Ampuls - 10 ml graduated (K-S70050)
4.5.5 Ampul Stoppers
105
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5.
4.6 Chromatographic Column - Chromaflex (400 mm long x 19 mm ID) with
coarse fritted plate on bottom and Teflon stopcock; 250 ml
reservoir bulb at top of column with flared out funnel shape at
top of bulb - a special order (Kontes K-420540-9011).
4
4.7 Chromatographic Column - Pyrex (approximately 400 mm long x 20 mm
ID) with coarse fritted plate on bottom.
4.8 Micro Syringes - 10, 25, 50 and 100 jjl.
4.9 Separatory Funnel - 2000 ml, with Teflon stopcock.
4.10 Blender - High speed, glass or stainless steel cup.
4.11 Florisil - PR Grade (60-80 mesh); purchase activated at 1250°F
and store in the dark in glass containers with glass stoppers or
foil-lined screw caps. Before use activate each batch overnight
at 130°C in foil-covered glass container. Determine lauric acid
value (see Appendix II).
Reagents, Solvents, and Standards
5.1 Sodium Chloride - (ACS) Saturated solution in distilled water
(pre-rinse Nad with hexane).
5.2 Sodium Hydroxide - (ACS) 10 N in distilled water.
5.3 Sodium Sulfate - (ACS) Granular, anhydrous (conditioned at 400* C
for 4 hrs.).
5.4 Sulfuric Acid - (ACS) Mix equal volumes of cone. HgSO^ with
distilled water.
5.5 Diethyl Ether - Nanograde, redistilled in glass, if necessary.
5.5.1 Must be free of peroxides as indicated by EM Quant test
strips. (Test strips are available from.EM Laboratories,
Inc., 500 Executive Blvd., Elmsford, N.Y. 10523,,)
106
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5,5:2 Procedures recommended for removal of peroxides are
provided with the test strips.
5.6 Hexane, Methanol, Methylene Chloride, Petroleum Ether - nanograde,
redistill in glass if necessary.
5.7 -Pesticide Standards - Reference grade.
5.9.1 TLC Standards - 0.100 ^ug/jul in chloroform.
5.8 Nitrous acid - prepare just before use fay mixing 1 g NaN02 with
20 ml 0.2 N HC1. '
5.9 Chromogenic agent - dissolve 1.0 g 1-Naphthol in 20 ml ethanol.
Prepare fresh daily.
6. Calibration
6.1 To insure even solvent travel up the layer, the tank used for
layer development must be thoroughly saturated with developing
solvent before it is used. This may be achieved by lining the
inner walls of the tank with chromatography paper and introducing
the solvent 1-2 hours before use.
6.2 Samples and standards should be introduced to the layer using a
syringe, micropipet or other suitable device that permits all the
spots to be about the same size and as small as possible. An air
stream directed on the layer during spotting will speed solvent
evaporation and help to maintain small spots.
6.3 For qualitative and quantitative work, spot a series representing
0.1-1.0 jug of a pesticide. Tables 1 and 2 present color responses
and R-. values for several solvent systems.
107
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TABLE 1
Rf VALUES OF N-ARYL CARBAMATE AND UREA PESTICIDES
IN SEVERAL SOLVENT SYSTEMS
Carbamates
Propham
Chloropropham
Barban
Swep
Urea
Fenuron
Fenuron-TCA
Monuron
Monuron-TCA
Diuron
Linuron
Neburon
Siduron
A
0.49
0.57
0.61
0.48
0.03
0.03
0.04
0.04
0.05
0.40
0.21
0.02
B
0.54
0.60
0.59
0.44
-
0.04
0.04
0.05
0.06
0.09
0.43
0.28
0.07
C
0.73
0.73
0.72
0.70
0.38
0.36
0.37
0.34
0.38
0.62
0.64
0.68
D
0.48
0.49
0.41
0.41
- 0.22
0.22
0.24
0.24
0.28
0.39
0.41
0.39
E
0.36
0.37
0.28
0.28
0.10
0.10
0.10
0.10
.a. 13
0.24
0.26
0.25
F
0.68
0.70
0.70
0.67
0.41
0.41
0.47
0.46
0.54
0.66 ,
0.68
0.62
G
0.69
0.73
0.74
0.66
0.30
0.30
0.34
0.34
0.44
0.64
0.65
0.55
Solvent Systems:
A. Methylene chloride
B. Chloroform
C. Ethyl Acetate
D. Hexane/acetone (2:1)
E. Hexane/acetone (4:1)
F. Chloroform/acetonitrile (2:1)
G. Chloroform/acetom'trile (5:1)
108
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TABLE 2
COLOR RESPONSES AND DETECTION LIMIT FOR THE N-ARYL CARBAMATES
AND UREAS
G-arbamates
Prop ham
Chlorpropham
Barb an
Swap
Ureas
Fenuron
Fenuron-TCA
Monuron
Monuron-TCA
Diuron
Linuron
Neburon
Siduron
Color Detection Limit (ug)
Red-purple
Purple
Purple
Blue-.purple
Red -purple
Red-purple
Pink -orange
Pink -orange
Blue-purple
Blue-purple
Blue-purple
Red-purple
0,2
0.1
0.05
0.2
0.05
0.1
0.05
0.1
0.1
0.1
0.1
0.05
109
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7. Quality Control
7.1 Duplicate and spiked sample analyses are recommended as quality
control checks. Quality control charts (1) should be developed
and used as a check on the analytical system. Quality control
check samples and performance evaluation samples should be
analyzed on a regular basis.
7.2 Each time a set of samples is extracted, a method blank is
determined on a volume of distilled water equivalent to that used
to dilute the sample.
8. Sample Preparation
8.1 Blend the sample if suspended matter is present and adjust pH to
near neutral (pH 6.5-7.5) with 50% sulfuric acid or 10 H sodium
hydroxide.
8.2 Quantitatively transfer a one-liter aliquot into a two-liter
separatory funnel.
9. Extraction ..
9.1 Add 60 ml of methylene chloride to the sample in the separatory
funnel,and shake vigorously for two minutes.
9.2 Allow the solvent to separate from the sample, draw the organic
layer into a 100-ml beaker, then pass the organic layer through a
chromatographic column containing 3-4 inches anhydrous sodium
sulfate, and collect it in a 500-ml K-D flask equipped with a
10-ml ampul. Add a second 60-ml volume of solvent to the
separatory funnel and complete the extraction procedure a second
time. Perform a third extraction in the same manner.
110
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9.3 Concentrate the extract to 10 ml in a K-D evaporator on a hot
water bath. Disconnect the Snyder column just long enough to add
10-ml hexane to the K-D flask and then continue the concentration
to about 5-6 ml. If the need for cleanup is indicated, continue
to Florisil Column Cleanup (10 below).
