ANALYSIS OF PESTICIDE RESIDUES IN
HUMAN AND ENVIRONMENTAL SAMPLES
A COMPILATION OF METHODS
SELECTED FOR USE IN
PESTICIDE MONITORING PROGRAMS
EDITED BY: J. F. THOMPSON
EDITORIAL COMMITTEE: Prepared by:
Primate & Pesticides Effects Laboratory
H. F. ENOS Environmental Protection Agency
F. J. BIROS Perrine, Florida 33157
M. T. SHAFIK
Revised in November, 1972
-------
Revised 11/1/72 Section 1
Page 1
IWI’RODUCrI (
The analytical methodology collected in this manual is intended for
use by EPA Laboratories conducting analyses for pesticides in various
segments of the environment and by the laboratories under contract with
EPA to conduct con nunity studies and the monitoring of pesticides in
people.
One of the primary objectives of the Connunity Studies and
Monitoring Laboratory program is to establish and maintain, in
collaboration with other Federal Agencies, a broad surveillance and
evaluation program concerned with the extent and significance of the
contamination of man and his environment by pesticides and their
metabolites. To accomplish this goal, data are continuously obtained on
the levels of pesticides and their metabolites in the human po ilation
and various elements of the environment. It is important that uniform
chemical methodology of utmost reproducibility and accuracy be used by
participating laboratories so that analytical results can be correlated
and directly compared between laboratories.
A prime responsibility of the Perrine Primate Research Laboratories
is to make new and inproved analytical procedures available to EPA and
related laboratories and to those of State and local agencies working to
assess pesticide residues in people and/or environmental media. Thus,
the Division serves as the primary reference source for analytical quality
control, and a research facility for sampling and analytical procedures,
in addition to a major center for training chemists in pesticide
analytical methodology.
The reconniende4 methods collected in this volume include those
which were presented at the Second Ccinmunity Studies Chemists’ Meeting,
Tucson, Arizona, April, 1968. Revisions and changes have been made in
the details of most of the procedures based on refinements and evaluatior s
substantially contributed by the several Community Studies Laboratories
as well as Perrine Laboratory research, development, and quality control.
The present state of development notwithstanding, pesticide residue
methodology is sufficiently dynamic to assure the need for additional
revisions and additions as present procedures are improved or new ones
are validated.
The analytical methodology compiled herein consists of both “nuilti-
residue” methods and “specific residue” i thods. Included also, are
miscellaneous topics covering a number of important activities such as the
cleaning of laboratory glassware, preparation of analytical standards, etc.
A numbering system has been initiated in this manual. Each page bears
a date and numbers and/or letters designating the identity of the section
arid subsection. Additions, deletions and revisions will be distributed to
manual holders as they are made available with each such issuance bearing
appropriate section identification and revision date.
-------
Revised 11/1/72 Section 1
-2-
The cooperation of chemists in laboratories using this manual is
solicited in helping to iirrprove and up-date the manual. Suggestions
for improvement in existing methods or for entirely new procedures
based on your experience will be welcomed. Such suggestions or proposed
new methods should be sent through your customary channels to
Dr. William F. Durham, Director
Perrine Primate Research Laboratory
P.O. Box 490
Perrine, Florida 33157
These suggestions and proposals will be reviewed and tested in the
laboratory if indicated, and the suggester will ‘be notified regarding
their disposal.
In addition to acknowledging the contributions made to this manual by
the scientists at Perrine and in the Community Pesticide Studies projects,
a special note of thanks is due Mrs. Barbara Elwert, secretary for the
Perrine Chemistry Branch who so graciously endured the many copy revisions.
EDITORIAL COMvlIrI’EE
-------
Revised 11/1/72 Page 1
TABLE OF CONTE TFS*
Section ibject
1. Introduction
2. Collection, preservation and storage of samples
I. General conuitents
II. Sample containers
III. Storage of samples
3. Miscellaneous information
A. Cleaning of laboratory glassware
B. Preparation, storage and use of pesticide
analytical standards.
C. General purity tests for solvents and reagents
D. Evaluation of quality of Florisil
E. Limits of Detectability
4. Gas-Liquid Chromatography
A. Electron Capture 1)ctection
(1) Description of instrument and accessories
(2) Columns
(3) Detector
(4) Chromatography of sample
(5) Quarititation and interpretation
(6) Figures and Tables
* alytical methods designated by a star have been subjected to
inter laboratory study.
-------
Revised 11/1/72 Table of Content
-2-
B. Flame Photometric Detection
(1) Description of component modules
(2) Columns
(3) Detector
(4) Q.iantitation and interpretation
(5) Figures and Tables
5. Chlorinated hydrocarbon pesticides and metabolites
A. In human tissVes and excreta
*(1) Adipose tissue - Modification of Mills,
Onley, Gaither method.
(2) Micro method
(a) Liver, Kidney, Bone Marrow, Adrenal, Gonads
(b) Brain
(3) Blood
* (a) Multiple chlorinated pesticides
(b) Pentachlorophenol
(4) Urine
(a) Pentachlorophenol
(b)DDA
(c) 2,4-D and 2,4,5-T
6. Organophosphorus pesticides and nietabo).ites
A. In human tissues and excreta
(1) General comments
(2) Urine
(a) Determination of metabolites or hydrolysis
products of organophosphorus pestic .des.
-------
Revised 11/1/72 Table of Contents
-3-
(b) Paranitrophenol
(3) Blood
* (a) Cholinesterase activity
7. Carbamate pesticides and metaholites
A. l-Naphthol in urine
8. The sanipling and analysis of air for pesticides
A. Sampling
*B. Analysis by GLC
C. Evaluation for purity of Ethylene Glycol
9. The polychiorinated biphenyls
A. Jntroduction
13.
C. Separation of PCB’s from organochlorine
pesticides
D. TLC method for semi-quantitative estimation
of PCB’s in adipose tissue
E. Typical chromatograms of the PCB’s
F. Tables of relative retention and response ratios
10. The sampling arid analysis of water for pesticides
A. Introduction
B. Sampling considerations
C. Analytical procedure
11. The analysis of soils, housedust and sediment
A. soils and housedust
B. Bottom sediment
-------
Revised 11/1/7 Table of Contents
-4-
12. Confirmatory procedures
A. Microcoulometry
B. Thin layer chromatography
C. p-values
D. Derivatization techniques
(1) Microscale alkali treatment for confirmation
and sample cleanup.
E. Infrared spectroscopy
13. Analysis for Metals and Other Elements
A. Determination of methyl mercury in fish,
blood and urine.
B. Determination of total mercury in water.
APPENDIX
Section Subject
I. Maintenance and repair of instruments.
II. Analytical Quality Control
A. Interlaboratory
B. Intralaboratory
VI. Block diagram of tentative tissue, excreta and method
selection for abnormal pesticide exposure cases.
VII. Pesticide standards repository.
The mention of specific supplier names and/or brands does not constitute an
endorsement of products or c npanies by the United States Government, but is
included only for purposes of illustration and equivalency.
-------
1/4/71 Section 3,A
Page 1
MISCELLANEOUS INFOR’4ATION
A. CLEANI OF LABORATflRY GLASSWA] E
In the pesticide laboratoiy involved in the analysis of samples
containing residues in the parts per billion range, the preparation of
scrupulously clean glassware is mandatory. Failure to do so can lead to a
myriad of problems in the interpretation of the final chromatograms due to
the presence of extraneous peaks resulting fran contamination. Particular
care must be taken with glassware such as Kudema-Danish flasks or
evaporative concentrator tubes - or any other glassware coming in contact with
an extract which will be evaporated to a lesser volune. The process of
concentrating the pesticide in this operation may similarly concentrate the
contaminating substance, resulting in extraneous chranatographic peaks which,
in extreme cases, may completely overlap and mask out the pesticide peak
pattern.
Chemists do not all agree on procedural details in the cleaning of
glassware. However, the basic cleaning steps do find the majority in agree-
ment. These are:
1. Removal of surface residuals immediately after use.
2. Hot soak to loosen and flotate most of soil.
3. Hot tap water rinse to flush away flotated soil.
4. Soak with deep penetrant or oxidizinc agent to destroy traces of
organic soil.
5. Hot tap water rinse to flush away materi a1s ] oosened by deep penetrant
soak.
6. Distilled water rinse to remove metallic deposits from the tap water.
7. Acetone rinse to flush off any final traces of organic material.
8. A preliminary flush of the glassware just before using with the same
solvent to be used in the analysis.
Each of these eight fundamental steps will be discussed in the order in
which they appear above.
1. As soon as possible after use of glassware coming in contact with
known pesticides, i.e., beakers, pipets, flasks or bottles used for
standards, the glassware should be acetone flushed before placing in
the hot detergent soak. If this is not done, the soak bath may
serve to contaminate all other glassware placed therein. Many
instances of widespread laboratory contamination with a given
pesticide are traceable to the glassware washing sink.
-------
Revised 11/1/72 Section 3, A
-2-
2. The hot soak 8 onsists of a bath of a suitable detergent in
water of 50 C or higher. The detergent, powder or liquid,
should be entirely synthetic and not a fatty acid base.
There are very few areas of the country where the water
hardness is sufficiently low to avoid the formation of sane
hard water scum resulting fran the reaction between calcium
and magnesium salts with a fat y acid soap. This hard water
scum or curd would have an affinity, particularly for the
chlorinated pesticides, and being almost wholiy water
insoluble, would deposit on all glassware in the bath in a
thin film.
There are many suitable detergents on the wholesale and retail
market. Most of the common liquid dishwashi.ng detergents sold
at retail are satisfactory but are more expensive than other
comparable products sold industrially. Alconox, in powder
or tablet form is manufactured by Alconox, Inc., New York and
is marketed by a number of laboratory supply firms. Cparkleen,
another powdered product, is distributed by Fisher Scientific
Company.
NOTE : Certain detergents, even in trace quantities,
may contain organics which will contribute
significant background contamination by E. C.
detection. For this reason any detergent
selected should be carefully checked to
insure freedom from such contamination. The
following procedure is recommended:
Add 25 ml dist. water previously checked for
background contaminants to a 250 ml sep funnel.
Add 1 drop of the liquid detergent or 50 mg
if in powder form, followed by 100 ml hexane.
Stopper funnel and shake vigorously for 2
minutes. Allow layer separation, draw off and
discard aqueous layer. Add a pinch of anhydrous
Na SO to the hexant extract and shake 1 minute.
Tr ns er extract to a Kuderna-Danish assembly
fitted with a 10 ml evaporative concentrator
tube containing one 3 mm glass bead. Reduce
extract volume to ca. 3 ml in a hot water bath.
Cool, rinse down joint and sides of tube with
hexane, diluting extract to exactly 5 ml.
Stopper tube and shake on Vortex mixer 1 minute.
Chranatograph by GLC, E.C. and evaluate
chromatogran for contaminant peaks.
-------
Revised 11/1/72 Section 3, A
-3-
3. No comments required.
4. The most conmon and highly effective oxidizing agent for removal
of traces of organic soils is the traditional chremic acid
solution made up of H SO and potas ii n or sodiijn dichranate.
For ma inizrn efficienc , he soak solution shañd he hot
(40-50 C). Safety precautions rust be igidly observed in the
handling of this solution. Prescribed safety gear should
include safety goggles, and rubber gloves and apron. The bench
area where this operation is conducted should he covered with
lead sheeting as spattering will inevitably disintegrate the
bench surface.
The potential hazards of using chromic sulfuric acid mixture
are great and have been well publicized. There are now
corinnercially available substitutes that posses the advantage
of safety in handling. These are biodegradable concentrates
that claim cleaning strength equal to the chronic acid solution.
They are alkaline, equivalent to ca. 0.1 N NaOH upon dilution
and are claimed to remove dried blood, silicone greases,
distillation residues, insoluble organic residues, etc. They
further claim to remove radioactive traces and will not attack
glass flOT exert a corrosive effect on skin or clothing. One
such product is Chem. Solv. 2157, manufactured by Mallinckrodt
and available through laboratory supply firms. Another
comparable product is “Detex’ t a product of Borer-Chemie,
Solothurn, Switzerland.
5, 6, and 7 - No comments required.
8. There is always a possibility that between the time of washing and
the next use, the glassware may pick up sane contamination,
airborne or from direct contact. To nsure against this, it
is good practice to flush the item irTu ediately before use with
sane of the same solvent that will be used in the analysis.
The drying and storage of the cleaned glassware is of great
importance or the beneficial effects of the scrupulous cleaning may be
nullified. Pegboard drying is not recommended as contaminants may be
introduced to the interior of the cleaned vessels. Nco rene coated metal
racks are suitable for such items as beakers, Flasks, chromatographic
tubes, etc., - any glassware then can be inverted and suspended to dry.
Small articles like stirring rods, glass stoppers, bottle caps, etc.,
can be wrapped in alumint.nn foil and dried a short time if oven space is
available. Under no ciraz nstance should such small items be left in the
open without protective covering . The dust cloud raised by the daily
sweeping of the laboratory floor can most effectively recontaminate the
clean glassware.
-------
Revised 11/1/72 Section 3,A
-4-
Pipet Washing
The efficient and effective washing of pipers offers some special prob-
leins. Self-contained equi.pment for the entire operation, while available
comercially, is quite expensive.
The basic cleaning steps are the same as those listed earlier for mis -
cellaneous glassware with the occasional exception of the chromic sulfuric
acid soak. If pipets are properly cleaned this step may be safely eliminated
for pipets except those which are to be used in a method involving concen-
tration of the extract by evaporation. In this case, the additional insurance
factor of the oxidizing action is recommended.
Hand washing performed by soaking in a pan or sink and then rinsing by
holding under running water is highly unsatisfactory, particularly as applied
to transfer or volumetric pipers. One simply cannot be sure of obtaining a
complete rinse of all surfaces inside the bulb. Therefore, an automatic or
semi-automatic washing system is strongly recommended. A complete unit is
offered by Scientific Glass Apparatus Co. under catalog ninnber P-6920 for $495
(1969 price). A modular system can be assembled to operate semi-automatically
for about half the price of the complete cc iinercia1 unit. Equipment required
for the assembly is as follows:
1. Electric timer w/automatic cut-off.
Fisher #6-656.
2. Electric heater, immersion, 66” length, 500 watts,
Fisher #11-463-SB.
3. Pipet washer, stainless steel,
Fisher #15-350-5.
4. Jars, cylindrical, 6” dia. x 18” height, Coming #6942,
1-1/2 gal. (2 required).
5. Pipet baskets (2) stainless steel, fabricated by local tin shop
per sketch in Figure 1. These are not available commercially.
One of the bell jars (Item 4) is filled with distilled water in which is
dissolved a very small amount of synthetic detergent to function primarily as a
wetting agent. After acetone rinsing the interior of the used pipets they are
placed in a stainless steel basket and ijiinersed in the weak detergent solution.
They will remain here until enough are collected to run through the complete
wash. The second jar is filled with a detergent solution. The same detergent
used in regular glassware washing is not recommended in this application.
Instead, a powdered detergent intended for use in automatic dishwashers is
recommended. Many of these are available at wholesale or retail.
-------
Revised 11/1/72 Section 3, D
Page 1
H I SCELLANE 1JS INFORMATION
P. EVALIJATJCN OF ( JALITY OF FLORISIL
1. INTRODUCTION :
Florisil is purchased in 200 or 400-lb. lots from the Floridin
l)ivision of the Pennsylvania Glass Sand Corp. To insure the procurement
of high quality material , direct contact is made with the plant engineer
at the Hancock, W. Virginia plant. He is advised of our purchase require-
ment and is requested to be on the lookout for an especially high quality
lot as indicated by the company’s quality control checks. When such a
batch is produced, 400 lbs., in 50-lb. fiber drums, is set aside, a
representative sample of ca 1 lb. is taken and mailed to Perrine as an
advance sample of the lot.
The advance sample is evaluated as described in the following sub-
section. If the quality indication is favorable, the company is so
advised and the entire lot is shipped to Perrine. Upon receipt, a repre-
sentative sample is taken and the quality evaluation is repeated as final
assurance of the quality of the lot.
Jriuiiediately upon completion of the evaluation, the material is trans-
ferred from the pnlyethylerie-lined fiber drums to specially cleaned wide-
mouth jars, each jar holding 2 lbs. of Florisil.
iT. S \MPLTN1
h ght fiber drums containing 50 lbs. each of Florisil comprise an
entire 41)0-lb. lot. Each druin is sampled by taking six plugs from top to
bottom with a 36” x 1” grain trier. The plug pattern is approximately as
shown in the following sketch:
a
C.
The trier plugs from all drums are placed in a single container,
mixed thoroughly, and a quantity transferred to a 500-ml Erlenmeyer flask.
At the same time, three elution cohmris are prepared as described in
Section ,A,(1), page 6. The flask and the prepared columns are placed
in a 131) C oven and held overnight or longer.
-------
Section 3,D
c -BHC 20
1 -BHC 20
Heptachlor 20
Aidrin 20
Dieldrin 40
Endrin 100
If the laboratory’s normal procedure is to pack the
co1un is imn diately before use, the prepacking of columns
for overnight activation may be vo ded, but the flask of
Florisil should be held in the 130 oven at least 24 hours
before use.
o,p’-DDT 50
p,p’-DDT 80
Diazinon 300
M. Parathion 250
Malathion 400
Trithion 240
Florisil check #2
-BHC 40
p,p’-DDE 60
Ilept. Epox. 40
Ronnel 100
E. Parathion 250
IV. FLORISIL ELUTION :
1. Remove the prepacked columns from the oven so
use.
2. Place a beaker or flask under each column and
50 ml of pet. ether.
NOTE :
they may cool down before
prewet the packing with
From this point and throughout the following elution
process, the solvent level should not be allowed to go
below the top of the Na 2 SO 4 layer.
3. Read and record the % relative humidity in the room.
4. With 5-mi volumetric pipets transfer 5 ml each of mixtures 1 and 2
onto separate columns and 5 ml of hexane onto the third column as a
control.
5. Place 150-mi beakers under each column and coninence elution with 100 rrLl
of 6% diethyl ether/pet, ether, the elution rate to be 5 ml per minute.
The 100 ml portion of elut ion solvent is measured in a graduate and
applied to the column when the prewetting liquid level just reaches
the top of the Na.,SOA layer. At the instant the liquid level of the
first 100 ml of etut ng solvent just reaches the top of the Na,SOA
layer, place a second beaker under the column and quickly add another
100 ml of eluting solvent. Allow this second 100 ml of solvent to
pass through the colunn.
Revised 11/1/72
NOTE :
III. STANDARD MIXTURES :
-2-
Prepare the two following standard mixtures with the concentration
shown as picograms per inicroliter:
Florisil check #1
-------
1 evised 11/1/72 Section 3,D
-3-
6. Continue elution with 200 ml of 15% diethyl ether/pet, ether into a
succession of two more beakers identified as 200-300 arid 300-400,
adding the eluting solvent in 100-mi portions as previously described.
7. Then continue the elution with 50% diethyl ether/pet, ether, follow-
ing the same procedure of collecting the two i00-ml increments which
are designated as 400-500 and 500-600 ml.
8. Place the 18 beakers containing the 100-mi eluate increments on a
35_ 400 water bath and evaporate under a nitrogen stream to ca 2-5 ml.
9. Add a pinch of arthydrous Na,SOA to each beaker and transfer extracts
to 10 ml grad. concentration ti bes, rinsing beakers with ca 5 ri]. of
hexane delivered by a syringe or 5-mi ‘!ohr pipet. Evaporate under
nitrogen stream to ca 3 ml, remove from bath, allow to cool and
dilute with hexane to exactly 5 ml.
10. Stopper evap. conc. tubes and mix on Vortex mixer 1 minute.
\‘. GAS C I ll 0 \T0GRAPI JY :
1. With a column of 1.5% OV-l7/i.95% QF-l installed in the instrument,
prime column as described in Section 4,i , and equilibrate instrument.
NOTE: Use only the column designated as the compounds in
the respective mixtures will produce miniivai peak
over] aps.
2. Hake 5-pi injections of the two original standard mixtures to obtain
peak height data for calculations of recoveries.
3. Iake a 5 -ul injection of each of the concentrated eluate increments.
In case of off-scale peaks or peaks of less than 10% f.s.d., make
appropriate attenuation adjestment for both standard and eluates.
However, the entire series should preferably be run at one attenuation.
VI. CALCULATIONS / ND RECORDATION :
1. £‘Ieasure all peak heights from original standards and eluate increments.
2. For each compound, and based on peak height measurement, calculate the
percentage of the compound appearing in each 100-mi increment and in
the original standard.
-------
Revised 11/1/72 Section 3,D
-4-
Example: Lindane, 0-100 eluate, peak ht. 30 mm.
100-200 eluate, peak ht. 60 mm.
Original standard 98 mm.
Percentages - - - In 0-100 = 30 x 100 = 33%
30+60
In 100-200 = 60 x 100 = 67%
30+60
3. Compute the elution recovery by dividing the sum of the combined
eluate peak heights by the peak height of the original standard.
Working from the same data given above, we would have: -
30+ 60 x 100 = 92% Recovery
With the possible exception of aidrin, the recoveries of the chlori-
nated compounds should fall in the range of 90-105%. Aidrin may not
exceed 80%. Some of the OGP compounds may not yield high recoveries.
For example, trithion may yield no higher than 40% recovery under
certain conditions as outlined in the MISC. N JfES of Section 5,A,1.
4. Recordation of results is made on a standard form and should appear
comparable to Table 1. The decision of acceptance or rejection of
each lot is based on a consideration of the elution pattern and on
the recovery efficiency of the pesticides of interest in the program.
VII. STORAGE OF FLORISIL :
It is imperative that the Florisil be transferred from the shipping
drums into glass jars as soon as possible after the lot is evaluated and
judged acceptable. The drums are lined with polyethylene which may contri-
bute unwanted contamination over a period of time.
Glass jars which have been found suitable for storage are available
from certain glass container distributors. The specific jar used bears
Owens-Illinois mold #C-3122, with 100-400 finish, packed in cartons of 6
jars. Metal screw caps with coated paper liners are used.
The jars are washed by mechanical dishwashers, then rinsed with dis-
tilled water and acetone. After thorough drying of the jars, the Florisil
is transferred with a 2-lb. aluminum sugar scoop previously washed and
acetone rinsed. The net contents of each jar when filled to 1/2-inch from
the rim with Florisil is ca 2 lbs. A square of aluminum foil is crimped
over the rim of the jar and the cap is screwed on tight. Each jar is
labelled with the lot number and now ready for distribution. A copy of
the compound elution pattern (Figure 1) is enclosed with each shipment to
-------
Revised 11/1/72 Section 3,D
-5-
the field laboratories.
VIII. NOtES :
1. Factors influencing the recovery efficiency, particularly of certain
organophosphoTous compounds, include (1) presence of impurities in
the pet. ether, and (2) presence of peroxides in the ethyl ether.
This j discussed in more detail in the MISC. NOTES of SectiQn 5,A,1.
2. Po arity of the elution solvents exerls a profound effect on the
selective elution of a nuniber of compounds, The ethyl ether must
contain 2% v/v of ethanol to obtain compound elution patterns
comparable to those shown in Table 1.
-------
Revieed 6/1/72
-6-
Section 3,D
TABLE 1.
EL1J1 ION PATTERNS AND RECOVERY DATA FOR FLORISIL, LOT # 285I
BY METHOD SECTION 5 ,A, (1)(MANUAL OF ANALYTICAL METHoDS)
FLORISIL-COLUMN PREPACKED AND HELD IN 130°C OVE N AT LEAST 214 URS BEFORE USE
RElATIVE HUMIDITY IN LABORATORY 65 $
ELUTION INCREMENTS(ml)
Recovery. %
Compound
0
— 100 100 — 200
6% Fraction 15% Fraction -- 5O%_Fraction
200 — 300 300 — 1400
1400 — 500 500 — 600
‘
cz-BHC
100
97
—BHC
100
95
Li ndarie
100
g
Heptachior
100
91
Aidrin
100
100
Hept. Epox.
78
22
105
Die ldrin
8
1
96
Endrin
89
11
99.6
p 1 p’-DDE
100
97
o p’-DDT
100
99 6
p,p’—DDT
100
90
Ronnel
100
93
Methyl
Parathion
147
53
103
Malathion
100
99
Ethyl
Parathion
78
22
.
96
Diazinon
100
83
Trithign
100
143
Numerical values represent the percentage of each compound eluting in the given cut.
-------
Revised 11/1/72 Section 4,A,(l)
Page 1
GAS CI fl Q\lATOGRAP11Y-ELECTRON AVI1JRL
INSTRUMENT
All references given presuppose that the gas chromatograph will be the
licro-Tek 220 manufactured by ‘I’racor, Inc., Austin, Texas. This unit may be
equipped with a detector with a radioactive source of Nickel 63 or 130 mc
tritiuni. The electrometer may be the single channel model E2 or the solid
state, dual channel unit, model SS.
In tins section the intent is to provide the operator with an outline of
the essentials for readying the instrument for routine use. Appendix I of
this manual should be consulted for troubleshooting instructions.
I. FLOW SYSTEM :
This consists of the entire system through which nitrogen gas will
flow, from the common point oF entry at the exit of the filter drier
branciung to (I) the purge 1 inc running through the purge rotameter and
flow controller thence through the detector, and (2) the carrier flow
line running through the rotameters, the flow control lers and through
the colunri, thence through the transfer line into the detector.
It is essential that no leaks exist anywhere in the f1ow systeni.
Even 6 a minute leak will result in erratic baselines with the 1-I detector.
The Ni detector will be even more seriously affected. Leaks can be
detected by the application oF “Snoop” at all connections or by spraying
the connection with Freon ‘iS-l80 with the instruniont operatuig and
observing For recorder response. Spray short squirts close to’the con-
nection. Do not spray around the detector or injection port.
IL DETECTOR :
Tins subject is covered in detail later in Section 4,A,(3).
III. ELECTRF \IETER :
To insure proper daily operation of the unit, set the attenuators
to OFF position and zero the recorder. Set the output attenuator at xl
and observe the baseline. A steady baseline with less than 1% noise is
considered good. A check should occasionally be made of the electrometer
electronic zero. Instructions for doing this may he obtained from the
Electronics Shop in Perrine.
Zero and bucking controls should operate “smoothly” and should not
cause erratic recorder response.
-------
Revised 11/1/72 Section 4,A,(l)
-2-
Check the “maximum” polarizing voltage available. If at least
-130 vdc is not available on the rear panel, it is quite possible that
the power printed circuit board (P03) is not functioning properly and
damage or noisy operation will result from continued use.
l v. TBv1IPERA11J1 E PROGRAMMER :
The operator should be certain this unit is functioning properly.
When the unit is operating properly, the column oven temperature should
not show appreciable deviation. If the temperature fluctuation is
excessive, baselines will cycle and, in all probability, retention
measurements will be erratic.
In an emergency situation, a 10 amp variable transformer (Variac or
Powerstat) may be used as a temporary measure. Constant use of this
device is not advised as it does not operate on temperature demand, but
simply supplies a fixed voltage to the heating elements. Therefore, oven
temperature will vary with any changes in line voltage and room tempera-
ture.
V. PYR( vfEFER :
The batteries of this unit should be checked monthly to be sure they
are delivering full voltage under load. This can be done easily with a
voltmeter set on the 3-volt range and shunting a 1 megohin resister across
the voltmeter leads to constitute a load. If the voltage under this test
situation falls below the rated voltage for the battery, replace battery.
The battery contacts should also be cleaned by spraying with Freon MS-l80
and wiping with dry cloth. To prevent shorts, it is recoumended that
electrical glass cloth tape be wound around each end of the battery at
positions where the clamps hold the battery in place.
A hint of inaccurate pyrometer operation may be obtained by switching
to one of the 8 nused sensors and observing the readout. If the reading
is more than 5 C from room temperature, faulty operation is suggested.
This is suggested as a daily check to prevent straying gradually into
grossly inaccurate temperature readings. Before final readings are
made, gently finger tap the pyrometer frame in the area around the set
screw.
vi. MISCELLANE(XJS :
A. Septums - there are a number of different types available, ranging
from the inexpensive plain black (or gray) silicone rubber to the
sophisticated “sandwich” type selling at a significantly higher
price.
-------
Revised 11/1/72 Section 4,A,(1)
-3-
Excellent results have been reported on the blue silicone rubber
material marketed by Applied Science Laboratories as their
series.
The 13 nini precut septums are available in lots of 100 for $27 under
catalog number W-13. The same material listed at “Type lv” is avail-
able in sheets of 12” x 12” selling for $55. About 400 13-mi
septums can be cut from this sheet with a No. 9 cork borer making
the price per hundred septums about half that of the precut septums.
The above prices are based on the 1972 catalog.
B. Column “0” Rings:
Very satisfactory results have been obtained with Viton “0” rings.
One source of supply is:
John C. Shelly Co., Inc.
16 Mica Lane
Wellesley Hills, Mass.
C. Prepurified nitrogen gas shall be used for the D.C. mode of opera-
tion. This is piped to the instrument through a filter drier of
molecular sieve, 1/16” pellets, Linde type 13X. When the filter
drier is charged with fresh molecular sieve, the interior of the
drier should be acetone rinsed and the drier unit placed in a 130 C
oven for at least an hour. The bronze frit should be aceton 8 rinsed
and flamed. After filling, the unit should be heated at 350 C for
4 hours with a nitrogen flow 01 ca 90 mi/minute passing through the
unit. If the activated unit is to be stored for a period of time
before use, the ends should be tightly capped.
-------
Revised 11/1/72 Section 4,A,(2)
Page 1
GAS CFil OMATOGRAPHY-ELECTRON CAPIURE
COLUMNS
I. SPECIFICATIONS :
Column material shall be of borosilicate glass, 6 feet long, 1/4 inch
o.d., 5/32 inch i.d. As off-column injection will be used, one side of
the column shall be 1 inch longer than the other. The Swagelok nut,
ferrule and silicone “0” ring are assembled as in Figure 4. Complete
column specifications are given in Figure 11.
II. COUJMN SELECTION :
Of the three columns listed below, A and B, made up from precoated
packings from Perrine, will be the working columns in laboratories con-
ducting analyses on miscellaneous sample substrates. If blood is the
predominant sample substrate, a preferable combination of working columns
might be A and C.
A. 1.5% OV-17/1.95% QF-l - liquid phases premixed and coated on
silanized support, 80/100 mesh.
B. 4% SE-30/6% iiW-2l0 - liquid phases premixed and coated on silanized
support, 80/100 mesh.
C. 10% OV-210 - coated on silanized support, 100/120 mesh.
The three precoated packings are purchased in bulk from a commercial
supplier under rigid performance specifications, and each batch is care-
fully evaluated upon receipt at Perrine. Each packing is available to the
Community Studies Laboratories from Perrine in 25 gram units. A copy of
the evaluation chromatograms will be included with each shipment to the
field laboratories.
III. PACKING THE COUJMN:
Make certain the coU.niin is actually 6 feet long. A paper template
tacked to the wall is a convenient and quick means of checking this. For
off-column injection in the Micro-Tek Model 220, one column leg should be
1 inch shorter than the other.
With a china marking pencil, place a mark on the long colum leg
2 inches from the end. Place a similar mark 1-1/8 inch from the end of
the short leg.
Add the packing to the column through a small funnel, ca 6 inches at
-------
Revised 11/1/72 Section 4,A,(2)
-2-
a time, and bounce the column repeatedly on a seinihard surface. Rapid
tapping up and down the column with a wooden pencil will promote settling
of the packing. The packing is added until it reaches the mark on each
leg and it is found that additional tapping will not produce any further
settling.
NOTE : This operation should be done with great care, tapping the
column a sufficient length of time to be certain that no
further settling is possible by manual vibration. The use
of mechanical vibrators is not advised as the packing can
be packed too densely, thus introducing the possibility of
an excessive pressure drop when carrier gas is applied.
Pack silanized glass wool into both ends of the column just tightly
enough to prevent dislodging when carrier flow is applied. The glass
wool should completely fill the space from the top of the packing to the
end of the column.
NGtE : If the glass wool is manipulated by hand, the hands should be
carefully prewashed with soap or detergent, rinsed and dried.
This minimizes the possibility of skin oil contamination of
the glass wool.
IV. COLUMN CONDITIONING :
The column is conditioned, or made ready for use, in two operations,
(1) by heat curing, and (2) by silylation treatment.
1. Heat Curing
A Swagelok fitting is attached to the inlet port at the top of
the oven. This is comprised of a 1/4-inch Swagelok to AN adapter,
part number 400-A-4ANF, connected to a 1/4- inch male union, part
number 400-6.
Before assembling, the bore of the union raist be drilled out
with a 1/4-inch drill and burnished with a rat-tailed file so that
it will accept the 1/4-inch o.d. column glass.
The short column leg is attached to the above fitting, with the
end of the long leg venting inside the oven. The nut, ferrule and
“0” ring is assembled as shown in Figure 4. Make sure the nut is
tight, as the “0” ring will shrink during the curing period, thus
allowing carrier gas to escape.
-------
Revised 11/1/72 Section 4,A,(2)
-3-
NOTE : The outlet ports leading to the transfer line should be
sealed off during the conditioning period to prevent
traces of column effluent from seeping through to the
detector. This is easily done by assembling a 1/4-inth
Swagelok nut on a short piece of 6-mm glass rod with
ferrule and “O” ring.
2. Silylating Treatment
Treatment with a silylating compound such as Silyl 8 serves to
block active adsorption sites, particularly prevalent in a new column,
thereby somewhat improving efficiency and resolution characteristics.
The most drastic effect is in the improvement of endrin response and
the near elimination of on-column breakdown of endrin. Silyl 8 is
available in 1 and 25-mi septum capped bottles from the Pierce
Chemical Company, P.O. Box 117, Roclcford, Illinois 61105.
At the end of the prescribed heat curing period, adjust the
oven teilipset and carrier gas flow controllers to the appropriate
settings to give the approximate recommended operating parameters
for the given column. While the temperature is dropping, open the
oven door and, wearing heavy gloves, retighten the Swagelok nut
which will invariably loosen during heat curing. Close door and
allow oven temperature to equilibrate. Make four consecutive in-
jections of 25 .i1 each of Silyl 8, spacing the injections ca 1/2
hour apart. Allow an hour for the final injection to elute off
the column before proceeding.
NOTE : Syringe used for Silyl 8 injections should be used for
no other purpose, and should be rinsed immediately
after use to avoid damage.
V. EVAUJATION OF COLUMN :
Shut down oven and carrier gas flow, remove column from special
fitting, remove fitting from inlet port, and connect column to detector,
making sure that nuts are securely tightened. Replace Vykor glass in-
jection insert with a clean one and install a fresh septum. Make certain
that the stainless steel retainer for the insert is reinstalled with the
slotted end up. Upside down installation will permit the escape of
carrier gas. After septum nut is screwed down by hand, a little further
tightening with pliers helps insure gas-tight septum installation. Raise
oven temperature and carrier gas flow to the exact values given in Table 1
for the appropriate column. The oven temperature must be monitored by
some means other than the built-in pyrometer, either a precalibrated dial
face thermometer with the stem inserted through the oven door, or a
-------
Revised 11/1/72 Section 4,A,(2)
-4-
mercury thermometer down through an unused injection port. DO NOT
RELY IVHOLLY (1 IHE INSTRUMENT v 9 c €rER .
Check the carrier gas flow rate using the sidearm buret device
sketched in Figure 4 (a) attached to the purge exit of the detector.
DO NOr RELY (}4 Il-tE INSTRUMENT ROFN4EtER in adjusting the carrier flow.
Allow an overnight period for complete equilibration of the column-
detector system at normal operating parameters of temperature and carrier
flow.
NJTE : If two columns are connected to the same detector,
the carrier flow to the column not in use should be
shut off while the flow rate through the column in
use is being measured. Likewise, the purge line
flow controller should be closed. The unused column
flow should also be kept at zero while determining
the background current.
After overnight equilibration, recheck the oven temperature and
carrier gas flow rate. You are now ready to assess the performance
characteristics of the column, and this should definitely be done before
attempting to use the column for routine work.
Run a background current profile at the normal operating parameters
for the given column, with the purge line flow controller set at 4.
Detailed instructions are given under Subsection 4,A,(3) DETECTOR. The
BGC profile is particularly important in providing an assessment of
detector behavior as affected by the column. It is presumed that a BGC
profile was run on the same detector within a few days from the time of
the present profile, so that the expected level of background current
may be compared to the level obtained in the present test. If the
present level falls far short of that expected, either the detector
itself is faulty or the column is exerting an adverse effect on the
detector. The cohnnn influence may be roughly determined by allowing
several hours n re for equilibration and repeating the BCC profile. If
an increase in BC current is obtained, additional checks are made until
no further increase is noted. A typical BGC profile is shown in Figure 5.
If the detector foil is new and the BC current is at a high level,
it is acceptable practice to set the polarizing voltage at 85% of the
full BGC profile. However, this practice is not reliable with an older,
partially fouled detector. A more reliable method is to i-wi a polariz-
ing voltage/response curve as described in Subsection 4,A, 3) OPTIMUM
RES1 )NSE VOLTAGE. A polarizing voltage/response curve is shown in
Figure 6.
-------
Revised 11/1/7 2
Section 4,A,(2)
-6-
minutes from the Perrine value, it is indicated that (1) one or
both operating parameters are off, or (2) column is not 6 feet
long, or (3) the density of the packing is not comparable.
a. If the absolute retention is less than the Perrine value
by n re than 2 minutes, the oven may be running too hot or
the carrier flow may be running too high, or both; the column
may be packed too loosely, offering less surface area of
coated support; the column may not be a full six feet in length.
b. Conversely, if the absolute retention value is high, the
indications may be: a low column temperature, a low carrier
flow, a column packed too densely, or sane combination of two
or nxre of these factors.