9.4 If further cleanup is not required, replace the Snyder column and
flask with a micro-Snyder column and continue the concentration to
0.5-1.0 ml. Analyze this final concentrate by thin-layer
chromatography (Section 11).
10. Florisil Column Cleanup
10.1 Adjust the sample extract to 10 ml with hexane.
10.2 Place a charge of activated Florisil (weight determined by lauric
acid value, see Appendix II) in a Chromaflex column. After
settling the Florisil by tapping the column, add about one-half
inch layer of anhydrous granular sodium sulfate to the top.
10.3 Pre-elute the column, after cooling,'with 50-60 ml of petroleum
ether. Discard the eluate and just prior to exposure of the
sulfate layer to air, quantitatively transfer the sample'extract
into the column by decantation and subsequent petroleum ether
washings. Adjust the elution rate to about 5 ml per minute and,
separately, collect up to four eluates in 500-ml K-D flasks
equipped with 10-ml ampuls. (See Eluate Composition, 10.3.1.)
Perform the first elution with 200 ml of 6% ethyl ether in
petroleum ether, and the second elution with 200 ml of 15% ethyl
ether in petroleum ether. Perform the third elution with 200 ml
111
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of 50% ethyl ether - petroleum ether and the fourth elution with
200 ml of 100% ethyl ether.
10.3.1 Eluate Composition - By using an equivalent quantity of any
batch of Florisil as determined by its lauric acid value,
the pesticides will be separated into the eluates indicated
below: ' . .
15% Eluate
Chlorpropham
Propham
Barban (95%)
50% Eluate
Barban (5%)'
Linuron
Neburon (8%)
100% Eluate
Neburon (92%)
Diuron
Moniuron
Siduron
CAUTION: Fenuron and Fenuron-TCA are not recovered from
the Florisil column.
10.4 Concentrate the eluates to 6-10 ml in the K-0 evaporator in a hot
water bath. Change to the micro-Snyder column and continue
concentration to 0.5-1.0 ml.
10.5 Analyze according to 11. below.
11. Separation and Detection
11.1 Carefully spot 10% of the extract on a thin layer. On the same
plate spot several pesticides or mixtures for screening purposes,
or a series of 1, 2, 4, 6, 8 and 10 ^1 of specific standards for
quantitative analysis.
11.2 Develop the layers 10 cm in a tank saturated with solvent vapors.
Remove the plate and allow it to dry.
11.3 Spray the layer rapidly and evenly with about 10-15 ml sulfuric
acid solution. Heat the layer in an oven at 110°C for 15
minutes.
-------
11.4 When the layer is cool, spray it with nitrous acid reagent and
allow 1t to dry. Spray the layer with 1-naphthol reagent and
allow it to dry again. The pesticides will appear as purple spots
(see Table 2). Identifications are made by comparison of colors
and R~ values. Quantitative estimates are made by visually
comparing the intensity and size of the spots with those of the
series of standard.
12. Calculation of Results
12.1 Determine the concentration of pesticide in a sample by comparing
the response in a sample to that of a quantity of standard treated
on the same layer. Divide the result, in micrograms, by the
fraction of extract spotted to convert to micrograms per liter.
13. Reporting Results
-13.1 Report results in micrograms per liter-without correction for
recovery data. When duplicate and spiked samples are analyzed all
data obtained should be reported.
113
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REFERENCES:
1. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories", Chapter 6, Section 6.4, U. S. Environmental Protection
Agency, National Environmental Research Center, Analytical Quality
Control Laboratory, Cincinnati, Ohio, 45268, 1972.
114
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METHOD FOR CHLOROPHENOXY ACID PESTICIDES IN WATER AND WASTEWATERS
1. Scope and Application
1.1 This method covers the determination of various chlorinated
phenoxy acid pesticides in water and wastewater.
1.2 The following pesticides may be determined individually by this
method:
Parameter . Storet No.
254-D
Dicamba
Si 1vex 39760
. . 2,4,5-T
1.3 Since these compounds may occur in water in various forms
(i.e., acid, salt, ester, etc.). a hydrolysis step is included
to permit the determination of the .active part of the herbicide.
2. Summary
2.1 Chlorinated, phenoxy acids and their esters are extracted from
the acidified water sample with ethyl ether. The esters are
hydrolyzed to acids and extraneous organic material is removed
by a solvent wash. The acids are converted to methyl esters
which are extracted from the aqueous phase. The extract is
cleaned by passing it through a micro-adsorption column.
Identification of the esters is made by selective gas
chromatographic separations and may be corroborated through the
use of two or more unlike columns. Detection and'measurement
is accomplished by electron capture, microcoulometric or
115
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electrolytic conductivity gas chromatography (1). Results are
reported in micrograms per liter.
2.2 This method is recommended for use only by experienced
pesticide analysts or under the close supervision of such
qualified persons.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing
hardware may yield discrete artifacts and/or elevated baselines
causing misinterpretation of gas chromatograms. All of these
materials must be demonstrated to'be free from interference
under the conditions of the analysis. Specific selection of
reagents and purification of solvents by distillation in
all-glass systems may be required. Refer to Appendix I-.
3.2 The interferences in industrial effluents are high and varied
and often pose great difficulty in obtaining accurate arid
precise measurement of chlorinated phenoxy acid herbicides.
Sample clean-up procedures are generally required and may
result in loss of certain of these herbicides. It is not
possible to describe procedures for overcoming all of the
interferences that may be encountered in industrial affluents.
3.3 .Organic acids, especially chlorinated acids, cause the most
direct interference with the determination. Phenols including
chlorophenols will also interfere with this procedure.
3.4 Alkaline hydrolysis and subsequent extraction eliminates many
of the predominant chlorinated insecticides which might
otherwise interfere with the test.
116
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3.5 The herbicides, being strong organic acids, react readily with
alkaline substances and may be lost during analysis. Glassware
and glass wool should be acid-rinsed and sodium sulfate should
be acidified with sulfuric acid to avoid this possibility.
4. Apparatus and Materials
4.1 Gas Chromatpgraph - Equipped with glass lined injection port.
4.2 Detector Options:
4.2.1 Electron Capture - Radioactive (tritium or nickel-63)
4.2.2 Microcoulometric Titration
4.2.3 Electrolytic Conductivity
4.3' Recorder - Potentiometric strip chart (10 in.) compatible with
the detector. .