N = 16
R 8x
R 4x
a T
x
z
(X
(at 1/4-inch chart speed)
(at 1/2-inch chart speed)
Peak A
(A].drin)
PeakB
(p,p’—DDT)
z
x
‘—. Injection Point
where
N = column efficiency in total theoretical plates.
-------
Revised 11/1/72 Section 4,A,(2)
-7-
Ra = absolute retention, in minutes, for peak B.
= retention ratio, relative to aidrin, for peak B.
x,y,z = measured in millimeters.
Based on the chromatograms of the evaluation mixture, a decision
generally can be made as to the potential quality of the column. If,
after making slight adjustments in the carrier gas flow rate, character-
istics of efficiency, absolute retention arKi peak resolution do not com-
pare reasonably well with the chromatograins and data furnished by Perrine,
it is inadvisable to proceed further with the column. For example, if an
efficiency value of over 2,700 theor. plates cannot be obtained on a new
column, it is unlikely that the column would ever improve to much over
3,000 T.P. On the other hand, if the new column yielded 3,000 T.P., it
is probable that it would improve to 3,300 or 3,500 T.P. after becoming
“seasoned”.
Assuming that a favorable indication is obtained from the mixture
chromatograms, the next evaluation step is to determine the compound
breakdown characteristics of the column. This may be done by injections
of puie p ,p’ -DDT in sufficient concentration and appropriate attenuation
to produce peak heights of 50 to 60% f.s.d. The DDT breakdown should not
exceed 3%. The endrin response and breakdown characteristics may be
determined similarly. This breakdown should not exceed 10%.
NOTE : This breakdown percentage is calculated by adding up the
peak areas of main peak and breakdown peak(s). This
value divided into the peak area value for the break-
down peak(s) x 100 is the breakdown percentage.
VI. MAINTENANCE AND USE OF COUJMN :
A column that is used and maintained properly should provide service
for many months. It is difficult to make precise time estimates because
of the variable in different laboratories. Data from a column performance
survey showed one laboratory using the two working columns 3-1/2 months
for an estimated nui±er of 1,300 injections of predominantly fat extract.
Another laboratory indicated their two columns to be at least a year old,
and each had been subjected to an estimated 3,000 injections of blood
extract. Neither laboratory’s columns gave any indication of deteriora-
tion. In fact, the laboratory injecting fat predominantly was included
in the group showing superior overall column performance.
The Vykor glass injection insert used iii off-column injections
serves as a trap to prevent a high percentage of dirty material from
-------
Revised 11/1/72 Section 4,A,(2)
-8-
befouling the front end of the column. However, if this insert is not
changed frequently, column performance characteristics can be signif i-
caritly altered. When a sufficient amount of residue collects in this
insert, lowered efficiency, compound breakdown, peak tailing and de-
pressed peak height response become evident. The changing of this
insert should be on a daily basis if sample extracts of any kind are
being injected.
The effects of Silyl 8 conditioning do not persist indefinitely.
Any laboratory with an interest in emirin detection may find that
resilylation may be necessary at intervals to be determined by weekly
monitoring for breakdown.
A certain amount of extraneous matter is eluted through the glass
insert am:! lodges in the glass wool plug at the column inlet. Indica-
tions from the survey mentioned above were that those laboratories
changing the glass insert daily could go for long periods of time
without changing the column plug. Daily compound conversion monitoring
provides a constant check on the need for changing the glass wool plug.
When the cohmin is idle overnight or over weekends, a low carrier
flow of ca 25 ml per minute through the column is advised. Simul-
taneously, a purge flow of ca 25-30 ml through the detector is also
advised. If a column is out of the instrument longer than two or three
days, reconditioning is advised wherein the column is not connected.
to the detector, but is allowed to vent into the oxen under a carrier
flow of ca 60 ml per minute at a temperature ca 25 above the prescribed
operating temperature.
An erratic and noisy baseline can indicate leaks in the column
connections, or at some other point in the flow system, starting at
the injection septum and on to the detector inlet. If the baseline
has been stable and first became erratic upon installation of a new
column, the probability is indicated of loose column connections.
If any laboratory has trouble obtaining performance characteristics
equal to those indicated by the chromatograins and data furnished with
each bottle of precoated packing, every effort should be made to pinpoint
the trouble and correct it. If the foregoing instructions are followed
with no deviations , trouble should not be experienced. If you are
unable to overcome the difficulty, write or call the Perrine Quality
Control group outlining your problem, including with your letter com-
pletely identified chromatograms of (1) the special evaluation mixture
and (2) a chromatograrn of pure p,p’ -DDT at a concentration and attenua-
tion producing a peak of 50-60% f.s.d. Give all instrumental and coltznn
operating parameters.
-------
Revised 11/1/72 Section 4,A,(2)
-9-
It should be borne in mind that before the precoated packing
material of any batch received in Perrine is mailed to the field
laboratories, a very thorough evaluation is conducted on a composite
sample of all bottle units received. It is therefore a near im-
possibility for a laboratory to receive poor column packing. Any
trouble experienced will almost certainly lie in some adverse con-
tribution made by the op’.ratofin preparing and/or conditioning and/or
operating the column.
VII. MISCELLANECLJS NOTES :
1. The carrier flow through the unused column should not be carried
any higher than is required for positive pressure. Detector response
is seriously affected by running both columns simultaneously at normal
operating velocity. For instance, in a series of observations with
a pair of nearly identical low-load columns in the oven, the peak
height response for aidrin is reduced ca 25 when the off-column is
carried at 70 mi/mm., the same flow at which the on-column is being
operated.
2. An obvious, but sometimes overlooked, point arises when only one
column is installed in the oven. The transfer line commonly used is the
dual type that conveys column effluent from the two-column outlet ports
to the single detector. When one column is removed, its outlet port
must be plugged or else a riassive leak will be created. One easy means
of doing this is to slip swagelok fittings and an “0” ring on the er I
of a short piece of 1/4” o.d. glass rod arid install in the unused outlet
port.
-------
Revised 11/1/72 Section 4,A,(3)
GAS CHR( V1ATOGRAPHY-ELECTRON ( AJ 11JRE Page 1
DETECTOR
Straight D.C. polarizirw voltage shall be supplied to the detector from
either an outboard power supply unit or from a strip on the back of the elec-
trometer. Provided the column and all electronic circuits in the various
modules of the instrument are functioning properly, the degree of sensitivity
in the electron capture mode relate most probably to the condition of the
interior of the detector. As radioactivity in the foil decreases, so does
sensitivity of the system. Measurement of the background current gives an
indication of the condition of the detector and should be run on a new or
overhauled detector. aibsequent periodic measurements should be made to
provide up-to-date information on the performance of the detector as in-
fluenced by the condition of the foil or by any other effects such as column
bleed or contaminated carrier gas.
I. BACKGRCXJND SIGNAL PROFILE :
1. Zero recorder arid electrometer in the normal manner.
2. With a well seasoned column such as OV-17/QF-l in the instrument,
set input attenuator on 10 and output attenuator on 256.
NOTE : The given attenuation values apply to electrometer
model E2. If the dual channel, solid tate unit is
used an equivalent setting would be 10 x 128.
3. Set column and detector temp. and carrier flow rate to the levels
prescribed for the column in use. Apply ca. 70 mi/mm. of purge gas.
4. Set CUTPUT POLARITY switch to the polarity opposite of that used in
normal operation.
5. Reduce polarizing voltage to zero by control on power supply unit
or in front of electrometer and adjust bucking control of electro-
meter to permit zeroing of pen on chart paper.
6. Set chart speed on 1/4 inch per minute, start cihart drive, and
allow about 1/2 inch horizontal trace.
7. Advance polarizng troltage control to 5 volts and allow sufficient
time for trace to level off, then repeat for 10, 15, 20, 25 volts
and so on, until a voltage value is reached which produced no
further recorder deflection.
Generally, a new detector or one with a new tritium foil should be expected
to produce a response of 60-80 full scale deflection. With aging, as the
response level approaches about 30% f.s. deflection, a replacement of the foil
-------
Revised 11/1/72 Section 4,A,(3)
-2-
is indicated. Figure 5 shows a background signal profile on a detector in
constant use for 2 months. At the time of original installation, the back-
ground signal profile produced 68% f.s. deflection.
II. OPTIMUM RESPONSE VOLTAGE :
It is important that a correct polarizing voltage be supplied to the
detector to achieve maximum peak response with minimal overshoot. An incorrect
voltage can result in (1) full potential sensitivity of the detector not being
utilized, or (2) a strong overshoot in the peak downstroke which makes for
difficult quantitation of peaks. The opti.mi.on response voltage is determined
as follows:
1. Upon completion of the background signal profile, reset ( JJTPUT
POLARI1Y switch back to normal operating position and set polar-
izing voltage control to the voltage that produced ca. 60% of
the total BGC profile.
NJTE : If you are fairly certain that the optimum polarizing
voltage will fall in some fairly high range, i.e.,
20-25 volts, time can be saved by starting about 7
volts under the expected optimum polarizing voltage.
2. Set oven and detector temperatures and carrier flow rate to the
prescribed operating levels for the column in use, and allow
system to equilibrate.
3. Set attenuators on the values appropriate for the condition of the
detector.
4. Adjust bucking control to zero recorder pen.
5. Inject an aidrin standard in quantity known from current operation
to produce a peak about 1/2 full scale at the attenuation being used.
? JTE : The volume injected must be carefully measured and
should notbe less than 5 i.’ 1 .
6. Repeat injection to obtain peaks from increments of 2.5 volts, i.e.,
15-17.5-20, etc., until two peaks show less height than that obtained
on the highest peak.
NOTE : C casiona1ly a new detector will require only around
7 volts, and it may be found that the 2.5 volt inter-
vals result in too much change in response. In this
case, it may be advisable to use 1 volt intervals to
set up the response curve.
-------
Revised 11/1/72 Section 4,A,(3)
7. Taking the exact peak height values, measured in millimeters,
plot a peak height vs voltage curve on linear graph paper (see
Figure 6). Usually the optimum polarizing voltage is that which
produces the next to greatest peak height. However, if appreciable
peak overshoot is evident at this voltage, it may prove desirable
to set polarizing voltage slightly higher to minimize overshoot
at some expense in response. Arrow in Figure 6 indicates the
voltage selected in this particular case.
III. DETECTOR LINBARIT ’ :
In making chromatographic runs for quantitati.on, it is mandatory that
compound concentration be within the linearity range of the detector. As this
characteristic may change with the age and use of the detector, standard curves
for pesticides of interest should be run periodically to provide up-to-date
linearity information. In most cases, operation at an output attenuation
setting of 10 x 8 (or 16) on the E2 electrometer of io2 x 8 (or 16) on the
SS will preclude the possibillcy of violating the linear range of the detector.
If samples are diluted so that quantifiable peaks are produced at these set-
tings, the large errors resulting from calculations based on non-linear
response can be avoided.
-------
Revised 11/1/72 Section 4, A, (4)
Page 1
GAS CI-1ROMATOGRAPHY-ELEO RON CAPTURE
CHRCi4ATOG1 APFW OF SAMPLE
When the chromatographic system has been idle for a number of hours,
such as overnight or over the weekend, it is generally necessary to “prime”
the column before quantitation may be attempted. The first early morning
standard injection will frequently show relatively poor response. The
second and third injections will usually improve the response to a constant
level. This “priming” may be done by successive injections of a dilute
working standard mixture or it may be accomplished by one injection of a
highly concentrated mixture. One laboratory has reported good success with
the latter system, and if other laboratories obtain comparable results,
considerable daily “priming” time may be saved. The suggested priming
mixture is given below and the concentration values in nanograms per
microliter.
Lindane 0.5 Dieldrin 1.0
-BHC 1 5 o,p’-DDT 1.5
Aidrin 0.5 p,p’-DDD 1.5
Hept. IEpox.----l.0 p,p’-DDT 1.5
p,p’-DDE 1.0
Forty microliters of this mixture is injected. If this one-shot system is
used, a special syringe should be set aside and used solely for this purpose.
Under no circumstances should the same syringe be used for routine injections.
in the early morning the priming may he conducted while the other daily
instrument checks are being made. If more than one column in the instrument
is to be used, the priming may be done simultaneously. After the priming
mixture has eluted of f t1 e columns, the carrier flow should be carefully
adjusted for the working column using the bubble device shown in Figure 4 (a).
The chromatogTaph should now be ready for the first working standard injection.
A sample extract concentration of 10 ml from a 5 gram sample contains the
tissue equivalent of 0.5 milligrams per microliter. A 5 iil injection of this
extract (2.5 milligrams of sample) into an F. C. detector of average sensi-
tivity should easily produce uantifiab1e peaks at pesticide concentrations
of at least 0.1 ppm provided that instrumental attenuation is appropriately
adjusted.
1. With the working column in the instrument, adjust column
and detector parameters as prescribed in Table 1. If
another column is in the oven, set a positive carrier gas
pressure of ca. 20 mi/imin., on this alternate column. Set
attenuation at an estimated appropriate sensitivity.
-------
Revised 11/1/72 Section 4, A, (4)
-2—
NOTE : The specified GLC instrument has a high sensitivity
potential provided that all modules are functioning
properly. It is i.mportant to take full advantage of
this potential by avoiding high output attenuation.
For instance, some workers atte]TqDt to operate the
electroneter at attenuations of 10 x 32 and 10 x 64,
adjusting their sample concentration and standards to
fit this attenuation range. With a new detector foil
this high attenuation may be necessary, but as the BCC
decreases, this practice, while resulting in a stable
looking baseline, requires injections of relatively high
sample concentration to produce quantifiable peaks.
This tends to promote faster fouling of column. and
detector than would result from injections containing
less sample material. This is particularly important
when injecting eluate from the 15% ethyl-ether/pet.
ether extract from fat. If all instrumental modules are
functioning properly it should be possible to obtain a
noise level not exceeding 2% of full scale at an
attenuator setting of 10 x 8. If 10 x 8 should not
meet this specification, then 10 x 16 should definitely
produce an acceptable noise level. In the event that
the electrometer noise level at 10 x 16 should exceed
2% full scale, the electronics shop at Perrine should
be contacted.
2. Inject 5 .i1 of sample extract for an exploratory run, followed
by one or more standard mixture runs. Evaluate the chromatogram
using the appropriate relative retention/column temp. table to
make tentative peak identifications.
NOTE : By using the table, the operator can determine at this
point whether the actual column temperature agrees with
that shown on the instnmient pyrometer readout. Com-
parison of the calculated RRR for p,p t -DDT with the values
given in the table will advise the operator if temperature
adjustment is needed.
3. If the data obtained from step 2 show one or more probable
pesticide peaks, it is desirable at this point to run a chromato-
gram of a quantitating standard mix known to contain compounds
of proper concentration to fall within the linearity range of
the detector and also produce peaks comparable in size to those
obtained from the sample chromatog-rain. This thromatograin should
provide information concerning the degree of additional dilution
of the sample extracLto produce peaks of the higher concentra-
tion pesticides approximating those of the standard mix. It may
also be possible at this point to quantitate some of the peaks
obtained in the exploratory run.
-------
Revised 11/1/72 Section 4, A, (4)
-3-
NOTES : 1. It is desirable to use standard mixtures with the
component pesticides at three concentration levels.
This will enable the operator to select a mix whose
concentrations will fall within the linear range of the
detector and have a peak size comparable to the unknown
peaks.
2. The height of sample and standard peaks should
preferably vary by not more than 25%. It is sometimes
alleged that this point is of no consequence provided
both standard and sample are within the linear range of
the detector. In theory this is true, but like many
theoretical postulations the fact does not necessarily
follow the theory. For example, the theory does not take
into consideration minor response variations arising from
injection error and/or instrumental sources. It can
be easily demonstrated that a response variation of as
little as 3 rTnn in peak height can result in a final error
of 20 to 25% when a 13 mm sample peak is calculated
against a 130 mm standard peak.
3. Electrometer attenuation should be adjusted to obtain
a minimum sensitivity level of a peak of 50% f.s.d.
resulting from the injection of 100 picograms of aidrin.
4. Prepare an estimated sample extract dilution and run another
chromatogram or. a S iil injection.
NOTE : It may be necessary to run several sample extract
chromatograms of various concentrations and/or injection
volumes to achieve reasonable approximation of peak sizes
with those of the standard mixture.
5. At this point, detailed evaluations are made of all diromato-
grams. If there is reason to suspect any peak identification
or quantitation, instrua nta1 controls should be switched over
to the alternate column for further scrutiny. The isomers of
BI-IC, o,p’-DDE, and o,p! DLTJ’ frequently pose identification
problems. If such identification problems are present and
cannot be confidently resolved by any of the three prescribed
columns, further confirmatory work is required by electrolytic
conductivity detection and/or by T.L.C.
-------
Revised 11/1/72 Section 4, A, (5)
Page 1
GftS Q-IRCNATOGRAPFW-ELECrRON CAPTURE
JANfITATION AND INTERPRETATION
There are several methods for quaiititating chroinatographic peaks.
While we are not partial to any particular method, it is desirable in a
syst n of laboratories providing data to a central point that some degree
of uniformity be specified.
The preferred method of calculation is sanewhat dependent on peak
shape. The major categories of peak shapes are: (1) Tall, narrow and
symmetrical, generally illustrated by a p ,p’ -DDT peak from a clean extract,
(3) Overlapping peaks where the overlap is estimated not to obscure the
peak height, (4) Unsy netrical peaks such as are cc muonly encountered in
an uncleaned extract.
Broadly speaking, quantitation methods recomnended for the various
types of peaks are:
I. PEAK IIEIGI-if :
A. Early eluting peaks, tall and narrow.
B. All peaks on the trace where there are no obscuring overlaps and
where peaks are tall, symmetrical and fairly narrow (See Figure 7).
II. PEAK HE I GIlT X WIDTH AT l-IALP HEIGHT :
A. Separated, syiimetrical and fairly wide peaks (See Figure 8).
III. TRIANGUlATION OR JJ’ffEGPATION :
A. Separated unsynnnetrical peaks, or peaks on sloping baseline
(Figure 9). Triangulation should not be attempted on very
narrow peaks. Extreme care must be taken in the construction
of the inflectional tangents and in measurements.
IV. INTERPRETATION :
Although this subject is listed last in this section devoted to
E.C., GLC, it is far from being the least important. An excellent
performance in all other areas may be nullified if the chromatograms
are not properly interpreted.
The electron capture detector, being non-specific, responds to any
electron capturing materials in addition to pesticides in the final
extract being chromatographed. For this reason, the task of interpre-
tation is one requiring careful study of the data and the application
of sound judgement. The presence of chromatographic peaks which pre-
cisely match the absolute and relative retention values of those of
-------
Revised 11/1/72 Section 4, A, (5)
-2-
certain pesticides does not necessarily indicate the indisputable
presence of those pesticides. For example, it is not uncommon to
observe peaks from human tissue extract with retention
characteristics precisely the sane as ci-EFIC and/or o,p’-DDE.
However, confirmation by ancillary techniques has never supported
the E. C. indications. In one instance n thyl parathion was
reported in a blood sample. Had the individual conducting the
interpretation exercised sound judgement, it should have been
inin diately apparent that the presence of parathion, per Se, in
body fluids other than gastro-intestinal would be a near
impossibility.
The chromatographer must recognize that quite often peaks are
obtained from a given sample substrate on one GLC column by E.C.
detection, the retentions of which strongly suggest certain
pesticidal compounds. If, based on experience, these particular
compounds are not likely to be present in the sample material,
song further confirmation is required. This may be done by (1) using
an alternate column via E. C. detection, (2) applying electrolytic
conductivity detection (3) thin-layer chromatography or (4) chemical
derivatization.
-------
Revised 11/1/72
Section ,A,(6)
.5% OV-17/1.95% QF- )
40/a SE-30/6°1 0 0v-210
10% OV-210
a
I .
I
w 0
00 — 0
0 0 0 0
I 0 I
Q. - a.
0
Figure 1
I)
I
a
C
W 0
0 0 C
0 0
0. ‘a. !
0
0
a.
a.
I-
0
a
a.
Figure 2
a
* Iu
-a.
—
0 0
3 0 0
0 ) 0
0 a. a.
_0. 0.
0
Figure 3
-------
Revised 11/1/72 Section 4, A, (6)
Fig. 4 — ColunTlto Port Assembly- Exploded View
0- RIng Rstaln.r
Glass Column
Polyeth. TubIng,1/8’ o.d.
Tygon TubIng
Rubbir Bulb , 30 ml —
Snoop”
Silicon. ORing
Back F.rru l. ____
Nut
Fig.4(a) — Bubble Flowmeter
Bur.t, 50 ml
-------
Rev1 d 11/1/72
Section 4, A, (6)
H H
‘vu—
I / b V-’4 . Cc/ ss .3oC
_______ .7 ;çp _.,I2o . ‘.:r(_
.
——
.
.
..
,
.
-;
$ - I - 0 - - -, - -
_ __
. k 7 ro d J / p#oA 4 .0
H-
H
_L _ ;j 1 f
—o ------
________-. 0—-—
-------
ReviSed 1IIiII Section 4, A, (6)
.C.
________
•0 3%OV-/
, ,o’ 7
-P 4. e
I. *
‘I
/ __ __ _ __
-. I _____ -
; ,
- -- -----—
Z$v /OV /lrr i r ..! 2O
______ __________
-------
Revised 11/1172
Tc’ction 4, t\, ( )
FIGURE 7
Peak height = CD
FIGURE 8
Peak Area - CD x AB
/
FIGURE 9
Triangulation
A = 1/2(FG)(JH)
-------
Revised 11/1/72
Section 4, A, (6)
SKSc..6 ef
Co/a, ,, ,‘, 1 o7’r-
C 0 / 11 , 2 lJeS/O#
4e cdboros;/IS 9/ 5S)
“c’ .d., %a s, ’. qç
M, . ,373OO oP
8a#jo, r/ n
S S ‘& e. d
7A , Z .s.# zec1 Z ac-
7 r “ hk N4. 7
1!
H3”H
3,
‘ S
2’ //
r/C. ,, 4P p’/ Pon 741
,‘cc
r 33 ,J_7
-------
Rcvisç d 11/1/72 Sectioi 4, , (6)
Table 1. Conditioning, Operation Parameters and I crfoniiance Lxpcctations for 0-ft. x 1/4-iiicn o.d. coliaiuis
of Precoated Packings. 3% DEGS Included Solely as a Confirmatory Coiwin, not for Routine USC.
1.5% OV-17 4% SE-30
Parameters - -210 iGo OV-210 3 o LthIJS
Liquid phase(s) Silicone OV-17 Silicone SL-30 (JV-210 IJEGS
Silicone DC QF-l Silicone DC QF-l Trifluoropropyl btabilizeci
(FS1265) (FS1265) Silicone Iiietnylene
Ulycol
Succina te
(Analabs L4)
Solid Support Chromosorb W, H.P.
100/120 mesh
Ghromosorb W, H.P.,
80/100 mesh
Lhroinosorb W, H.P. Gas-Cflrom P
100/120 mesh 0/100 inesn
Heat Curing Temp °C 245
Time Hours 48 (minimum)
245
72 (minimum)
245
48 (minijm m)
235
20 (exact)
O perat ng
Temp., C 200
200
280
195
tector Temp
C (tritium) 205
205
205
205
Carrier Flow mi/mm. 50-70
70-90
45-60
70-90
Elution Time for p,p t -DLff
Approx (nun) 16-20
16-20
1c-20
16-20
Fxpected Minimum
Efficiency (Total theor. 3000 3000 3000 2800
plates in 6-ft.. column
basis p,p -DDT)
-------
I I I I I I I
170 174 178 182 186 190 194 198 202 204
Retention ratios relative to aldrin. of 48 pesticides on a column of 1 5%O V-li/i 95%OF -i at temperatures
from 170 to 204°C, support of Chromosorb W,H P - 100/120 mesh, election capture detector, tritium
source, parallel plate, all absolute retentions measured from injection point Arrow indicates optimum
lumn operating temperature with carrier flow at 60 ml per minute
l/ij/71 Table 2(a) Sect3on Li,A,(6)
1.5%OV-17/1.95% QF-1
Column Temperature, °C.
170 174 178 182
I I I
186 190
I
194
I
i
198
i
202
I
204
i
Phosdrin
032 032 032 032 032 032 032 032
032 032 033 033
033
033
033
033
033
033
044 045 045 045 045 045 045 046
046 046 046 046
04b
047
047
047
047
047
2.4 D(ME)
048 048 048 049 049 049 050 050
050 050 051 051
051
051
052
052
052
052
Thimet
048 048 049 049 050 050 050 051
051 052 052 052
053
051
054
054
054
055
a-BHC
054 054 054 054 055 055 055 055
055 055 055 055
056
056
056
056
056
056
CDEC
056 056 056 056 056 056 056 056
056 056 058 056
056
056
056
058
056
056
2.4-D( IPE)
070 010 070 070 069 069 069 069
069 068 068 068
068
067
067
067
067
067
Simazine
069 069 069 068 068 068 068 068
068 068 067 067
067
067
067
067
067
066
Atrazine
067 067 066 066 066 066 066 065
065 065 065 065
065
064
064
064
064
064
Diazinon
066 067 067 067 067 067 067 068
068 068 068 068
068
0b9
069
069
069
069
Lindane
076 076 076 076 075 075 075 075
075 074 074 074
074
073
073
073
073
073
2 4.5-1) ME)
082 082 082 082 081 081 081 081
081 081 081 060
080
080
080
080
080
080
f3BHC
086 085 Oab 084 084 084 083 083
082 082 081 081
080
080
079
079
078
078
2.4-D(BE)l
097 097 097 096 096 096 095 095
094 094 094 093
093
093
092
092
092
091
6BHC
082082 082 082 082 082 082 082
094 094 093 092 092 091 090 090
082 082 082 082
089 088 088 087
082
087
082
086
082
085
082
085
082
084
082
083
Heptachlor
2,4.5.T(IPE)
103102 101 101 100 099 099 098
097 096 096 095
094
093
093
092
091
090
2.4-D(BE)uI
1 02 1 02 1 01 101 I 01 1 01 1 01 1 00
I 00 1 00 1 00 1 00
099
099
099
099
099
098
Dichlone
117 116 115 114 113 111 110 109
108 107 106 105
103
102
1 01
100
099
098
Dimethoate
100 100 100 100 100 100 100 100
1QQ 100 100 100
100
100
100
100
100
100
Aldrin (REFERENCE)
117 116 116 115 114 114 113 112
112 111 110 109
109
108
107
107
106
105
Ronnel
1 41 1 40 1 39 1 38 1 36 1 35 1 34 1 33
1 32 1 31 I 30 1 29
1 28
1 27
1 26
1 25
1 24
1 23
1 Hydroxychlordene
171 169 167 166 164 162 160 1 59
157 1 55 153 152
150
1 48
1 47
145
1 43
1 41
M Parathion
1 70 1 69 1 68 1 67 1 66 1 65 I 64 1 63
1 62 1 61 1 59 1 58
1 5/
1 56
1 55
1 54
1 53
I 52
Heptachlor Epoxide
2 07 2 04 2 01 1 98 1 95 1 92 1 89 1 87
1 84 1 81 1 78 1 75
1 72
1 69
1 66
1 63
1 60
1 57
Malathion
182 1 80 1 781 76 — 1 74 1 72 1 70 1 68
1 66 64 1 62 1 60
1 68
1 56
1 54
1 52
1 50
1 48
Dacthal
215 213 211 209 207 205 202 200
198 196 194 191
189
187
185
183
181
179
Dyrene
214 212209 207 205 203 201 198
196 194 192 190
188
186
1 84
182
1 79
1 77
o .p’-DDE
220 2 18 2 16 2 14 2 12 2 10 208 206
205 203 201 1 99
1 97
1 95
1 93
1 91
1 89
1 87
Chlorobenside
232 228 225 222 2 19 2 16 13 209
220218 216 2 15 213 211 210 208
206 203 200 - 197
206 205 203 201
- 1 93
200
1 90
198
1 87
1 97
1 84
195
1 81
1 93
1 78
1 91
E Parathion
Thiodan I
275 272 268 264 261 258 254 251
247 243 240 237
233
230
227
223
220
217
P.P”DDE
297 293 288 284 279 275 271 266
262 257 253 249
244
240
235
231
227
222
DDA(ME)
327 322 3 18 3 13 307 304 300 295
291 286 281 277
273
208
263
259
254
250
Captan
3 32 3 27 3 23 3 18 3 13 308 3 04 2 99
2 95 2 91 2 87 2 82
2 77
2 73
2 68
2 64
2 59
2 55
Folpet
280 277 275 272 269 267 264 261
259 256 253 251
248
245
2 43
240
237
2 35
Dieldrin
352 346 341 335 330 3_24 319 314
334 329 325 320 315 311 306 301
308 303 298 292
297 292 288 283
287
277
282
274
276
269
271
265
266
260
260
256
Perthane
o.p’DDD
398 394 388 383 377 371 366 360
354 348 343 338
332
327
321
316
310
304
o,p’-DDT
347 34.3 340 3 36 333 329 3 26 3 22
326 323 3 19 3 16 3 13 309 306 3 03
3 18 3 15 3 12 3 08
300 296 293 290
304
287
301
283
2 97
280
293
277
290
274
287
2 70
Endrin
Kepone
465 457449 441 433 426 418 410
402 394 387379
371
364
361
348
340
332
p,p’DDD
445 439 434 428 423 417 411 405
399 394 388 382
376
371
365
359
354
348
Thiodan II
61 597 585 573 561 549 536 524
512 600 488 476
464
452
440
428
416
404
Ethion
557 548 539 529 520 511 501 492
483 474 464 455
448
436
427
418
409
400
p,p’ DDT
64 62 61 599 568 576 564 552
540 528 516 504
492
480
466
456
444
432
Trithion
8079 77 75 74 72 71 69
67 66 64 63
61
595
560
562
548
534
Dulan I
77 76 75 73 73 71 70 69
124 121 118 116 113 110 107 104
94 92 90 88 86 64 82 80
68 67 66 65
101 98 95 93
78 76 73 71
64
90
68
63
87
67
62
84
65
61
81
63
60
78
61
585
75
588
Mirex
Methoxychlor
Dilan II
169 165 161 157 153 149 145 141
137 133 129 125
121
117
113
109
105
102
Tedion
240 234 228 222 216 210 204 198
192 186 180 174
168
162
156
150
144
138
Guthion
-------
Retention ratios, relative to aidrin, of 48 pesticides on a column of 4%SE 30/6%OF 1 at temperatures
from 170 to 204°C, support of Chromosorb W,H P , 80/100 mesh, electron capture detector, trutium
urce, parallel plate all absolute retentions measured from injection point Arrow indicates optimum
lumn operating temperature with carrier flow at 70 ml per minute
1/ it/ il
Table 2(b)
4%SE-30/6%OV -2J0
Column Temperature, °C.
Section Lj,A,(b)
182
I I
029 029 030
040 041 041
04.4 045 045
044 045 045
046 046 047
55_ 55 055
053 053 053
056 056 056
059 059 059
058 056 057
065 065 065
058 059 059
082 082 082
066 067 067
170 174 178
027 028 028 028 029
039 039 039 040 040
043 043 043 044044
042 043 043 044 044
044 044 045 045 046
054 054 054 054 0.54
052 052 052 05,3053
055 055 055 055 055
060 060 060 060 059
0 4 054 055 0.55 055
066066 066 066 065
057057 057 058 058
085 084 084 0.83 083
065 065 066066 066
t 40 080 081081 081 081 081 082
089 089 088 088 087 087 087 086
098 097 096 095 095 094 093 093
082082082082 082 082 082 082
096 095 094 094 093 093 092 091
186 190 194 198 202 204
I I
030 030 030 031 031 031 032 032 032 033
042 042 042 043 043 043 044 044 044 045
045 045 046 046 046 046 047 047 047 048
046 046 047 047 048 048 048 049 049 050
047 048 048 048 049 049 050 050 050 051
055 055 055 055 055 055 055 055 055
053 053 053 054 054 054 054 054 054 054
056 056 056 056 057 057 057 057 057 057
059 059 059 058 058 058 058 058 058 058
057 057 058 058 059 059 059 060 080 061
065 065 065 065 064 064 084 064 064 064
059 059 060 060 060 060 061 061 061 061
081 081 080 080 079 079 079 078 078 077
067 067 068 068 068 069 069 069 069 070
082 082 082 082 082 083 083 083 084 083
086 085 085 085 084 084 083 083 083 082
092 091 091 090 089 088 088 087 086 086
082 082 083 083 083 083 083 083
091 090 089 089 088 087 087 086
100 100 100 100 100 100 100 100
096 095 094094093 092 092 091
103 103 103 103 102 102 102 102
100 100 100 100 100 100 100 100
098 097 096
103 103 103
101 10100 099 098
104 104 104 103 103
1 491 48 1 47 1 46 1 45
1 53 1 53 1 52 151 I 50
1 70 1 68 166 164 1 63
164163 161 160 159
153 152 151 150 149
167 166 165 163 162
162 161 159 158 157
209 207 205 202 200
199196 197 195 194
216 214 231 209 207
227 225 222 219 216
227 225 223 221 219
222 220 218 216 214
243 241 239 237 235 233 231 229 227
243 240 237 233 230227 224 220 217
083 083
085 085
100 100
090 090
102 102
133 132
142 142
140 138
143 1 41
1 35 134
145 143
144 143
174 172
178 176
180 177
181 178
192 190
144 143 142 141 140 139 138 137 136 135 134
150 149 148 148147146 146 145 144 144 143
161 159 157 155 153151 149 148 146 144 142
157 156 154 153 152 151 149 148 147 145 144
148 146 145 144 143 142 141 139 138 137 136
160 159 157 156 155 153 152 150 149 147 146
156 155 154 153 152 151 149 148 147 146 145
198 196 194 191 189 187 185 183 180 178 176
1 93 1 91 1 90 1 89 187 1 86 1 85 1 83 1 82 1 80 1 79
205 202 200 198 195 193 191 189 lSb 184 182
213 210 20? 204 201 198 196 193 190 187 184
216 214 212 210 207 205 203 201 199 196 194
212 210 208 206204202 199 197
Phoedrin
2,4 D(ME)
Thimet
a BHC
CDEC
2,4 D(IPE)
Sumac me
Atrazine
Diazinon
Lindane
2,4,5 .T(ME)
/3 BHC
2,4-O(BE)l
6.BHC
Heptachior
2,4,5-T( IPE)
2,4-D(BE)ll
Dichlone
Dimethoate
Aldrin (REFERENCEI
Ronnel
1 Hydroxychlordene
M Parathion
Heptachlor Epoxide
Malathion
Dactha l
Dyrene
o,p’ DOE
Ch lorobens ude
E Parathion
Thuodan I
p,p’ DDE
DDA(ME)
Captan
Folpet
Dieldrin
Perthane
o,p’-DDD
o p’•DDT
Endrin
Kepone
p,p’ DOD
Thuodan II
Ethion
p.p’-DOT
Truth ion
Dilan I
Mirex
Meihoxychior
Dilan II
Teduon
Guthuon
234 2.31 2.29 227 224 222 219
195 193 191 189 187
225 222 220 218 216 214 212 210 208
— 214 211 207 204 201 197 194 191187
212 210 207 205 203 200 198 196 193
264 280 256 252 247 243 239 235 231
302
2 76
2 97
3.22
3 19
4,08
404
408
61
67
74
316
2 97
2 73
294
3 17
316
4 02
398
40 2
61
60
65
72
113
293 288 284
271 269 267
291 2.89 286
313 308 304
313 310 307
396 389 38
392 3.86 380
396 3.90 3.83
598 5,83 572
596 587 578
64 63 61
71 69 68
111 108 106
280 276
264 262
283 280
298 294
304 300
376 368
373 367
378 372
560 549
568 560
598 584
66 65
103 101
217 215
273 268
260 258
278 27 i
290 286
297 294
360 353
361 354
366 359
538 527
552 543
570 557
63 62
98 96
255 253
272 269
282 277
291 288
347 340
348 343
352 347
517 502
533 524
542 529
60 587
93 90
251 249 246 244 242 240 237
267 264 261 259 256 253 250
273 268 264 259 255 251 246
285 281278 275 272 269 266
332 327 320 313 305 298 290
336 330 324 318 312 305 288
340 334 328 322 316 310 303
490 479 468 456 443 432 420
515 506 497 488 479 470 462
516 501 488 473 460 446 432
571 557 541 528 511 498 481
88 85 83 80 78 75 73
94 92 89 86 84 81 78
TTTT
194 198 202 204
375
12.2 119 117 114 111 108 106 103 100 97
I
I
I I
I I I I I
170 174 178 182 186 190
-------
1/ It/ il Table 2 (c) Section It,A,(6)
5%OV-210
Column Temperature, °C.
170 174 178 182 188 190 194 198 202 204
I I I I I i I i I I
068 068 066 068 066 066 068 086 088 066 066 066 066 066 066 086066086 Phosdrin
069 069 069 069 069 069 069 069 069 069 069 069 069 069 069 069 069 069 2,4 -D(ME)
083 064 084 064 064 064 064 065 085 085 065 065 065 066 068 068 066 066 Thimet
062 082 062 063 063064064 065 065065 066 066 067 067 068 068 068 069 a.BHC
060 069 069 069 069 069 069 069 069 070 070 070 070 070 070 070 070 070 CDEC
088 087 087 087 088 088 086 085 085 085 085 084 084 084 083 083 083 082 2.4-DUPE)
087 086 086 086 085 - 085 085 084 084 084 084 083 083 083 082 082 082081 Simazine
088 088 087 087 086 088 086 085 085 084 084 084 083 083 082 082 081 081 Atrazlne
76 075 075 075 075 075 075 074 074 074 074 074 074 073 073 073 073 073 Diazinon
080 080 080080 080 081 081 081 081 082 082 082 082 083 083 083 083 084 Lindane
108 108107 107106 105 105 104 103 103 102 101101 100 100099 098 098 2 ,4 ,5-T(ME)
097 097 096 096 096 096 096 095 095 095 095 095095094 094 094 094 094 /3BHC
130 129 128 127 126 125 123 122 121 120 119 118 116 115 114 113 112 111 2,4-D(BE)I
107 107 106 lOB 105 105 105 104 100 103 103 103 102 102 101 101 101 100 5.BHC
087 087 087 087 087 087 087 087 087 087 088 088 088 088 088 088 088 088 Heptachlor
1 36 1 34 1 33 1 32 I 30 1 29 127 1 26 1 25 1 23 1 22 1 20 1 19 118 116 115 114 1 12 2 ,4,5-T(IPE)
1 47 1 46 1 44 1 43 I 41 1 40 1 38 1 38 1 35 1 33 1 32 I 30 1 29 1 27 1 25 1 24 1 22 1 21 2,4-D(BE)ll
1 51 1 51 1 50 1 49 1 48 1 48 1 47 1 46 1 45 1 45 1 44 1 43 1 42 1 42 1 41 1 40 1 39 I 39 Duchlone
218 2 16 2 12 209 208 204 201 1 98 1 95 192 1 89 1 86 1 83 180 1 78 1 75 1 72 169 Dumethoato
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Aldrun (REFERENCE)
141 140 139 138 137 136 135 134 133 132 131 130 129 128 127 126 125 124 Ronnel
143 142 141 140 139 138 138 137 135 135 134 133 133 132 131 130 129 128 1-Hydroxychlordene
317 312 307 302 297292 288 283 278 273 268 263 258 254 249 244 239 234 M Parathion
202 200 1 88 1 97 1 95 1 93 1 91 1 89 1 87 1 85 183 1 81 1 79 1 77 1 75 1 73 1 71 169 Heptachlor Epoxide
320 314 308302 296291 285 279 273 267 261 255 249 244 238 232 228220 rv Iathion
284 280 275 271 267 263 259 254 250 246 241 237 233 229 224 220 216 212 Dacthal
206 204 202 199 197 194 191 189 187 184 181 179 177 174 171 169 166 164 Dyrene
1 87 1 65 1 63 1 62 1 60 1 59 I 57 1 55 I 53 1 52 1 50 I 49 1 47 1 45 1 43 1 42 1 40 1 39 o,p ’-DDE
197 1 95 1 93 1 90 1 88 186 1 84 1 82 - 180 1 78 1 75 1 73 1 71 169 167 165 1 63161 Chlorobenside
424 417 409402 394387378371 363 356 348 340333325317310303 295 EParathuon
263 260 •257 254 261 248 - 245 _242 239 236 233 230 227 224 221 218 215 212 Thiodan I
224 221 218 215 212 210 207 204 201 198 195 192 190 187 184 181 178 175 p.p’-DDE
310 306 300 294 289 284 279 274 269 264 259 254 248 243 238 233 228 223 DDA(ME)
447 440 432 424 417 409 401 393 385 377 369 362 354 346338 330 322314 Captan
404 398 392 385 378 372 365 359 353 346 339 333 326 319 313 306 299 293 Folpet
321 317 313 309304 300 296 292 288 283 279 275 271 268 262 258 254 249 Dieldrun
207 204 201 198 195 192 189 186 183 180 177 174 171 168 165 182 169 156 Perthane
274 270 287 283 259 255 251 247 243 239 235 231 227 223 219 215 211 207 o .p ”DDD
292 2 87 2 83 2 79 2 74 2 70 2 65 261 2 57 2 52 2 48 2 43 2 39 2 35 2 30 2 26 2 21 2 17 o,p ’-DDT
382 377372 367 361 356 350 345339 334 328323 318 313 307 302 288 291 Endrin
278 273 270 267 264 261 258 255 252 249 246 243 240 237 234 231 228 225 Kepone
410 403 396 389 382 375 368 361 353 346 338332 325318 311303 296 289 p,p’-ODD
498 491 483 475 467 459 451 443 435 427 419 411 403 395 387 379 371 363 Thuodanll
60 589 578564 551 637 525 512 499 486 473 460 448 435 422 409 396 383 Ethion
447 439 431 423 415 407 398 390 382 374 366 358 349 341 333 325 317 309 p ,p ’ -DDT
529 5 19 509499 489 478 468 458 448 437 427 417 406 396 386 376 3 16306 Trithion
123 120 117 114 111 108 104 101 98 95 92 88 86 82 79 76 73 70 Dunn I
406401 396 389 384 378 373 368 382 357 352 346 340 335 329 324 319 313 Mirox
74 72 70 88 87 66 83 61 594 576 569 541 523 506 488 470 453 435 Methoxychlov
142 138 134 130 126 123 119 115 111 107 103 100 96 92 88 84 81 77 Dulan II
211 206 200 195 189 184 179 173 187 162 156 151 145 140 134 129 123 118 Tedion
247 240 233 226219 212 208 199 192 185 178 171 164 157 150 143 136 129 Guthion
I I I I I I I I
170 174 178 182 186 190 194 198 202 204
Retention ratios, relative to alcfrmn, of 48 pesticides on a column of 5%OV-210 at temperatures
from 170 to 204°C. support of Chromosorb W,H P. 100/120 mesh, electron capture detector,
tritium source, parallel plate, all absolute retentions measured from Injection point Arrow
indicates optimum column operating temperature with carrier flow at 60 ml per minute
-------
11/1/72 Section 4,B,(1)
Page 1
GAS CHRcMATOGRAPHY-FLANE PHOT ETRIC
INSTPIJ [ ENT
See Section 4,A,(l). This ui -ut may be equipped with a Melpar Flame
Photometric detector (F.P.D.).
I. FLOW SYSTHI :
See Section 4,A,(1), I. Flow systems should be tight but the F.P.D.
is not as sensitive to leaks as electron capture detection.
In addition to the carrier gas, the F.P.I1L requires Hydrogen, Oxygen and
possibly air to operate. Leaks in these systems can be hazardous from
the explosion standpoint.
II. DETECTOR :
This subject is covered in detail later in Section 4,B,(3).
III. ELECrRavfflTER :
See Section 4,A,(l), III. The electrometer for the F.P.D., must deliver
at least 750 VDC 6 The electrometer should also be capable of delivering
at least 1 x l0 amperes bucking current.
1V. TR1PERA11JT E PROGRAMvIER :
See Section 4,A,(l), l v.
V. PYRCNETER :
See Section 4,A,(l), V.
VI. MISCELLANEOUS :
See Section 4,A,(l), VI.
-------
11/1/72 Section 4,B,(2)
Page 1
GAS CHR( ATOGRAPFW-FLAME PHOTCMETRIC
COLUMNS
I. SPECIFICATIONS :
See Section 4,A,(2), I.
II. COLUMN SELECTION :
A. 4% SE-30/6% QF-l - liquid phases premixed and coated on silanized
support, 80/100 mesh.
B. 5% OV-210 - coated on silanized support, 100/120 mesh. See
Section 4,A,(2), II.
III. PACKING THE COUJMN :
See Section 4,A,(2), III.
RI. COLUMN CONDITIONING :
1. Heat condition a 6-foot column of 4% SE-30/6% O\T-210 (QF-l)
according to Section 4,A,(2), IV, 1.
2. Carbowa.x treai ient - Place a small wad (ca 1/2”) silanized glass
wool in one end of a 3” length of 1/4” o.d. glass tubing. Pack
loosely with 2 inches of 10% Carbowax and place another wad of
glass wool in the other end of the tube (See Fig. 1).
Place Swagelok nut, ferrule and “0” ring on each end of the packed
tube. Pass one end of the tube half way through the male union
and attach Swagelok nut to union as tight as possible.
Attach other end of tube to the column inlet port in the oven,
tightening Swagelok nut as much as possible. Place 1/4” nut on
inlet end of the 6-ft. GLC column (previously heat conditioned)
and insert into the male union until it touches bottom end of 3”
tube, then slack off very slightly to prevent glass ends from
grinding together when nut is tightened. (See Fig. 2).
Thoroughly tighten Swagelok nut to attach GLC column to male
union and place some object on the floor of the oven to function
as a retainer in case GLC column should slip out of the union
during conditioning per od. A littlejack works nicely. Bring
oven heat up to 230-235 C arid apply a carrier gas flow of 20 mi/mm.
Hold for a 17-hour period .
-------
11/1/72 Section’ 4VB, (2)
-2-
NOTES : 1. The combined parameters of temperature, time and
carrier flow are critical in the assurance of uni-
formity of vapor phase deposition as affecting ultimate
retention characteristics.
2. Special materials needed include:
(a) A 3-inch length of borosilicate glass tubing,
1/4” o.d. x 5/32” id. (or 6 mm x 4 mm).
(b) Silariized glass wool.
(c) 10% Carbowax 2 1 on a silanized support,
80/100 mesh.
(d) Swagelok male union #400-6, 1/4”. This must
be drilled out to accommodate the 1/4” o.d.
tubing.
3. DO NJT USE A SILYLATED COLIThIN WI lT- I F.P.D . The
Silyl-8 will bleed into the F.P.D. and fog the heat
shield excessively.
V. EVALUATION OF COLUMN :
See Section 4,A,(2), V.
A mixture containing the following should give approximately equal peak
heights.
Compcvni ng/5i.il injection
Ethyl Parathion 0.50
Methyl Parathion 0.38
Diazinon 0.17
Ronnel 0.25
Malathion 0.51
Trithion 1.22
Ethion 0.58
NCTrE : A drastic reduction in the peak height of malathion can be
an indication of a poor column, provided that the rest of
the system is known to be operating properly.
VI. MAINT {ANCE AND USE OF COJII4N :
See Section 4,A,(2), VI.
The effects of the vapor phase deposition from Carbowax appear to
persist at least three nths with a slow decrease iii response becoming
evident, depending on the particular column and the amount and type of use.
-------
L1/l/72 Section 4,B, (2)
-3-
Each operator should monitor the response characteristics in relationship
to the column just after treatment.
NJTES : 1. Response will sometimes drop rapidly for several days
after treatment, then stabilize.
2. Retreatment of the same columns appears to rejuvenate
the response, but will shift the RRR-p-values. Retreatment
of the column is not recommeixled because the table of
RRR-p-values would lose its usefulness.
-------
Revised 11/1/72 Section 4,B,(3)
Page 1
GAS CF]RGvIATOGPAPHY-FLAME PHOT METRIC
DETECTOR
D. . voltage shall be supplied to the detector from either an outboard
power supply or from a strip on the back of the electrometer. Provided the
column ajid all electronic circuits in the various modules of the instrument
ar functio iing properly, the degree of sensitivity in the flame photometric
mode relate to four factors: (1) condition of the photoniultiplier (P.M.)
tube; (2) voltage applied to P.M. tube; (3) flow rates of hydrogen, oxygen
and 4r; nd (4) condition of interior of detector.
T. OPTTh1IJM FLOW PATES :
A. Set hydrogen flow rate at 150-200 mi/mm.
B. Maxiiium response by varying the oxygen flow with air flow at zero.
C. i1axiiiiize response by varying the air flow with oxygen set at
optimum flow rate.
NOTES : (1) Some detectors respond best with no air flow.
(2) An increase in flow rates, while increasing
response, may increase the baseline noise.
Signal to noise ratio is a more accurate
definition of sensitivity than response alone.
&iggested operating parameters for the phosphorus mode are as follows:
Temperatures Flow Pates (ml/min. )
Column 200°C 2 Purge 70-80
Injection Block 225°C Carrier 70-80
Detector 165-200°C Hydrogen 150-200
0
transfer Line 2 5 C Oxygen 10-30
2 itching Valve 235°C Air 0-100
I3 Do not operate above 170°C unless Eitted with heat shield.
(2) Appropriate only if using a Valco vent valve.
-------
Revised 11/1/72 Section 4B,(3)
-2-
II. OFF IMUM RESPONSE VOLTAGE :
In order to determine the optimum response voltage for the P.M. ,t be,
a variable power supply is necessary which allows the voltage to.be ifl
creased with little increase in electronic noise. Increasing the vpltage
from the electrometer will increase the electronic noise inordinately.
A. With flows at optimum, set power supply at 750 V.D.C. Inject
enough Ethyl Parathion to give 35-60 f.s.d.
B. Reset to 850 V. Inj ect the sante amount of Ethyl Parathion . s
before.
C. Repeat in 100 V increments until the signal to noise ratio reaches
maximum and starts to decrease. (See Fig. 4).
NOTES : (1) It should be necessary to attenuate to keep on
scale. It is therefore mandatory to check the
linearity of the electrometer at different
attenuations.
(2) Comparison with a P.M. tube of known sensitivity
will give indication of condition of P.M. tube.
III. DETEC1DR LINEARITY :
The F.P.D. has a broad range of linearity. Excluding any effect from the
instrument electronics, the effective range is from 1 to 50 times the minimum
acceptable level of Ethyl Parathion (0.5 ng to 25 ng). The appropriate atten-
uation will be dependent upon the sensitivity of the particular system used.
However, it is best to operate at the minimum detection level and dilute the
sample when necessary.
IV. PHOSPHORUS MODE :
When the detector is fitted with a 526 mp filter, it becomes selective
for phosphorus. Large amounts of sulfur will give a response in this mode.
V. SULFUR MODE :
When the detector is fitted with a 394 mp filter, it becomes selective
for sulfur. Sensitivity for sulfur is usually an order of magnitude less
than for phosphorus since sulfur response increases approximately a the
square of the concentration.
-------
11/1/72 Section 4,B,(4)
Page 1
GAS CHR(]vIATOGRAPHY-FLt%JvIE PHOTCNETRIC
SAMPLE QUANTITATION AND INTERPRETATION
I. See Section 4,A,(4J .
The priming mixture below is given in nanograins per microliter.
Ethyl Parathion 1.0 Malathion 1.0
Methyl Parathion 1.0 Ethion 1.0
Ronnel 0.5 Trithion 2.0
Diazinon 0.5
Forty microliters of this mixture is injected. Do not inject with the
same syringe used for routine work.
II. Peak Height :
See S ction 4,A,(5),I.
III Peak Height x width at half height :
See Section 4,A,(5),II.
NOTE : Both I. and II. presuppose that the absolute retention
of standard and sample are the same.
IV. Triangulation or Integration :
See Section 4,A,(5),III.
V. Interpretation :
Because of the selectivity of the filters, interpretation is greatly
simplified.
Identification of a thiophosphate can be accomplished in the following
manner:
1. Retention ratio on a given column matched with a standard.
2. Suspect compound in the correct Florisil elution.
3. Response on a selective detector.
4. Sulfur to phosphorus ratio matched against a standard.
-------
11/1/72
Section 4,B,(5)
FIGURE 1
Carbowax Tube section
1O°o Carbowax
-t1
V ’ooI
FIGURE 2
Cutaway View of Column with Carbowcix Assembly
-------
FIItTRE 3
Section 4,B,(5)
Chromatograms of a mixture of organophosphorous pesticides
on an untreated column of 4% SE-30/6% QF-1 (Fig.1),and on the
same column treated with Carbowax(Fig.2)
Column:4% SE-30/6% QF-1;amps,full scale 0.8 xlO -8 ;voltage 850 V.
TEMP., C.
OPERATING
Column
200
Carrier
60
Inlet
225
Vent
60
Detector
195
Oxygen
30
Transf.line
235
Hydrogen
‘
180
Vent
235
Air
40
z
2
x
4
4 2
x_ 1
-I
).
I
•
g gC
o
WI WIN
o o
z
-I
— z
1
1
0 -
0
• S
C
WI
2;
11/1/72
PARAMETERS
FLOW RATES,mI/min.
I
01
x2
S.
NW!
p .o
be
WI
p.
0
WI
be
WI
p.
U
WI
0
1
0
WI
be
WI
I
0
I
t
WI
S
C
N
N
1
0
I
N
C
WI
VI
0
1
z 0
o
— I ..
4
WI
14
4 0
a.
. 41
4W
hi
S
C S
C
WI
00
-. 0
1 -
P4
o 5
— 0
• S
C C
II
N
o 0
0
WI
be
a
be
VI
P .
N
0
WI
P .
N
-------
tV7
5%CP L:p4bsE. qRllo
x L’1 ( ‘ )( tO 8 A. s.)
0.11
• V7.tv
j L
c ’s’
fDAf TH iorJ
1.6 P’4 v’4
‘7,’-
i’1.3 SIc JAL,’JotsE IQAIID
X
—
NoiS t 1’O
lo 3 X1 Cl(o Kto ArnPS
- O.1lv
(F . L. )
FULL SCJ LEj)
Il wi
G SELI,’1E.
I
I
-------
11/1/72 Section 4,B,(5)
TABLE I. RF1 IQN AND RBS1 ]NSE RATIOS, RElATIVE TO EThYL PARAThION ON
COUlIN, OF 4% SE-30/6% QF-1
RRR-PY RPH-P.Y
TEPP 0.08 5.0
Dichiorvos .10 5.0
Deineton-Thiono .22 2.0
1 1ed .28 0.02
Phorate .28 4.0
&ilfotepp .28 5.2
Diazinon .35 2.5
Deneton-thiolo .37 2.0
Dioxathion .38 0.5
Disyston .40 3.8
Diazinon-oxygen analog .42 1.0
Dimethoate .49 0.50
Azodrin .54 0.08
Ronnel .57 1.42
Ronnel-oxygen analog .58 0.25
Lkxrsban .68 1.4
Fenthion .72 1.6
Methyl Parathion .75 0.71
Malathion .81 0.71
Methyl Parathion-oxygen analog .83 0.10
Malathion-oxygen analog .85 0.063
&unithion .85 0.80
Ethyl Parathion (reference) 1. 00 1.00
Phosphamidon 1.02 0.16
Ethyl Parathion-oxygen analog 1.10 0.50
Merphos 1.23 0.35
DEF 1.25 0.80
Trithion-oxygen analog 1.78 0.12
Ethion 1.83 0.71
Trithion 1.90 0.36
Phenlcapton 3.04 0.20
Dasanit 3.16 0.03
Imidan 3.91 0.02
EPN 3.95 0.134
Guthion 6.03 0.044
Coumaphos 11.84 0.20
1/ Retention ratio, relative to ethyl parathion. Retention measurements
frxt injection point.
2/ Peak hei t ratio, relative to ethyl parathion.
-------
Revised 11/1/72 Section 5,A,(l)
Page 1
ANALYSIS OF 1-RIMAN OR ANIMAL ADIPOSE TIS JE*
(MODIFIED MILLS, ONLEY, GAITHER PROCEDURE)
I. E JIR ENT :
1. Aluminum foil, household type.
2. Beakers, 250 ml, stainless steel or heavy duty glass.
3. Beakers, 250 ml, Griffin low form.
4. Stirring rods, glass 10 mm diameter.
5. Water bath with temperature adjusthent of 90-100°C.
6. Filter p iper - Whatman No. 1, 15 cm diameter.
7. Funnels, glass, ca 60 ml diameter.
8. Separatory funnels - 125 ml and 1 liter, Kimble 29048-F, or equiv.
9. Chromatographic columns - 25 urn o.d. x 300 inn long, with Teflon
stopcocks, without fritted glass plates, Kontes 420530, Size 241.
10. Filter tubes, 150 x 24 mm, such as Coming 1i9480.
11. Erleriuieyer flasks - 500 ml capacity.
12. &iderna-Danish concentrator fitted with grad. evaporative con-
centrator tube. Available from the Kontes Glass Company, each
component bearing the following stock numbers:
a. Flask, 500 ml, stock #K-570001
b. Snyder column, 3-ball, stock #K-503000
c. Steel springs, 1/2”, stock #K-662750
d. Concentrator tubes, 10 ml, size 1025, stock #K-570050
13. Modified micro-Snyder columns, 19/22, Kontes K-56925l.
14. Glass beads, 3 mm plain, Fisher #11 - 312 or equivalent.
* This method, with appropriate n difications, may be used for the analysis of
other tissues if original sample size is adequate.
-------
Revised 11/1/72 Section 5,A,(l)
-2-
15. Glass wool - Corning #3950 or equivalent.
II. REM TS :
1. Petrg1e .mi ether - Pesticide Qiality, redistilled in glass, b.p. 3Ø0
- 60 C. (See Note 7, p. 10).
2. Diethyl ether - AR grade, peroxide free, Mallinckrodt #0850 or the
equivalent. The ether must contain 2% (v/v) absolute ethanol. Some
of the AR grade ethers contain 2% ethanol, added as a stabilizer,
and it is therefore unnecessary to add ethanol unless peroxides are
found and renx)ved.
)TE : To determine the absence of peroxides in the ether,
add 1 ml of freshly prepared 10% 1(1 solution to 10 ml
of ether in a clean 25-ml cylinder previously rinsed
with the ether. Shake and let stand 1 minute. A
yellow color in ether layer indicates the presence of
peroxides which must be removed before using. See
Misc. Note 4 at end of procedure. The peroxide test
should be repeated at weekly intervals on any single
bottle or can as it is possible for peroxides to form
from repeated opening of the container.
3. Eluting mixture, 6% (6+94) - purified diethyl ether 60 ml is diluted
to 1000 ml with redistilled petroleum ether and anhydrous sodium
sulfate (10-25 g) is added to remove moisture.
4. Eluting mixture, 15% (15+85) - purified diethyl ether 150 ml is
diluted to 1000 ml with redistilled petroleum ether and dried as
described above.
Kfl’E : Neither of the eluting mixliires should be held longer
than 24 hours after mixing.
5. Florisil, 60/100 mesh, PR grade, to be stored at 130°C until used.
}DTh : (1) In a high humidity room, the column may pick up
enough moisture during packing to influence the elution
pattern. To insure uniformity of the Florisil
fractionation, it is recoimnended to those laboratories
with sufficiently large drying ovens that the columns
be packed ahead of time and held (at least overnight)
at 130 C until used.
(2) Florisil furnished to the contract laboratories
by the Perrine laboratory on order, has been activated
-------
Revised 11/1/72 Section 5,A,(l)
-3-
by the manufacturer, and elutioii pattern data is
included, with each shipment. However, each laboratory
should determine their own pesticide recovery and
elution pattern on each new lot received, as environ-
mental conditions in the various laboratories may
differ somewhat from that in Perrine. Each new batch
should be tested by the procedure described in
Section 3,D for assurance that the operator can obtain
recoveries arid compound elution patterns comparable to
the data given on the accompanying table.
6. Acetonitrile, reagent grade, saturated with pet. ether.
M TB : Occasional lots of C1-L cN are impure and require re-
distillation. Generally, vapors from impure aceton-
itrile will turn litmus paper blue when the moistened
paper is held over the mouth of the bottle.
7. Anhydrous sodium sulfate, reagent grade granular, Mallinkrodt stock
#8024 or the equivalent.
NOTE : When each new bottle is opened, it should be tested
for contaminants that will produce peaks by Electron
Capture Gas Liquid Chromatography. This may be done
by transferring ca 10 grams to a 125 ml Erlenmeyer
flask, adding 50 ml pet. ether, stoppering and shaking
vigorously for 1 minute. Decant extract into a 100-mi
beaker and evaporate down to ca 5 ml. Inject 5 p1
into the Gas Liquid Chromatograph and observe chroma-
togram for contaminants. When impurities are found,
it is necessary to remove them by extraction. This
may be done by using hexane in a continuously cycling
Soxhlet extraction apparatus or by several successive
rinses with hexane in a beaker. The material is then
dried in an oven and kept in a glass-stoppered container.
8. Sodium Chloride solution, 2%, from reagent grade NaCl.
1’flTE : See ‘IJTh for sodium sulfate, Step 7, above.
9. Sand, quartz, which has been acid washed and extracted with hexane
to produce a zero background in the determinative step.
10. MgO-Celite mixture (1:1) weigh equal parts of reagent grade MgO
and Celite 545 and mix thoroughly.
-------
Revised 11/1/72 Section 5,A,(l)
-4-
11. Hexane, redistilled, pesticide quality.
III. SAMPLE PREPARATION ECTRACTION :
1. On a cupped sheet of lightweight ali.nninuin foil, weigh 5 gratis of
the previously minced fat. Transfer entire cup to a 250 ml
stainless steel or heavy duty glass beaker.
2. Add ca 10 grains of clean, sharp sand, ca 10 grains of anhydrous
and 1.0 ml of hexane solution containing 200 rianogralns
of alcirin.
M)TE : The aidrin is added here for the dual purpose of
(1) providing a built-in retention marker for direct
peak identification on all chromatograins of the first
fraction extract, and (2) as a quantitative recovery
check for the procedure. This inoculation should, of
course, not be made if aldrin is suspected to be in
the substrate.
3. Grind the mixture with a heavy glass rod and continue adding portions
of to give a uniform, dry granular mass.
4. Add 50 ml of pet. ether and warm carefully on a water bath with con-
tinuous stirring until solvent boils gently .
5. Place Whatman No. 1 filter paper in glass funnel and rinse several
times with pet. ether. Place funnel over previously tared 250-ml
beaker and transfer extract to funnel by decantation.
6. ctract the contents of the first beaker with t nxre 50-mi portions
of pet. ether as described in steps 4 and 5.
7. Transfer insoluble material to the filter paper and rinse beaker and
paper with a final 10 ml of pet. ether.
8. Place beaker on a 40°C water bath and evaporate just to dryness
under a stream of nitrogen. Check odor to be sure all solvent is
rem)ved and allow to cool to room temperature in a dessicator.
9. Weigh beaker arid record for calculation of percent fat in the sample.
10. Pccurately weigh between 2.8 and 3.0 grams of the fat obtained in
Step 9 into a l25- n1 separator. Add 12 in]. of pet. ether previously
saturated with acetonitrile.
-------
Revised 11/1/72 Section 5,A,(l)
-5-
NOTE : In the case of highly saturated animal fat, it will
be necessary to add 17 ml of pet. ether to the
separator. In such a case the an unt of acetonitrile
used in the partitioning step should be increased
to 40 ml.
1V. LI( JID-LIQUID PARTITIONING :
1. Add 30 nil of acetonitrile, previously saturated with pet. ether.
Stopper funnel and shake vigorously for 2 minutes.
2. Allow phases to separate and draw off the acetonitrile layer into
a 1-liter separator containing 700 nil of a 2% solution of NaC1 and
100 nil of pet. ether.
3. Similarly extract the pet. ether layer in the 125-nil separator
three n re times with 30-mi portions of acetonitrile, combining all
acetonitrile extracts in the 1-liter separator.
4. Stopper, invert 1-liter separator, vent off pressure and mix by
shaking for two minutes, releasing pressure as required.
5. Allow the layers to separate and drain aqueous layer into a second
1-liter separator.
6. Add 100 ml pet. ether to second separator, and after a 30-second
vigorous shaking, discard aqueous phase and transfer pet. ether
phase into first 1-liter separator.
7. Wash pet. ether with two 100-mi portions 2% NaCl and discard the
aqueous washings.
8. Prepare a 2-inch column of anhydrous, granular Na 2 SOA in a 150 x 24
nun filter tube and position over a 500-mi K-D evaporator fitted with
a 10-mi grad. concentrator tube containing one glass bead. Dry the
pet. ether by filtering through this column. Rinse the separator
twice with 10-mi portions of pet. ether aixi finally rinse down sides
of the filter tube with 10 ml pet. ether.
9. Attach a 3-ball SByder column to the top of the K-D evaporator and
place in a 90-100 C water bath. Approximately 1-1/2 inches of the
concentrator tube should be below the surface of the water.
10. Concentrate the extract to ca 5 nil, rinse down the sides of K-D
evaporator and the ground glass joint with a total of 3 ml pet.
ether. Reconcentrate extract to ca S nil under a gentle stream of
nitrogen at room temperature.
-------
Revised 11/1/72 Section 5,A,(1)
-6-
V. FLORISIL RACTI(} ATION :
1. Prepare a chromatographic column containing 4 inches (after
settling) of activated Florisil topped with 1/2 inch of anhydrous,
granular Na,S0 4 . A small wad of glass wool, preextracted with pet.
ether, is placed at the bottom of the column to retain the Florisil.
N.)T.ES : (1) If the oven is of sufficient size, the columns
may be prepacked and stored in the over, withdrawing
columns a few minutes before use.
(2) The aimunt of Florisil needed for proper elution
should be determined for each lot of Florisil.
2. Place a 500-nil Erlenmeyer flask under the column and prewet the
packing with pet. ether (40-50 ml, or a sufficient volume to com-
pletely cover the Na 2 SO 4 layer).
N)TE : From this point am:1 through the elution process, the
solvent level should never be allowed to go below
the top of the Na ,SO 4 layer. If air is introduced,
channeling may oc±ur, making for an inefficient column.
3. Using a 5-mi Mohr or a long disposable pipet, ininediately transfer
the tissue extract (ca 5 ml) from the evaporator tube onto the
column and permit it to percolate through.
4. Rinse tube with two successive 5-mi port ions of pet. ether, carefully
transferring each portion to the column with the pipet.
NOTE : Use of the Mohr or disposable pipet to deliver the
extract directly onto the column precludes the need
to rinse down the sides of the column.
5. Prepare two 1 iderna-Danish evaporative assemblies complete with
10 ml graduated evaporative concentrator tubes. Place one glass
bead in each concentrator tube.
6. Replace the 500-mi Erlenmeyer flask under each column with a 500-mi
Xüderna-Danish assembly and commence elution with 200 ml of 6%
diethyl ether in pet. ether (Fraction I). The elution rate should
be S ml per minute. lVhen the last of the eluting solvent reaches
the top of the Na 2 50 4 layer, place a second 500-mi Kuderna-Daflish
assembly under the column and continue elution with 200 ml of 15%
diethyl ether in pet. ether
-------
Revised 11/1/72 Section 5,A,(1)
-7-
7. I’o the second fraction only , add 1.0 ml of hexane containing 200
nanogra .ms of aldrin, place both Kudema-Danish evaporator assein-
blies in a water bath and concentrate extract until ca 5 ml
r nain in the tube.
8. Remove assemblies frciii bath, and cool to ambient temperature.
9. Disconnect collection tube from Kudema-Danish flask and carefully
rinse joint with a little hexane.
10. Attach modified micro-Snyder column to collection tubes, place tubes
back in water bath and concentrate extracts to 1 ml. If preferred,
this may be done at room temperature under a stream of nitrogen.
11. Remove from bath, and cool to ambient temperature. Disconnect
tubes and rinse joints with a little hexane.
NOTE : The extent of dilution or concentration of the
extract at this point is dependent on the pesticide
concentration in the substrate being analyzed and the
sensitivity and linear range of the Electron Capture
Detector being used in the analysis. See Section 4,A
in this manual.
12. Should it prove necessary to conduct further cleanup on the 15%
fraction, transfer 10 grams MgO-Celite mixture to a chromatographic
column using vacuum to pack. Prewash with ca 40 ml pet. ether,
discard prewash and place a Kuderna-Danish receiver under column.
Transfer concentrated Florisil eluate to column using small portions
of pet. ether. Force sample and washings into the MgO-Celite
mixture by slight air pressure and elute column with 100 ml pet.
ether. Concentrate to a suitable volume and proceed with Gas
Liquid Chromatography.
NOTE : Standard Recoveries should be made through column
to insure quantitative recoveries.
VI. ASSESSvIENT OF EXTRACT C0NCB TRATION :
1. Inject 5 p1 of each fraction into the gas chromatograph for the
purpose of determining the final dilution. If all peaks are
on-scale and quantifiable, it will not be necessary to proceed
with any further adjustment in concentration. With human fat,
however, it is probable that there will be several sizable
on-scale peaks arid one or more off-scale peaks in the 6% fraction.
2. If off-scale peaks are obtained in either fraction it will be
necessary to dilute volumetrically with hexane to obtain a con-
-------
Revised 11/1/72 Section 5,A, (1)
-8-
centration that will permit quantitation of those peaks from a
5 iii injection.
? Th : A 5-mi dilution of a 3.0 grain sample containing .01
ppm of a given pesticide will yield 30 picogranis of
the pesticide per 5-microliter injection. Provided
the detector is operating at average sensitivity, it
should be possible to obtain quantifiable peaks of
nost compounds likely to be present at this con-
centration.
VII. MISCELLANBWS N)TES :
1. The two fractions from the Florisil column should never be com-
bined for examination by Gas Liquid Chromatography. By so doing,
a valuable identification tool is voided.
2. Meticulous cleaning of glassware is absolutely essential for success
with this procedure. All reagents and solvents nust be pretested
to insure that they are free of contamination by electron capturing
materials at the highest extract concentration levels. Reagent
blanks should be run with each set of samples.
3. The method, as described, is known to be capable of producing
recoveries of most of the chlorinated pesticides of from 85-100%.
Each laboratory should conduct their own recovery studies to make
certain of their capability to achieve this recovery range. A
clue may be obtained from the recovery of the aldrin spike. The
recovery of this compound should not be less than 70%.
4. For the removal of peroxides from the ethyl ether, place an appro-
priate volume in a separatory funnel and wash it twice with portions
of water equal to about 1/2 the volume of ether. The washed ether
is shaken with 50-100 nil of saturated NaC1 solution and all of the
aqueous layer is discarded. The ether is then transferred to a
flask containing a large excess of anhydrous sodium sulphate and
shaken vigorously on a mechanical shaker for 15 minutes. This
treatment should not be attempted on ether containing ethanol, as
the amount of ethanol that would remain is indeterminate.
5. If the presence of malathion is suspected, it is necessary to pass
200 ml of 50% diethyl ether in pet. ether through the Florisil
cohmtn into a third K-D evaporator ass nb1y, concentrating the
eluate as described for the 6% and 15% eluates.
6. Table 1 gives the elution pattern for a number of comnon pesticides.
On occasion it may be observed that a portion of a given compound
-------
Revised 11/1/72 Section 5,A,(1)
-9-
may elute into a different fraction than the one given. For
example, some operators have difficulty eluting all the dieldrin
in the l5 o fraction. This is generally due to either moisture
in the system or the use of solvents of different polarity than
those specified in the reagent list. For example, it is essential
that the diethyl ether contain 2 9 o (v/v) ethanol. Ether without
the ethanol or w]th too much would expectedly result in an altered
elution pattern.
7. If this method is used for the detection and quantitation of
organophosphorous compounds, s e special factors must be con-
sidered. The presence of any peroxides in the ethyl ether and/or
impurities in the pet. ether can result in extremely low recoveries.
Recovery efficiency should be predetermined on standard mixtures
containing the specific compounds of interest. If low recoveries
are obtained, it may be necessary to try an alternate manufacturer’s
pet. ether.
-------
Revised 11/1/72 Section 5,A,(1)
- 10 -
TABLE I. EUJTION PA 1TERN OF S(NE CCN C}4 PESTICIDES
FR(}1 FLORISIL PARTITIONING
6% Eluate
15% Eluate
50% Eluate
c -BHC
X
-B}
X
1 -BHC
X
Heptachior
X
Hept. Epoxide
X
Aidrin
X
o,p’-DDE
X
p,p’-DDE
X
Die ldrin
X
Endrin
X
o,p’-DDD
x
p,p’-DDD
X
o,p’-DDT
X
p,p’-DDT
X
Perthane
X
Methoxychlor
X
Chiordane
X
Chiorobenside
X
chlorobenzi late
X(80%)
X
2,4,5 -T, iscoctyl estet
X
-------
Revised 11/1/72 Section 5,A,(l)
- 11 -
TABLE I. ELUTION PA1TERN OF S(ME CC!+ION PESTICIDES
FR(}1 FLORISIL PARTITIONING (CONTINUED)
6% Eluate
15% Eluate
50% Eluate
2,4,5-T,isopropyl ester
x
2,4,5-T, n-butyl ester
X
X
2,4-D, isobut. ester
X
2,4-D, isooctyl ester
X(80%)
2,4-D, isopropyl ester
X
Tedion
X
Erxiosulfan I
X
Endosulfan II
X
Dilan
X
Ronnel
X
Diazinon
X
Methyl Parathion
X
Ethyl Parathion
X
Malathion
X
Ethion
X
Trithion
X
-------
Revised 11/1/72 SeCtion 5,A(2),(a) b)
Page 1
MICRO METhOD FOR WE DETEI MINATION OF Q-ILORINATED
PESTICIDES IN HUMAN TISSUE
I. MATERIALS AND REAGENTS :
1. MICROCOLIJMN :
Place a iiia1l loose plug of glass wool in the tip of a size “B”
Chromaflex column. (Kontes Cat. No. 42100 Size 22-7 mm.).
Pack the column with 1.6 gms of 60-100 mesh Fl 8 risil which
has been activated by the manufacturer at 1200 F. (PR grade
Florisil should be used for this method). The column packing
is added in increments followed by a gentle tapping. Add 1.6
gms of sodium sulfate, granular, to t 1 ie top of the column.
Wash the column with 50 ml of Nanograde hexane fol1owe by 50 ml
of Nanograde methanol. Dry and store co ,un1ns in a 130 C oven.
The columns should 1 e conditioned at 130 C at least over night
before using. For routine work it is convenient to prepare a
large number of columns at one tijite.
2. SODIUM SULFATE, ANff DROUS, GRANULAR :
Store in glass stoppered bottles in an oven at 130°C. Extract
a portion of the sodium sulfate, equivalent to the amount used
in the Florisil column, with hexane. Concentrate the extract to
50 microliters and inject 5 imicroliters into the gas chroinatograph.
The results will indicate whether it is necessary to extract the
batch of sodium sulfate with hexarie and methanol prior to storing
in the oven.
3. NANOGRADE’ SOLVENTS :
Hexane, acetonitrile, methanol.
4. DISTILLED WATER :
Extract 8.0 ml. with hexane. Concentrate the extract to 300 .il,
and mi ect 5 l into the gas chromatograph. If extraneous peaks
result then the distilled water must be extracted with hexane
prior to use.
5. TISSUE GRINDER :
Ilial tissue grinder Size 22 or 23 (Kontes Cat. No. K-885450).