4.4 Gas Chromatographic Column Materials:
4.4.1 Tubing - Pyrex (180 on long X 4 mm ID)
4.4..2 Glass Wool - Silanized
4.4.3 Solid Support - Gas-Chrom-Q (100-120 mesh)
4.4.4 Liquid Phases - Expressed as weight percent coated on
solid support.
4.4.4.1 OV-210, 5%
4.4.4 2 OV-17, 1.556 plus QF-1 or OV-210, 1.95%
4.5 Kuderna-Oanish (K-D) Glassware
4.5.1 Snyder Column - three ball (macro) and two ball
(micro)
4.5.2 Evaporative Flasks - 250 ml
117
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4.5.3 Receiver Ampuls - 10 ml, graduated
4.5.4 Ampul Stoppers
4.6 Blender - High speed, glass or stainless steel cup.
4.7 Graduated cylinders - 100 and 250 ml.
4.8 . Erlenmeyer flasks - 125 ml, 250 ml ground glass 3 24/40
4.9 Microsyringes - 10, 25, 50 and 100 1.
" 4.10 Pipets - Pasteur, glass disposable (140 mm long X 5 mm I'D).
4.11 Separatory Funnels - 60 ml and 2000 ml with Teflon stopcock.
4.12 Glass wool - Filtering grade, acid washed.
4.13 Diazald Kit - Recommended for the generation of diazomethane
' (available from Aldrich Chemical Co., Cat. #210,025-2).
5. Reagents, Solvents and Standards
5.1 Boron Trifluoride-Methanol-esterification-reagent, 14 percent
boron trifluoride by weight.
5.2 N-methyl-N-nitroso-p-toluenesulfonamide (Diazald) - High
purity, melting point range 60-62^C. Precursor for the
generation of diazomethane (see Appendix IV).
5.3 Potassium Hydroxide Solution - A 37 percent aqueous solution
prepared from reagent grade potassium hydroxide pellets and
reagent water.
5.4 Sodium Chloride - (ACS) Saturated solution (pre-rinse NaCl with
hexane) in distilled water.
5.5 Sodium Hydroxide - (ACS) 10 N in distilled water.
118.
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5.6 So'dium Sulfate, Acidified - (ACS) granular sodium sulfate,
treated as follows: Add 0.1 ml of cone, suIfuric acid to lOOg
of sodium sulfate slurried with enough ethyl ether to just
cover the solid. Remove the ether with the vacuum. Mix 1 g of
the resulting solid with 5 ml of reagent water and ensure the
mixture to have a pH below 4. Store at 130*C.
5.7 Sulfuric acid - (ACS) concentrated, Sp. Gr. 1.84.
5.8 Florisil - PR grade (60-100 mesh) purchased activated at 1250°F
and stored at 130°C.
5.9 Carbitol (diethylene glycol monoethyl ether).
5.10 Diethyl Ether - Nanograde, redistilled in glass, if necessary.
5.1.0.1 Must be free of peroxides as indicated by EM Quant test
strips. (Test strips are available from EM
Laboratories, Inc., 500 Executive Blvd., Elmsford, N.Y.
10523.)
5.10.2 Procedures recommended for 'removal of peroxides are
provided with the test strips.
5.11 Benzene Hexane - Nanograde, redistilled in glass, if necessary.
5.12 Pesticide Standards - Acids and Methyl Esters, reference grade.
5.12.1 Stock standard solutions - Dissolve 100 mg of each
herbicjde in 60 ml ethyl ether; then make to 100 ml with
redistilled hexane. Solution contains 1 mg/ml.
5.12.2 Working standard - Pipet 1.0 ml of each stock solution .
into a single 100 ml volumetric flask. Make to volume
with a mixture of ethyl ether and hexane (1:1).
Solution contains 10 jug/ml of each standard.
119
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5.12.3 Standard for Chromatography (Diazomethane Procedure) -
Pipet 1.0 ml of the working standard into a glass
stoppered test tube and evaporate the solvent using a
steam b.ath. Add 2 ml diazomethane to the residue. Let
stand 10 minutes with occasional shaking, then allow the
solvent to evaporate spontaneously. Dissolve the
residue in 200 ^1 of hexane for gas chromatography.
5.12.4 Standard for Chromatgraphy (Boron Trifluoride Proce-
dure) - Pipet 1.0 ml of the working standard into a
glass stoppered test tube. Add 0.5 ml of benzene and
evaporate to 0.4 ml using a two-ball Snyder microcolumn
and a steam bath. Proceed as in 11.3.1. Esters are
then ready for gas chromatography.
6. Calibration -
6.1 Gas chromatographic operating conditions are considered
acceptable if the response to dicapthon is at least 50% of full
scale when < 0.06 ng is injected for electron capture detection
and < 100 ng is injected for microcoulometric or electrolytic
conductivity detection. For all quantitative measurements, the
detector must be operated within its linear response range and
the detector noise level should be less than .2% of full scale.
6.2 Standards, prepared from methyl esters of phenoxy acid
herbicides calculated as the acid equivalent, are injected
frequently as a check on the stability of operating
conditions. Gas chromatograms of several chlorophenoxys are
shown in Figure 1.
120
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_L
6 4 2-0
RETENTION TIME IN MINUTES
Fig. I Column: 1.5% 0V -17 * 1.95 % QF- I,
Carrier Gas : Argon (5%) /Methane: 70mt/min.s
Column Temp. 185 C, Detector: Electron Capture .
121
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8.
6.3 The elution order and retention ratios of methyl esters of
chlorinated phenoxy acid herbicides are provided in Table 1, as
a guide.
Quality Control
7.1 Duplicate and spiked sample analyses are recommended as quality
control checks. Quality control charts (2) should be developed
and used as a check on the analytical system. Quality control
check samples and performance evaluation samples should be
analyzed on a regular basis.
7.2 Each time a set of samples is extracted, a method blank is
determined on a volume of distilled water equivalent to that
used to dilute the sample.
Sample Preparation
8.1 The sample size taken for analysis is dependent on the type of
sample and the sensitivity required for the purpose at hand.
Background information on the pesticide levels previously
detected at a given sampling site will assist in determining
the sample size required, as well as the final volume to which
the extract needs to be concentrated. A 1-liter sample is
usually taken for drinking water and ambient water analysis to
provide a detection limit of 0.050 to 0.100^jg/l. One-hundred
milliliters is usually adequate to provide a detection limit of
1 ;jg/l for industrial effluents.
122
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Table 1
RETENTION RATIOS FOR METHYL ESTERS OF SOME CHLORINATED
PHENOXY ACID HERBICIDES RELATIVE TO 2,4-D
Liquid Phase1
Column Temp.