6. MIXER :
Vortex Junior or ec 1 uivalent.
7. CENTRIFUGE :
-------
Revised 11/1/72
Section 5,A,(2) ,(a) b)
-2-
8. EVAR)RATIVE CONCEWrRkTOR :
Complete with modified micro Snyder column, ‘ joint 19/22,
Kontes #K-569250.
9. CONCEWFRATOR TUBE :
Size 1025 Kontes Cat. No. K-570050.
10. CONGENTRA1DR TuBE :
Size 2525 Special Order - Kontes Cat. No. K-570050.
11. TEST luBE :
25 ml cap. with ‘ 19/22, joint with hooks Special Order
Kontes Cat. No. K-897900.
II. SAMPLE PREPARATION - LIVER, KIDNEY, BONE MARROW, ADRENAL, GONADS :
1. Extract a 500-mg sample of tissue in a size 22 or 23 dual tissue
grinder with 2.5 ml of acetonitrile . Add 20 nanograms of aidrin,
in 0.1 ml of hexane, to the tissue grinder. This will serve as
a recovery check as well as a marker for relative retention time.
NOTE : Pun a complete reagent blank with each set of samples.
2. Centrifuge and pour supernatant into a SO-ml round bottom test tube.
Repeat extraction twice more, collecting supernatants in the test
tube.
3. Add 25m1 of 2% aqueous sodium sulfate to the test tube and mix
the contents with the aid of a Vortex mixer.
4. Extract the aqueous acetonitrile mixture with one 5-mi and two 2-nil
portions of hexane. Combine the extracts in a 10-mi evaporative
concentrator.
5. Concentrate the extract to 300 microliters with the aid of a
modified micro Snyder column* and a 3-mm glass bead in the tube.
6. Proceed o Subsection III.
* J. Burke et al., J.A.O.A.C. 49 (5), 999-1003, 1966.
-------
Revised 11/1/72 Section S,A,(2),(a) b)
-3-
III. FLORISIL FIC ATIC :
1. Remove a Florisil column from the oven and allow it to cool
to room temperature.
2. Pre-wet the column with 10 ml of hexane and discard the
eluate.
3. Transfer the 0.3 ml of extract remaining after step (5), to
the top of the Florisil column with the aid of a dispo ’ab1e
pipet fitted with a rubber bulb. Begin immediate collection
of eluate in a 25 ml. capacity concentrator tube.
4. Rinse the 10 ml concentrator tube with 0.25 ml of hexane
transferring this to the top of the column. Repeat this
step a second time.
5. Proceed with the elution and collection using a total of 12 ml
of hexane followed by 12 ml of 1% methanol in hexane. This
24 ml represents fraction one , and will contain heptachlor,
aidrin, p,p’-DDF, o,p’-DDT and p,p’-D]Jf.
6. Collect a second fraction by eluting with a second 12 ml portion
of 1% methanol in hexane. This fraction will contain dieldrin,
heptachior epoxide, endrin, BHC*, Lindane*, and p,p? DDD.*
See Table 1.
*
A small amount of -BHC, Lindane, and/or p,p’-DDD
may appear in the first fraction.
7. Add 20 nanograins of aldrin in 0.1 ml of hexane to fraction two,
evaporate both fractions using a modified micro Snyder column
and a 3 mm glass bead in the tube.
8. Adjust the volumes in fractions (1) and (2) to 500 and 300
microliters, respectively, and proceed with the GLC portion
as outlined under ELECFRON CAI I1JRE GAS CI-IROMATOGRAPHY , diluting
basic concentrates as required to obtain quantifiable peaks
with minimal tissue concentration.
IV. ANALY ’IS OF BRAIN :
Proceed with steps (1) thru (4) as described under II. Sample
Preparation .
-------
Revised 11/1/72 secti 5,A,(2),(a) (b)
-4-
5. Concentrate the combined hexane extracts to 500 p1 in a 25 ml
test tube fitted with a modified micro Snyder column and
using a 3 rrm i glass bead in the tube.
6. Add 0.3 ml Acetic Anhydr de and 0.3 ml pyridine and incubate
in a water bath at 60-65 C for 1/2 hour.
7. Add 9 ml of 2% Na 2 SO 4 and extract with 2-3 ml portions of
hexane.
8. Concentrate the combined extracts to 300 ml in a 10 ml
evaporative concentrator fitted with a modified micro Snyder
column using a glass bead for a boiling chip.
9. Proceed as described under III. FLOPJ$ IL FRACTIONATION .
Table 1. Elution pattern for microflorisil column.
Fraction 1 12 ml hexane followed by 12 ml 1% methanol in hexane
aidrin
heptachior
o,p’ -DDE
r, ’ -DDE
o,p’-DIYF
p,p’ -DDT
heptachior Split between Fraction 1 and Fraction 2
polychiorinated biphenyls ct-BHC
lindane
diazinon
o,p’ -DDD
p,p’ -DDD
TOK
toxaphene (mostly in fraction 1)
Fraction 2 12 ml 1% methanol in hexane
DDA (methyl ester)
dieldrin
endrin
ethion (recovery variable with Florisil)
ethyl parathion
- BHC
6- BHC
heptachior epoxide
l-hydroxychlordene (80-90%)
malathion
methoxychlor
methyl parathion
ronnell Neither Fraction 1 nor Fraction 2
thiodan I and II . .
tritiii .on
paradichlorobenzophenone
-------
Revised 11/1/72 Section 5,A,(3),(a)
Page 1
ANALYSIS OF HtIvIAN BLOOD OR SERUM
I. Because of its availability and probable diagnostic value with regard
to extent of both chronic and acute exposure to chlorinated hydrocarbon
and other classes of pesticides, blood specimens present a convenient
tissue for study, providing meaningful data pertinent to the Community
Study and Monitoring laboratory program. Of several methods available
in the literature, the Dale, et al., (1966) method provided some desirable
features in rapidity, simplicity and sensitivity for the determination
of chlorinated insecticides and related materials in blood. A method
including these features is essential in the monitoring situation in-
volving analyses of large nini ibers of samples. The following procedure
utilizes the direct solvent extraction principle of the Dale, et al.
method. It is to be considered a general survey method for the deter-
mination of chlorinated hydrocarbon pesticide levels in blood, particu-
larly Wr and its metabolites. For an in-depth study of total
pesticide residue levels in this tissue, it is recommended that a cleanup
method for the determination of chlorinated pesticides in human tissue,
[ i.e., Section 5,A,(l) in this manual] be applied, together with con-
firmatory determination such as TLC and chemical derivatization techniques.
REFERENCE: Dale, W. E., A.Curley, and C. Cueto, (1966),
Hexane Extractable Chlorinated Insecticides
in Human Blood, Life Sciences 5: 47.
II. PRINCIPLE :
A 2-mi aliquot of serum is extracted with 6 millilliters of hexane
in a round-bottom tube. The extraction is conducted for 2 hours on a
slow-speed rotating mixer. The formation of emulsion is unlikely, but
if it should occur, centrifugation may be used to effect separation of
the layers. A 5-mi aliquot of the hexane layer is quantitatively trans-
ferred to an evaporative concentrator tube to which is affixed a nxdified
micro-Snyder column. The extract is concentrated in a water or steam
bath and the final volume is adjusted to correspond to the expected
concentration of the pesticide residue. A suitable aliquot is analyzed
by electron capture gas chromatography.
-------
1/4/71 Section 5, A,(3),(a)
-2—
APPARATUS AND REMENTS
1. Rotary mixing device, “Roto-Rack ”, Fisher Scientific Company,
Catalog #14-057.
2. Tubes, Culture, 16 x 125 nun., fitted with screw caps, size 15-415
with Teflon-faced rubber liners, Corning #9826.
3. Micro-Snyder column, modified, with 19/22 ‘ joint, Kontes #K-569251.
4. Concentrator tube, 10 ml, grad. 0 to 1 x 0.1 and 2 to 10 x 1,
19/22 joint, size 1025, Kontes #K-570050.
5. Syringe, 100 p1, Hamilton #710 or equivalent.
6. Vortex Genie mixer.
7. Pipet, Mohr type, 1 ml in 0.01 ml grad., Corning #7063 or
equivalent.
8. Pipets, transfer, 2, 5 and 6 ml Corning #7100 or the equivalent.
9. Beads, solid, glass, 3 mu., Corning #7268 or the equivalent.
10. Six-place tube carrier, stnls. steel. May be fabricated at local
tin shop per attached sketch.
11. Water bath capable of holding temp. of 95 to 100°C.
12. Centrifuge with head to accomodate the Corning #9826 tube,
capable of speed of 2,000 imp.
13. Hexane, distilled in glass, pesticide grade.
III. PROCEDURE:
1. Mix blood serum sample thoroughly and, with a volumetric pipet,
transfer 2 ml to a 15 ml round bottom culture tube.
2. Add 6 ml hexarie from a volumetric pipet. Tightly stopper the
culture tube with a Teflon-lined screw cap. Place tube on
rotator.
3. Set rotator speed at 50 rpn and rotate for 2 hours.
NCTI’ES : (1) This speed may vary from 50 to 55 rpn but
should be confined to this range.
-------
—7 - ,
Revised 11/1/72
Figure 1. ROTO_RACKR Mixer, variable speed
Section ,A,(3),(a)
Figure 2. Evaporative concentrator tube holder, 6—place, stainless steel
‘ .4 ,
11. T
j t;’.•
2 ’J
(\0 - 0 \
\ L) (o /
- p ----i
- ,y. ,-
I
-------
Revised 11/1/72 Section 5,A,(3),(b)
-5-
This should pose no problem on the other two columns used in the
program as the RR values at 200°C are:
SE-30/QF-1 OV-210
2,4-D(ME) 0.44 0.09
PCP(ME) 0.63 0.56
4. All reagents including the distilled water used in the method must
be extracted with hexane before use as they may be contaminated with
PCP or other materials which may cause interferences. Glassware
should be washed with dilute NaOI! solution followed by deionized
water and acetone rinsed. Care should be taken not to permit con-
tact between wooden or paper materials and glassware as peg boards
and some brands of absorbent paper products have been found to
contain PCP.
5. If the recommended volumes are used calcualtions are simplified and
are as follows:
PCP (in ppb) in serum = pg/ui injected X 10.
-------
Revised 11/1/72 Section 5,A,(3),(b)
-6-
Table 1. Percent recovery of PCP from samples fortified before extraction
Sample PCP Found ppm PCP Added, ppm PCP Recovered, ppm Recovery %
Blood Plasma 0.19 0.50 0.67 96
0.65 92
0.68 98
5.00 4.58 88
4.70 90
4.70 90
4.70 90
50.0 46.9 93
48.5 97
42.0 84
Mean 92 b
IJrine 0 01 a 0.50 0.46 90
0.50 98
0.48 94
5.00 4.86 97
4.7 ( 1 94
4.89 98
50.0 49.0 98
49.8 100
49.8 100
Mean 96C
aLjmit of detectability for blood and urine.
hStandard deviation, blood plasma --- + 4.5%
CStandard deviation, urine --- + 3.5% —
-------
Revised 11/1/72 Section 5,A,(4),(c)
Page 1
D ERMINATION OF 2,4-D /‘J’ID 2,4,5-1 IN URINE
I. INTRODUCTION :
A number of derivatives of 2,4-dichlorophenoxyacetic acid (2,4-D)
and 2,4,5-trichiorophenoxyacetic acid (2,4,5-T) are applied extensively
as selective herbicides in the control of terrestrial and aquatic broad-
leaf plants. Because of their widespread use and relatively lengthy
persistence, particularly in treated lakes and streams, potential human
exposure to these materials may occur via several routes. These include
consumption of contaminated edible plants, livestock, and water, as well
as direct exposure by agricultural spraymen and herbicide formulators.
Thus, rapid, sensitive procedures for the detection of the free acids
and chlorinated phenol degradation products in human and animal urine
assumes an important role in the toxicological and environmental moni-
toring of these herbicidal compounds.
REFERENCE : A Method for Determination of Low Levels of Exposure
to 2,4-D and 2,4,5-T, Shafik, M. T., Sullivan, H. C.
and Enos, H. F., Journal of Environmental Analytical
Chemistry, 1971, Vol. 1 pp 23-33.
II. PRINCIPLE :
The phenolic conjugates are subjected to acid hydrolysis, the free
phenols and acids are extracted and ethylated with diazoethane. Cleanup
of the derivatized 1)roducts is carried out on a silica gel column, the
resulting eluate is concentrated to an appropriate extent and subjected
to analysis by electron capture GLC, chromatographing on a column of
4% SE-30/6% QF-l.
III. EQUIFMENT :
1. - Gas chromatograph with E.C. detector fitted with a glass column
6’ x l/4”o.d. packed with 4% SE-30/6% QF-l. Column operating
parameters are those prescribed in Section 4 of this manual.
Injection port, transfer line and detector as maintained in normal
operation.
2. Chromatographic columns, Size 22, Kontes #420100.
3. Boiling water or steam bath.
4. Distilling column (condenser), 200 mm jacket, fitted with tight
glass stopper at top, Kontes #286810.
-------
Revised 11/1/72
Section 5,A, (4), (c)
-2-
5. Circulating water pumps
6. Vortex mini-mixer.
7. Evaporative concentrator tubes, grad., 25 ml 19/22, hontes #570050.
8. Conical centrifuge tubes, conical, grad., 15 ml with stoppers,
Corning #8084 or the equivalent.
9. Disposable pipets, Pasteur, 9 inch.
10. Dry nitrogen. Tank fitted with 2-stage pressure regulator.
11. Volumetric flasks, 50 and 100 ml.
12. Mohr pipets, 0.2, 0.5 and 5 ml.
13. Transfer (vol) pipets, 1 through 5 ml.
14. An exhaust hood with a minimum draft of 150 linear feet per minute.
IV. REAGENTS :
1. Benzene, pesticide quality.
2. Hexane, pesticide quality.
3. Hydrochloric Acid, conc., A.R. Grade.
4. Silica gel, Woelm, Activity Grade I.
NOTE : Dry adsorbent for 48 hours at 170°C and store in a
desiccator. On day of use deactivate the silica gel
by adding 15 microliters of water and 1 gram of silica
gel to a 125 ml Erleniiieyer flask. Stopper and rotate
until the wateT is evenly distributed throughput the
adsorbent. Allow to equilibrate for 2 to 3 hours with
periodic shaking. Prepare the chromatographic columns
just prior to use.
5. N-ethyl-N’ -nitro-N-nitrosoguanidine, Aldrich Chemical Co.
6. Distilled water. All distilled water used throughout procedure
must be benzene extracted.
-------
Revised 11/1/72 Section 5,A,(4),(c)
-3-
7. Ethylating Reagent, Preparation:
a. In a 125 ml Erlennieyer flask dissolve 2.3 grams of K I, A.R.
grade in 2.3 ml of distilled water. When solution is complete
allow to cool to room temperature.
b. Add 25 ml hexane and cool flask in a -18°C freezer for 15 mm.
c. In a VERY HIGH DRAFT hood, add 1.6 grains of N-ethyl-N’-nitro-
N-nitrosoguanidine in small portions at a time, mixing contents
of flask after each addition.
d. Decant the hexane layer into a bottle with a Teflon-lined
scr 8 w cap. This may be stored for periods up to a week at
-18 C.
‘.IOTES : (1) Because of demonstrated carcinogenicity
and toxicity, do not allow the nitrosoguanidine
or the diazoethane to come in contact with
the skin . Disposable gloves and safety
goggles should always be worn when handling.
(2) Do not use ground glass stoppered bottles
or bottles with visible interior etching.
8. Analytical grade standards for 2,4-D and 2,4,5-T. Available from
the Perrine Repository.
9. Preparation of ethylated standard mixtures;
a. Weigh 20 mg of each of the two analytical standards into
separate 100-mi vol. flasks, dissolve, and make to volume
with benzene. These concentrated stock solutions will contain
200 ng/pl of the respective compounds.
b. Transfer aliquots from each of the concentrated stock solutions
into a single SO-mi vol. flask in the following volumes:
2,4-D 1.0 ml 2,4,5-T 0.5 ml
c. Add diazoethane dropwise with a disposable pipet until a
definite yellow color persists.
-------
Revised 11/1/72 Section 5, A,(4),(c)
-4-
d. Allow solution to stand 15 minutes, then bubble nitrogen
through the solution until yellow color disappears
(ca 5-10 minutes). ThIS OPER 1T( N ‘llJ: I BL DONE IN A IIll 1L
DRAFT 1-LOOD . Dilute to volume with ben:cne. This is thc
alkylated stock standard ruxtinc’ oF the Following concen-
trations in nanograms per nucio [ jter:
2,4-1) 4 2,4,5-T 2
e. Prepare an ethylated working standard mixture of highest
usable concentration by pipetting 5 ml of the aflcylated
stock mixture (d. above) into a 50-mi vol. flask and make
to volume with benzene. This will yield a dilute mixture
of the following concentrations:
tdkylated 2,4-D 400 pg/id
Alkylated 2,4,5-T 200 pg/pI
Injection of 5 pl of this mix into ttie gas d romatograph
will provide information on the F]nal concentration range
needed for further diluted standards.
NOTE : These alkylated standards should he stored at
-18 C when not in use and discarded after one
month.
V. PPQGELXJRE :
Extraction arid A11;yintion
A control sample of urine from an unexposed donor should he carried
through the entire procedure parallel with the saniple(s being tested.
1. Pipet 1 to 5 ml of urine into a 25-mi evap. conc. tube.
NOTE: The precise volume is predicated on the expected residue
level.
2. Add dropwise a volume of conc. HC1 equal to 1/5 the volume of
urine and mix well.
3. Fit a stoppered reflux condenser to the tube and heat in boiling
water bath for one hour, cooling the condenser with circulating
ice water .
4. Remove frc ii bath, cool and rinse inside walls and condenser til)
with 3 ml benzene.
-------
Revised 11/1/72 Section 5,A,(4),(c)
-5-
5. Mix contents of tube for two minutes on a Vortex mixer set at
high speed and centrifuge.
6. By means of a disposable pipet, carefully transfer the benzene
(upper) layer to a 15-mi centrifuge tube taking special care not
to transfer any water.
7. Repeat the extraction with another 3-mi portion of benzene, adding
the second benzene to the centrifuge tube.
8. Add diazoethane reagent dropwise with a disposable pipet until the
yellow color persists (ca 2 ml).
9. Allow tuDe to stand 15 minutes, then bubble nitrogen through the
solution to remove excess reagent.
10. Concentrate the ethylated extract to ca 0.3 ml at room temperature
or on a 40 C water bath under a gentle stream of nitrogen.
Silica Gel Elution Pattern
The elution pattern of the ethylated compounds must be determined before
using the silica gel column for cleanup of the ethylated urine extracts. The
column preparation and elution pattern evaluation is outlined in the Following
Steps
a. Place a small vad of gJass wool at the bottom of a Diromaflex
column and add 1 gram of the partially deactivated silica gel.
Top this with 1/2 inch of arthydrous, granular Na 2 SO 4 .
b. Pre ash the column with 10 ml of hexane and discard the eluate.
c. lVhen the surface level of the hexane reaches a point on the column
ca 2 an from the top of the Na SOA, add 0.3 ml of the alkylated
stock standard mixture (subsectioi IV,9,c) to the column. Elute
successively with 10 ml of each of the solvent systems listed in
the following table, collecting each fraction separately. Inject
from 5 to 10 p1 from each fraction into the gas chrc natograph
and calculate the percent of each compound present in the fraction.
-------
Revised 11/1/72 Section 5,A,(4),(c)
-6-
A typical elution pattern is shown in the table:
Elutin g Solvents 2,4-D 2,4,5-T
5pg 2pg
20% Bçnzene-Hexane 0 0
40% Benzene-Hexane 0 0
60% Benzene-Hexane 0-2 o 20-25%
80% Benzene-Hexane 98-100% 75-80%
100% Benzene 0 0
Silica Gel Sample Cleanup
1. Prepare a chroniatographic column of silica gel as described on the
previous page and prewash column with 10 ml of hexane exactly as
described, discarding the eluate.
2. Transfer the concentrated extract to the column, rinsing centrifuge
tube with two successive portions of 5 ml each of 20% benzene/hexane,
collecting the eluate.
NOTE : If chlorinated phenols are present they should eluate
in this fraction.
3. Finally, add 10 ml of 60% benzene/hexane followed by 10 ml of 80%
benzene/hexane, collecting both these fractions in a single tube.
The ethylated 2,4-D and 2,4,5-T are contained in these fractions.
NOTE : If the individual analyst has determined that his
elution pattern differs from that given in the author’s
table and ]ie is able to obtain a consistent altered
pattern, s e appropriate revision in the eluate
collection instructions may be indicated.
Gas Chromatography
Inject into the gas chromatograph 5-10 p 1 of the 20% fraction for the
determination of the phenols and 5-10 p1 of the combined 60-80% fraction for
the determination of the chiorophenoxyacetic acids. Injections of 5-10 p1
-------
Revised 11/1/72 Section 5,A,(4),(c)
-7-
can also be made from fractions which have been concentrated to 5 nil, if
necessary, in case of lower levels of exposure. The elution pattern of
the 2 compounds extracted from a fortified urine sample must be established
as described below. The limits of detectability for 2,4-D and 2,4,5-1 are
0.05 and 0.01 ppm, respectively. Quantitation is conducted by mathematical
comparison of sample peaks against peaks resulting from the injection of
working standard (subsection JV,9,e).
The retention values, rela ive to aldrin, of the two ethylated compounds
on the SE-30/QF-l column at 200 C are:
2,4-D -0.51
2,4,5-T-——-—-----0.78
Recovery Determination
Recovery rims are essential for the operator to detenTline the efficiency
of alkylation and cleanup. From the concentrated stock standard of IV,9,a,
transfer the aliquots specified in Step 9,b to a single 50-mi vol. flask.
Dilute to volume with benzene without ethylating. Transfer a 2-mi aliquot to
a 15-mi grad. centrifuge tube and add an equal volume of 1 N NaOH. Mix well
and all to stand for 10 minutes, agitating from tii to time. Centrifuge
5 minutes at 2,000 rpm and discard benzene (upper) layer. Fortify 5 ml of
control urine with aliquots of 0.1 to 1 ml of the aqueous extract and proceed
as described in subsection V starting at Step 2.
Partitioning
NOTE : Because of differences in ambient temperature and relative
humidity from one laboratory to another, it is imperative
that each laboratory establishes silica gel elution patterns
under local conditions. Should the compounds of interest
elute in a later fraction (i.e., in 100% benzene instead of
60% or 80% benzene-hexane) the percent water added to the
silica gel must be increased by 1% increments until desired
elution pattern is established. If the compounds of interest
elute in an earlier fraction (i.e., in 20% B-H instead of
60% or 80% B-H), the amount of water initially added to silica
gel mist be decreased (use spiked control urine, not standard
compounds to determine pattern).
-------
Revised 11/1/72 Section 6,A,(2),(a)
Page 1
ME1}DD FOR DETERMINATION OF METABOLITES OR HYDROLYSIS PPUtUCTS OF
ORGANOr OSfl-1ORUS PESTICIDES IN HUMAN URINE
I. DTFROIXJCIICN :
The metabolism and urinary hydrolysis of organophosphorus pesticides
in manniials results in the excretion of a variety of alkyl phosphates.
These include the salts of dimethyl or diethyl phosphate, phosphoro-
thioate and phosphorodithioate. The gas chromatographic separation and
quantitation of such products in urine may be of value in estimating
the extent of exposure to the parent organophosphorus pesticide. The
procedure permits the determination of six major metabolites and hydrolysis
products of organophosphorus pesticides. In addition, the method pos-
sesses high sensitivity and allows for elimination of interferences from
inorganic phosphates.
REFERENCES :
1. Bowman, N. C., and Beroza, N., 1968, Gas Chromatographic
Detector for Simultaneous Sensing of Phosphorus and Sulfur-
Containing Compounds for Flame Photometry, Anal. Chem. 40;
1448. —
2. Shafik, M. T. and Enos, H. F., 1969. Determination of Meta-
bolites and Hydrolysis Products of Organophosphorus Pesticides
in Human Blood or Urine. J. Ag. Food them. 17 (6); 1186.
3. Shafik, Ivl. T., Bradway, D., Biros, F. J. and Enos, H. F.,
1970. Characterization of Alkylation Products of Diethyl
Phosphorothioate. J. Ag Food them., 18 (6); 1174.
II. PRINCIPLES
Organophosphate metabolites or hydrolysis products in urine are
extracted with a 1:1 (v/v) mixture of acetonitri and diethyl ether
after acidification with 6 N HC1. An aliquot of the extraction solvent
containing the dialkyl phosphates is treated with diazopentane to con-
vert the dialkyl phosphates to the more volatile and gas chromatograph-
able trialkyl phosphates. It is necessary to clean up the alkyl
phosphate derivatives using silica gel chromatography to eliminate
interferences arising from inorganic phosphate and other materials and
to obtain separations of couqxRlnds producing closely adjacent peaks.
Determination of trialkyl phosphates is accomplished by gas chroma-
tography using flame photometric detection with a phosphorus filter
(526 mp). The presence of sulfur-containing trialkyl phosphates may
-------
Revised 11/1/72 Section 6,A,(2),(a)
-2-
be confirmed by use of the flame photometric detector with a sulfur
filter (394 mu).
III. APPARA11JS :
1. Dual flame photometric detector, Tracor, Inc., Austin, Texas,
equipped with 526 mu filters for the determination of sulfur and
phosphorus compounds, i-espectively. This detection system was
fitted to a [ ‘licro-Tek I rF-22O gas chromatograph for the initial
development of this method, but can be adapted for use with any
as chromatograph.
2. Gas chromatograph modifications. The following modifications are
suggested for operation of the gas chromatograph with flame photo-
metric detection and are generally applicable to any gas chroma-
tograph.
(a) Buck-out contrcl on electrometer type E-2 equipped W] th a
20 niegohm VlctoTeen resistor to reduce detector signal to a
level acceptable to electrometer circuitry.
(b) Valco switdiing valve #CV 4 HT interfaced between gas chroma-
tographic column and flame photometric detector. The valve
is mounted in a mod Lfied block and is coupled with R 100
dead volume Swagelok unions. The heater in the block heats
both the transfer lines and the valve. The valve permits
interchange of column effluent and nitrogen purge. The purge
flow rate is adjusted to equal the flow from the GLC column
so that when an interchange of flows is made for the purpose
of venting solvent, no change is observed in the recorder
baseline. Thi s arrangement prevents the flame from being
extinguished when injections are made.
3. Gas chroniatographic column - Borosihcate glass, 6’ x 1/4” o.d.
packed with OV-2l0 on Ghromosorb W, H.P., 80/100 mesh.
Prepare and condition column as follows:
Fill each column leg to a point 1-3/4” from the end and insert
small plugs of s lane-treated glass wool. Heat condition cohnmi
overnight at 240 C with an applied carrier gas flow of ca 30
mi/mm. Cool column and add at inlet end 1-3/4” of 10% Carbowax
20M coated on Chrornosorb I V, h.P. Insert another small plug of
glass wool to retain Carbowax. Place co1ui n back in oven for
another heat conditioning overnight at 225 C with an applied
carrier flow of ca 10 mi/mm. Upon completion of this period,
cool column and dump out Carbowax by carefully tapping inverted
-------
Revised 11/1/72 Section 6,A,(2),(a)
-3-
column. Fill the void left by the Carbowax removal with silane-
treated glass wool and. install column in the operating nxde.
N(YrE : An alternate column, which may be used for con-
firmation of identity of peaks, is a 6’ x 1/4” o.d.
borosilicate glass column packed with 4% SE-30/6%
OV-210 on 80/100 Gaschrom Q. Condition as described
above.
4, Centrifuge tubes - 15 ml capacity, grad., conical, with ‘ ground
glass stoppers.
5. Pipets - Disposable glass, Pasteur type, (9” length) fitted with
rubber bulbs (Fisher Scientific Co.).
6. Column, chromatographic, Size 23, (Kontes #420100).
7, Vortex-Genie Mixer - Model K-550-G or equiv. (Scientific
Industries, Inc., Springfield, Mass.).
8. Evaporative concentrator tube, 25 ml, (Kontes #570050).
9. Bottles - Reagent, narrow mouth, capacity 1 oz., complete with
polyseal screw type caps. (A. H. Thomas Co., Phila., Pa., Cat.
No. 2203-C, bottles; and 2849-B, polyseal caps.)
10. Exhaust hood with a minimum draft of 150 linear feet per minute.
IV. REP4 TS :
1. Acetonitrile - “Pesticide Grade” solvent, distilled from all-glass
apparatus (Mallinckrodt Nanograde solvent or equiv.).
2. Diethyl ether - AR, or USP grade with 2% ethanol, Mallinckrodt,
Cat. No. 0850 or equivalent.
NOTE: Under no conditions should anhydrous ether be used
for the preparation of diazopentane because of the
danger of explosion.
3. Methylene chloride, dist. from all-glass apparatus.
4. Silica gel, Woe1 , activity grade I (Waters Associates, Inc.),
activated at 130 C for 48 hours and stored in desiccator.
-------
Revised 11/1/72 Section 6,A,(2),(a)
-4-
• Potassiinii hydroxide, pellets, AR grade.
• Sodium chloride, AR grade.
7• N-amyl-N’-nitro-N-nitrosoguanidine - Aldrich Chemical Co., Inc.,
Milwaukee, Wis.
8. Extracting solvent - 1:1 (v/v) mixture 1 ‘cc’tonitrile and anhy-
drous diethyl ether.
• Formic acid reagent - make up a 1% solntio 1 i of formic acid in
benzene.
10. Diazopentane reagent - Preparation:
a. Dissolve 2.3 grams of KC 1 in 2.3 nil oF dist. H 2 0 in a 125-mi
Erlenmeyer flask. mien solution i complete, cool in a
freezer for 30 minutes.
b. Add 25 ml of cold diethyl ether (flallinc rodt #0850), cover
flask mouth with foil, and cool in a -18 C Freezer 15 minutes.
c. In a VERY HIGH DRAFT hood, add 2.1 gTnIII of N-arnyl-N’-nitro-
N-nitrosoguanidine to the flask iii small portions over a
period of a few minutes, swirling the Flask vigorously after
each addition.
d. Decant the ether layer into a i-oz. reagent hottle fitted 0
with a Teflon - lined s crew cap. ]1 is may 1 )C stored at -20 C
for periods up to a week.
NOTES: (I) Because of the demonstrated carcino-
genicity and skin irritating diaracteristics,
iJO NOT ALLOW TIlL NTTRO 1)GtJANJDINE OR ThEE
I)IAZOALK TO CC ’ff TN (C)NTAO’ WITh THE SKIN .
Wear cI posahic vinyl gloves and safety
goggles whIle handl]ng. Avoid breathing
vapors.
(2) Un not use ground glass stoppered bottles
or bottJ Lti1 asib1e interior etching.
Avoid strong light.
13. Standards
For brevity’s sake, the names of various phosphate compounds
will be abbreviated from this point on. Ihc ] entit3es of the
-------
Revised 11/1/72 Section 6,A,(2),(a)
-5-
abbreviations are given in Table 3. The standard solutions are
prepared as described in the following:
a. In 15-mi grad., glass stoppered centr. tubes, accurately
weigh c . 10 milligrams of each of the following dialkyl
phosphate standards:
(1) Sodiuin* dimethyl phosphate
(2) Potassium dimethyl phosphorothionate
(3) Potassium climetliyl phosphorodithioate
(4) Diethyl* phosphate
(5) Potassium die thyl phosphorothionate
(6) Potassium diethyl phosphorodithioate
*For consistency, convert the weights of these compounds to the weight of an
equivalent amount of the potassium salt For sodium dimethyl phosphate the
conversion factor is 1.109; for diethyl phosphate it is 1.247.
b. The alkylation of these standards is carried out as follows:
(1) To each tube add 2 drops of 6 N HC1.
(2) Add suffident diazopentane to produce a persistent
yellow color (2 to 5 ml usually suffices), mix and
allow to stand for 20 minutes.
(3) Remove excess reagent by adding the formic acid!
benzene reagent dropwise until the yellow color
just disappears. Avoid an excess of this reagent.
NOTE : The procedure, up to this point, must be
carried through without interruption.
Solutions of dialkyl phosphates are unstable,
especially in an acidic medium. Therefore,
the length of time in contact with HC1 must
be kept to a minimum. After alkylation, the
trialkyl derivatives are stable indefinitely.
(4) Dilute the solution(s) exactly to 10 ml with benzene,
stopper and mix thoroughly. Store in the freezer in
glass stoppered containers when not in use.
c. These alkylated concentrated stock standards may be diluted
individually or more generally prepared as mixtures at an
intermediary concentration range. For the mixtures, pipet
aliquots of appropriate voli.m s into three volumetric flasks
-------
Revised 11/1/72 Section 6,A,(2),(a)
-6-
to obtain the concentrations given in the fo1lo ing, in
nanograms per microliter:
Mixture 1 Mixture 2 Mixture 3
JMTP 10 1M TP 5 rMP 11)
DETP 10 DEIJFP 5 DEP 10
NOTE: Because of the scarcity of the potassium salts
—— of the dialkyl phosphates, the Fernnc Laboratory
will supply the Conmmnnty Studies Laboratories
with sealed ampoules of ho iurcrniediary con-
centration mixtures listed aiove. All that will
he required is a dilution rith hcnzcne in a
1:50 ratio.
d. tVorking Standard 1ixtures:
(1) Dilute each of the intermediary ,tock mixtures in a
1:50 ratio with henzene.
(2) To establish that the working tLnuard mixtures arc in
a proper concentration range, ol)scrvc the recorder
response resulting from the inj :t ion of 5 ] of each
into the gas chromatograph.
Photometric tubes vary somewhat in sensitivity and it
may prove necessary to either furthier d] ‘ute or to
prepare higher concentrat ions o I tilC i or1.i ng standards.
Injection volumes may be varied Iroii S ti 25 il.
V. SAMPLE PREPARATION, EXTRACTION AND ALKYLATION :
1. Store urine samples in a freezer i mti1 ready for i.na1ysis. Do not
store acidified samples. when urine samp c is thawed, centrifuge
and discard solids. Pipet a 2-mi aliquot into 15-ml ccntr. tube.
NOTE : At this point, a sample of control urine from an
iuiexposed donor should be started and carried
through the entire procedure. The donor should he an
individual lmown to have no contact i . .ii i orgario-
phosphorus pesticides fo at least -i week.
2. If necessary, pr’s-extract urine samples with two 5-mi portions of
diethyl ether. Centrifuge foT one minute to obtain separation of
phases and discard extracts.
-------
Revised 11/1/72 Section 6,A,(2),(a)
-7-
NOTE: Pre-extraction of urine is usually necessary only
when the urine has been contaminated with feces as
would be the case with urine collected from experi-
mental animals housed in cages. The pre-extraction
step does not remove any of the metabolites deter-
mined by the method.
3. Add 2 grains of NaCl and pipette 4.0 ml of extracting solvent (IV,8)
to the centrifuge tubes.
4. Add 1 ml of 6 N HC1, stopper tubes arid mix on a Vortex mixer for
pne minute. It is advisable to do samples in pairs from this
point until diazopentane is added.
S. çentrifug9 at 2,000 rpn for one minute.
NOTE : No interruptions nor delays are permissible until
Step 7 has been completed.
6. With a disposable pipet, transfer 2.0 ml of the organic solvent
1aye to a grad., 15 ml, glass-stoppered centr. tube.
7. Wprking in a high draft hood, add ca 2 ml of diazopentane. A
pronounced orange color should persist after mixing. If necessary,
add slightly more of the alkylatjing reagent until the color
persists. Allow to stand for 20 minutes.
8. Concentrate solution to 0.2 to 0.3 ml under a g 8 ntle stream of
nitrogen, either at room temperature or on a 40 C water bath.
Dilute to 5 ml with dist. water, add ca S gm of NaCl and 3 ml of
hexane. Stopper and mix vigorously on Vortex mixer for 1 minute.
Allow layers to separate and centrifuge in case of interfacial
emulsion.
9. Prepare silica gel as follows: Partially deactivate 10 gm of silica
gel by shaking 2 hours with 2.0 ml dist. water. Transfer 2.4 gm to
a size 23 chroniatographic column with a small wad of glass wool at the
bottom. Top the column with ca 2 gin of anhydrous Na 2 SO 4 and prewash
with 10 ml of hexane.
NOTE : Elution patterns may vary from one laboratory to
another depending on the temperature and relative
humidity. This dictates the need for each laboratory
to establish an elution pattern of standards and
spiked control urine samples under local conditions
before attempting to analyze samples.
-------
Revised 11/1/72 Section 6,A,(2),(a)
-8-
10. Transfer hexane extract (Step 8 above) to the silica gel column.
Re-extract a.jueous layer with 2 ml of hexane and add to column.
Extract with another 2 ml of hexane and add to column. All eluate
to this point is discarded.
11. Place a 25-mi concentrator tube under column and add 15 ml of
methylene ditoride to the column. Th;is fraction contains I14TP,
DEW, DMDTP dnd DEDTP.
12. Add 15 ml of 1% acetone in methylene chloride and discard this eluaje.
NOTE : This eluate contains most of the triantyl phos-
phate and other interfering substances.
13. Place another 25-mi concentrator tube under column and add 20 ml of
3 acetone in methylene chloride. This fraction contains LMP and DEP.
VI. GAS Q-IRCIvIATOGRAPHIC DETERMINATION :
1. Inject 5-25 .il aliquots of the silica gel column fractions and work-
ing standard mixtures into the gas chromatograph. Dilution or con-
centration of the extract will be governed by chromatographic response
from the initial injections.
2. Suggested operating condition.s:
0 0
Column temperature 165 -175 C
Injection block temperature 200 0 C
Detector temperature 200 C
Transfer line temperature 200 C
Switching valve temperature 200 C
Nitrogen (carrier) flow rate 30-40 mi/mm.
Nitrogen (purge) flow rate 30-40 mi/mm.
Hydrogen flow rate 180 mi/mm.
Air flow rate 80 mi/mm.
Oxygen flow rate 10 ml/min.
NOTE : It may be necessary to adjust air and oxygen
flow rates to obtain optimum response.
3. Exanune the two eluates by gas chromatography. Quantitate sample
alkyl phosphate peaks by mathematical comparison with peaks
obtained from working standards using the phosphonis response.
-------
Revised 11/1/72 Section 6 A,(2) ,(.j)
-9-
‘4OTE : F1 1TP and IJETP isomerize upon alkyl at ion produc Lfl
tinonates and thiolates. QuantLtatlon is based on
the U”LATP and DEATP.
4. Confirm the presence oF sulfur-containing alkyl phosphates by use
of the flame photometric detector with sulfur Ii iter (394 mU).
See Figure 1 for chromatograms of the ainyl derivatives of d a1kyl phosphates.
This figure was obtained using the phosphorus Filter (526 ruJ).
Table 2 lists the relative retention times, and detector sensitivity for the
amyl derivatives of dialkyl phosphates using the phosphorus filter.
L not inject Silyl 8 on a column connected to the FPD. If column needs
reconditiening, disconnect exit end on column from the detector and condi-
tion as required.
NOTES : (1) Often after extended use of the gas chromato-
graphic system, extraneous peaks may appear.
They arise chiefly from the accumulation of
underivatized compounds on the column. Since
the appearance of the extraneous peaks is not
obvious, it is recommended that the operator
inject about 1 p1 of diazopentane solution
periodically (2 weeks). If i.mderivatized com-
pounds are present in the column, they show as
peaks following the diazopentane injection. If
this is the case, recondition the column.
(2) Quantitation is based on the amyl der ivatives,
1I’IAP, DEAP, DI LATP, DEATP, DMADTP, z nd IJEAIJFP.
Some inorganic phosphate is extracted which is
converted to TAP.
(3) Confirmation and Specificity.
a. The ability to interchange the sulfur and 1)h0S
phorus filters in the single detector, or the
use of the base assembly for dual phototube
operation with both filters (Bowiian and Beroza
1968), greatly enhances the specificity of tins
method. Suspected thiophosphate can be con-
firmed using the sulfur filter by simply in-
creasing the concentration of the compound
injected into the gas chromatograph by a
factor of 5 to 10.
-------
Rcv scd 11/1/72 Section 6,A,(2),(,:)
- 10 -
Ii. Confirmation of any particular COmpOund can
be accomplished by [ )rcpanng the liexyl de-
rivative. The method described for diazo-
pentane is followed exccpt that N-hcxyl-N’-
nitro-N-nitrosoguanithnc’ is used as the
diazoalkane precursor.
c. Purther confirmation is achieved using the
silica gel colunri uhen the sulfur-containing
derivatives are eluted in the methylcne
chloride fraction and the non-sulfur de-
rivatives eluted in the ethyl acetate fraction.
(4) Before attempting to analyze any samples, the
diemist must run fortified urine for assurance of
satisfactory recoveries. A spike of 0.1 ppm is
suggested of both sodium dimethyl phosphate and
diethyl phosphate. Proceed as follows:
a. To 5-ml aliquots of control urine in separate
15-mi centrifuge tubes add 0.5 microgram (in
aqueous solutions) of NaIIMP to one and like
amount of DEP to the other, converting the
weights to the potassium salt equivalents (see
subsection IV,ll,a).
b. Proceed with extraction and alkylation as
described in subsection V,3-13 and the GLC
as outlined in subsection VI.
Table 1. Amyl derivatives of dialkyl phosphates. Concentration ranges, in
nanograms, of standards which produce linear detector response
for normal GLC operating conditions.
Phosphorus Filter (526 mu )
IIvIAP 0.1-25 ng
DEAP 0.1-50 ng
LT IATP 0.1-50 ng
DEATP 0.1-50 ng
IIvIADTP 0.1-50 ng
DEA]JTP 0.1-50 ng
-------
Revised 11/1/72 Section 6,A (2),(a)
- 11 -
Table 2 Data from isothermal gas ch oiiatography of ria1kyl phosphates
with phosphorous f1a e photometric detection system. Co1w u +
5% QV-2i0, Temp. 173 C, carrier flow 40 mi/nan. of N 2 , noise -1%.
Relative
Ret. Value Response Sensitivity (ng)
Compour .d Retention (Mm) (Basis IMADTP) % fsd/ng 4:1 signal/noise
JjvIATP 1.46 0.59 37.2 0.11
DEATP 1.81 0.73 27.5 0.14
IMAP 2.36 0.95 57.0 0.07
IIvIAUFP 2.48 1.00 67,8 0.06
DEAP 3.03 1.22 45.3 0.09
DEAUI’P 3.11 1.25 58.7 0.07
IMAPTh 3.46 1.40
DEAPTh 4.37 1.76
TAP 16.6 6.69
Table 3. Compound identifications for bbr viations used in text:
IlviP 0,0-Dimethyl phosphate
DEP 0,0-Diethyl phosphate
Thfl’P O,O-Din thy1 phosphQrothionate
DEW O,O-Diethyi phosphorothionate
DELTI’P 0,0-Diethyl phosphorodithioate
LIvIDTP 0,0-Dimethyl phosphorodithioate
LTVIAP O,O-Dimethyl-O-amyl phosphate
-------
Revised 11/1/72 Section 6,A,(2),(a)
- 12 -
Table 3. Compound identifications for abbreviations used in text (Cont’d)
DEAP O,O-Diethyl-O-ainyl phosphate
LNATP O,O-Diniethyl -O-aniyl phosphorothionate
DEATP 0,0-Diethyl -0-anlyl phosphorothionate
‘ vI A.PTh O,O-Din thy1 -S-amyl phosphorothiolate
DEAPTh O,O-Diethyl-S-aniyl phosphorothiolate
LMAUFP O,O-Dimethyl-S-amyl phosphoxodithioate
DEADT P O,O-Diethyl-S-amyl phosphorodithioate
TAP O,O,O-Triainyl phosphate
-------
Figure 1. Chromatograms of standard solutions.
C—DiADTP—O.63 ng, DEADTP—O.54 ng.
A DNAP-O.59 ng, DEAP-O.53 ng, B DMATP—l.13 ng, DEATP—1.OO rig,
C
2
A
B
(D
a)
(D
- 4
I- ’
U I
—a
‘ -I
E-4
P i
• S
o 2
Minutes
S
0
•
2
cn
r)
0
a’
F’.)
Minutea
S S
60
•
-4
Minutes
-------
Revised 11/1/72 Section 6,A,(3),(a)
* Page 1
CHOLINESTERASE ACTIVITY IN BLOOD
I. INTRODUCTION :
The cholinesterase enzyme in blood is inhibited in varying degrees
by organophosphate pesticides and is loosely correleated with the de-
crease of acetyicholinesterase activity in the nervous system which in
turn is accompanied by an increase in the concentration of acetyl-
choline. Therefore, a scheme for measuring the level of activity of
the cholinesterase is one means of establishing possible exposure.
The technique of continuous titration of the acetic acid released
from acetyicholine by the enzyme cholinesterase overcomes many of the
widesirable features of other methods. This method does not utilize
buffers, is temperature and atmospher:Lcally controlled, and has easily
calculated units. In addition, the substrate and enzyme concentrations
can be adjusted and maintained at levels which allow optiinal enzyme
activity.
REFERENCES :
1. Michel, H. 0. An electrometric method for the determination
of red blood cell and plasma cholinesterase activity. J. Lab
Clin. Med. 34; 1964 (1949).
2. Nabb, D. P. and F. Whitfield. Determination of cholinesterase
by an automated pH-stat method. Arch. Environmental Health
15:147 (1967).
3. Pearson, J. R. and G. F. Walker. Conversion of acetylcholin-
esterase activity values from the Michel to the pH-stat scales.
Arch. Environmental Health, 16:809 (1968).
II. PRINCIPLES :
The whole blood sample is separated into the plasma and RBC (red
blood cells). Each fraction is placed in the reaction vessel of a
pH-stat and mixed with an excess of acetylcholine iodide. The cholin-
esterase present in the blood fraction reacts with the AChI releasing
acetic acid as illustrated in the following:
C C
C-’--N-C-C-O-C-C C+-N-C-C-OH + C-C-OH
C C
Acethycholine choline Acetic Acid
( ) HA
-------
Revised 11/1/72 Section 6,A,(3),(a)
-2-
A standardized solution of dilute NaOH is used as the titrant for
the released HA. The automatic titrator records the amount of titrant
delivered to the reaction in a given time period. A feedback signal
from the pH measuring electrodes controls the titrant delivery rate.
A high ChE activity produces a large hydrogen ion release in a fixed
time period, calling for a faster titrant delivery rate than will a
lower ChE activity.
III. EQUIPMENT :
1. *pH.stat, recording, complete with micro glass reference combination
electrode, thennistor temperature sensing element, a buret assembly
for delivery of 0.5 and 2.5 ml, reaction vessels.
2. Vortex mixer.
3. Centrifuge capable of spin velocity of 2000 r.p.m.
4. Aspirator for connection to suction pipet.
5. Pipet, volumetric, 2 ml.
6. Pipets, measuring, 0.2, 0.5 arid 5.0 ml, Corning 7064 or the
equivalent.
7. Centrifuge tubes, 5 ml, glass stoppered, Corning 8061 or the
equivalent.
IV. REAGENTS :
1. Sodium chloride, reag. grade.
Prepare 0.9% solution by dissolving 9.0 grams and diluting to a
liter with dist. water.
2. Potassium acid phthalate (P.A.T.) - analytical pri ary standard,
available from the National Bureau of Standards, Washington, D.C.
*M. ufacturers of equip. applicable for automatic pH titration are:
Burkland Scientific, 919 North Michigan Avenue, Chicago, Illinois.
Fisher Scientific, 711 Forbes Avenue, Pittsburgh, Pennsylvania.
Joseph Kaye, 737 Concord Avenue, Cambridge, Massachusetts.
Metrohm-Brinkmann, Cantiague Road, Westbury, New York.
Precision Scientific, 3737 West Cortland, Chicago, Illinois.
Radiometer-London, 811 Sharon Drive, Westlake, Ohio.
E. H. Sargent, 4647 West Foster Avenue, Chicago, Illinois.
-------
Revised 11/1/72 Section 6,A,(3),Iu)
-3-
Standard Solution - Dry standard P.A.T. in 105°C oven at least 4
hours before use. Store dried salt in desiccator. Weigh exactly
0.20423 grams and transfer to a i-liter vol. flask, dissolving and
making to volume in freshly boiled dist. water.
2. Titrant standard solutions prepared from sodium hydroxide, reag.
grade, Fisher S-3l8 or the equivalent, 98.7% NaOI-I.
a, Stock solution (1.0 N MaC3M): Weigh 4.053 grains MaUI- i, dissolve
in freshly boiled dist. water, cool and dilute to 100 ml. Store
in Pyrex bottle with neoprene rubber stopper.
b. Working solution (0.01 N Nail !): Pipet 1.0 ml of the 1.0 N
solution into a 100-mi vol. flask and make to volume with
freshly boiled dist. water. Standardize using potassium acid
phthalate as the primary reference.
3. Standardization of titrant working solution.
This solution should be restandardized each tine that a series’ ‘of
samples is run. Triplicate standardizations are run to obtain an
average, with the deviation between replicates no greater than 2 RU
(recorder units). It is not mandatory that the titrant working
solution be exactly 0.01 N as long as the exact nomialitj isknbwn,
but the value should fall in the range of 0.0095 to 0.0105.
The standard solution of P.A.T. contains 0.001 meq/ml. The neutrali-
zation of each ml of the P.A.T. solution will require 0.001 meq of
NaIJH. The Nail! working solution contains 0.01 meq/ml. Therefore,
0.1 ml of the working titrant 2’ 1.00 ml of the P.A.T. solution.
a. With a vol. pipet, transfer 2 ml of the standard P.A.T. solution
into a clean titration vessel and normalize instrument operating
temperature to 37 C.
b. Set titrant delivery to appropriate position for 2.5 ml de-
livered full scale, set pH to 7.0, position pen at zero and
turn on function switch.
c. The end point is reached when pen levels off at a certain RU
level. Record this level and compute the titrant normality
as follows:
N - 2.0 ml x 0.001 meg/ml = 0.4 me ml
- RUx O.005m 1/R IJ RU q
-------
Revised 11/1/72 Section 6,A,i3),( fl
-4-
NOTE : Total syringe delivery in the assumed system is
0.50 ml, indicated by 100 RU, or 100% of full
syringe travel. It is not convenient to use the
entire syringe volume for a titrant standardiza-
tion or assay. For other syringe delivery
capacities, such as 0.25 or 1.0 ml, adjust the
RU value accordingly.
4. Acetyicholine iodide available from Calbiochem Company, P.O. Box
54282, Los Angeles, California 90054.
Substrate solution - Weigh 0.7510 grams of AChI into a 25-mi vol.
flask. Dissolve and make to volume at room temperature. Store in
an amber bottle in the refrigerator and hold no longer than 2 weeks.
NOTE : Weigh sample without delay since all salts of acetyl-
choline are hygroscopic and weight will change rapidly.
V. SAMPLE HANDLING AND PREPARATION :
The sample preparation and analysis of blood should be carried out
as soon as possible after drawing sai p1e. If a few hours delay is un-
avoidable, keep samples refrigerated. If the delay will be overnight or
longer, blood should be centrifuged and plasma separated from RB cells
and the latter taken through the following steps (a through f) before
storage.
a. Place blood tube in centrifuge and spin for 20 minutes at 2000 r.p.m.
b. Pipet plasma into clean tubes for storage.
NOTE : If a part of this sample will eventually be analyzed
by GLC, E.C., the tube cap should be ground glass or
screw cap with Teflon or foil liner.
c. Using vacuum pipet, remove and discard fluffy layer.
d. Resuspend red cell mass in an equal volume of 0.9% NaCl solution.
This is done by gently inverting tube.
e. Centrifuge again for 10 minutes and remove supernatant NaCl solution
by vacuum pipet.
f. Repeat steps d. and e. The RBC mass is now ready for assay.
-------
Revised 11/1/72 Section 6,A,(3),(a)
-5-
VI. PREPARATION OF RBC I-LFIvULYSATE :
1. Pipet 1.8 ml of thst. water into a 5-mi conical centrifuge tube and
then pipet in 0.2 ml of packed red cells.
NOTE : Care must be taken to wipe the tip of the pipet with
tissue while still retaining the full 0.2 ml contents.
This may require some practice.
2. With a rubber bulb attached to the pipet, draw the hemolysate solu-
tion up into the bore of the pipet repeatedly until all adhering
cells are washed into the water.
3. Stopper tube arid mix on Vortex mixer about 30 seconds or until all
cells have hemolyzed. The hemolysate so prepared may remain up to
20 minutes at room temperature before assaying. For longer periods,
hold in refrigerator.
VII. CHOLINESTERASE ASSAY :
1. Calibrate instrument with reference buffers, following manufacturer’s
instructions. This is best done by using two buffers, one bel 8 w
arid one slightly above pH 8. Normalize the instrument to a 37 C
operating temperature.
2. Into a clean titration vessel pipet 4.2 ml of 0.9% NaC1 solution
and 0.15 ml of plasma, (or 4.2 ml of 0.9% NaC1 and 0.50 ml of RBC).
3. Place titration vessel on instrument and be sure that instrument
end point is set at pH 8.0. As the initial pH of sample and NaCl
solution will nearly always be a little low, it is necessary to
adjust the pH to 8.0 with the 0.01 N NaOH titrant.
4. Check to be sure that recorder and titrant delivery systems are set
at zero.
5. Add substrate to mixture in titration vessel:
a. If plasma assay, add 0.6 ml of acetylcholine iodide solution.
b. If RBC assay, add 0.10 ml of AChI solution.
6. Start recorder and titrant flow and allow titration to proceed until
recorder describes a line with constant slope, then mark off the
start and end of a 3-minute period of titration. See Figure 1.
-------
Revised 11/1/72
Section 6,A, (3) , (a)
-6-
NOTE : None of the line included should be nonlinear. It is
good practice to allow titration to proceed for a
minute or so before counting the RU.
0.600 0.600
From formula (1), N = 53.961 = 3.961 = 0.011 N
2. Calculation of ChE activity.
A. Factor, from Table 1, for plasma ChE, based on 0.011 N titrant,
is 0.1222. The observed RU value is multiplied by this factor:
Plasma 1: 46.5 x 0.1222 = 5.682 i.iN/nun/ml (first replicate)
48.5 x 0.1222 5.927 iM/min/ml (second replicate)
Plasma 2: 30.5 x 0.1222 3.727 pM/mm/nil. (only one replicate shown)
B. Alternative method, without using factors:
Subs ti tuted:
RU xO.005 ml , = mi/minute = 46.5 X 0.005 = 0.0775
utes
mi/minute x Normality = meq/minute = 0.0775 x 0.011
= 0.000853 meq/minute
meq/minute x 1000 .dv1/meq = pM/minute = 0.000853 x 1000
pM/meq
= 0.853 pM/minute
sa ’ me = pM/mii m1 = _____ = 5.687 1 . M/min/m1
VIII . SAMPLE CALCULATIONS :
Refer to Figure 1:
1. Standardization of titrant.
Average of three replicate titrations:
54.0 RU
54.0
53.9
3)161.9 = 53.961 RU
Plasma 1:
-------
Revised 11/1/72 Section 6,A,(3),(a)
-7-
IX. MISCELLANEOUS NOTES :
1. Because of the variety of instruments available, operating instruc-
tions are not given for pH-stat equipment. Detailed instructions
furnished by the manufacturer will govern operating details. A step-
by-step outline of sample preparation, reagent preparation and stan-
dardization, plus the scheme for performing calculations are outlined.
2. Heparin is preferable to other anticoagulants, because oxalate,
citrate and EDTA will sequester calcium and magnesium, which are
required co-factors for chE. In an emergency, these anticoagularits
can be used, but ChE assay results are likely to be lower than if
heparin were used.
3. Considering the low pipetting volumes, meticulous care must be taken
to obtain reproducible aliquots. This is particularly true with
packed red blood cells.
4. Centrifugation speeds and times should be consistent from one sample
lot to the next. Formation of packed red cell mass after final
saline wash is critical. Each successive red cell pack should be
the same, otherwise differences in density will yield different
results from sample to sample.
5. Be Gentle . Hemolysis of red cells in contact with plasma will
liberate acetylcholinesterase into plasma, thus altering the true
enzyme activity of the latter. Mix red cells with plasma or saline
solution by gentle inversion of tubes, or by gentle stirring with
glass rod or wooden applicator stick.
6. The approximate lower limits of normal ChE activity for human blood
assayed by the present method are:
2.0 .iM/min/rnl - Plasma
8.0 pN/min/ml - Red Cells
An individual laboratory should, of course, establish its own normal
ranges based on experience with the method.
7. Assay results obtained by the pH-stat method are expressed as:
micromoles (acetic acid liberated) /minute/ml sample (either packed
red cells or plasma). Abbreviated: pM/mm/mi.
8. Standard sources of enzyme are available from Sigma and are useful
in intralaboratory quality control.
-------
ktevised ff117 2
—8-
Section 6,A,(3),(a)
Figure 1. Sample of strip chart record of pH—stat assay of cholinesterase
activity.
80
70
60
50
LLO
30
20
Standardization of
0.01 N NaOH against
potassium acid
phthalate, three
replications
. — ‘ 5 .O RU’
-------
Revised 11/1/72 Section 6,A,(3),(a)
-9-
Table 1. Factors for Three Minute Titrations
Normality NaOH
Solution Factor Plasma ChE Factor
0. 0. 0.
0095 3167 1055
0096 3200 1067
0097 3233 1078
0098 3267 1089
0099 3300 1100
0100 3333 1111
0101 3367 1122
0102 3400 1133
0103 3433 1144
0104 3467 1156
0105 3500 1167
These factors are derived as follows:
Red cell factor mi/mm x meg/mi x 1000 pN/meg
0.05
Plasma factor = mi/mm x meg/mi x 1000 pM/meg
0.15
These factors are valid only if a 0.5-mi syringe is used for titrant delivery,
so that the mi/mm factor in the equation becomes 0.005 , or 0.00167 mi/mm.
3
-------
Revised 11/1/72 Section 7,A
Page 1
DETERMINATION OF l-NAPHThOL IN URINE
I. INTRODUCTION :
Humans exposed industrially to the insecticide carbaryl (l-naphthyl-
N-methyl carbainate) excrete relatively large quantities of l-naphthol,
conjugated either as the sulfate or glucuronide. Quantitative deter-
n .ination of J naphthol in human urine has been genei a .1y accomplished
using a colorimetric procedure. This method lacks both the sensitivity
and specificity necessary for determining the relatively small amounts
of l-naphthol excreted in the urine of agricultural workers exposed to
low levels of this insecticide.
The l-naphthol resulting from the hydrolysis of carbaryl has been
used as an indirect measure of the residue level of the parent insecti-
cide on a variety of agricultural crops. Argauer has described a
procedure for chloroacetylating phenols and 1-naphthol for subsequent
detection by electron capture gas chromatography. In a recent publica-
tion this procedure was utilized to determine a number of carbamate
insecticides. However, it was indicated that further medification
would be necessary if the method was to be extended to carbaryl.
The method described in this section utilizes the enhanced electron
capture characteristics of the monochioroacetate derivative. This,
coupled with a silica gel cleanup results in a method sensitivity down
to 20 ppb of 1-naphthol.
REFERENCES :
1. Shafik, N. 1., Sullivan, H. C., Enos, H. F., ul1. of
Envir. Containin. Toxic., Vol. 6, No. 1, 1971 pp 34-39.
II. PRINCIPLES :
A small sample of urine is subjected to acid hydrolysis. The
l-naphthol present is extracted in benzene and derivatized with chioro-
acetate anhydride solution. After silica gel cleanup, the resulting
l-naphthyl chioroacetate is quantitatively determined by E.C., ( IC,
comparing sample peaks against peaks obtained from pure l-naphthol
standard, similarly derivatized.
III. APPARATUS :
1. Gas chromatograph equipped with E.C. detector and fitted with a
6’ x 1/4” o.d. column of 1.5 OV-17/l.95% QF-l. Operating para-
meters for the column are those prescribed in Section 4,B of this
manual.
-------
Revised 11/1/72 Section 7,A
-2-
III. APPARATUS (Continued)
2. ChromatogTaphic column (Chromaflex), size 22, Kontes #420100.
3. Evap. concentrator tubes grad., glass stoppered, 25 ml, ‘ 19/22,
Kontes #57O( 5O.
4. Distilling column (condensor), 200 mm jacket, Kontes #286810,
fitted with tight glass stopper at top.
5. Centrifuge capable of 2000 r.p.m. that will accept metal shield,
I.E. Company No. 367.
6. Volumetric flasks, 10 and 100 ml.
1• Pipets, Mohr, 0.2, 0.5 and 10 ml, Corning 7064 or the equivalent.
8. Pipets, transfer, 2, 3 arid 5 ml, Corning 7100 or the equivalent.
9. Vortex-Genie mixer.
10. Disposable pipets, Pasteur, 9 inch.
11. Graduated conical centrifuge tubes, 15 ml, with ‘ glass stoppers,
Corning 8084 or the equivalent.
12. Circulating wnter pump.
13. Bailing water or steam bath.
-------
Revised 11/1/72 Section 7,A
-3-
IV. SOLVENTS AND REAGENTS :
1. Benzene, pesticide quality.
2. Hexane, pesticide quality.
3. Pyridine, Spectro Grade, Eastman #13098.
4. Chioroacetic anhydride, Eastman /t335 - prepare a 2% solution in
benzene and hold no ]onger than one week.
NOTE : The chioroacetic anhydride must he dry and
therefore should be stored in a desiccator as it
is extremely hygroscopic.
5. Hydrochloric Acid, reag., conc,
6. Sodium sulphate, anhydrous, granular - preextract on a Soxhlet
with benzene for 0 about 50 discharge cycles, remove excess solvent
and store in 130 C oven.
7. Sodium sulphate, 3% solution in dist. water. Use preextracted Na 2 SO 4 .
NOTE : De]onized or distilled water, preextracted with benzene,
is used throughout the procedure.
8. Mixtures of benzene/hexane as follows: 20/80, 40/60 and 80/20.
9. 0.1 N arid 1.0 N NaOH solutions.
10. Silica ge], Woelm, activity grade T, Waters Associates, Inc., DO
NOT SUBSTITUTE . —
11. Preparation of sdica gel.
Dry adsorbent for 48 hours at 170°C and store in the same oven.
On day of use, cool the silica gel in a desiccator and deactivate
with 1.59 water in the following manner: Add the necessary volume
of water to a 125-mi glass stoppered Erlenmeyer flask, rotating
the flask to coat the sides with water. Add the weighed amount of
silica gel, stopper and mix until the water is evenly d .stributed
throughout the adsorbent. Allow to equilibrate for 2 to 3 hours
with periodic shaking. chromatographic columns are prepared just
prior to use.
-------
1/4/71 Section 7,A
-4-
12. 1-Naphthol, Eastman #170.
13. Preparation of standard solutions:
a. Stock Standard I. Weigh 20 mg of l-naphthol into a 100-mi vol.
flask, dissolve and dilute to volume with benzene. This is th 8
conc. stock of 200 ng/Ml and may be held several months at -18 C.
b. Stock Standard II. With a 0.5-mi Mohr pipet, transfer 0.25 ml
of stock standard I to a 50-mi vol. flask and make to volume
with benzene. This intermediary stock standard of 1 ng/ul is
used for preparation of working standards and sodium naphthoxide
for recovery studies (See Misc. Note, 1, a).
c. Prepare reagent blank and working standards by transferring
aliquots of stock standard II of 0, 0.1 and 0.5 ml to separate
25 ml evap. concentrator tubes. Dilute each to 5 ml with
benzene.
(1) Add 2 ml of 2% chloroacetic anhydride and 0.2 ml of pyridine
to each tube. Stopper and mix vigorously on Vortex mixer
for 2 minutes. Allow to stand 10 minutes.
(2) Add 5 ml of dist. water, stopper and reagitate on Vortex
for one minute.
(3) Allow layers to separate and, with a disposable pipet,
c irefully remove and discard as much as possible of the
lower (aqueous) layer.
(4) Repeat water wash (Steps (2) and (3) above) twice more.
(5) Place tubes in centrifuge, spin 5 minutes at 2,000 r.p.m.
and remove any final traces of water from bottom of tubes.
Dilute to exactly 10 ml with benzene and mix thoroughly.
The three tubes will contain concentrations of l-naphthyl
chioroacetate of 0, 10 and 50 pg/i.il.
d. Prepare another intermdeiary stock standard (III) of derivatized
1-naphthol (l-naphthyl chioroacetate) by transferring 0.5 ml of
stock standard I to a 25-mi evap. concentrator tube and dilute to
S ml with benzene. Proceed with derivatization as described above
in c, (1), (2), (3), (4) and (5) but instead of diluting to 10 ml
as described in step (5), transfer extract through a glass funnel
into a 100-mi vol. flask, rinsing tube with several portions of
benzene and finally making to volume with henzene. From this
derivatized stock of 1 ng/i.il concentration of l-naphthyl chioro-
acetate, dilutions may be made and used to check the derivatized
working standards finalized in step c, (5) above.
-------
1/1/71 )C’(1 11)1) 1, i\
-5-
NO’ft : The derivatized standards arc relatively stable.
Assuming no solvent losses from repeated opening
of the storage bottles, it is probable that the
standards could be held for 6 months, storing in
the refrigerator when not in use.
V. PROCLIXJRL :
Hydrolysis, Extraction and Derivatization
When the following irocedure is started there should be no interruption
until the final derivatized extract is obtained in Step 10. It is highl
desirable that a sample of control urine from an unexposed donor be carried
along parallel with the sample(s) under test.
1. Pipet S ml of urine into a 25 ml evap. concentrator tube, add
1 ml of conc. HC1, stopper and mix on the Vortex mixer 2 minutes.
2. Fit concentrator tube with a glass stoppered condenser (distilling
column) and reflux mixture in a hot water or steam bath 90 minutes,
cooling the condenser with circulating ice water.
NOTE : The top of the condenser should he tightly stoppered.
3. After re!noval from the bath and cooling, wash down bore and condenser
tip with 2 ml of 0.1 N NaOH followed by 3 ml of benzene.
4. Stopper and mix vigorously for 2 minutes on the Vortex mixer.
5. Place conc. tube in centrifuge and spin for 10 minutes at 2,000 r.p.m.
6. Carefully transfer as much of the benzene (upper) layer as possible
to a clean 25 ml concentrator tube, using a disposable pipet fitted
with a rubber bulb.
7. Add 3 ml more of benzene and repeat Steps 4, 5 and 6.
NOTE : Extreme care should be taken to prevent water
from being transferred as this would seriously
affect derivatization efficiency.
-------
1/4/7 1 SoctLnzI 7, /\
-6-
8. nash benzene extract with two 3 ml portions ol N t SO solution,
centrifuging and discarding each successive aqueous ay r.
9. To the combined benzene extract add 2 ml of 2% diloroacetic an1i dride
solution and 0.2 ml of pyridine. Stopper tube, mix on Vortex mixer
for 2 minutes and allow to stand 10 minutes at room temperature.
10. Refer to and follow identically Steps (2),(3),(4), and (5) of
su section l iv., 13, C but placing tubes of derivatized extract in a
40 C bath and evaporating to 0.5 ml under a dry nitrogen stream.
NOTE : Under no circiunstances should final volume
be permitted to go below 0.3 ml.
Silica Gel Cleanup
1. Place a small wad of glass wool at the bottom of a Chromaflex
column and add 1 grain of the partially deactivated silica gel. Top
this with ca 1/2-inch of anhydrous, granular Na 2 SO 4 .
2. Prewash the column with 10 ml of hexane, discarding the eluate.
3. When the surface level of the hexane reaches a point on the column
ca 2 an from the top of the Na 2 SO 4 fransfer the concentrated benzene
extract to the column with a disposable pipet and rinse tube with two
portions of 0.5 ml of 20/80 benzene/hexane solvent applied with
another disposable pipet, directing stream so as to wash down walls
of tube. Follow this with 8.5 ml of 20/80 benzene/hexane solvent,
discarding all eluates up to this point.
4. Place a 15 ml grad, conical centrifuge tube under column and add
10 ml of 60/40 benzene/hexane solvent, collecting this fraction
which contains the l-naphthyl chloroacetate derivative. Finally,
adjust volume of extract to exactly 10 ml with benzene.
NOTE : 1. The foregoing instructions are based on the
premise that the operator obtains an elution pattern
identical to that obtained by the authors. However,
this should not be assumed until the operator has
confirmed it by actual trial. This may be readily
done by applying 0.5 ml of the derivatized 200 ng/uJ.
stock standard to a silica gel column in the manner
described in Steps 1, 2 and 3 but collecting the
10 ml of hexane prewash and the 20/80 benzene/hexane
eluates instead of discarding them. In addition to
this, 10 ml each of benzene/hexane mixtures of 40/60
60/40, 80/20 and 100% benzene should be eluted
through the column, collecting each of the cuts for
-------
Revised 11/1/72 Section 7, \
-7-
examination by GLC to determine ‘h er any of thc
derivatized compo md is elutint out ot place. If so,
it may prove necessary For the ndlvidual operator t”
make appropriate revisions in the cluate collection
instructions.
2. At rio time during the eL t i.i liould the liquid
level in the column be allowed to go below the top oF
the Na 2 SO 4 layer.
Gas Chromatography
After adjust]ng operating parameters of the gas chromatograph to
the values prescribed in Section 4,B of this manual, commence injections
of derivatized sample and standard extracts. Assuming an average hacI-
groi md current, it should be possible to quantify as little as 50
picograms of the derivatized l-naphthol Using the 1.5% OV-17/l.95%
QF-l column, the relative retention value for l-naphthyl chloroacetnte
should be ca 0.92 with respect to aldrin.
VI. MISCELLANEOUS NOTES :
1. Elution patterns may vary from one laboratory to another depending
on the temperature and relative humidity. This emphasizes the
need for establishing an elution pattern of standards and spiked
control urine samples under local conditions before attempting
to analyze samples. The procedure for ‘spiked control urine samples
is as follows:
a. In a 15-in], conical grad. centrifuge tubc mix 2 ml of 1.0 N NaOH
and 2 ml of the diluted, underivatized standard described in
the NOTE in Step h, of subsection IV, 13.
b. Stopper tube and mix 2 minutes on :i Vort2x mixcr. Allow to
stand 10 minutes and centrifuge 5 minutes at 2,000 r.p.m.
c. Pipet aliquots of 0.1, 0.25 and 0.5 from the aqueous layer
(sodium naphthoxide solution) into separate 25-mi grad. evap.
concentrator ti ibcs.
d. From this point on, conduct the hydrolysis and dcrivatization
as previously described in subsection V, starting with Step 1,
under Hydrolysis, Extraction and Derivntizntion, ending at
Step 4 under Silica Gel Cleanup. The Final extracts contained
in the three 15-mi centrif. tubes should have concentrations
of 10, 25 and 50 pg/uI of derivatized l-naphthyl chloroacetatc.
Recovery data is obtained by chromatographing these extracts
against the derivati zed working standards.
-------
Revised 11/1/72 section 8,B
Page 1
SAMPLING AND ANALYSIS OF PESTICIDES IN AIR
B. ANALYSIS
I. INTROWCTIC :
The analytical method described presupposes the collection of the
air sample with the sampling device developed by Mid West Research
Institute* utilizing a vacuum pump which draws air through ethylene
glycol contained in glass impingers. A customary sample consists of
400 ml of ethylene glycol representing the contents of four impingers
of 100 ml each, two of which are operated simultaneously for 12 hours,
and then two more for another 12 hours. The total air volume sampled
in the 24-hour period is generally ca 80 cubic meters.
II. PRINCIPLE :
The ethylene glycol contents of four 100-mi impingers are transferred
to a large separatory funnel and diluted with water. Pesticides are ex-
t±acted with hexane and transferred to a Kuderna-Danish evaporator.
After concentration to a suitable volume, a preliminary gas chromato-
graphic evaluation is made using electron capture detection for organo-
chlorine compounds and flame photometric detection for the organophos-
phates.
The extract is then subjected to column cleanup with Florisil,
partitioning into two or three eluate fractions, each of which is re-
concentrated to a suitable volume and subjected to final gas chromato-
graphic analysis.
*stan]ey et al., Envir. Sci. Technol., 5, (5) 430 (1971).
-------
Revised 11/1/72 Section 8,B
-2-
III. EQUIPM T :
1. Chromatographic columns 22 miii i.d. x fl0 mm lencth, Teflon
stopcocks, without filter frits.
2. Separatory funnel - 1 and 2-liter capacity, Teflon stopcocks.
3. Hot water bath - 95-100°C.
4. Glass beads 3 imi plain, Fisher #11-312 or equivalent.
5. Glass wool - angel hair, fine.
6. Kuderna-Danish concentrator.
Evaporative concentrator flask - Kontes Catalog No. K-570001, 500
and 1,000 ml, lower joint 19/22 , upper joint 24/40 .
Snyder column - 3 ball, lower joint 19/22 ‘, upper joint 24/40 ‘ ,
Kontes Cat. No. K-503000.
Tube - 10 ml grad., 19/22 ‘ joint, Kontes No. K-570050, Size 1025.
7. Gas Chroriatographic Columns.
1.5% OV-17/1.95% QF-l, 4% SE-30/6% QF-i mid 10% OV-210, preparation,
conditioning and operating parameters as outlined in Section 4,A.
iv. REAGENTS :
1. Et 1 ylene Glycol, pesticide quality. Must meet the criteria
describec’ in Section 8,C.
2. Hexane, reaistilled in glass. Must be evaluated for background
interference by gas chromatography, electron capture, of a 1-mi
concentrate from 180 ml. of original solvent.
3. ?etro euun ether - pesticido quality, redistilled in glass, E.P.
30-60 C.
4. Ethyl ether - AR grade, peroxide free.
The above solvents should be free of materials that give a
response to the eleLtron capture detector. Preparation of
peroxide-free ethyl ether is given in Section 5,A,(l).
-------
Revised 11/1/72 Section 8,B
- 3_
v REAGENTS (CONTD.)
5. Distilled water - check for interferences. If necessary, extract
with benzene and pet. ether as described in Section 8,C for
glycol extraction.
6. Florisil, P. R. grade. See MISCELLANEOUS NOTE 4, Page 7.
7. Anhydrous sodium sulfate.*
8. Eluting mixture (6+94 ether in pet. ether). (Used for Fraction I.)*
9. Eluting mixture (15+85 ethyl ether in pet. ether). (Used for
Fraction IJ.)*
10. Eluting mixture (50+50 ethyl ether in pet. ether). Prepare same
as other ether mixtures. (Used for Fraction III.) *
v. PROCEDURE :
All glassware to be used must be scnipulously cleaned and given a
final hexane rinse immediately before use.
1. Combine the impinger contents representing the collection of 81
of air (presumably 4-100 ml increments of glycol).
2. Transfer the 400 ml of E.G. to a 2-liter sep. funnel (No. 1). Wash
the Sample bottle with a measured amount of dist. water and add
washings to sep. funnel. Dilute with a total of 1400 ml of water.
NOTE : All procedural instnictions from here on assume the
extraction of 400 ml of E.G. Should a smaller volume
be extracted, the glassware sizes and reagent volumes
should be scaled down proportionately.