Argon /Methane
Carrier Flow
Herbicide
dicamba
2,4-D
si 1 vex
2,4, 5-T
2,4-D
(minutes absolute)
1.5% OV-17
1.95% QF-12
185°C
70 ml/min
RR
0.60
1 = 00
1.34
1.72
2.00
5% OV-210
185°C
70 ml/min
RR
0.61
1.00
1.22
1.51
1.62
columns glass, 180 cm x 4- mm ID, solid support
Gas Chrom Q (100/120 mesh)
2OV-210 may be substituted
123
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8.2 Quantitatively transfer the proper aliquot of sample from the
sample container into a two-liter separatory funnel. If less
than 800 ml is analyzed, dilute to one liter with interference
free distilled water.
9. Extraction
9.1 Add 150 ml of ether to the sample in the separatory funnel and
shake vigorously for one minute.
9.2 Allow the contents to separate for at least ten minutes., After
the layers have separated, drain the water phase into a 1-liter
Erlenmeyer flask. Then collect the extract in a 250-ml
ground-glass Erlenmeyer flask containing 2 ml of 37 percent
.aqueous potassium hydroxide.
9.3 Extract the sample two more times"using 50 ml of ether each-
time, and combine the extracts in the Erlenmeyer flask. (Rinse
the 1-liter flask with each additional aliquot of extracting
solvent.) -
10. Hydrolysis
10.1 Add 15 ml of distilled water and a small boiling stone to the
flask containing the ether extract, and fit the flask with a
3-ball Snyder column. Evaporate the ether on a steam bath and
continue heating for a total of 60 minutes.
10.2 Transfer the concentrate to a 60-ml separatory funnel. Extract
the basic solution two times with 20 ml of ether and discard
the ether layers. The herbicides remain in the aqueous phase.
124
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10.3 Acidify the contents of the separatory funnel by adding 2 ml of
cold (4°C) 25 percent sulfuric acid (5.9). Extract the
herbicides once with 20 ml of ether and twice with 10 ml of
ether. Collect the extracts in a 125-ml Erlenmeyer flask
containing about 0.5 g of acidified anhydrous sodium sulfate
(5.8). Allow the extract to remain in contact the the sodium
sulfate for approximately two hours.
11- Esterification (3,4)
11.1 Transfer the ether extract, through a funnel plugged with glass
wool, into a Kuderna-Danish flask equipped with a 10-ml
graduated ampul. Use liberal washings of ether. Using a glass
rod, crush any caked sodium sulfate during the transfer.
11.1.1 If esterification is to be done with diazomethane,
evaporate to approximately 4 ml on a steam bath (do not
immerse the ampul in water) and proceed as directed in
Section 11.2. Prepare diazomethane as directed in
Appendix IV.
11.1.2 If esterification is to be done with boron trifluoride,
add 0.5 ml benzene and evaporate to about 5 ml on a
steam bath. Remove the ampul from the flask and further
concentrate the extract to 0.4 ml using a two-ball
Snyder microcolumn and proceed as in 11.3.
11.2 Diazomethane Esterification
11.2.1 Disconnect the ampul from the K-D flask and place in a
hood away from steam bath. Adjust volume to 4 ml with
125
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ether, add 2 ml diazomethane, and let stand 10 minutes
with occasional swirling.
11.2.2 Rinse inside wall of ampul with several hundred
microliters of ethyl ether. Take sample to
approximately 2 ml to remove excess diazomethane by
allowing solvent to evaporate spontaneously (room
temperature.
11.2.3 Dissolve residue in 5 ml of hexane. Analyze by gas
chromatography.
fl.2.4 If further clean-up of the sample is required, proceed
as in 11.3.4 substituting hexane for benzene.
11.3 Boron Trifluoride Esterification
11.3.1 After the benzene solution in the ampul has cooled, add
0.5 ml of borontrifluoride-methanol reagent. Use the
two-ball Snyder microcolumn as an air-cooled condenser
and hold the contents of .the ampul at 50°C for 30
minutes on the steam bath.
11.3.2 Cool and add about 4.5 ml of a neutral 5 percent aqueous
sodium sulfate solution so that the benzene-water
interface is in the neck of the Kuderna-Oanish ampul.
Seal the flask with a ground glass stopper and shake
vigorously for about one minute. Allow to stand for
three minutes for phase separation. '
11.3.4 Pipet the solvent layer from the ampul to the top of a
small column prepared by plugging a disposable Pasteur
126
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pipet with glass wool and packing with 2.0 cm of sodium
sulfate over 1.5 cm of Florisil adsorbent. Collect the
eluate in a graduated ampul. Complete the transfer by
repeatedly rinsing the ampul with small quantities of
benzene and passing the rinses through the column until
a final volume of 5.0 ml of eluate is obtained. Analyze
by gas chromatography.
12. Calculation of Results
12.1 Determine the methyl ester concentration by using the absolute
calibration procedure described below or the relative calibra-
tion procedure described in Appendix III.
(1) . Mi crograms/ liter = (A) (B^
(V-f) (s
A = nq standard
standard area
B = Sample aliquot area
. V-| = Volume of extract injected
V-t= Volume of total extract Cul)
Vs= Volume of water extracted (ml)
12.2 Molecular weights for the calculation of methyl esters as the
acid equivalents.
2,4-D 222.0 Dicamba 221.0
2,4-D methyl ester 236.0 Dicamba methyl ester 236.1
Silvex 269.5 2S4,5-T 255.5
SiIvex methyl ester 283.5 2,4,5-T methyl ester 269.5
127
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13. Reporting Results
13.1 Report results in micrograms per liter as the acid equivalent
without correction for recovery data. When duplicate and
spiked samples are analyzed all data obtained should be
reported.
128
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REFERENCES:
1. Goerlitz, D. G., and Lamar, W. L., "Determination of Phenoxy and
Herbicides in Water by Electron-Capture and Microcoulometric Gas
Chromatography", U. S.- Geol. Survey Water-Supply Paper 1817-C (1967).
2 "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories" (1972), U. S. Environmental Protection Agency, National
Environmental Research Center, Analytical Quality Control Laboratory,
Cincinnati, Ohio, 45268.
3. Metcalf, L. D. and Schmitz, A. A., "The Rapid Preparation of Fatty Acid
Esters for Gas Chromatographic Analysis", Analytical Chemistry, 33,
363 (1961). .
4. Schlenk, H. and Gellerman, J. L., "Esterifi cation of Fatty Acids with
Diazomethane on a Small Scale", Analytical Chemistry, 32, 1412 (1960).
129
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METHOD FOR VOLATILE CHLORINATED ORGANIC -COMPOUNDS IN WATER AND WASTEWATERS
1. Scope and Application
1.1 This method covers the determination of various chlorinated organic
compounds in water and wastewater.