3. Transfer the prefilter glass cloths to a 400-mi beaker with a clean
pair of forceps. Add 240 ml of hexane, stopper and shake funnel
vigorously for 2 minutes. If emulsions are formed at this point,
add 10 ml of a saturated NaC1 solution to the separatory funnel
(NaC1 and distilled H 2 O should be free of interferences - pre-
extract if necessary).
* Instructions and comments re purity of these materials may be found in
Section 5,A,(l).
-------
Revised 11/1/72 Section 8,B
-4-
V. PROCEDURE (CONTD.)
4. Transfer the aqueous layer to a second 2-L sep. funnel (No. 2), and
the hexane layer to a l-L sep. funnel (No. 3). Add another 240-mi
portion of hexane to funnel No. 2, stoDper and shake vigorously for
2 minutes. Allow a few minutes for layer separation and transfer
aqueous layer back into funnel No. 1. Combine the hexane layer
with that already in funnel No. 3. Add a final 240-mi portion of
hexane to funnel No. 1 and repeat shaking and settling. Discard
aqueous layer and transfer hexane layer to funnel No. 3.
5. Add 200 ml of dist. water to S.F. No. 3, stopper and shake vigorously
1 minute. Discard aqueous layer and repeat extraction with more of
the 200-mi portion of dist. water, again discarding aqueous layer.
6. Place a glass wool retaining plug in the bottom of a 300-mm chromato-
graphic column, add 2 inches of Na SO and pass the combined hexane
extract through the column, collec in eluate in a 1,000-mi K-D
flask which has been fitted to a 10-mi evaporative concentrator tube
containing one 3-mm glass bead.
7. When extract has eluted from the column, rinse the sep. funnel with
three consecutive portions of 10 ml each of hexane, washing down
the walls of the column. Allow each rinse to elute off the column
before adding the next. Finally, rinse the column walls with two
more 10-mi portions o. hexane.
8. Place K-D assembly in a boiling water bath and concentrate extract
to ca 5 ml. Remove from bath, cool, disconnect tube from K-D flask,
rinsing joint with a little hexane. Place tube under a nitrogen
stream at room temp., and further concentrate extract to ca 0.5 ml.
Rinse down tube sidewalls with hexane delivered from a 100-ui
syringe, diluting extract back up to exactly 1.0 ml. Stopper and
mix on Vortex 30 seconds.
9. Make exploratory injections into the gas chromatograph via FPD
detection. In the event that no peaks appear with relative reten-
tion values suggestive of organophosphorus compounds, it may be
concluded that the sample contains none of these compounds, or that
any compounds present are below the minimum detection levels. (See
Section 4,B for sensitivity guidelines.) If peaks of greater than
10% f.s.d. appear by FPD, and their identities can be detennined
as organophosphorous pesticides, quantitations should be conducted
at this point.
-------
Revised 11/1/72 Section 8,B
-5-
V. PROCEDURE (CONTD.)
1’ JTE : It is probable that at this extract concentration
ratio (400:1), background interference would make
electron capture appraisal difficult or impossible.
However, if the detectors are so arranged in one
ins trument that an E . C. chromatogram can be obtained
siinultancously with the FPD chromatograin, some useful,
preliminary information may be obtained.
Florisil Partitioning
1. Dilute extract up to ca 5 ml with hexarie and transfer to a Florisil
column. [ Prepare column as described in Section 5,A,(l).] Begin
eluate collection immediately on adding hexane to column and collect
the eluate in a 500-mi Kiderna-Danish concentrator fitted with a
10-mi grad. concentrator tube containing one glass bead. When
hexane reaches the Na.,SOA layer, wash the tube with 3- to 5-al
portions of pet. ethet’, insing the column walls each time, then
wash column walls with 1- to 10-mi portion of pet. ether. When
final wash reaches Na,S0 4 level, elute column with 200 ml of the 6%
mixed ethers and coll&t until ethers reach Na 2 SO 4 layer (Fraction I).
2. Change Kuderna-Danish concentrators and elute with 200 ml of the
15% mixed ethers in the same manner (Fraction II).
3. Change Kuderna-Danish concentrators and elute with 200 ml of the
50% mixed ethers (Fraction III).
4. Attach Snyder columns on the K-D flasks, immerse tubes in hot water
bath and concentrate eluates to slightly less than 5 ml. Cool and
detach tubes from K-il flasks, rinsing joints with a little hexane.
5. Adjust the volume of the Fraction I arid II extracts to exactly 5.0
ml and analyze by electron capture detection.
NOTE : If the 1-mi concentrate of the unpartitioned extract
was examined via electron capture, it may be possible
to estimate the degree of dilution necessary to bring
peaks of chlorinated pesticides into quantifiable
range by electron capture or electrolytic conductivity
detection.
-------
Revised 11/1/72 Section 8,B
v. PROCEDURE (CONTD.)
6. After completing GLC examination of Fractions I and II, continue
extract concentration, along with Fraction III, down to ca 0.5 ml
under a nitrogen stream at room temperature. Rinse down sides of
tube with hexane delivered from a 100 -Ml syringe, diluting extract
to exactly 1.0 ml. Stopper and shake on Vortex 30 seconds.
NOTES : (1) Do not use air for blow down and do not permit
evaporation to dryness. Organophosp1i e com-
pounds may be seriously affected by either of
these practices.
(2) If predetermined Florisil elution patterns have
indicated that any chlorinated pesticides are
eluting in Fraction III (endrin or dieldrin are
sometimes troublesome), the volume of this
fraction should also be adjusted to exactly 5 ml,
and examination by electron capture conducted.
7. Analyze the l-ml extract concentrates of all three fractions by FPD,
carefully observing sensitivity guidlines set forth in Section 4,B.
NOTE : The FPD analysis on the partitioned extract is run
for assurance of identifications obtained on unparti-
tioned extract. There will probably be no need for
requantitating these 0. G. P. compounds except in cases
of distorted or overlapped peaks in the original
chromatograms.
v i. MISCELLANEOUS NOTES :
1. Use the Ov-17/QF-l and SE-30/QF-l columns for electron capture GLC,
and the SE-30/QF-l, Carbowax-treated, for all FPD work. An alter-
nate column that may prove useful is the OV-210. At this point in
time, relative retention and response data given in Section 4,B,
Table 1 is restricted to the SE-30/QF-l Carbowax-treated column.
Data will eventually be forthcoming for the columns of 1.5%
OV-17/l.95% and 10% OV-210.
2. The pesticides of interest may be in any of the three fractions and
can be identified on the electron capture or flame photometric
detector systems as needed. (i.e., Fraction I contains organo-
chlorine pesticides; Fraction II contains organochlorine, chloro-
phenoxy acid esters and organophosphate pesticides; and Fraction III
-------
Pevised 11/1/72 Section 8,B
- 7_
v i. MISCELLANEOUS NOTES (CONTD.)
contains malathion.) Information on any other organochiorine or
organophosphate pesticide of particular interest may be character-
ized by n nütoring the appropriate fraction with either the
electron capture or flame photometric detector.
3. As a reiteration, the miscellaneous notes discussed in the Section
5,A,(1), under the “Analysis of Adipose Tissue”, are very pertinent
to this methodology and should be thoroughly understood.
4. The analyst must know from predetermined tests of recoveries and
compound elution patterns which compounds will appear in each
Florisil fraction eluate of the particular lot of Florisil being
used. Those laboratories operating under contract with EPA are
supplied with standardized Florisil of known adsorptive character-
istics. It may prove necessary to use nxre or less than the
4 inches of Florisil specified to achieve the partitioning indicated
in Table 1, this section or in Table 1 of Section 5,A,(l).
5. Occasions arise when positive compound identification by electron
capture is not possible. This situation is not unusual with certain
of the organochlorine compounds at low concentration levels. One
confirmation technique involving minimum effort is TLC. This is
described in Section 12,B, pp 7 and 8. Another relatively rapid
technique is the p-value procedure described in Section 12,C.
Those laboratories equipped with electrolytic conductivity or
microcoulometric detectors can utilize this equipment for both
qualitation and quantitation.
-------
Revised 11/1/72 Section 8,B
TABLE 1. FLORISIL ELIJTION PAIFERNS, RE )VERY EFFICIENCY,
AND LOWER LIMITS OF DETECFION OF SOME CO?vM)N
PESTICIDES EXTRAcTED FROM ETHYLENE GLYCOL BY
PROCELXJRE C JTLINED IN SUBSECTION III.
Elution Lower Detection Liini.ts (ng/m 3 )
Compound Fraction 4% SE-30/6% QF-1 1.5% OV-17/1.95% QF-1
c -BHC I 0.2 0.2
Diazinon II .4 -
B-BHC I .8 1.2
Lindane I .4 .4
6-BHC I .4 .4
Heptachior I .4 .4
Ronnel I .6
Aidrin I .6 .6
Heptachior Epoxide I .8 .8
Die ldrin II 1.2 1.8
Endrin II 2.6 4.0
o,p t -DDE I 1.4 2.0
thiorobenside I 1.6 1.2
p,p’-DDE I 1.0 1.2
Thiodan I II - -
o,p’-DDD I 2.0 2.8
*perthane I 167. 167.
p,p’-DDD I 1.6 2.0
o,p’-DDT I 2.0 2.6
Thiodan II III - -
p,p’-DDT I 2.0 1.5
Tedion II 25.
Ethyl Parathion II 1.3
Methyl Parathion II .9
Malathion III 1.3
Ethion I 1.5
ch lordane I -
Carbophenothion I 3.1
Aroclor 1254 I -
2,4-D esters II
*BE 13.6 10.2
*IOE 50. 44.
IPE 6.2 9.
43. 22.
*PGBEE 19. 15.
-------
Revised 11/1/72 Section 8,B
-9-
TABLE 1 (CONTD.)
Elution Lower Detection Limits (ng/m 3 )
Compound Fraction 4% SE-30/6% QF-1 1.5% OV-17/1.95% QF-1
2,4,5-T esters II
*IOE 18. 7.6
*IpE 1.4 1.6
*n..Buty l
Lower detection limits of organophosphorous compounds determined by flame
photometric detection; all other compounds by electron capture.
*P very efficiencies not established for these compounds. Recoveries of
85% or better determined on aLL other c npounds listed.
-------
Revised 11/1/72 Section 8.B
- 10
TABLE 2. EL1! ION AND RECOVERY OF ORGANOB-IOSPHOROUS
PESTICIDES BY M)G MEI1-LOD
Copied from F.D.A. Release
Elution from Florisil
Pesticide Nair % Recove y 6% 15% 50%
Abatea -
Azinphosethyl 50% -
Azinphosmethy l 0
Azodrin 0
Bensulidea
Bidrin 0
Bomy l 0 -
Br phos ethyl 102% +
Carbophenothion Oxygen Analog 0 -
Ciodrin 0
Compotmd 4072 0
Couinaphos 0 - -
Def 91% + +
Deineton 0 -
Dicapthan a 76% - + +
Dichiorvos - -
Dimethioate Oxygen Analog 0
Diinethioate 0
Dioxathion 0 -
Disulfoton 51% +
Disulfoton Oxygen Analog 0 -
Disulfoton Sulfone 0 -
Dyfona e 91% + -
Falone - -
Fainphur 0 - -
Fenthion 45% + +
Fensulfathion 0 - -
Fensulfathion Oxygen Analog 0 -
Fensulfathion Sulfone 0
Gardona 0
Gophacidea - -
Hercules 14503 95% +
IlTLidan 0
Malath.i. n Oxygen Analog 0
Maretin -
M erphos 133% +
Methyl Parathion Oxygen Analog 0 - -
Methyl Trithion 82% + -
Mevinphos 0 -
Mecap 55% +
-------
Revised 11/1/72 Section 8,B
- 11 -
TABLE 2 (CONTD.)
Elution from Florisil
Pesticide Name % Recovery 6% 15% 50%
Naled 0 -
Nemacide 110% +
avIPA 0
Parathion Oxygen Analog 0 -
Phenkaptan 95% + -
Phorate Sulfone Oxygen Analog 0 - -
Phosalone 83% - +
Phosphamidon 0 - -
Phostex 67% +
Ronnel Oxygen Analog 0 - -
Ruelene 0 -
SD 7438 c 98% - + -
Sulfatepp 85% + - -
Sumithion 81% - + -
Supracide a 33% - +
Trichiorfon 0 - -
Zinophos 59% - +
Zytron 100% +
aRecovery of pesticide not determined because the coinpo .uid was not
satisfactorily chromatographed using the GLC conditions of this experiment.
(6 ft. x 4 m 10% DC 200 on 80/100 Gas chroni Q; C.T. = 200°C, D.T. = 205 C,
Inlet = 225 C; N 2 flow = 120 mi/mm; KC1TD detector.)
b% recovery determined by averaging results of 3 determinations. The
majority of the pesticide was obtained in the 15% elution; however, a
portion was obtained in the 50% fraction in one determination and in the
6% fraction in two of the determinations.
CValue listed for SD 7438 is the result of a single determination. Except for
this pesticide and for Merphos, all recovery values which are listed are the
average of two determinations.
-------
Revised 11/1/72
Section 8,B
- 12 -
Figure 1. Flow Sheet Diagram of Analysis of Air Sample
COLLECT IN K-D
FLASKS AND
EVAPORATE ‘10
lm l
INJECT G.C.
WITh F.P.D.
ELUTE WITh 6%
MIXED ETHERS
(FRACTIc 4 I)
(XJLLECT IN
K-D
FLASK
EVAPORATE
ID
5.0 ml
DILUTE UP TO 5
AND PASS THRU
FLORISIL COLUMN
COLLECT IN
K-D
FLASK
EVAPORATE
TO
5.0 ml
INJECT G.C.
WI’fl1 PC. fl
INJECT G.C.
WITH F.P.D.
COMBINED I
II XANEWIThH 2 OI
ELUTE WITh 50%
MIXED ETHERS
(FRACTION III)
COLLECT IN
K-D
F9SK
EVAPORATE
ID
5.0 ml
I I
INJECT G.C. EVAPORATE ID
WITH E.C.D. 1.0 ml
COMBINE SAMPLES
FILTER CLOTH
TRANSFER ID BEAKER
EXTRACT 3 TIMES WITH HEXANE
ETHYLENE GLYCOL
TRANSFER 10 SEPARA1DRY FUM’JEL
DILUTE WITH H 2 O
EXTRACT WITh SAME
COMBINE HEXANE IN
SEPARATORY RJN _
PASS TI-IRU
Na 2 SO 4 COLUMN
n ]
ELUTE WITH 15%
MIXED E1}IERS
( FRACTION II )
-
INJECT G.C.
WITH E.C.D.
EVAPORATE FURTHER
TO 1.0 ml
EVAPORATE TO
1.0 ml
INJECT G.C.
WITh F.P.D.
INJECT G.C.
WITH F.P.D.
-------
Revised 11/1/72 Section 8,C
Page 1
SAMPLING AND ANALYSIS OF PESTICIDES IN AIR
C. EVALUATION OF EThYLENE GLYCOL
I. INTROI JCFION :
Ethylene Glycol (hereinafter referred to as E.G.) is purchased in
4-kg glass bottles. Each lot received must be sampled and subjected to
analysis by GLC, E.C. and FPD, to insure that the material does not
contain contaminants which will result in chrornatographic peaks that
interfere with peaks resulting from pesticides. The following procedure
is designed to provide this infomation.
II. APPARA11JS REAGENTS :
1. Erlenmeyer flask, 500 ml with glass stopper.
2. Grad. cylinders, 50 and 500 ml.
3. Separatory funnels, with Teflon stopcocks, 250 and 500 ml.
4. thromatographic columns, 300 umi column length x 22 nnn i.d. (no frit).
Kontes #420530, size 241, or the equivalent.
5. Evaporative concentrator tubes, 10 ml, Kontes #570050, size 1025,
or the equivalent.
6. Kuderna-Danish flasks, 500 ml, Kontes #570001 or equivalent.
7. Snyder columns, size 121, Kontes #503000 or Kjeldahl bulbs, 24/40,
Ace Glass #5230-1.
8. Hexane, pesticide grade, distilled in glass, prechecked for zero
background by GLC, E.C. at a concentration of 180:1.
9. Distilled water, prechecked for contaminants by GLC, E.C. (See
NOTE in III., 4, next page).
10. Sodium Sulphate, anhydrous, A.R. grade. Precthecked for con-
taminants and, if found, extracted as described in Section 5,A,(l).
11. Glass wool, pre-extracted with hexane.
-------
Revised 11/1/72 Section 8,C
-2-
III. PROCEDURE :
All glassware used must be scrupulously cleaned and given a final
hexane rinse inmediately before use.
1. The E.G. will be contained in 4-kg or 1-gal. amber glass bottles
with Teflon-lined screw caps. One composite sample of each manu-
facturer’s lot will be taken for evaluation. The number of
bottles to be sampled depends on the size of the lot. If the lot
consists of less than a dozen bottles, the composite should be
comprised of some fluid from each individual bottle. If the lot
is 12 to 24 bottles, half the bottles should be sampled. From 24
to 48, a quarter of the total bottles in the lot should be sampled.
2. Shake each bottle by inverting end for end about 15 times and pour
25 ml into a grad. cylinder. Transfer each 25 ml into a 500-mi
Erl. flask. Stopper flask and shake ca 50 times to insure complete
mixture of composite.
3. Carefully measure 80 ml of the E.G. composite into a 100-mi grad.
cylinder and retransfer this to a 500-mi sep. funnel.
4. Measure 350 ml of dist. water into a 500-mi cylinder and transfer to
the sep. funnel containing the E.G., rinsing the 100-mi cylinder
used to measure the E.G. composite with several portions of the
water.
NOTh : At this point, prepare a 350-mi water blank
and carry through all following steps.
5. Add 60 ml of hexane, stopper and shake vigorously two minutes.
6. Allow layers to separate, draw off lower aqueous layer into a
second 500-mi sep. funnel (No. 2), and the upper hexane layer into
a 250-mi sep. funnel (No. 3).
7. Add 60 ml of hexane to the aqueous solution in Funnel 2 and again
shake vigorously two minutes.
8. Draw off lower aqueous layer into Funnel 1 and hexane layer into
Funnel 3.
9. Repeat the extraction a third time, discarding the final aqueous
layer and combining all three hexane extracts in Funnel 3.
-------
Revised 11/1/72 Section 8,C
-3-
III. PROCEDURE (CONTI).)
10. Add 50 ml of H 0 to Funnel 3, stopper and shake 1 minute, discard
aqueous layer nd repeat scrubbing with another 50-mi portion of
water also discarding the final aqueous layer.
11. Place a glass wool retaining plug in the bottom of a 300-mm
chrcm atographic column, add 2 inches of Na 2 SOA and pass the
combined hexane extract through the column, collecting eluate in
a 500-mi K-D flask which has been fitted to a 10-mi evaporative
concentrator tube containing one 3-mm glass bead.
12. When extract has eluted from the column, rinse the sep. funnel
with three consecutive portions of 10 ml each of hexane, washing
down the walls of the column. Allow each rinse to elute off the
column before adding the next. Finally, rinse the column walls
with two nxre 10-mi portions of hexane.
13. Place K-D assembly in a boiling water bath and evaporate to 5 ml.
Remove the tube from K-D flask rinsing joint with a little hexane.
Concentrate to exactly 1 ml under a nitrogen stream at room temp.
14. Conduct GLC, E.C. with the 1.5% OV-17/1.95% QF-l column, following
instructions given in Section 4,A of this manual.
15. At room temperature and under a nitrogen stream, evaporate extract
to exactly 0.2 ml and conduct GLC, FPD using the 4% SE-30/6% QF-1,
Carbowax-treated column (See Section 4,B).
IV. INTERPRETATION OF C C ’4ATOGRAI -IIC DATA :
1. It is necessary that the contaminant peaks contributed by the water
be subtracted from the peak structure obtained in the sample
chromatograin. If the water blank yields troublesome contaminant
peaks, it may be necessary to purify the water by multiple extrac-
tions with benzene and hexane.
2. Generally, if the contaminant peaks from the E.G. do not exceed
10% f.s.d. and whose R.R. ratios do not indicate an overlap with
any pestici peaks, the E.G. would be considered acceptable. If
such peaks match the R.R. ratios of any of the organophosphate
compounds via E .C. but are not detected by flame photometric, the
E.G. would be considered acceptable.
If contaminant peaks are produced of less than 10% f.s.d. which
match the R.R. ratios of any of the common chlorinated pesticides,
-------
Revised 11/1/72 Section 8,C
-4-
IV. INThRPRETATI( OF CHROMA1OGRAWIC DATA (CONTD.)
the SE-30/QF-1 column should be tried. Should this also produce
overlaps, consideration may be given to rejection of the lot
depending on the size and location of the contaminant peak(s).
3. If contaminant peaks greater than 10% f.s.d. are obtained by
E.C. but do not produce R.R. ratios overlapping any of the
pesticides, acceptance of the sample would probably be indicated.
One possible exception to this would be an extremely late eluting
peak which, although not interfering with any pesticide, might
entail excessive delays between injections.
-------
Revised 11/1/72 Section 9,C
Page 1
SEPARATI(T J OF SOME POLYCHLORINATED BIPHENYLS FROM CERTAIN
ORGANOCHLORINE PESTICIDES
I. IWrRODUCr ION :
Polychiorinated biphenyls (PCB) are a group of chemicals ith indus-
trial applications. They are stable (resistant to alkali and acid) and
persistent; their residues have been found in wild life. Commercially,
PCB are manufactured in this country by Monsanto and sold as a series of
products tradenanied Aroclors. Most Aroclors actually consist of many
different chiorobiphenyls, although some, partially or totally, consist
of members of another group of compounds, chloroterphenyls.
The various co iponcnts of P B residues are partially or completely
recovered through niultiresidue methodology for organochlorine pesticides;
they are eluted from the Florisil column by the 6% ethyl ether/petr ether
eluant. PCB residues exhibit complex gas chromatographic patterns because
of the various components represented. These peaks appear in and beyond
the retention time region of the organochlorine pesticides. If present
in high enough concentration, relative to pesticides present, PCB can
interfere with the determination of some organochlorine pesticides. Like-
wise, the presence of certain organochiorine pesticides can interfere
with the determination of PCB.
NOTE : The polythiorinated terphenyls (PCF) are also recovered
through the multiresidue methodology used for the
analysis of organochlorine pesticides and PCB. However,
the PCI elute from the GLC column much more slowly than
either the pesticides or PGB and so do not interfere
with the determination. In order to analyze for the
PCT, it is necessary to use a GLC column and operating
parameters which permit much more rapid elution and
greater sensitivity for the chloroterphenyl components.
A number of procedures have been proposed for dealing with the
various pesticide PCB combinations encountered. These include the silicic
acid column chromatographic separation technique presented in detail
here and several other published approaches, some of which are noted in
Subsection VII. The residue analyst must make judicious use of the
available techniques in order to obtain accurate results. The proper
course of action in the determination of residues of PCB and pesticides
found together depends on the suspected identity of each and ,on the•
estimated amounts of each. Some combinations will permit quantitation
of both pesticides and PCB without their separation from one another on
the silicic acid column. Other combinations of PCB and pesticides must
-------
Revised 11/1/72 Section 9,C
-2-
be separated before quantitation; still other combinations cannot be
separated by silicic acid, yet cannot be determined in the presence of
one another. The relative an unts of residues of pesticides and PCB
may also influence the decision on whether or not to perform silicic
acid separation prior to quantitation. Even when the residues will not
be caupletely separated by this technique, its use may be the best
n ans of achieving quantitative estimation of the residues when one
chemical is present in much larger amounts than the other.
REFERENCES:
1. Arn ur, J., and Burke, J., JADAC 53, 761-767 (1970).
2. Masi noto, H. T., JAOAC, in press.
3. Pesticide Analytical Manual, Vol. 1, Section 251, U. S. Food
Drug Adm.in.
II. PRINCIPLES:
The silicic acid column chromatographic procedure given here permits
separation of DDT and its analogs from some of the P03, including those
with which they interfere. The silicic acid is standardized before use
by addition of enough water to effect the best possible separation be-
tween p,p’-DDE and Aroclor 1254. The interfering PCB are eluted with
petroleum ether from a column of the standardized silicic acid. The DIJF
compounds, most other organochlorine pesticides, and some other PCB are
then eluted from the column with a mixture of hexane, methylene chloride,
and acetonitrile. See Table I for list of chemicals eluting in each of
the two eluates.
The method is applicable to the 6% Florisil eluate [ Sect. 6,A,(1)]
obtained in the analysis of fatty tissues or to extracts cleaned up by
other methods for gas chromatography. Extracts in polar solvents must
be transferred to nonpolar solvents prior to separation.
III. APPARA11JS :
1. Chromatographic column 400 x 22 mn i.d., with 24/40 outer joint
with coarse fritted plate and Teflon stopcock, Kontes No. K-420550,
C-4, or the equivalent.
2. Grad. Cylinder, 250 ml.
-------
Revised 11/1/72 Section 9,C
-3-
3. Kt erna-Danish Assembly as follows:
Evaporative concentrator flask - Kontes Catalog No. K-570000,
500-mi capacity, lower joint 19/22 , upper joint 24/40 !; Snyder
column - 3 ball, lower joint 19/22 , upper joint 24/40 ; Tube -
10 or 15 ml capacity, 19/22 ‘ upper joint.
4. Air pressure regulator - for pressure reduction to deliver ca 1 lb.
psig; air must be clean and dry.
5. Separatory funnel - Used for column eluant reservoir, 250 ml, with
Teflon stopcock, 24/40 joint at top, Kontes No. K-633030, or the
equivalent.
6. Hot water bath adjustable to temp. of 90-100°C.
IV. REAGENTS :
1. Petroleum ether, acetonitrile, hexane, and methylene chloride, all
pesticide quality.
2. Celite 545, Johns-Manville.
3. Silicic acid, Mallinckrodt, 100 mesh powder; “specially prepared for
chromatographic analysis by the method of Ramsey and Patterson,”
Analyt. Reagent No. 2847.
V. PREPARATION OF SPECIAL REAGENTS :
1. Celite 545 - must be dry and free of electron capturing substances.
If electron capturing substances are extracted by petroleum ether,
treat as follows: Slurry Celite with 1 + 1 hydrochloric acid -
H O while heating on steainbath; wash with H,O until neutral; wash
s&cessively with several portions each of iflethanol and acetone (to
remove H O); then ethyl acetate and petr ether. Remove solvents by
suc ion nd air drying. Hold a 1- to 2-inch layer of Celite in
130 C oven for at least seven hours to remove water and other
volatile substances. After washing treatment and/or drying, store
Celite in closed glass container.
2. Eluant for Pesticides - 1% acetonitrile, 19% hexane, 80% methylene
chloride (v/v/v). Pipette 10 ml acetonitrile into 1-liter volumetric
flask, add 190 ml hexane, and fill to volume with methylene chloride.
-------
Revised 11/1/72 Section 9,C
-4-
3. Silicic acid - Place silicic acid to depth of about 1 inch in open
beaker and heat for a mini.mum of 7 hours, but preferably up to 24
hours, in 130 C oven to remove water. Mter heating, place beaker
in desiccator and allow to cool to room temperature. ( iick1y weigh
silicic acid into glass-stoppered bottle and add 3% H,O by pipette
(97 g silicic acid + 3 ml H ,O = 3% H O). Stopper bottle tightly
and seal with tape to insure that cofftainer is air tight. Shake
well until all H O is absorbed; make sure that no lumps remain.
Place sealed container in desiccator and allow to equilibrate for
15 hours.
To determine the separation achieved with the treated silicic acid,
prepare a column as described in VI, below, and add to it a standard
solution containing 40 iig Aroclor 1254 and 3 g p,p’-II)E in hexane.
Elute as described and determine recoveries in each eluate. made-
quate separation of PCB from p,p-DDE will require that further test-
ing be done with other heated batches of silicic acid, treated with
different amounts of H 2 0 as needed to achieve the desired separation.
Increments of 0.25% or 0.5% more or less H 0 are recommended for the
testing. vbre H,O is required when the initial test results show
PCB eluting in the polar solvent with the p,p’-DDE; less H 2 0 when
p,p’-DDE elutes in the petr ether fraction.
This testing and standardization is required for each new lot of
silicic acid obtained from the manufacturer.
Once a batch of standardized silicic acid is prepared, it should be
stofed in a desiccator between uses. Desired activity remains for
about 5 days.
VI. PROCEDURE :
SEPARATIC OF PCB FROM ORGANOCHLORINE PESTICIDES
1. Weigh 5 g Celite, then 20 g activated silicic acid and conibinein
250-mi beaker. Lmnediately slurry with 80 ml petr ether, mixing
well.
2. Pour slurry into a chromatographic column with coarse frit, keeping
stopcock open. Complete transfer of silicic acid - Celite mixture
by rinsing beaker with small portions of petr ether.
-------
Revised 11/1/72 Section 9,C
-5-
NOTE : Apply air pressure to top of column as much as
necessary to force enough petr ether from column
to allow space for all silicic acid - Celite.
3. Stir material in column with long glass rod to remove air bubbles,
applying air Iressure to settle adsorbent and to force petr ether
through column. Continue application of air pressure until petr
ether level is ca 3 mm above surface of gel.
NOrE : Do not allow column to go dry or to crack at any
tiineLring the procedure. Close stopcock when air
pressure is not being applied. At this point, column
of adsorbent should be fiim and should not lose its
shape if tipped.
4. Place 250-mi grad. cylinder under column for collection of eluate
and take a suitable aliquot of 6% Florisil extract for addition to
the column.
NOTE : Large amounts of PCB and pesticides placed on column
may result in incomplete separation. Choose aliquot
to contain amounts of PCB and pesticides required for
determination. The weight of sample equivalent
placed on the column may also affect separation by
causing p,p’-DDE to appear in the petr ether eluate.
Should this occur, an amount of extract equivalent to
a smaller weight of sample should be used. In
analysis of samples by this procedure, it is suggested
that no more than 0.3-0.4 g fat equivalent be placed
on the silicic acid column.
5. Md aliquot carefully to column being careful not to disturb top of
adsorbent.
6. Apply slight air pressure until solvent level is ca 3 nun above
adsorbent and then complete transfer of sample extract to column
using small portions of petr ether and again applying slight air
pressure until solvent level again reaches the 3 irun point above
adsorbent.
7. Position a 250-mi sep. funnel containing 250 ml of petr ether on
top of column. Open funnel stopcock and slowly apply air pressure
to reservoir until an elution rate of ca 5 mi/mm. is established.
Continue elution until eluate volume in the graduate is exactly
250 ml.
-------
Revised 11/1/72 Section 9,C
8. ( iantitative1y transfer eluate to a 500-mi Kuderna-Danish evaporator
fitted with a 5-mi evap. concentrator tube. Rinse graduate with
small portions of pet. ether.
9. Place a second 500-mi K-D flask assembly under the column for
collection of any remaining petr ether eluant and second eluate
described in Step 10.
10. Apply air pressure until petr ether eluant level is ca 3 nun above
adsorbent and add 200 ml of G -hexane-Q-L,CL, (1:19:80) eluant
to upper reservoir. Open stop ock and slowty teapply air pressure,
continuing elution until all of eluant passes through column into
the K-D concentrator.
11. Place Snyder columns on both K-D assemblies, place in a hot water
bath and reduce eluate volumes to 5 ml in preparation for explora-
tory GLC analyses.
NOTE : The first (petr ether) eluate should contain the PCB’s
and the combined solvent eluate should contain the
chlorinated pesticides.
DETERMINATION OF POLYCI-LLORINATED BIPHENYLS
Determine quantity of PCB in the sample by electron capture GLC or
by halogen specific microcoulometric or electrolytic conductivity GLC.
Compare the total area of response for the residue to the total area of
response for a known weight of the Aroclor(s) reference with most similar
( IC pattern(s). The pattern of GLC peaks for a sample containing PCB is
often not exactly like that from any of the Aroclor standards. This is
probably due to a combination of circumstances, e.g., weathering and/or
metabolism of the residue; and perhaps slight variation in the recovery
of the different PCB components through the methodology. Sometimes the
GLC curve clearly indicates the presence of components of mere than one
Aroclor. In this case, quantitate the PCB residues separately if possible,
using the appropriate Aroclor references for the respective portions of
the GLC curve. Choosing the appropriate Aroclor reference(s) against
which to measure a residue requires good jud nent on the part of the
analyst.
GLC with halogen specific microcoulometric or electrolytic conducti-
vity detection is often preferable as means of quantitation. This type
of detection system also provides confirmatory evidence for the identif i-
cation of the residue as PCB.
-------
Ravlasd U/1/72 Section 9,C
-7-
VII. REF ES 10 ADDITIONAL P1 )CELURES :
The discussion under INT1 )IXJCTION indicates the complexity involved
in analysis of samples containing combinations of PG and pesticides,
including combinations of n re than one Aroclor. The following procedures
can be used to provide additional information on the identification and
quantitation of residues found by the GLC determination. In certain cases,
use of one or more of these techniques may be the only way to determine
the residues. Knowledge of the behavior of compounds of interest in each
of these procedures is necessaty in making a decision on the best approach
to a particular analysis.
The papers cited here do not always contain such complete information,
so it may be necessary for the residue analyst to generate iditional
recovery data before using a method.
1. Alkaline hydrolysis. Young, S. J. V., and Burke, J. A., Micro Scale
Alkali Theatment for Use in Pesticide Residue Confirmation and Sample
Cleanup, Bull. Environ. Contain. Toxicol., in press. Vol. 7, No. 1
(1972); Krause, R. T., Quantitative Dehydrochiorination of Perthane
Residues and Effect of Alcoholic Potassium Hydroxide on Other
Pesticides, JAOAC, in press.
The stability of PG to alkali permits the use of alkaline hydrol-
ysis as a test for confirming the identity of PG residues. At the
same time, the conversion of DDT to DDE by alkali treatment provides
a means of ren ving some interferences from the quantitative measure-
ment of PCB. Alkaline hydrolysis also provides an additional cleanup
for many sample types. An appropriate sequence of determination
step(s) and alkaline hydrolysis can be chosen to permit quantitation
and confirmation of both PG and the D1Yf compounds.
2. Oxidative treatment. (a) Muihern, B. M., Cromartie, E., Reichel,
W. L., and Belisle, A. A., Semiquantitative Determination of Poly-
chlorinated Biphenyls in Tissue Samples by Thin Layer Chromatography,
JADAC, 54, 548-550 (1970).
Cleaned up sample extracts are reacted with a chromic acid-acetic
acid mixture to convert DDE to 4,4’-dichlorobenzophenone. The
authors follow this reaction with UC in which the PG migrate as
a single spot and are not separated from DDE, but are separated from
the benzophenone. Sequential use of the alkaline hydrolysis arid
oxidation on a sample containing PG, DDE, and DDT would result in
the conversion of both o,p’- and p,p’-DDT and o,p’- and p,p’-DDE to
the respective chlorobenzophenone while leaving the PG unchanged.
-------
Revised 11/1/72 Section 9,C
-8-
(b) This manual, Section 9,D, Senii-Quantita-
tive Estimation of Polychiorinated Biphenyls in Adipose Tissue.
3. Two-dimensional TLC. Fehringer, N. V., and Westall, J. E., Separa-
tion and Identification of DDT analogs in the Presence of Polychlori-
nated Biphenyl Compounds by Two-Dimensional Thin-Layer Chromatography,
J. Chromatography 57, 397-405 (1971).
Thin-layer thrcmiatography is used in a manner designed to separate
PCB from DDT and its analogs. After an initial developiient in
n-heptane, the UC place is rotated, spotted with additional
standards and developed with 2% acetone in n-heptane in a direction
perpendicular to that of the first development. The pattern of
spots indicates the identity of residues in the sample.
4. Reversed-phase partition TLC. de Vos, R. H., and Peet, E. W., Thin-
Layer Chromatography of Polychiorinated Biphenyls, Bulletin Environ.
Contam. Toxicol. 6, 164-170 (1971).
A stationary phase of liquid paraffin on Kieselguhr G adsorbent is
utilized in a TLC system which involves three successive developments
of the spotted plate with a mixthre of acetonitrile, acetone, methanol,
and water. Visualization is by the usual silver nitrate spray and
UV irradiation. These parameters permit separation by TLC or DDE,
DDT and several other organochiorine pesticides from the components
of Aroclor 1260, and cause the PCB to appear as a number of distinct
spots rather than a long streak as sometimes occurs with other TLC
systems.
[ Editor’s Note: The ability of this TLC system to separate Aroclor
1260 into several spots suggests the possibility
that further work would show the system capable
of distinguishing among the different Aroclors.]
-------
Revised 11/1/72 Section 9,C
-9-
Table 1. Pesticides and Other Chemicals Recovered Through Silicic Acid
Column Chromatographic Separation of Some Polychiorinated
Biphenyls (PCB) from Certain Organochiorine Pesticides.a
Acetonitrile, Methylene Chloride,
Petroleum Ether Eluate Hexane Eluate
Aidrin b Aroclor 1221
Aroclor 1221 b Aroclor
Aroclor 1242 b Aroclor
Aroclor 1248 Aroclor 5442 d
Aroclor 1254 Aroclor 5460 c,
Aroclor 1260 BHC (all isomers)
Aroclor 1262 chiordane (technical)
Aroclor 4465 c d p,p’-DDE
Aroclor 5460 ‘ o,p’-DDT
hexac lorbenzene p,p’ -DDT
mirex die ldrin
octachloro-dibenzo-p-dioxin f endrin
polychiorinated naphthalenes heptachior
2,3,7, 8-tetrachloro-dibenzo-p-dioxins heptachior epoxide
1 indane
p,p’-TDE
toxaphene
aMethod tested only with chemicals listed.
bDivi s between the two eluates. The earliest (GLC) eluting peaks in any of
these Aroclors are the most likely to elute in the polar eluate.
cDivides between the two eluates.
dArociors 5442 and 5460 are composed of polychlorinated terphenyls and must be
chromatographed on a GLC column that permits rapid elution; e.g., 1% OV-101
on 100/120 Gas Ghrom Q at 240°C, 120 ml N 2 /min. (Wieneke, W., private com-
munication, Jan. 1972).
eMirex may be separated from Arociors 1260 and 1254 by collecting the first
100 ml petr ether separately. This fraction will contain the mirex. (Gaul,
J., private comniunication, July 13, 1971).
iethod tested with commercial polychiorinated naphthalenes: Halowaxes 1014,
1099 (Armour, J., and Burke, J., JADAC 54, 175-177 (1971).
R. T., in press. JAQAC.
-------
Revised 11/1/72
4%SE-30/6%OV-21 0
Section 9, r
Page 1
Chromatograins of three AROCLORS on column of
14% SEi.30 / 6% OV.210. Coli rnn temp. 200°C.,
carrier flow 60 mi/mm., -‘H detector, electrom.
attenuation on an E-2 10 x 16; dotted line a
mixture of chlorinated pesticides, identity and
Injection concentration given below:
3..
2.
3.
14.
5.
Diazinon
Heptachior
Aidrin
Hept.Epox.
p,p’...DDE
--
—
——
—-
--
1.5 rig
0.03
.0145
.09
.09
7.
8.
9.
10.
II.
o,p’- DDT —
p,p’—DDD
p,p’-DDT —
Dilan —-
Methoxychior
0.214 rig
.214
.30
.75
.60
6..
Dieldrin
—
.12
AROCLOR
7r. injsc$on
1232
AROCLOR 22
6 ng injection
0
0
©
2
6
2 • 4 • 6
8 • 10 12
AROCIOR 1242
bus imsctiOu
I C 12
-------
Revised 11/1/72 - 2 -
Section 9, E
4%SE .-30/6%OV-210
Chromatograms of three AROCLORS on column of
14% SE-30 / 6% OV—2l0. Column temp. 200°C.,
carrier flow 60 mi/mm., 3 H detector, electrom.
attenuation on an E ..2 10 x 16; dotted line a
mixture of chlorinated pesticides, identity and
injection concentration given below:
1. Diazinon —— 1.5 rig 7. o,p’—DDT — 0.2I rig
2. Heptachlor -— 0.03 8. p,p’-.DDD —— .2 1 4
3. Aidriri -— oOk5 9. p,p’—DrT .30
I . Hept.Epox. -— .09 10. Dilan -— .75
5. p,p’.-DDE -- .09 11. Methoxychior .60
6. Dieldrin — .12
AROCLOR 1260
4 ny injection
AROCLOR 12.
I
I . t 2 6 20 24 • 28
AROCLOR 1248
6 ng injection
8
12
16
8
16 • 24 • 32
-------
Revised 11/1/72
3
Section 9, [
Chromatograrns of three AROCLORS on column of
5% OV-17 / 1.95% QF-.1, Column temp. 200°C.,
...arrier flow 60 n1/min., 3 F1 detector, electrometer
atten. 10 x 16 on an E-2; dotted line a mixture
oI chlorinated pesticide compounds, identity and
injection concentration given below:
1. Diazinon -- 1.5 ng 7. o,p’—DDT 0.2L ng
2. Heptachior —— 0.03 8, p,p’—DDD —
3. Aidrin -— 01 5 9. p,p’—DDT -— .30
14. Hept.Epox. —— .09 10. Dilan .75
5, p p t .DDE -- .09 II. Methoxychior .60
6. Dieldrin -— .12
AROCLOR 1232
7ng injection
1.5°/a 0V17/1.95% QF1
AROCLOR 1242
5 ng injection
I n
I n
II
AROCLOR 1221
6 ng injection
0
‘I
It
0
I I
II
II
ji
II
It
SI
IS
I
• 2
2 • 4 • 6 • 8 • 10 12
• 4
S
12 • 16
-------
Revised 11/1/72
1.5°/o OV-17/1.95% QF-1
Chrornatograms of three AROCLORS on column of
1.5% OV-.17 / 1.95 (F- 1. Column temp. 200°C.,
carrier flow 60 mi/mm., 3 H detector, electroieter
atten. 10 x 16 on an E—2; dotted line a mixture
of chlorinated pesticide compounds, identity and
injection concentration given below:
1.
Diazinon
——
1.5 rig
7.
o,p’-.DDT -—
0.2L rig
2.
Hept ch1or
-—
0.03
8.
p,p’-.DDD —
.2t
3.
Aidrin
——
0I 5
9.
p,p’-LLT ——
.30
I .
5.
Hept.Epox.
p,pLtDE
--
--
.09
.09
10.
11.
lilian — —
Methoxychior
.75
.60
6.
Dieldrin
——
.12
AROCLOR 1254
Sng , I. tion
-4-
AROCLOR 1248
4 ng injection
Sectinn 9, E
8 12 • 16
AROCLOR 1260
3 ng injection
• 8 • I ? • 16
20 • 24
28
E )
!
t ! 1
A
( ) H
1
(
4
12
20 - 28 • 36
-------
1/4/7 I
Se_tIoII ), I
Page
Retention \ a 1ue , ReLit i ye to ALdrLn and Response Va1ne’ , Relative to the
Major Peak 0! ci the Aroclor Compounds (poly-diloi iiiated Iiiphciiykj
Lohmin. Pyrex glass , h-ft., 4 mn. i.d., l.5° 0V-l7/l.O5 Ql-l,
200°C Lohnnn leap. Carrier flow 60 nil/mm.
Detectot Liectron capture, H. parallel plate, 210°C .
I
ii
ii —r
I
.
Misc.’
p est icld os
Phosdrin
0.32
111221
•RR
RPH
fll 32
I RN!
111242
RRR
RN!
111248
RRR I RPII
/ l j__
RRR I RPII
1/124(J
RRR 1
T
I
0.271
.34
0.19
.03
0.27 0.17
.33 I .03
I
I
I
Thnnet
Diazinon
Fleptachlox
.50
.63
*
—
.83
.40;
.43
.49 I
.62
I
I
.82
.05
.27
1.00
.05
.04
.40 .07
.43 I .27
.47 1.00
.611 .38
.73 .20
.80 I .85
I
0.48
.631
.74
I
.82 I
0.34
.41
.22
1.00
I
0.48 0.08
.63 I .34
.74 l .15
.83 I 1.00
I
I
I
I_-
L—
I
6-BFIC
.92
.911
.02
.90 .33
.90
.38
.92 .31
.95 I .26
.971
.32
.981 .24
I
I
4.ldrin
1.00
I
1.03 .13
1.05 I
,
.23
1.06 .68
I
1.04 0.23
- I
-i—-
1.43
I
1.22 I .20
1.24 I
.23
1.26 I .62
1.24 I .14
I
1.30 .19
1.33
.21
1.34 .56
1.3 1 .08
irat1iton
1.44 I .13
1.49 I
.15
1.50 I .42
1,48 I .07
— I —
I-k’pt.
!a1athion
P
Parathion
- - -
j i
*
1.81
2.23
2.40
I
1
I
1.54 .27
I
1.83 .19
I
1.57
I
1
1.88 I
I
.29
.21
158 .96
I
I
1.88 .65
I
1.56 .29
1.62 I ,33
I
1.80 .58
1.97 I .13
1 61 l j•Q9
I -
1.78 .lo
—
p,p’-DDE
Dieldrin
2.26 .03
I
2.43 .05
I
2.52
.03
2.21 .13
2.32 I .17
2.50 .24
2.20 .19
2.29 I .36
2.47 .68
- I
2.53 .39
Endrin
2.93
2.78 I .07
2.85 I
.04
2.85 I .32
2.82 I 1.00
2.81 I .42
o,p’-DDI’
3.17
I
3.13 .02
‘
.
3.18 .24
3.16 .53
-
p.p’-DDD
p 1 p’-DLIT
4.18
I
I
3,571 .04
4.04 .03
3.67 1
.02
3.66 I .16
4.12 .05
3.60 I .50
4.08 .66
3.50 V.52
4.10
Ethion
D1an1
4.44
—
5.7
I
;
I
I
I
I
I
I
I
i
I
I
I
t
I
I
r
I
I
I
i
4,42 I .08
.
I
3.28 I .11
I
S.95I .0°
4.38 I .58
SM
5.4 I .38
I
s __L±
I
1 -l
I I
I
I
I
go
S.4
_ _L
given for some common pesticides for comparison
2 RRR - Means the retention relative to aidrin.
5 Rl’II - Means the peak height response relative to that of the tallest heal’ hots’ii LII
itoli.,cs.
l id I 1t . ijilordanc pc,ik 1 clot i ng in the appropriate area.
Dilan IT
6.4
-
c , ji -
8.5
6.4
‘Relative
8.4
.05
retention ratios
-------
1/4/71 - 2 - ,eLtion 0, 1
Retcntto Values, Rclatwe to Aidrin and Response V,ilucs, ReI,itive o the
Major Peak of Six of the AroLlor C npounds (poly—thloriiiated hi 1 Iieiiyls)
Column: Pyrex glass, 6-ft., 4 m. i.d., 4 SL-30/b a QI -1, 200°(. Column tdllip.
Cjrricr flow 70 mI/mm.
1 tcctor: Liectron caprtire, II, parallel plate, 210°C.
—
‘ii. sc
1
1
H
Pesticides
Phosdrin
.RRR
0.32
.44
.47
f7 1221
rm
RR RPM
#1J232 —
RRR I RPM —
//1242
RRR I RPM
01248
RRR I RPM
01 54
RRR I RPM
#1260
F RR I or
0.24 I 0.37
.32 .07
.34 .13
.39 .40
.44 I 1.00
I
I
0.25 0.26
.32 I .07
.35 .13
.39 I .33
I— — ———————.—
I
0.34 0.04
.38 I .08
I
I
1
I
I
I
—
I
I ——
I
I_.._— .__
I
J
I
2,4-D(ME)
Thiiiiet
.43 1.00
I
.43 .52
.47 I .03
0.44 0.14
i
.——
Siniazmne
Lmndane
5-BHC
2,4-D(BE)
—
.S4
.60
.69
—
.78
.SS I .04
.60 .05
.73 .06
I
.55 .37
.62 i .28
.72 .70
.79 I .31
•53 •54
.61 I .38
.72 1.00
.77 I .43
•S5 .42
.62 .25
• 73 I 99
.80I .34
I
I
I
—1—
I
I
Heptachior
I
—
.82 .27
.81 .36
.83; .36
I
I
Ronnell
.91
I
.90 1 .14
.89 I .18
.90i .61
0.90 1 0.38
I
Aidrmn
1.00
I
1.02 .16
1.00 .22
1.03; .66
i.02 .25
‘
1.09 I .20
1.07 .27
1.101 .68
1.081 .14
1
N
Parathion
Hept
Epo ada....
p,p’-DCE
1.34
*
1.43
1.82
I
I
I
1.16 .03
1.31 I .24
1.48 [ .19
1.77 I .03
1.15 .02
1.30 1 .30
1.47 23
1.77 I .03
1.18 .10
1.321 1.00
1.SO: .78
1.801 .19
‘
I
1.291 .79
1.48: .95
1.75I .53
I——
!
1.30 2.10
1.52 .17
1.80 .07
Captan
p’-DDD
Dieldrmn
1.94
1.98
2.12
—
2.39
2.42
.*—
2.5
1
I
I
I
I
1.96 .04
I
‘
I
1.90 .03
2.02 I .03
••
1.92: .25
I
I
1.87: .83
2.001 .09
2.14 .28
1.93
2.02 I .36
2.18
1
I
2.241 .07
2.2 1( 1.00
J
o,o ’-DlJf
Endrmn
p,p’-DDD
I
I
I
I
2.27 : .05
I
I
2 27 .02
I
I
I
2.30 .27
I
I
‘
I
2.511 .78
.62 .38
I —
I
2.59 1.00
Thiodan 1
2.7
—
I
I
1
.
I
‘
I
2.69 I .03
I
I
2.731 .18
I
i
I
—
i
I
I
2.81.
p,p’-DDT
Trithion
—
3.1
3.2
I
I
I
I
I
1
i
3.12I .06
I
2.991 .91
I
I
3.10 I .67
—
—
I
I
•
I
I
I
I
,
_
3.55I .12
I
I
3.71 I
39 5 T
Dilan I
etho
thior
d 4(
.f
j
I
I
I
I
—
I
4.201 .10
4.60 1 .12
—
I
4.83 I
-—
i _u_.l_ J
I
j
5•54 1 .05
5.77
..! J ____________ _____________ IL _________ _IE ____________ 6.2
‘Pellative retention ratios given for some cannon pesticides for ulllparlson purposes.
2 RP .R - Means the retentIon relative to aldrui.
‘ RPII - Means the peak height response relative to that of the tallest peak ‘,Iiown in
italics.
*Indicates chlçrdane peaks eluting in the approximate area.
-------
11/1/72 Section 9,F
-3-
Retention values, relative to aidrin and response values, relative to the major peak, of six
of the Aroclor compounds (polychlorinated biphenyls).
Coltm n: Pyrex glass, 5 ft., 5/32” i.d., 5% OV-210, 200°C column temp., carrier flow 45 ml/nu.n.
Detector Electron capture, 3 H, parallel plate, 205°C.
1
#1221
#1232
#1242
#1248
#1254
#1260
RRR
—
2 f flj RPl-l
0.33 0.27
.41: .10
—
.46: .31
RRR RFH
0.33 ‘0.19
.42 .07
.46 : .24
RRR RPI-I
‘
0.47 0.01
RRR RPH
RRR RPH
RRR RPH
I
I —
I
—
.51: 1.00
.61: .02
.52 1.00
.62 .30
.53 .43
.62 ‘ .50
0.52 1 0.08
.60: .23
I
0.73
—
.72: .06
.791 .08
.70 : .20
.80 .84
.71 ‘ .22
.81 1.00
.69 .11
.80 .58
,
I
.88! .03
.88 ‘ .13
.88 .16
.861 .05
‘
‘
.9
—
1-1-lydroxychiorden 1.3(
—
Epoxide 1.T
.92: .03
I
:
I
I
:
1
-_______
.93 1
1.12 .29
1.29 i .27
1.41 .54
I
1.62 I .15
: .66
1.12 .29
1.29 .17
1.41 I .52
,
1.62 I .17
‘
I
I
.92’ .68
1.08’ .54
1.28: .49
1.3?’ 1.00
1.47; .06
1.59 24
I
1.771 .10
I
0.91’ 0.25
I
1.08’ .13
1.27: .38
1.39 I .26
1.46’ 1.00
‘
‘
1.77 .41
1
1.28’ 0.05
‘
1.49 .20
I
1.72.04
I
I
i
I
—
I
I
1.7
1.8
‘
p
1.83 I .03
1.96 .07
I
1.97 : .04
I
1.92: .22
I
1.92: .75
1.86 .44
i
I —
2.2
2.3
—
2.4
I
,
‘
I
I
2.32 .13
I
2.32’ .08
I
I
2.28L .32
I
‘
2.28 .77
2 06. .48
,
I
I
I
2.40 .57
2.44 .89
‘
I
2.82 1 .05
I
2.86 .04
2 . 78 .19
I
2.b3i .11
2.79 .20
2.66 1 .22
,
3.2
—
I
2.99 .07
‘
‘
I
I
2.96’ .08
I —
3.60’ .02
2.93’ .80
,
3.63 1 .21
2.98 1.00
3 . 58 i .43
4.5
I
I
‘
‘
- :
4.32, .02
4.291 .13
I
I
4.4l .76
—
‘
I
I
—_________
‘
I
I
I
‘
•
,
—
‘
I
I
5.32 .0
‘
S.38 .34
6.5 .05
8.4
‘
‘
‘
I
I
8.0 .10
I
—
—
I
,
,
,
—
I
.oi4
‘
I
,
I
I
I
i.O2L.
I
4 pj
.0097
I
: .02
.02
I
: .025
—
I
I
— I
‘
‘
I
‘
—
I
‘Relative retention ratios given for some conuion pesticides for comparison purposes.
2 lndicates the retention ratio, relative to aldrin.
3 lndicates the peak height response relative to that of the tallest peak shown 1 n italics.
4 lndicates the peak height response of the tallest peak relative to that obtained fiom an
equivalent amount of aldrin.
-------
Revised 11/1/72 Section 11,A
Page 1
SAMPLE PREPARATION AND ANALYSIS OF SOILS AND I{)USE DUST
I. INTRODUCTION :
The analytical method described below is similar in principle to
the method presented in the Analytical Manual distributed at the annual
Chemist’s Meeting in Tucson in 1968. The main difference lies in the
incorporation of the standard Mills, Onley, Gaither Florisil cleanup
technique for which all laboratories have equipment and a degree of
expertise in manipulation. This is preceded by percolation through an
alumina column for further removal of contaminants.
II. PRINCIPLES :
Organochlorine pesticides, together with other lipid-soluble sub-
stances, are extracted from homogenized samples by continuous Soxhlet
extraction with acetone-hexane. Bulk of solvent is removed by evapora-
tion in Kuderna-Danish equipment. Interfering lipid-soluble materials
are then partially removed from the extracts by successive cleanup on
aluminum oxide and Florisil columns. Extracts are adjusted to appro-
priate concentration for determinative analysis by E.C. and F.P.D.,
confirming as needed by M.C. and/or T.L.C.
III. EQUIPMENT :
1. Soxhiet extraction apparatus, complete with 125-mi 24/40 flask,
extraction tube with 24/40 lower and 34/45 upper joints and
Friedrichs condenser with 34/45 joint. Kimble #24010 or the
equivalent for the entire assembly.
2. Soxhiet extraction thimbles, paper, Whatman, 25 x 80 mm, Fisher
#9-656-c or the equivalent.
3. Sieves, U. S. Standard, #10 mesh, #18 mesh and #60 mesh with top
covers and bottom pans, 8” dia. x 2” depth, stainless steel.
4. Chromatographic columns, 22 x 300 nun with Teflon stopcock, without
glass frit. Size #241, Kontes #420530 or the equivalent.
5. Kuderna-Danish concentrator fitted with grad. evaporative concen-
trator tube. Available from the Kontes Glass Company, each com-
ponent bearing the following stock numbers:
a. Flask, 250 and 500 ml, stock #K-57000l.
b. Snyder column, 3 ball, stock #K-503000.
-------
Revised 11/1/72 Section ll,A
-2-
c. Steel springs, 1/2”, stock #K-662750.
d. Concentrator tubes, 10 ml grad., size 1025, stock #K-570050.
6. Modified micro-Snyder columns, 19/22, Kontes K-56925l.
7. Glass beads, 3 nun plain, Fisher #11 - 312 or equivalent.
8. Evap. concentrator tubes, grad., 25 ml, ‘ 19/22, Kontes #570050.
9. Water or steam bath.
10. Glas Col heating mantles with variable autotransformers, size to
match 125-mi Soxhiet flasks.
11. Filter paper, Whatman No. 1, 15 an.
IV. REAGENTS AND SOLVENTS :
1. Acetone, pesticide quality.
2. Hexane, pesticide quality.
NOTE : Both solvents must be carefully checked for
background contaminants as outlined in Section 3,C
of this manual.
3. Extraction mixture - acetone/hexane, 1:1.
4. Aluminum oxide, Merck reagent grade, stock #71695 acid-washed.
Prepare for use by shaking with 10% distilled water (w/w) for
partial deactivation. Shelf life of 10 days if stoppered tight.
NOTE : The distilled water must be prechecked for contam-
inant background. If any interferences are detected,
the water must be hexane extracted before use.
5. Diethyl ether - AR grade, peroxide free. The ether must contain
2% (v/v) absolute ethanol. Most of the AR grade ethyl ether con-
tains 2% ethanol, added as a stabilizer, and it is therefore un-
necessary to add ethanol unless peroxides are found and removed.
NOTE : To determine the absence of peroxide s in the ether,
-------
Revised 11/1/72 section ll,A
-3-
add 1 ml of ether in a clean 25-mi cylinder pre-
viously rinsed with ether. Shake and let stand
1 minute. A yellow color in either layer indicates
the presence of peroxides which iiust be removed
before using. See Misc. Note 4 at end of procedure.
The peroxide test should be repeated at weekly
intervals on any single bottle or can as it is possible
for peroxi.des to form from repeated opening of the
container.
6. fluting mixture, 6% (6+94)-purified diethyl ether - 60 ml is diluted
to 1000 ml with rethstilled petroleum ether,and anhydrous sodium
sulfate (10-25 g) is added to remove moisture.
7. Eluting mixture, 15% (15+85)-purified diethyl ether - 150 ml is
diluted to 1000 ml with redistilled petroleum ether, and dried as
described above.
NCTrE : Neither of the eluting mixtures should be held
longer than 24 hours after mixing.
8. Florisil, 60/100 mesh, PR grade, to be stored at 130°C until used.
Furnished by Perrine on order.
NOTE : (1) In a high humidity room, the colurmi may pick up
enough moisture during packing to influence the
elution pattern. To insure uniformity of the Florisil
fractionation, it is recommended to those laboratories
with sufficiently large drying ovens that the columns
be pac ed ahead of time and held (at least overnight)
at 130 C until used.
(2) Florisil furnished by the Perrine Laboratory has been
activated by the manufacturer, and elution pattern
data is included with each shipment. However, each
laboratory should determine their own pesticide
recovery and elution pattern on each new lot received,
as environmental conditions in the various labora-
tories may differ somewhat from that in Perrine.
Each new batch should be tested with a mixture of
8-BHC, aidrin, heptachlor epoxide, dieldrin, p,p’-DDE,
p,p t -DDD, and p ,p’ -DDT, eluting the standard mixture
as described in Section 5,A, (1) of this manual.
Dieldrin should elute entirely in the 15% diethyl
ether fraction, whereas all other c upounds should be
in the 6% fraction.
9. Anhydrous sodium sulfate, reagent grade granular, Mailinckrodt
-------
Revised 11/1/72 Section ll,A
-4-
Stock #8024 or the equivalent.
NOTE : When each new bottle is opened, it should be tested
for contanu.nants that will produce peaks by Electron
Capture Gas Liquid Chromatography. This may be done
by transferring ca 10 grains to a 125-mi Erlenmeyer
flask, adding 50 ml pet. ether, stoppering and shaking
vigorously for 1 minute. Decant extract into a
100-mi beaker and evaporate down to ca 5 ml. Inject
5 ii into the Gas Liquid Chromatograph and observe
chromatogram for contaminants. When impurities are
found, it is necessary to remove them by extraction.
This may be done using hexane in a continuously
cycling Soxhlet extraction apparatus or by several
successive rinses with hexane in a beaker. The
material is then dried in an oven and kept in a
glass - s toppered container.
V. PROCE [ IJRE :
Sample Preparation
1. Soils and vacuum cleaner bag dusts are analyzed in the air-dry state.
If a soil sample is obviously damp, it is allowed to equilibrate its
moisture content with room air before handling. Trials have shown
that house dust screenings generally contain approximately 0.1%
moisture, possibly more in areas of high relative humidity.
2. Vacuum cleaner bag contents are sieved on U. S. Standard #10 and #60
sieves to remove hair, fibers and large particles. The resulting
“fines” are separated into sealed glass jars until analyzed. Soils
are sifted on a U. S. Standard #18 sieve to remove stones and other
foreign material. Store the sieved soil in a sealed glass jar until
analyzed.
3. The 15-cm filter paper and the Soxhlet extraction thimbles should be
preextracted with the ace tone/hexane extraction solvent prior to use.
This may be conveniently done by folding several sheets of filter
paper and placing in the Soxhiet extractor. Allow to cycle ca 2 hours,
remove and dry. Wrap in aluminum foil and store in desiccator. The
thimble is similarly preextracted and may be used repeatedly with no
need for reextraction as long as it remains in good physical shape.
-------
Revised 11/1/72
Section ll,A
-5-
Sample Extraction
1. Weigh sample (2 grains of soil or 1 gram of dust) onto a sheet of
15-an filter paper. Carefully fold paper to form a half-circle with
the sample in the center (along the diameter line). Fold in the ends
of the half-circle towards the center, the total resulting length to
be ca 70 nun; then, starting at the diameter line, roll into an
approximately cylindrical shape and insert into the extraction thimble.
2. As a recovery check, another portion of the same dust (or soil)
should be spiked and carried through the entire procedure. This is
done as follows:
a. Weigh exactly 3.0 grams of the soil or 2.0 grains of dust into
an evaporating dish. Add sufficient hexane to make a slurry.
b. Prepare a standard mixture of the following compounds, the
concentration expressed in micrograms per mllliliter:*
Lindane 5.0 Die ldrin 7.5
Hept. Epoxide 5.0 p,p’-DDD 10.0
A ldri.n 5.0 o,p’-DDT 10.0
p,p’-DDE 7.5 p,p’-D [ ff 10.0
*In case your testing program indicates the presence of any
other compounds or metabolites, standards of these should be
included.
c. Add 1.5 ml of this mixture to the soil sample or 1.0 ml to
dust. 0 Mix gently with a glass rod and evaporate the solvent
at 40 C under a nitrogen stream, stirring from time to time.
d. After renx)val of the solvent, allow the spiked sample to
equilibrate to room temperature and humidity, and weigh the
sample for extraction as outlined above in Step 1.
3. At this point, a reagent blank should be initiated, starting with
the folded filter paper and carrying through the entire extraction,
cleanup and determinative procedures.
4. Place the sample, reagent blank and spiked sample thimbles into
separate Soxhlet extractors. Fill the boiling flasks, each con-
taining six glass beads, about half full with the 1:1 acetone/hexane
co-solvent, assemble the extraction apparatus, position in the
heating mantles and start extraction.
-------
Revised 11/1/72 Section 11,A
-6-
NOTE : Each laboratory will need to determine the setting
of their voltage controller. There should be
sufficient heat to result in 1 discharge cycle
about every 5 minutes, or ca 60 syphon discharges
in a S-hour period. This should be an adequate
number of cycles to insure complete extraction.
5. At the completion of the extraction period disassemble the extraction
apparatus, rinsing the joint between flask and extractor with a
few ml of hexane.
6. Assemble a Kuderna-Danish evaporator with the 250-mi K-D flask
attached to a 10-mi evap. concentrator tube containing one 3-mm
glass bead.
7. Transfer the extract from the l25-ml Soxhiet flask to the K-D flask,
rinsing the Soxhlet flask with 3 portions of 5 ml each of hexane.
Attach the Snyder column and immerse evap. concentrator tube about
1-1/2 inches into the boiling water bath. Evaporate extract down
to ca 3 ml, remove from bath and cool. Extract is now ready for
cleanup.
Alumina and Florisil Cleanup
1. Prepare an alumina column as follows:
a. Place a small wad of prerinsed glass wool at the bottom of a
22 x 300 nun chromatographic column.
b. Add preextracted anhydrous Na 2 SO 4 to a depth of 1/2 inch.
c. Close stopcock and fill column with hexane.
d. In a SO-mi grad. beaker, fill exactly to the 30 ml mark with
alumina (this should be ca 30 grams). Add this slowly to the
column, allowing all the alumina to settle to the bottom.
Top this witi a 1-inch layer of Na,SOA. When settling is
complete, open stopcock and allow th&hexane to elute through
the column down to a point ca 1/8 inch above the top of the
upper Na 2 SO 4 layer, then close stopcock.
NOTE : This column packing technique minimizes the
density that may be obtained in dry packing.
The volume of hexane specified provides suff i-
cient column prerinse.
-------
Revised 11/1/72 Section 11,A
-7-
2. Position a second K-D flask fitted with 10-mi evap. concentrator
tube under column.
3. Transfer the 3 ml of concentrated extract from the first K-D evapor-
ation to the column. Rinse tube with three portions of 3 ml each
of hexane transferring the rinsings to the column.
4. Open stopcock and add 85 nil of hexane to the column, open stopcock
wide and elute into the K-D flask.
5. Concentration of the eluate from the alumina column is conducted
exactly the same as outlined above in Step 7 under Sample Extraction ,
taking extract down to 3 ml. This extract is now ready for Florisil
partitioning.
6. Florisil column: Prepare the column as described in Section 5,A,(l)
of this manual under FLORISIL FRACTIONATION, Steps 1 and 2, sub-
stituting hexane for pet. ether.
7. Assemble two more K-D apparatus but with 500-mi flasks and position
the flask of one assembly under the Florisil column. However, at
this point use 25-mi grad. evap. concentrator tubes instead of the
lO-iril size for previous concentrations.
8. Using a 5-mi Mohr or a long disposable pipet, iiamediately transfer
the extract from the evaporator tube in Step 5, above, onto the
column and permit it to percolate through. Rinse tube with two
successive 5-mi portions of hexane, carefully transferring each
portion to the column with the pipet.
NOTE : Use of the vbhr or disposable pipet to deliver
the extract directly onto the column precludes
the need to rinse down sides of the column.
9. Commence elution with 200 ml of 6% diethyl ether in pet. ether
(Fraction I). The elution rate should be ca 5 ml per minute. When
the last of the eluting solvent reaches a point ca 1/8 inch from
the top of the Na )A layer, place the second 500-mi Kuderna-Danish
assembly under the column and continue elution with 200 ml of 15%
diethyl ether in pet. ether (Fraction II). Place both Kuderna-
Danish evaporator assemblies in a water bath and concentrate extract
to ca 20 ml.
NOTE : If there is reason to suspect the presence of mala-
thion in the sample, have a third 500-mi K-D assembly
-------
Revised 11/1/72 Section l1,A
-8-
ready. At the end of the 15% fraction elution,
add 200 ml of 50% diethyl ether in pet. ether
(Fraction III), evaporating the eluate in the
same manner.
10. Remove K-D assemblies from bath, cool and rinse joint between tube
and flask with a little pet. ether. Finally, dilute both extracts
to exactly 25 ml and proceed with the GLC determinative step.
NOTE : A relatively high dilution is suggested as it has
been observed that residues are generally suffi-
ciently high to warrant this. Furthermore, the
concentration of contaminants remaining after
cleanup is hereby reduced.
Determinative by GLC
1. Inject 5 il of each fraction extract into the gas chromatograph
(E.G. mode) primarily to determine whether the extracts will re-
quire further adjustmont by dilution or concentration.
2. Mien appropriate dilution adjustments have been made in the extracts
and column oven is set to a laiown temperature, the relative reten-
tion values of the peaks on the chromatograns should be calculated.
Mien these values are compared with the values in the printed table
for the appropriate column, the operator should be able to make
tentative compound identifications. Microcouloinetry and/or UC
may be required for positive confirmation of some of the suspect
chlorinated compounds, whereas FPD may be utilized for the organophos-
phate suspects.
-------
Revised 11/1/72 section 11,A
-9-
‘IYPICAL RE OVER ’ DATA - Soxhiet Method
-Pesticides -
Lindene Hep. Epox. p,p’-DDE Dieldrin p,p’-TDE p,p’-DDT
SOILS:
Mean percent 85.25 87.83 83.08 88.25 91.17 94.17
recovery
S.D. : 5.446 9.446 6.345 6.210 6.886 8.922
n: 12 12 12 12 12 12
HOUSE DUSTS:
percent 87.33 80.58 82.92 86.27 90.00 87.78
recovery
S.D. : 6.997 12.85 6.345 12.89 9.715 20.20
n: 12 12 12 11 12 9
-------
11/1/72 Section ll,B
Page 1
SAMPLE PPEPARATION AND ANALYSIS OF BOTI1JM SEDJI4ENT
I. INTRODUCTION :
The examination of sediment from the bottom of a stream or lake
provides information concerning the degree of pollution resulting from
pesticides, particularly the organochiorine compounds which are not
readily biodegradable. This information combined with residue data
obtained by analysis of the water and tissues from resident marine life
contribute in the development of an overall profile of the pesticidal
contamination of a given body of water.
RIFERENCES:
1. Column Extraction of Pesticides from Fish, Fish Food and Mud,
Hesselberg, R. J. and Johnson, J. L., in press.
2. Sediment Extraction Procedure, Southeast Water Laboratory,
EPA, Athens, Georgia, Method Number SP-8/71.
II. PRINCIPLES :
The sediment sample is partially dried and extracted by column
elution with a mixture of 1:1 acetone/hexane. The extract is washed
with water to remove the acetone and then the pesticides are extracted
from the water with 15% CH 2 CL, in hexane. The extract is dehydrated,
concentrated to a suitable volume, subjected to Florisil partitioning,
desulfurized if necessary, and analyzed by gas chromatography.
III. EQUIPMENT AND REAGENTS :
1. Pans, approximately 14” x 10” x 2-1/2”.
2. Oven, drying.
3. Muffle furnace.
4. Desiccator.
5. Crucibles, porcelain, squat form, size 2.
6. Omru. or Sorvall mixer with chamber of ca 400 ml.
7. Chromatographic columns, 300 mm x 22 mi i i with Teflon stopcock.
-------
11/1/72 cc cn
-2-
8. Separatory funnels, 500 ml and 250 ml with Teflon stopcocks.
9. Filter tube, 180 mm x 25 mm.
10. Kuderna-Danish concentrator fitted with grad. evaporative concen-
trator tube. Available from the Kontes Glass Company, each com-
ponent bearing the followmg stock numbers:
a. Flask, 250 ml, stock I K-570001.
b. Snyder column, 3 ball, stock #K-503000.
c. Steel springs, 1/2”, stock #K-662750.
d. Concentrator tubes, 10 ml, size 1025, stock #K-570050.
11. Pyrex glass wool - preextracted with methylene chloride in a
Soxhiet extractor.
12. Hot water bath, temp. controllable at 80°C.
13. Sodium sulfate, anhydrous, Baker, prerinsed or Soxhiet extracted
with methylcnc chloride.
14. n-Hexane, pesticide quality.
15. Acetone, pesticide quality.
16. Methylene chloride, pesticide quality.
17. Acetone-hexane, 1:1.
18. Diethyl ether, pesticide quality, free of peroxides.
19. Distilled water, suitable for pesticide residue analysis.
20. Sodium sulfate solution, saturated.
21. Methylene chioride-hexane, 15% v/v.
IV. PROCEDURE :
1. Decant and dis card the water layer over the sediment. Mix the
sediment to obtain as homogeneous a sample as possible and transfer
-------
11/1/72 Section l1,B
-3-
to a pan to partially air dry for about 3 days at ambient tempera-
tires.
N(YIE : Drying tiii varies considerably depending on soil
type and drying conditions. Sandy soil will be
sufficiently dry in one day, whereas nuick requires
at least three days. The silt and nu.ick sedin nt
is sufficiently dry when the surface starts to
split, but there should be no dry spots. Moisture
content will be 50-80% at this point.
2. Weigh 50 gin of the partially dried sample into a 400-mi Omni-Mixer
chamber. Add 50 gin of anhydrous sodium sulfate and mix well with a
large spatula. Allow to stand with occasional. stirring for approx-
iinately one hour.
NOTh : As the final calculations will be made on a “bone
dry” basis, it is necessary at this point to initiate
the test for percent total solids in the sample being
extracted for pesticide evaluation. Immediately
after weighing the 50gm sample for extraction, weigh
ca 5 gin of the partially dried sediment into a tared
crucible. Deter ine the percent solids by drying
overnight at 103 C. Allow to cool in a desiccator
for half an hour before weighing. Determine the
percent volatile solids by placing the oven-drie
sample into a nuffle furnace and igniting at 550 C
for 60 minutes. Allow to cool in a desiccator before
weighing.
3. Attach the 400-mi chamber to art irii or Sorvall mixer and blend for
about 20 seconds. The sample should be fairly free flowing at this
point.
4. Carefully transfer the sample to a chromatographic column. Rinse the
mixer chamber with small portions of hexane adding the rinsings to
the column.
5. Elute the column with 250 ml of 1:1 acetone-hexarte at a flow rate of
3-5 mi/mm into a 400-mi beaker.
6. Concentrate the sample extract to about 0 l0O ml wider a nitrogen
stream and at a temp. no higher than 55 C. Transfer to a 500-mi
separatory funnel containing 300 ml of distilled water and 25 ml
of saturated sodium sulfate solution. Shake the separatory funnel
for two minutes.
-------
11/1/72 Section 11,11
-4-
7. Drain the water layer into a clean beaker and the hexane layer into
a clean 250-nil separatory funnel.
8. Transfer the water layer back into the 500-mi separatory funnel and
reextract with 20 ml of 15% methylene chloride in hexane, again
shaking the separatory funnel for two minutes. Allow the layers to
separate. Discard the water layer and combine the solvent extracts
in the 250-nil separatory funnel.
9. Wash the combined solvent extract by shaking with 100 ml of dis-
tilled water for 30 seconds. Discard the wash water and rewash the
extract with an additional 100 ml of distilled water, again dis-
carding the wash water.
10. Attach 10 ml evap. concentrator tube to a 250-mi Kuderna-Danish
flask and place under a filter comprised of a small wad of glass
wool and ca 1/2 inch of anhydrous Na 2 SO 4 in filter tube.
11. Pass the solvent extract through the drying Filter into the K-D
flask, rinsing with 3 portions of ca 5 ml each of hexarie.
12. Attach Snyder column to top joint of K-fl flask, immerse tubo in 80°C
water bath and concentiate extract to S ml.
13. Remove tube, rinsing joint with small volume of hexane. The sample
is now ready for F]oris 1 partitioning. Ihis is conducted as
described on page 3, Section 8,B of this manual.
V. CALCULATIONS :
1. Percent Dry Solids
gin of dried sample x 100 = % Dry Solids
gin of sample
2. Percent Volatile Solids
gm of dried sample - gin of ignited sample = m of volatile solids
gin of vol,tile solids x 100 = 9 o Volatile Solids
gin of sample
-------
11/1/72 Section 11,B
-5-
3. Concentration of Pesticide in Sediment
% dry solids x 50 gin = gin of dry sample extracted
1 of samRle extract injected x gin of dry sample extracted = gin of
1 of sample extract dry sample injected
ng of pesticide = ppb of pesticide
gin of dry sample injected
V I. SULFUR INThRFERENCE :
Elemental sulfur is encountered in most sediment samples, marine
algae and some industrial wastes. The solubility of sulfur in various
solvents is very similar to the organochlorine and organophosphate
pesticides; therefore, the sulfur interference follows along with the
pesticides through the normal extraction and cleanup techniques. The
sulfur will be quite evident in gas chromatograins obtained from electron
capture detectors, flame photometric detectors operated in the sulfur or
phosphorus mode, and Coulson electrolytic conductivity detectors. If
the gas chromatograph is operated at the normal conditions for pesticide
analysis, the sulfur interference can completely mask the region from the
solvent peak through aidrin.