1.2 The following chlorinated organic compounds may be determined
individually by this method:
Parameter
Benzylchloride
Carbon tetrachloride
Chlorobenzene
Chloroform
Epichlorohydrin
Methylene Chloride
1,1,2,2-Tetrachloroethane
Tetrach1oroethy1ene
1,2,4-Trichlorobenzene
1,1,2-Trich1oroethane
Storet No.
32102
34301
32106
34423
34475
2. Summary
2.1 If the sample is turbid, it is initially centrifuged or filtered
through a fiber glass filter in order to remove suspended matter.
A three to ten micro liter aliquot of the sample is injected into
the gas chromatograph equipped with a halogen specific detector.
The resulting chromatogram is used to identify and quantitate
specific components in the sample. Results are reported in
micrograms per liter. Confirmation of qualitative identifications
are made using two or more dissimilar columns.
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3. Interferences
3.1 The use of a halogen specific detector minimizes the possibility of
interference from compounds not containing chlorine, bromine, or
iodine. Compounds containing bromine or iodine will interfere with
the determination of organochlorine compounds. The use of two
dissimilar chromatographic columns helps to eliminate this
interference and, in addition, this procedure helps to verify all
qualitative identifications. When concentrations are sufficiently
high, unequivocal identifications can be made using infrared or
mass spectroscopy. Though non-specific, the flame ionization
detector may be used for known systems where interferences are not
a problem.
3.2 Ghosting is usually attributed to the history of the
chromatographic system. Each time a sample is injected, small
amounts of various compounds are adsorbed on active sites in the
inlet and at the head of the column. Subsequent injections of
water tend to steam clean these sites resulting in
non-representative peaks or displacement of the baseline. This
phenomenon normally occurs when an analysis of a series of highly
concentrated samples is followed by a low level analysis. The
system should be checked for ghost peaks prior to each quantitative
analysis by injecting distilled water in a manner identical to the
sample analysis (1). If excessive ghosting occurs, the following
corrective measures should be applied, as required, in the order
listed:
1) Multiple flushes with distilled water
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2) Clean or replace the glass Injector liner
3) Replace the chromatographic column.
4. Apparatus and Materials
4.1 Gas Chromatograph - Equipped with programmed oven temperature
controls and glass-lined injection port. The oven should be
equipped with a column exit port and heated transfer line for
convenient attachment to the halogen specific detector.
4.2 Detector Options:
4.2.1 Microcoulometric Titration
4.2.2 Electrolytic Conductivity
4.2.3 Flame lonization
4.3 Recorder - Potentiometric strip chart recorder (10 in) compatible
with the detector.
4.4 Syringes - 1 ^il, lOjjl, and SOjul.
4.5 BOO type bottle or 40 ml screw cap vials sealed.with Teflon faced
silicone septa.
4.6 Volumetric Flasks - 500 ml, 1000 ml. ;
4.7 Syringe - Hypodermic Lur-lock type (30 ml).
4.8 Filter glass fiber filter - Type A (13 mm).
4.9 Filter holder - Swinny-type hypodermic adapter (13.mm).
4.10 Glass stoppered ampuls - 10 ml
4.11 Chromatographic columns
4.11.1 Moderately-Polar Column - 23 ft x 0.1 in ID x 0.125 in OD
stainless steel column #304 packed with 5% Carbowax 20 M
on Chromosorb-W (60-80 mesh).
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4.11.2 Highly-Polar Column - 23 ft x 0,1 in ID x 0.125 in OD
stainless steel #304 packed with 5% l,2s3-Tris-(2-cyano-
ethoxy) propane on Chromosorb-W (60-80 mesh).
4.11.3 Porous Polymer Column - 6 ft- x 0.1 in ID x 0.125 in 00
stainless steel #304 packed with Chromosorb-101 (60-80
mesh).
4.11.4 Carbopack Column - 8 ft x 0.1 in ID x 0.125 in OD
stainless steel #304 packed with Carbopack-C (80-100
mesh) + 0.2% Carbowax 1500.
5. Reagents
5.1 Chlorinated hydrocarbon reference standards
5.1.1 Prepare standard. mixtures in volumetric flasks using
contaminant-free distilled water as solvent. Add a
known amount of the chlorinated compounds with a
micro liter syringe. Calculate the concentration of
each component as follows:
mg/1 = (Density of Compound) (pi -injected) v - ^-^ - /
(Dilution Volume (ml))
6. Quality Control
6.1 Duplicate quantitative analysis on dissimilar columns should be
performed. The duplicate quantitative data should agree within
experimental error (+6 percent). If not, analysis on a third
dissimilar column should be performed. Spiked sample analyses
should be routinely performed to insure the integrity of the method.
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7. Selection Gas Chromatographic Column
7.1 No single column can efficiently resolve all chlorinated
hydrocarbons. Therefore, a specific column must be selected to
perform a given analysis. Columns providing only partially or
non-resolved peaks are useful only for confirmatory
identifications. If the qualitative nature of the sample is known,
an efficient column selection can be made by reviewing the
literature (2). In doing this, one must remember that injection of
large volumes of water can cause two serious problems not normally
noted using common gas Chromatographic techniques:
1) Water can cause early column failure due to liquid phase
displacement.
2) Water passing through the column causes retention times and
orders to change when compared to common sample solvent media,
i.e., hexane or air.
For these reasons, column life and the separations obtained by
direct aqueous injection may not be identical to those suggested in
literature.
8. Sample Collection and Handling
8.1 The sample containers should have a total volume in excess of 25 to
40 ml , although larqer narrow-mouth bottles may be used.
8.1.1 Narrow mouth screw cap bottles with the TFE fluorocarbon
face silicone septa cap-liners are strongly recommended.
Crimp-seal serum vials with TFE fluorocarbon faced septa or
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ground glass stoppered bottles are acceptable if the seal is
properly made and maintained during shipment.
8.2 Sample Bottle Preparation
8.2.1 Wash all sample bottles and TFE seals in detergent. Rinse
with tap water and finally with distilled water.
8.2.2 'Allow the bottles and seals to air dry at room temperature.
:8.2.3 Place the bottle in a 200°C oven for one hour, then allow
to cool in an area known to be free of organics.
8.2.4 When cool, seal the bottles using the TFE seals that will be
used for sealing the samples.
8.3 The sample is best preserved by protecting it from phase
separation. Since the majority of the chlorinated solvents are
volatile and relatively insoluble in water, it is important that
the sample bottle be filled completely to minimize air spaca over
the sample. Acidification will minimize the formation of
nonvolatile salts formed from chloro'brganic acids and certain
chlorophenols. However, it may interfere with the detection of
acid degradable compounds such as chloroesters. Therefore, the
sample history must be known before any chemical or physical
preservation steps can be applied. To insure sample integrity, it
is best to analyze the sample within 1 hour of collection.
8.4 Collect all samples in duplicate.
8.5 Fill the sample bottles in such a manner that no air bubbles pass
through the sample as the bottle is filled.
/
8.6 Seal the bottles so that no air bubbles are entrapped in it.
8.7 Maintain the hermetic seal on the sample bottle until analysis.
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8.8 Sampling from a water tap.
8.8.1 Turn on water and allow the system to flush. When the
temperature of the water has stabilized, adjust the flow to
about 500-ml/minute and collect duplicate samples from the
flowing stream.
8.9 Sampling from an open body of water.
8.9.1 Fill a 1-quart wide-mouth bottle with sample from a repre-
sentative area. Carefully fill duplicate 25 to 40 mi-sample
bottles from the 1-quart bottle.
9. Sample Preparation
9.1 If the sample is turbid, it should be filtered or centrifuged to
prevent syringe plugging or excessive ghosting problems. Filtering
the sample is accomplished by filling a 30-mT hypodermic syringe
with sample and attaching the Swinny-type hypodermic filter adaptor
with a glass fiber filter "Type A" installed. Discard the first 5..
ml of sample then collect the filtered sample in a glass stoppered
sample filled to the top. (One should occasionally .analyze the
non-filtered sample to insure that the filtering technique does not
adversely affect the sample).
10. Method of Analysis
10.1 Daily, analyze a standard containing 10.0 mg/1 of each compound to
be analyzed as a quality check sample before any samples are
analyzed. Instrument status checks and lower limit of detection
estimations based upon response factor calculations at two times
the signal to noise ratio are obtained from these dflta. In
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addition, response factor data obtained from this standard can be
used to estimate the concentration of the unknowns.
10.2 Analyze the filtered sample of unknown composition by injecting 3 to
10 ^il into the gas chromatograph. Record the injection volume and
detector sensitivity.
10.3 Prepare a.standard mixture consisting of the same compounds in
concentrations approximately equal to those detected in the sample.
Chromatograph the standard mixture under condition's identical to the
unknown.
11. Calculation or Results
11.1 Measure the area of each unknown peak and each reference standard
peak as follows:
Area = (Peak Height)(Width of Peak at 1/2 Height)
11.2 Calculate the concentration of each unknown as follows:
(Area of Sample peak)Cul of Standard Injectedj(Conc'n of Standard)
mg/1 = (jul of Sample injected)(Area of Standard Peak)
12. Reporting Results
12.1 Report results in mg/1. If a result is negative, report the minimum
detectable limit (see 10.1). When duplicate and spiked samples are
analyzed, all data obtained should be reported.
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CS
as e»
t I I I ! I! Illltllf t)
RETENTION TIME IN MINUTES
Figure 1. Column: Cliromosorls-101, Temperature Program: 125 C
for 4 min then 4C/min up to 280 C., Carrier Gas: Nitrogen at
36ml/min, Detector: Microcoulometric.
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REFERENCES: .
1. Dressman, R. C., "Elimination of Memory Peaks Encountered in Aqueous-
Injection Gas Chromatography", Journal of Chroma to graphic Science s 8.,
265 (1970).
2. "Gas Chromatography Abstracts", Knapman, C, E. H.s Editor, Institute of
Petroleum,. 61 New Cavendish Street, London W1M8AR, Annually 1958 to date,
since 1970, also includes Liquid Chromatography Abstracts.
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METHOD FOR PENTACHLOROPHENOL IN WATER AND WASTEWATER
1. Scope and Application
1.1 This method covers the determination of pentachlorophenol (PCP) in
water and wastewater.
2. Summary .
2.1 Pentachlorophenol is extracted from the acidified water sample (pH
3) with toluene, methylated with diazomethane, and analyzed by
electron-capture gas chromatography, using the columns listed in
the organochlorine pesticide method. (Page 7, this manual)
2.2 Further identification of pentachlorophenol is made with a mass
spectrometer.
3. Interferences
3.1 Chlorinated pesticides and ot.her high boiling chlorinated organic
compounds may interfere with the analysis of PCP.
3.2 Injections of samples not treated with diazonmethane indicate, to a
certain degree, whether interfering substances are present.
4. Precision and Accuracy
4.1 Single laboratory accuracy and precision reported for this method
when analyzing five replicates of tap water spiked with 0.05 to
0.07 ;ig/l of PCP is as follows:
Recovery - mean 95.9%, range 88.1 to 100.2%
'Standard Deviation - 6.0%
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REFERENCE:
"Analysis of Pentachlorophenol Residues in Soil, Water and Fish," Stark,
Ac, Agricultural and Food Chemistry, 17, 871 (July/August 1969).
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APPENDIX I
CONSIDERATIONS FOR GLASSWARE AND REAGENTS USED IN ORGANIC ANALYSIS*
1. Glassware
1.1 Cleaning Procedure - It is particularly important that glassware
used in trace organic analyses be scrupulously cleaned before
initial use as well as after each analysis. The glassware should
be cleaned as soon as possible after use, first rinsing with water
or the solvent that was last used in it. This should be followed
by washing with hot soap water, rinsing with tap water, distilled
water, redistilled acetone and finally with pesticide quality
hexane. Heavily contaminated glassware may require muffling at
e
400C for 15-to 30-minutes. High boiling materials, such as some of
the polychlorinated biphenyls (PCBs) may not be eliminated by such
heat treatment. NOTE: Volumetric ware should not be muffled. The
glassware should be stored immediately after drying to prevent
accumulation of dust or other contaminants. Store inverted or
cover mouth with foil.
1.2 Calibration - Individual Kuderna-Danish concentrator tubes and/or
centrifuge tubes used for final concentration of extracts must be
*Methods for Organic Pesticides in Water and Wastewater," 1971,
Environmental Protection Agency, National Environmental Research Center,
Cincinnati, Ohio, 45268
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accurately calibrated at the working volume- This is especially
important at volumes below 1 ml* Calibration should be made using
a precision microsyringe, recording the volume required to bring
the liquid level to the individual graduation marks. Glass A
volumetric ware should be used for preparing all standard solutions,
2. Standards, Reagents and Solvents
2.1 Analytical Standards and Other Chemicals - Analytical reference
grade standards should be used whenever available. They should be
stored according to the manufacturer's instructions. Standards and
reagents sensitive to light should be stored in dark bottles and/or
in a cool dark place. Those requiring refrigeration should be
'allowed to come to room temperature before opening. Storing of
such standards under nitrogen is advisable.
2.1.1 Stock Standards - Pesticide stock standards solutions
should be prepared in 1 jjg'/^ul concentrations by
dissolving 0.100-grams of the standard in pesticide-
quality hexane or other appropriate solvent (Acetone
should not be used since some pesticides degrade on
standing in this solvent) and diluting to volume in a 100
ml ground glass stoppered volumetric flask. The stock
solution is transferred to ground glass stoppered reagent
bottles. These standards should be checked frequently
for signs of degradation and concentration, especially
just prior to preparing working standards from them.
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2.1.2 Working Standards - Pesticide working standards are
prepared from the stock solutions using a micro syringe
preferably equipped with a Chaney adapter. The
concentration of the working standards will vary
depending on the detection system employed and the level
of pesticide in the samples to be analyzed. A typical
concentration (0.1 ng/jul) may be prepared by diluting 1
jul of the 1 jug/ul stock to volume in a 10-ml ground glass
stoppered volumetric flask. The standard solutions
should be transferred to ground glass stoppered reagent
bottles. Preparation of a fresh working standard each
day will minimize concentration through evaporation of
solvent. These standards should be stored in the same
manner as the stock solutions.
2.1.3 Identification of Reagents - All stock and working
standards should be labeled as follows: name of
compound, concentration, date prepared, solvent used, and
name of person who prepared it.
2.1.4 Anhydrous sodium sulfate used as a drying agent for
solvent extracts should be prewashed with the solvent or
solvents that it comes in contact with in order to remove
any interferences that may be present.
2.1.5 Glass wool used at the top of the sodium sulfate column
must be pre-extracted for about 40-hours in soxhlet using
the appropriate solvent.
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2.2 Solvents - Organic solvents must be of pesticide quality and-
demonstrated to be free of interferences,in a manner compatible
with whatever analytical operation is to be performed. Solvents
can be checked by'analyzing a volume equivalent to that used in the
analysis and concentrated to the minimum final volume.
Interferences are note.d in terms or gas chromatographic response -
relative retention time, peak geometry, peak intensity and width of
solvent response. Interferences noted under these conditions can
be considered maximum. If necessary, a solvent must be redistilled
in glass using a high efficiency distillation system. A 60-cm
column packed with 1/8 inch glass helices is effective.
2.2.1 Ethyl Ether - Hexane - It is particularly important that
these two solvents, use'd for extraction of organochlorine
pesticides from water, be checked for interferences just
prior to use. Ethyl ether, in particular, can produce
troublesome interferences.' (NOTE: The formation of
peroxides in ethyl ether creates a potential explosion
hazard. Therefore it must be checked for peroxides
before use.) It is recommended that the solvents be
mixed just prior to use and only in the amount required
for immediate use since build-up of interferences often
occurs on standing.
2.2.2 The great sens-itivity of the electron capture detector
requires that al-1 solvents used for the analysis be of
pesticide quality. Even these solvents sometimes require
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redistillation in an all glass system prior to use. The
quality of the solvents may vary from lot to lot and even
within the same lot., so that each bottle of solvent must
be checked before use.
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APPENDIX II
STANDARDIZATION OF FLORISIL COLUMN BY WEIGHT ADJUSTMENT BASED ON
ADSORPTION OF LAURIC ACID
1. Scope
T.I A rapid method for determining adsorptive capacity of
Florisil is based on adsorption of lauric acid from hexane
solution. An excess of lauric acid is used and amount not
adsorbed is measured by alkali titration. Weight of lauric
acid adsorbed is used to calculate, by simple proportion,
equivalent quantities of Florisil for batches having
different adsorptive capacities.
2<, Apparatus
2.1 Buret 25 ml with 1/10 ml graduations.
2.2 Erlenmeyer flasks 125 ml narrow mouth and 25 ml,-glass
stoppered.
2.3 Pipet ~ 10 and 20 ml transfer.
2.4 Volumetric flasks 500 ml.
3. Reagents and Solvents
3.1 Alcohol, ethyl. ~ USP or absolute, neutralized to
phenolphthalein.
3.2 Hexane Distilled from all glass apparatus.
3.3 Lauric acid Purified, CP.
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3.4 Laurie acid solution - Transfer 10.000 g lauric acid to 500
ml volumetric flask, dissolve in hexane, and dilute to 500 ml
(1 ml = 20 mg).
3.5 Phenolphthalein Indicator - Dissolve 1 g in alcohol and
dilute to 100 ml. I
3.6 Sodium hydroxide. - Dissolve 20 g NaOH (pellets, reagent
grade) in water and dilute to 500 ml (IN.). Dilute 25 ml IN.
NaOH to 500 ml with water (O.OSJ^). Standardize as follows:
Weigh 100-200 mg lauric acid into 1250 ml Erlenmeyer flask.
Add 50 ml neutralized ethyl alcohol and 3 drops ;
phenolphthalein indicator; titrate to permanent end point.
Calculate mg lauric acid/ml 0.05, jN NaOH (about 10 rag/ml).
4. Procedure . .
4.1 Transfer 2.000 g Florisil to 25 ml glass stoppered Erlenmeyer
flasks. Cover loosely with aluminum foil and heat overnight
at 130°C. Stopper, cool to room temperature, add 20.0 ml
lauric acid solution (400 mg), stopper, and shake
occasionally for 15 min. Let adsorbent settle and pipet 10.0
ml of supernatant into 125 ml Erlenmeyer flask. Avoid
inclusion of any Florisil.
4.2 Add 50-ml neutral alcohol and 3 drops indicator solution;
titrate with O.OSjN to a permanent end point.
5. Calculation of Lauric Acid Value and Adjustment of Column Weight
5.1 Calculate amount of lauric acid adsorbed on Florisil as
follows:
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Laurie Acid value = mg lauric acid/g Florisil = 200 - (ml
required for titration X mg lauric acid/ml O.OBN^ NaOH).
5.2 To obtain an equivalent quantity of any batch of Florisi-1,
. divide 110 by lauric acid value for that batch and multiply
fay 20 g. Verify proper elution of pesticides by 6.
6. Test for Proper Elution Pattern and Recovery of Pesticides
6.T Prepare a test mixture containing aldrin, heptachlor epoxide,
p,p'-DDE, dieldrin, Parathion and malathion. Dieldrin and
Parathion should elute in the 15% eluate; all but a trace of
malathion in the 50% eluate and others in the 6% eluate.
7. References
7.1 "Pesticide Analytical Manual," U«S. Department of Health,
Education and Welfare, Food and Drug Administration,
Washington, D.C.
7.2 Mills, P.A., "Variation of Florisil Activity: Simple Method
for Measuring Adsorbent Capacity and- Its Use in Standardizing
Florisil Columns," Journal of the Association of Official
Analytical Chemists, 5J, 29 (1968).
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APPENDIX III '
CHROMATOGRAPHIC CALIBRATION TECHNIQUE
Relative" Calibration (Internal Standardization):
A relative calibration curve is prepared by simultaneously chromatographing
mixtures of the previously identified sample constituent and a reference
standard in known weight ratios and plotting the weight ratios against- area
ratios. An accurately known amount of the reference material is then added
to the sample and the mixture chromatographed. The area ratios are
calculated and the weight ratio is read from the curve. Since the amount of
reference material added is known, the amount of the sample constituent can
be calculated as follows:
Rw x Ws
micrograms/liter = Vs
Rw = Weight ratio of component to standard
obtained from calibration curve
Ws = Weight of internal standard added to '
sample in nanograms
Vs = Volume of sample in milliliters
Using this method, injection volumes need not be accurately measured the
detector response need not remain constant since changes in response will
not alter the ratio. This method is preferred when the internal standard
meets the following conditions:
a^ well-resolved from other peaks
b) elutes close to peaks of interest
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c) approximates concentration of unknown
d) structurally similar to unknown.
"Methods for Organic Pesticides in Water and Wastewater," U.S.
Environmental Protection Agency, National Environmental Research
Center, Cincinnati, Ohio 45268
151
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APPENDIX IV
PREPARATION OF DIAZOMETHANE IN ETHER
1. Scope
1.1 Diazomethane is prepared by reaction of Carhitol and Diazald in the
presence of KOH. Solutions of diazomethane decompose rapidly in
the presence of solid material such as copper powder, calcium
chloride, boiling stones, etc.' These solid materials cause solid
.polymethylene and nitrogen gas to form.
2. Apparatus .
2.1 .Distilling flask with condenser, 125 ml, long neck with dropping
funnel.
2.2 Erlenmeyer flasks - 500 ml and 125 ml.
2.3 Water bath.
3. Reagents .and Solvents
3.1 Ether
3.2 Potassium hydroxide pellets.
3.3 Carbitol (diethylene glycol monoethyl ether).
3.4 Diazald in ether. Dissolve 21.5 g of Diazald in 140 ml ether.
4, Procedure
/
4.1 Use a we 11-ventilated hood and cork stoppers for all connections.
Fit a 125-ml long-neck distilling flask with a dropping funnel and
an efficient condenser set downward for distillation. Connect the
condenser to two receiving flasks in a series - a 500-ml Erlenmeyer
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followed by a 125-ml Erlenmeyer containing jO ml ether. The inlet
to the 125-ml Erlenmeyer should,dip below the ether. Cool both
receivers to 0°C. As a water bath for the distilling flask, set
up a 2-liter beaker on a stirplate (hot plate and stirreH,
maintaining temperature at 70°C.
4.2 Dissolve 6-g KOH in 10 ml water in the distilling flask (no heat).
Ad 35 ml Carbitol (diethylene glycol monoethyl ether), stirring
bar, and another 10 ml ether. Connect the distilling flask to the
condenser and immerse distilling flask in water bath., By means of
the dropping funnel, add a .solution of 21.5 g Diazald in 140 ml
ether over a period of 20 minutes. After distillation is
apparently complete, add another 20 ml ether and continue
distilling until distillate is colorless. Combine the contents of
the two receivers in a glass bottle (WITHOUT ground glass neck),
stopper with cork, and freeze overnight. Decant the diazomethane
from the ice crystals into a glass bpttle, .stopper with cork, and
store in freezer until ready for use. The final solution may be
stored up to six months without marked deterioration. The 21.5 g
of .Diazald reacted in this manner produce about 3 g of Diazomethane,
5. Cautions
5.1 Diazomethane is very toxic. It can explode under certain
conditions. The following precautions should be observed.
5.1.1 Use only in we 11-ventilated hood.
5.1.2 Use safety screen.
5.1.3 Do not pipette solution of diazomethane by mouth.
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5.1.4' For pouring solutions of diazomethane, use of gloves is
optional.
5.1.5 Do not heat solutions at 100°C (EXPLOSIONS).
5.1.6 Store solutions of gas at low temperatures (freezer
compartment of explosion-proof refrigerators),,
5.1.7 Avoid ground glass apparatus, glass stirrers and sleeve
bearings where grinding may occur (EXPLOSIONS).
5.1.8 Keep solutions away from alkali metals (EXPLOSIONS).
6. Reference
6.1 "Pesticide Analytical Manual," U.S. Department of Health, Education
and Welfare, Food and Drug Administration, Washington, D;C.
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BIBLIOGRAPHY
1. "Analysis of Pesticide Residues in Human and Environmental
Samples," U.S. Environmental Protection Agency, Perrine Primate
Research Laboratories, Perrine, Florida, 33157, 1971.
2. Mills, P;A., "Variation of Florisil Activity: Simple Method for
Measuring Adsorbent Capacity and Its Use in Standardizing Florisil
Columns," Journal of the Association of Official Analytical
' Chemists, 51., 29 (1968).
3. Goerlitz,, O.F. and Brown, E., "Methods for Analysis of Organic
Substances in Water," Techniques of Water Resources Investigations
of the United States Geological Survey, Book 5, Chapter A3, U.S.
Department of the Interior, Geological Survey, Washington,. D.C.
20242, 1972, pp. 24-40.
4. Steere, N»V., editors "Handbook of Laboratory Safety," Chemical
Rubber Company, 18901 Cranwood Parkway, Cleveland, Ohio, 44128,
1971, pp. 250-254.
3. Cochrane, W. P. and Wilson, B. P., "Electrolytic conductivity
detection of some nitrogen-containing herbicides," Journal of
Chromatography, 63, 364 (1971).,
4. "Standard Practice for Measuring Volatile Organic Matter in Water
by Aqueous - Injection Gas Chromatography," 02908 Annual Book of
ASTM Standards, Part 31, Water; American Society for Testing and
Materials, 1916 Race Street, Philadelphia, PA, 19103.
5. Gas-liquid Chromatographic Techniques for Petro Chemical
Wastewater Analysis, Sugar, J.W. and Conway, R.A., Journal of
WPCF, 40, (Annual Conference Issue) 1622 (1968).
6. "Handbook of Chemistry and Physics," 48th Edition, the Chemical
Rubber Company, 18901 Cranwood Parkway, Cleveland, Ohio, 44128.
(1967-1968).
U. S. GOVERNMENT PRINTING OFFICE: 1979 657-060/1624
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