This technique eliminates sulfur by the formation of copper sulfide
on the surface of the copper. There are two critical steps that nust be
followed to remove all the sulfur: (1) the copper nn.ist be highly reactive;
therefore, all oxides must be removed so that the copper has a shiny,
bright appearance; and (2) the sample extract must be vigorously agitated
with the reactive copper for at least one minute.
it will probably be necessary to treat both the 6% and 15% Florisil
eluates with copper if sulfur crystallizes out upon concentration of the
6% eluate.
Certain pesticides will also be degraded by this technique, such as
the organophosphates, chlorobenzilate and heptachlor (see Table 1).
However, these pesticides are not likely to be found in routine sediment
samples because they are readily degraded in the aquatic environment.
If the presence of sulfur is indicated by an exploratory injection
from the final extract concentrate (presumably 5 ml) into the gas chroma-
tograph, proceed with removal as follows:
1. Under a nitrogen stream at ambient temp., concentrate the extract
in the concentrator tube to exactly 1.0 ml.
-------
11/1/72 Section ll,B
-6-
2. If the sulfur concentration is such that crystallization occurs,
carefully transfer, by syringe, 500 p1 of the supernatant extract
(or a lesser volume if sulfur deposit is too heavy) into a glass-
stoppered, 12-mi grad., conical centrifuge tube. “.dd 500 i ii of
iso-octone.
3. Add ca 2 pg of bright copper powder, stopper and mix vigorously
1 minute on a ‘vortex Genie mixer.
NOfE : The copper powder as received from the supplier
must be treated for removal of surface oxides with
6N HNOZ. After about 30 seconds of exposure, decant
off add, rinse several times with dist. water and
finally with acetone. Dry under a nitrogen stream.
4. Carefully transfer 500 p1 of the supernatant-treated extract into a
10-mi grad. evap. concentrator tube. An exploratory injection into
the gas chromatograph at this point will provide information as to
whether further quantitative dilution of the extract is required.
NOTE : If the volume transfers given above are followed,
a ñnal extract volume of 1.0 ml will be of equa]
sample concentration to a 4-mi concentrate of the
Florisil cleanup fraction.
Table 1. Effect of Exposure of Pesticides to Mercury and Copper
Percentage Recovery Based on Mean
of Duplicate Tests
Compound Mercury Copper
BHC 81.2 98.1
Lindane 75.7 94.8
Heptachlor 39.8 5.4
Aidrin 95.5 93.3
Uept. Epoxide 69.1 96.6
p,p’-DDE 92.1 102.9
Dieldrin 79.1 94.9
Endrin 90.8 89.3
DDT 79.8 85.1
Ch lorobenzilato 7.1 0
Aroclor 1254 97.1 104.3
Malathion, diazinon, 0 0
Parathion, Ethion,
Trithion
-------
Revised 11/1/72 Section 12, B
Page 1
C( FIRMA TORY PROCEDU1 ES
B. ThIN- LAYER DIATOGRAPHY
I. INTROWCTI(Th
Thin-layer chromatography is primarily a qualitative tool which
is useful in the identification of pesticides. It can be used to
advantage as a confirmatory method in conjunction with gas chroma-
tography. Thin-layer chromatography introduces a second physical
basis for separation, that of adsorption chromatography.
Additional advantages of this technique, include simplicity,
rapidity, low man-hour consumption, and its utility as a resolving,
and cleanup procedure, for use with other methods of analysis.
The method is, in general, somewhat less sensitive than micro-
coulometry, being limited to about 10 Ng for easy visual inspection
of mest chlorinated pesticides and about 50 Ng of wost organothio-
phosphates. Consequently, a macrosample is required. A stringent
sample cleanup procedure is also required.
REFEPENCES:
1. Kovacs, Martin, F., JAQAC. 46 (1963).
2. Kovacs, lvlartin, F., Ibid, 47 (1964).
3. Kovacs, Martin, F., Ibid, 48 (1965).
4. Kovacs, Martin, F., Ibid, 49 (1966).
5. Kovacs, Martin, F., Private Communication (1968).
6. Moseman, Robert, Private Communication (1968).
7. Pesticide Analytical Manual, U. S. Food Drug
Administration, Volume I, Sect. 410.
II. APPARA11JS :
1. 8” x 8” glass plates, double strength window glass (Pittsburgh
Plate Glass).
2. 3-1/4” x 4” clinical microslides (Arthur H. Thomas Co.).
-------
Revised 11/1/72 Section 12, B
-2-
3. Developing tank, Thomas-Mitchell, 8-1/2” x 4-1/2” x 8-1/2” deep
(Arthur H. Thomas Co.).
4. Desaga/Brinkmann standard counting board.
S. Chromatographic chamber, 800 ml beaker.
6. Desaga/Brinkmasm standard model applicator.
7. Desaga/Brinkmann drying rack, holds 10, 8” x 8” plates.
8. Spotting pipettes, 1, 5, and 10 .il, Kontes 763800.
9. Spray bottle, 8 oz., Thomas Co. #9186-R2.
10. Desaga/Brinkmann glass vacuum desiccator.
‘11. Desk blotter paper.
12. Ultra violet light source: 4-15 watt C. E. germicide lamps,
shielded to protect operator, General Llectric Co. G 15 T 8.
III. REAGENTS :
1. Aluminum oxide C. (Brinkmann or Warner-Chilcott).
2. N-heptane, chromatographic grade.
3. Methycyclohexane, practical, B.P. 100.5 - 101.5°C. (Matheson,
Coleman, and Bell).
4. Tetrabromophenolphthalein ethyl ester (Eastman Organic Chemicals
#6810).
5. Acetone, Reagent.
6. Silver Nitrate, Reagent.
7. Tetraethylenepentamine.
8. Citric Acid, granular, Reagent.
9. p-Nitrobenzyl pyridine.
10. Hydrogen peroxide, 30%, Reagent.
-------
Revised 11/1/72 Section 12, B
-3-
11. Ethyl ether, Reagent.
12. Acetonitrile, chromatographic grade.
13. Dimethylformaiiiide, Reagent.
14. Preparation of reagent solutions.
A. Developing solvents
(1) For organochiorines
(a) 2% acetone in N-heptane (v/v) (mobile solvent).
(b) N-heptane (mobile solvent).
(2) For thio and nonthio organophosphates.
(a) Nethylcyclohexane (niobi]e solvent).
(b) 15% or 20% dimethylformanu.de (v/v) in ether
(Immobile solvent).
B. Chromogenic reagents.
(1) For organochlorines.
(a) Dilute 0.1 gm of silver nitrate and 20 ml of
2-phenoxyethanol to 200 ml with acetone.
Immethately add 3 drops of 3Q9 hydrogen peroxide
and nax. Keep stored in a cool dark place not
longer than 1 week. Dnrk solutions should be
discarded.
(2) For organothiophosphates.
(a) Stock dye solution.
Dissolve 1 gin of tetrabromophenolphthalem
ethyl ester in 100 ml of acetone.
(b) Use concentration dye solution.
Dilute 10 ml of stock solution to 50 ml with
acetone.
-------
Revised 11/1/72 Section 12, B
-4-
(c) Silver nitrate solution.
Dissolve 0.5 AgNO 3 gm in 25 ml of distilled water
and dilute to 100 ml with acetone.
(d) Citric acid solution.
Dissolve 5 gm citric acid in 50 ml of distilled
water and dilute to 100 ml with acetone.
(3) For thio and nonthio organophosphates.
(a) 2% p-Nitrobenzyl pyridine in acetone (w/v).
(b) 10% Tetraethylenepentamine in acetone (v/v).
IV. PPEPARATION OFT.L.C. PLATES :
8” x 8” plates
1. Add 30 g alumin aTt oxide G to 50 ml distilled water and shake for
30-45 seconds.
2. Pour into applicator and spread a 250 micron layer on glass plates.
Make an arrow in corner of plate indicating direction of appli-
cation.
3. Air dry for 15 minutes, then at 80°C for 45 minutes in a forced
draft oven.
4. Remove, cool, and store plates in a desiccator.
3-1/4” x 4” micro slide plates
1. Preparation of adsorbent layer - Select five 8” x 8” and one 4”
x 8” (calculated to cover the entire surface of the applicator
board) photo-glass plates of uniform width and thickness. Wet
the surface of the applicator board with a few ml of distilled
water delivered from an eye dropper in the form of the letter ‘ix’,,
approximately the size of the plate to be mounted. Press each
plate snugly into position on the applicator board, forcing out
excess water under the plate to insure a tight fit. (Add enough
water to the applicator board to prevent the appearance of air
bubbles under the plate after it has been pressed into position).
-------
Revised 11/1/72 Section 12, B
-5-
2. Examine each 3-1/3” x 4” micro slide carefully by looking down
each edge. To insure flatness of plates, use only slides
that are visibly straight along all edges.
3. Mount the micro slides individually on the surface of the photo-
glass plates with their long axis perpendicular to the direction
of layer application. With an eye dropper, place a few ml dis-
tilled water on the surface of the photoglass plate and mount
micro slides. Force out the excess water so that no large air
bubbles remain under the slide. The presence of a large air
bubble indicates slide “bowing” due to an irregularity in the
slide. 1 hen “bowing” is noted, discard the slide, and position
another in its place.
4. Repeatedly slide an empty applicator across the series of mounted
slides to force out excess water, and wipe surface of slides dry
each time with a tissue. The empty applicator must ride smoothly
and without effort across the series of slides. If not, re-
examine the uniformity and positioning of micro slides.
5. To remove any remaining water, wipe the surface with a dry tissue,
then with one soaked in 95% ethanol, and let dry.
6. Weigh 30 g Al,0 G or MN-silica gel C-HR into a 250 ml Erlenmeyer
flask. Add SO l distilled water to Al 03 C or 60 ml to MN-silica
gel G-HR, stopper flask, shake moderate’y for 15 to 20 seconds,
and immediately pour slurry into applicator chamber. The time
required for actual application should be approximately 10 seconds.
Immediately after coating, grasp the applicator board at its ends,
raise a few inches, then drop. This procedure which is repeated
a few times, smooths out slight ripples or imperfections in the
wet coating.
7. Let coated plates dry in position on the mounting board f 9 k 20
minutes. Mark each micro plate on the 3-1/4” edge farthest from
the longitudinal center of the applicator board. This edge
represents the top of each micro plate during subsequent develop-
ment. The 3-1/4” edge of each micro plate at the center of the
applicator board represents the end to be spotted. This is done
because most of the coating irregularity occurs on the outer
3-1/4” edge of the plates.
-------
Revised 11/1/72 Section 12, B
-6-
8. Remove each plate individually with a spatula, and wipe the back
side dry with a tissue. Place 4 micro slides on the surface of
on 8 8” x 8” plate and place the plates in a rack for drying at
80 C for 1 hour in a forced draft oven.
9. After heating, cool the micro plates, and examine each individually
in strong transmitted light for possible gross irregularities in
the uniformity of the coating. Discard plate if gross
irregularities are observed. Place 4 micro plates on the surface
of each 8” x 8” plate, slide into a drying rack, and store in a
desiccating storage cabinet until needed.
10. Sample spotting - Make a pencil mark at each side 1/2” above the
bottom edge of the slide. The imaginary line between these points
serves as the sample “spotting line.” Draw an actual line across
the slide 2-3/8” (about 6 cin) above the “spotting line.” The
actual line serves to mark the solvent front after development.
Draw a pencil line along each side 1/4” in from the edge to
prevent distortion of the solvent front during development.
11. Spot samples at 1/4” intervals along the imaginary “spotting line.”
Each micro plate will accomlm3date 10 application points as compared
to 18 on a norma]. 8” x 8” plate. Spot samples and standards on
the micro slide in the same manner as described later under
SPOTTING.
V. SANPLE PPEPARATION :
1. The sample must be of sufficient size that when the extract from
Florisil cleanup is concentrated to an appropriate volume, a 10 iii
spotting volume will produce detectable compound spots. A serum
extract from 50 grains concentrated to 100 l should produce a
visible spot of 2 ppb. An adipose tissue extract from 5 grams
concentrated to 500 pl should give a readable spot at 10 ppb.
These values assume the detection of chlorinated pesticides.
2. The extract from the 15% diethyl ether fraction contains far more
lipids than are present in the 6% fraction. For this reason, some
further cleanup is required. This is conveniently accomplished by
spotting the equivalent of 5 grams of blood or 0. 5 grams of fat on
a 3-1/4” x 4” micro plate, developing with acetonitrile, and
scraping off the alumina from the area at the solvent front. This
is extracted in hexane and the resulting extract respotted on a
standard 8” x 8” TLC plate.
-------
Revised 11/1/72 Section 12, B
-7—
VI. SPOITING AND DEVELOPING :
1. Provide an imaginary spotting line across the plate by making a
pencil mark 1-1/2” from the bottom edge of the plate on both sides.
2. Provide an imaginary solvent front by i a1dng a pencil mark 5-1/2”
from the bottom edge of the plate on both sides.
3. With a micropipette, transfer a suitable amount of the extract
to one of the spots, with repeated applications.
4. Spot standard solutions on the same plate. Standard concentrations
should bracket the calculated amount of residue in sample.
5. Prepare chromatographic tank by placing 50 ml of developing solvent
in the trough and 75 ml in the bottom of the tank.
6. Seal the tank and develop to the line scribed on the plate.
7. Remove plate and air dry in the hood.
v i i. C IP YJND DETECrION :
1. Organochiorines
a. Immediately after drying, spray plates with the chroinogcnic
reagent.
b. Par dry plates for 15 minutes.
c. Expose plates to ultraviolet until the lowest concentrations
of standards are visible.
2. Organothiophosphates
a. Immediately after drying, spray plates with the “use concen-
tration ’ 1 dye solution. Spray moderately heavy.
b. Overspray lightly with the silver-nitrate solution.
c. After 2 minutes overspray the plate moderately with citric
acid solution.
-------
Revised 11/1/72 Section 12, B
-8-
3. Thio and nothio organophosphates
a. Mter drying, spray plate with 0 p-Nitrohenzyl pyridine chromo-
genic solution and heat at 110 C for 10 minutes.
b. Cool and overspray plate with tetraethylenepentamine solution.
NOTES : 1. The color of solid p-Nitrobenzyl pyridine should be
yellow. If there is any purple color, recrystallize from
acetone. Oxidized solution will cause high background color
and will reduce sensitivity.
2. The tetraethylenepentaiinne should not have a deep
color. If it does, decolorize and purify with charcoal.
4. Interpret results by comparing Rf value5 of sample spots against
those of standard spots on the sane plate.
\r I I. GAS 0-IROMATOGRAPHY (E . C.) CONFIPMATION OF R. F. VALUES :
At tines there may be reason to question the validity of a spot
because of a slight shift in the position of the R. F. site or because
of spot diffusion or a very faint appearing spot. 1 hen such doubt
exists, GLC, EC examination of the material from the questionable R. F.
site can serve to either confirm or negate the presence of the
suspected compound.
Any of the pesticidal compounds (organochiorine) of lowest con-
centration in blood or fatty tissue such as -BLIC, heptachior epoxide,
o,p’-Dtff and p,p’-DDD are frequently the most difficult to identify by
customary GLC, EC. These compounds, if present, are generally in such
low concentration that an extract aliquot equivalent to 5.0 grams of
blood and 0.5 grams of fat is needed for spotting on the TLC plates.
1. Spot the TLC plate with 200 nanogranis each of standards of the
suspect compounds. Also spot the 6 o diethyl ether fraction of the
unknown extract in two places on the plate.
2. Develop the plate with n-Hexane until the solvent front has migrated
10 cm.
3. Cover with a small glass plate a portion of the plate containing one
of the sample applications and spray the plate with the AgNO 3 /2-
phenoxyethanol reagent.
-------
Revised 11/1/72 Section 12, B
-9-
4. Develop the spots in the sprayed area in the usual manner and
compute the Rf values for the standards.
5. Utilizing the standard R values, pin-point the elution sites for
these compounds along th imaginary migration line of the unsprayed
sample.
6. Scrape each sample elution with a flat edge spatula and transfer
the alumina to separate centrifuge tubes.
NOTE : Scrape another spot from an area of the plate lying
outside the region of samples and standards and extract
identically to the sample spots. This will serve as a
reagent blank.
7. Add 1 ml of hexane which has been previously examined for assurance
that it is free of contaminants which niight contribute artifact
peaks.
8. Stopper tube and shake vigorously one minute on a Vortex mixer.
9. Inject 5 p1 of this extract into the gas chromatograph and observe
the chromatograin for the presence or absence of the suspect com-
pound peak. Adjustment of the injection volume may be required
based on peak height resulting from the initial injection, provided
of course, that there are any peaks.
IX. MISCELLANEOUS NOI’ES :
1. Thorough sample and extract cleanup must be employed.
2. Plates must be thoroughly washed.
3. All solvents, except ethyl ether, must be redistilled.
4. Prevent even minor contamination.
5. Isooctane tolerates more oil in the sample than other developing
solvents.
6. For the detection of nanogram quantities it is imperative to use a
source of U. V. radiation at least as intense as that provided by
the specified equipment.
7. Always spray the chromogenic agent in a direction perpendicular to
the direction of solvent flow (side to side).
-------
11/1/72 Section 12, 3
• 10 -
n—Heptane Solvent System
Adsorbent A1,O, G(Merck), 230 p thIck, air dried 72 hr at room
temperature
Plate Size 8”x8”
Front Travel 10 cm
pp’-TDE Travel 3.9 cm
Developing Tank 9”x9”x3. 5”, saturated
Visualization AgNO,, UV exposure
Temperature 24-26°C
Amount Spotted 80-200 ng
Pesticide
Hexachlorobenzene 2.7
A ldr ln 2.1
pp’-DDE 2.0
Heptachlor 2.0
Chiordane (tech) 2.0, 1.8, 1.4, 1.2’
o,p’-DDT 1.9
PCNB 1.8
Perthane olefln 1.8
p,p’-TDE olefin 1.8
TCNB 1.7
Telodrin 1.7
Toxaphene 1.7, 1.2’
Strobane 1.7, 1.2’
p,p’-DDT 1.6
o,p’-TDE olefin 1.6
Chiorbenside 1.3 (grey)
BHC (tech) 1.1, 0.27, 0.10
-BHC 1.3
Perthane 1.3
Lindane 1.1
o,p’-TDE 1.1
p,p’-TDE 1.0
Endosulfan Qjj. 0.07
Ronnel 0.85
Heptachlor epoxide 0.71
Endrln 0.71
DIeldr ln 0.52
Carbophenothion 0.42 (yellow)
Methoxychlor 0.33, 0.27
$-BHC 0.27
Ovex 0.18
Dichione 0.16
Dyrene 0.15 (grey)
-------
11/1/72 Section 12, B
- 11 -
pesticide _____
Tetrad lfon 0.11
6 -‘BHC 0.10
Delta Keto H153U 0.09
Kdlthane 0.06
Su1 henone 0.00 (large and fuzz y)
Captan 0.00 (sharp edged grey)
ChIorobenzilate 0.00 (light)
Monuron 0.00 (large and dark)
Dluron 0.00
Endrin aldehyde 0.00 (Very small)
Endrin alcohol 0.00
Most Intense spot underlined
‘Leave8 a streak with these major spots
-------
11/1/72 Secti 13,
- 12 -
2% Acetone th n-Heptane Solvent System
Adsorbent A1,O, G (Merck), 250 p thIck, air dried 72 hr at room
temperature
Plate Size 8”x8”
Front Travel 10 cm
p,p’-TDE Travel 5.7 cm
Developing Tank 9’x9”x3.5”, saturated
Visualization AgNO, UV exposure
Temperature 24-26°C
Amount Spotted 80-200 ng
Pesticide R nn
Hexachlorobenzene 1 7
Perthane olefin 1.4
PCNB 1.4
Aldrtn 1.4
pp’-DDE 1.4
Chiordane (tech) 4, 1.3, 1.2, 1.1’
p,p’-TDE olefin 1.4
Telodrln 1.4
Heptachior 1.4
TCNB 1.3
o,p’-TDE olefin 1.3
Toxaphene 1.3, 1.2
Strobane 1.3, i.2
o,p’-DDT 1.3
p,p’-DDT 1.2
Chiorbenside 1.2 (fuzzy grey)
Perthane 1.2
BHC (tech) Jj,, 0.92, 0.72, 0.25
a -BHC 1.1
Ronnel 1.1
Endrin 1.0
Carbophenothion 1.0 (fuzzy yellow)
Heptachior epoxlde 1.0
p,p’-TDE 1.0
o,p’-TDE 0.95
Lindane 0.92
Endosultan 0.92, 0.24
Die ldrin 0.90
Tetradifon 0.82
Methoxychlor 0.79
Ovex 0.76
$ -BHC 0.72
Dichione Q , 0.00
-------
11/1/72 Section 12, B
- 13 -
Pesticide R iD
Dyrene 0.51
Sulphertone 0.31
Kefthane 0.28
J-BBC 0.25
Delta Keto “153’ 0.23 (very small)
Captan 0.09
Chlorobenz l late 005
Monuron 0.00
Diuron 0.00 (dark spot)
Endrin aldehyde 0.00 (very 8mall)
Endrin alcohol 000
Most intense spot underlined
Leaves a streak with these major spots
-------
11/1/72 Section 12, B
- 14 -
Rf Values
Adsorbent A120$ G
Mobile solvent Methylcyclohexafle
Pesticide R Value
Immobile Solvent
15% DMF 20% DMF
DUnethoate 0.01 0.01
Azlnphosmethyl (GuthIon) 0.09 0.06
Imldan 009 0.07
Methyl parathion 0.17 0.11
Coumaphos 0.23 0.15
Malathion 034 0.22
Dloxathion 0.37 0.24
Parathion 041 0.27
Demeton (thiOl) 0.44 0.32
EPN 0.49 0.33
Methyl carbophenothion 0.50 0.36
Sulfotepp 0.69 0.55
Carbophenothion 0.74 0.59
Ronnel 0.76 0.62
Ethion 0.77 0.63
Demeton (thiono) 0.79 0.67
Phorate 0.81 0.71
Disulfoton 0.82 0.7
Dlazthofl 0.86 0.78
(1) Presence of chloride in adsorbent layer reacts with AgNO, and prevents coupling
of dye and pesticide to form characteristic blue or purple spot. Some aluminum oxide
coatings do not have tobeprewashedtoremove chloride. If, however, maximum com-
pound sensitivities of 0.05 pg cannot be achieved with unwashed Al ,O, coating, pre-
washing is recommended.
(2) Chromogenic spray reacts only with sulfur—containing phosphate esters. The fol-
lowing compounds do not react; oxygen analog of parathion, dichlorvos naled,
mevlnphos, Phoephamidon and trichlorfon.
(3) The following minimum amounts of sulfur-containing phosphate esters can be
detected: 0.05 p g diazinon, demeton (th.Iono), carbophenothion, parathion, malathion,
ronnel, dioxathion, EPN, coumaphos, sulfotepp, and ethion; 0.1 pg azlnphosmethyl,
methyl parathion, and derneton (thiol). The lower limits of detectability of dimethoate,
Irnidan, methyl carbophenothion, phorate, and disulloton were not determined. At 0.5
g, or greater, the thio-phoaphate esters vary as to color produced with the chromo-
genic reagents. Carbophenothion, parathion, EPN, coumaphos and diazinon appear
vivid blue. Ethion, azinphosmethyl, sulfotepp, dioxathion, and malathion appear
purple. Ronnel and methyl parathion appear dull blue while both thiol and thiono
demeton appear bluish purple.
-------
11/1/72 Section 12,D,(1)
Page 1
MICRO SCALE ALKALI fl EAThEN FOR USE IN PESTICIDE RESIDUE
C( FI1 M TION I N1) SAMPLE CLEANUP
(Reproduction of manuscript in press, 1972 by
Susan J. V. Young and Jerry A. Burke
Division of Chemical Technology
Bureau of Foods
Food Drug Administration)
Procedures involving alkali treatment for dehydrochiorination of certain
organochlorine pesticides and saponification of fats have long been employed
in pesticide residue analysis. In 1942 Brand and Busse-Sunderman (1), and in
1946 Soloway et al. (2), studied rates of dehydrochlorination of DDT. Mills
(3) used refluxing alcoholic KOH in the cleanup of fatty foods for paper
chromatographic detection of alkali-stable organochlorine pesticides. Klein
and Watts (4) used alcoholic NaOH to dehydrochlorinate o,p t - and p,p t -DDT,
p,p’-TDE, and Perthane prior to gas chromatographic separation of the respec-
tive olefins. These investigators called attention to several earlier uses of
alkali dehydrochiorination in pesticide residue chemistry. Recent literature
contains nui rous references to application of this treatment in pesticide
residue analyses, including an adaptation for use in a pre-gas chromatographic
(GLC) column (5).
In spite of the knowledge of this reaction, we have observed that it is
not fully and effectively utilized by residue laboratories for forming deriva-
tives, gaining information for identity confirmation, or obtaining better
cleanup of troublesome extracts. This is probably because the procedure has
not been described in detail for simple micro scale application in multi-
residue analysis.
The purpose of this work was (1) to arrive at optimum and convenient para-
meters of the alkali treatment in order to obtain rapid dehydrochlorination
resulting in product solutions suitable for analysis by GLC; (2) to obtain
complete yield and recovery of olefins from a number of bis (phenyl) chloro-
ethane pesticides; (3) to detennine the effect of the treatment on several
Important pesticide and industrial chemicals; (4) to describe in detail the
procedure for routine application in the residue analytical laboratory.
METhOD
Reagents and Apparatus
(a) Potassium hydroxide - Anhydrous pellets.
(b) Ethanol - DSP 95%.
-------
11/1/72 Section 12,D,(l)
-2-
(c) Hexane - Suitable for use with electron capture gas chromatography
(Burdick and Jackson Laboratories, Inc. 1953 S. Harvey St.,
Muskegon, Mich. 49442).
(d) Micro condenser - 19/22 ‘; K-569250 (Kontes Glass Company, Vineland,
N. J. 08360).
(e) Concentrator tube - Mills type, 19/22 ‘ ‘ with stopper, 10 ml graduated
in 0.1 ml up to 1.0 ml; K-570050 (Kontes Glass Company).
(f) Alkali dehydrochiorination reagent - Dissolve 2 g KOH in 100 ml
ethanol.
(g) Ethanol - water, 1+1 - Combine equal parts by volume of distilled
water and ethanol.
(h) Gas chromatograp - Equipped with electron capture detector and
61 x 4 mm id glass column containing either (1) 10% DC-200 or (2)
1:1 mixture of 15% QF-1 + 10% DC-200 on 80-100 mesh Chromosorb W(1-LP).
Operating conditions : N 2 flgw 120 rnljrriin; temperatures, column and
detector 200 C, injector 225 C; concentric design electron capture
detector operated at DC voltage to cause 1/2 full scale recorder
deflection for 1 ng heptachior epoxide when full scale deflection
is 1 x lO amp.
Procedure
Accurately pipet, into a 10-ml Mills tube, 2 ml of a petroleum ether solution
of sample extract [ 6% or 15% Florisil eluate (6)] containing concentrations of
pesticides suitable for subsequent GLC analysis. Add 1 ml of 2% ethanolic KOH
and a few carborundum chips and fit the tube with a micro condenser. [ Note:
Avoid getting alkali on the ground glass joint: light greasing of joint with
silicone lubricant may prevent sticking.] With a test tube clamp, hold the
tube over an opening in the steam bath in such a manner that gentle boiling
occurs. When the volume has been reduced to about 1 ml, insert the tube com-
pletely into the steam bath opening and heat vigorously for 15 minutes or until
the volume reaches 0. 2 ml. Remove tube from the steam. If a precipitate has
formed, as is often the case with extracts containing fatty substances, add a
few drops of 2% ethanolic KOH and warm gently in steam with swirling until the
precipitate dissolves. After the solution has cooled slightly, add about 2 ml
ethanol-f-{ 2 0 (1 + 1). Allow solution to reach room temperature and pipet 1 or 2
ml hexane into tube. Stopper tube with ground glass, invert, shake vigorously
for about 30 seconds, and allow solvent layer to separate sharply. With micro-
liter syringe, carefully withdraw aliquot of upper layer for determination by
GLC. [ Note: Separation of phases should be sharp so that solution withdrawn
for GLC analysis will be free from alkali.]
-------
11/1/72 Section 12,D,(l)
-3-
Discussion
Development of Method
Initial experimentation was performed to establish the reaction conditions which
would give complete and rapid dehydrochiorination of ,p’ -DLTr, o,p’ -DDT,
£.‘E’ -TDE, o,p’ -TDE, methoxychlor, and Perthane and which would permit complete
recovery 0 r €he respective olefins. Work was done to determine the effect on
olefin formation of fatty substances not removed during sample cleanup and the
capacity of the reaction to eliminate fatty substances.
Several considerations found necessary for practical and reliable use of the
alkali treatment have been incorporated into the method and are briefly dis-
cussed. The steam bath was chosen as a source of heat because of its ready
availability and convenience. The Mills reaction tube was fitted with a micro
condenser to eliminate losses due to volatilization, which often occurred in
the absence of the condenser. Both K(1-I and NaOH have been used to the satis-
faction of previous investigators. The more frequent use of KOH by other
workers and its higher solubility in ethanol made it the choice for this work.
An alkali concentration of 2% has been widely used, and was found ideal for
treatment of aliquots of cleaned-up sample extracts containing quantities of
bis(phenyl) chioroethane pesticides ranging from a few nanogranis to 100 g.
In order to provide sufficient reflux time and temperature, it was necessary
that the initial volume of ethanol be in excess of 0.5 ml. When smaller vol-
umes were used, dehydrochiorination was usually incomplete. Emulsions often
occurred during extraction of the olef in into hexane after saponification of
fatty substances. The use of ethanol + water (1+1) instead of water as the
di .luent resulted in a sharp separation of hexane and aqueous layers. Recoveries
of olefins were not adversely affected if the ethanol content was less than
about 70%. Less than 30% ethanol did not adequately enhance separation of the
two layers. The non-volatile fatty substance transferred to acetonitrile by
partitioning a petroleum ether solution of butterfat with acetonitrile was used
in tests to evaluate the effects of fatty substances on the reaction. Experi-
ments in which varying volumes of 2% ethanohc KOH were used to saponify 350-mg
portions of this butterfat showed that each 1 ml of 2% KOH would saponify about
50 mg of the fat. When the weight of fatty substances exceeded about 50 mg,
complete dehydrochiorination of Perthane (40 i.’g) and methoxychlor (4.0 jig) was
not obtained. However, E,2’-DDT (8.0 i.’g) was completely dehydrochiorinated in
the presence of 100-120 mg of fat. Additional experimentation showed that com-
plete dehydrochlorination of methoxychlor and Perthane did not occur until the
fat was completely saponified; these were the bis(phenyl) chloroethanes most
resistent to dehydrochiorination. Quantities of Perthane and a, ’-DDT up to
100 i .tg in the presence of not n re than 50 mg butterfat were readily dehydro-
chlorinated with 1 ml of 2% KOH at steam bath temperature; dehydrochiorination
of larger amounts of pesticide was not attempted. Hexane, because of its
higher boiling point and greater ease of drawing into a microsyTinge, was used
-------
11/1/72 Section 12,D,(l)
-4-
instead of petroleum ether, to extract the olef in after reaction in order to
avoid possible errors in quantitation.
Effect on Selected chemicals
The dehydrohalogenation reaction, as described under “Method”, was applied to
fl, 2 ’-DDT (0.8 i g), o, -DDT (0.8 pg), p,a’-IDE (2.0 iig), o,p’-IDE (0.4 jig),
methoxychlor (4.0 ‘j’), and Perthane (40 .‘g). Quantities Thown in parentheses
were chosen for ease in GLC determination. The pesticides were treated indi-
vidually In petroleum ether, in 6% ethyl ether/petroleum ether Florisil eluates
(6) containing the equivalent of 6 g of kale, and in petroleum ether containing
30-60 mg of fatty substances extracted from butter by partitioning (6) between
petroleum ether and acetonitrile. Gas chromatography with electron capture
detection, operated as described under “Method t1 , was used for all determinations.
Each pesticide was completely altered in each of the solution types, i.e.,
none of the parent compound remained after treatment as described under
“Method”. Percent recoveries of the respective olefins were calculated
according to the following equation:
wt. olefin compound determined by GLC x
wt. parent compound represented in aliquot to GLC
wt. parent campound/mol. wt . 100
wt. olefin compound/nol. wt.
Recoveries of olefins approximated 100% and ranged from 86% for ,p ’-DDE and
o, ’-ThE olef in in petroleum ether to 110% for , ‘-DDE in the extract from
Butter. I o olefin derivatives, the cis and trans isomers, are formed from
o, -TDE (7). These have identical retention times on the two GLC columns
used in this work and were quantitated as a single compound.
Several additional coniiion organochiorine pesticides and polychiorinated bi-
phenyls (PCB) were subjected to the described alkali treatment. All tests
were made with petroleum ether solutions of the chemical under study. The
quantity of each chemical was chosen for ease of determination by GLC and is
given in parentheses.
Polychiorinated biphenyls ranging from 21 to 60% average chlorine content were
stable to this treatment. Complete recoveries were obtained for the commercial
PCB mixtures, Aroclors 1221 (16 jig), 1232 (16 jig), 1242 (16 jig), 1254 (8 jig),
and 1260 (8 jig).
Recoveries of unchanged aidrin (0.4 jig), dieldrin (0.4 jig), and endrin (0.4 jig)
ranged from 70 to 90%. No alteration products were detected.
-------
11/1/72 Section 12,D,(l)
-5-
Heptachlor (0.4 iig) and heptachior epoxide (0.4 iig) were markedly affected;
recoveries of the original compound ranged from 30 to 50%. Minor GLC peaks
were observed on the 10% DC-200 column at retention times relative to aldrin
of 1.63 after treatment of heptachlor epoxide and 0.59 and 0.93 after treat-
ment of heptachior.
The alkali treatment completely eliminated lindane (0. 2 jig) and the alpha
(0. 2 jig), beta (0. 2 Mg), and delta (0. 2 Mg) isomers of BHC. Following the
reaction, only small early eluting gas chromatographic peaks, presumably from
trichlorobenzenes, were observed.
About 40% of mirex (4.0 Mg) remained unchanged after reaction; sometimes a
minor GLC peak appeared at a retention time relative to aldrin of 1.83 on
the 10% DC-200 column.
Endosulfan I and II, treated separately, were completely eliminated. Each
isomer gave a single alteration product with retention time relative to aldrin
of 1.82 on the 10% DC-200 column and 2.23 on the 1:1 10% DC-200/l5% QF-l
column. The peak height of the alteration product was approximately one-tenth
the peak height of the pdrent compound. A structure for this derivative has
been proposed (8).
Endosulfan sulfate also was completely eliminated. Two alteration products
were obtained with retention times relative to aldrin of 0.28 and 0.38 on the
10% DC-200 column.
Dicofol (1.0 Mg) was completely eliminated, but only 65% of the major altera-
tion product, 4,4’-dichlorobenzophenone, was recovered. A minor peak was
observed in the chromatograni at a retention time relative to aldrin of 1. 71
on the 10% DC-200 column. The 4,4’-dichlorobensophenone (2.0 Mg) was not
affected by treatment with alkali.
The products resulting from alkali treatment of toxaphene (10.0 Mg) gave a
rnulticomponent chromatograni but consisting of components with earlier reten-
tion times than toxaphene itself.
The electron capture GLC responses to sulfur (20 Mg), frequently encountered in
residue analysis at retention times relative to aldrin of 0.23, 0.55, and 1.13
on the 10% DC-200 column, were eliminated by the alkali treatment.
Application of Method
The mest obvious use of the alkali treatment is to form the olefins of
bis (phenyl) chloroethane pesticides for confirmation of residue identity. The
complete yield and recovery of the olefin derivative makes possible quantitative
-------
11/1/72 Section 12,D,(l)
-6-
confirmation of a residue of the parent pesticide. For example, a residue of
-DDT can be quantitated before alkali treatment and verified by quantita-
tion as , ‘-DDE after treatment.
In addition, the GLC retention time region of the reacted compound can be
examined for presence of peaks from unreacted and presumably interfering sub-
stances. We have found the alkali treatment especially useful in connection
with the real or suspected presence of residues of P03. In this case the
characteristic olefin derivatives of ,p’-DDT,o, ’-DDT, and ,p ’-TDE can be
formed and the GLC retention time region underTying the parent compounds can
be examined. The stability of PCB to alkali, with no change in the GLC pattern,
is a characteristic which can be readily utilized in confirmation of the iden-
tity of this complex residue. The high recovery of unreacted dieldrin, endrin,
and aldrin following alkali treatment likewise can be of value in the con-
firmation of identity of these pesticides.
Cleaned-up extracts of some samples may contain non-pesticidal substances
which give rise to electron capture response. Other extracts may contain
fatty substances, not removed by the cleanup, which can prohibit application
of some tests, e.g., thin layer chromatography. This is particularly true of
the 15% ethyl ether/petroleum ether Florisil column eluate (6) for non-fatty
samples such as carrots and fatty samples such as some fish. Electron captur-
ing substances present in carrots must be eliminated before determination of
dieldrin and/or endrin residues. Treatment with alkali serves well for this
purpose. Extracts of fatty samples may require treatment to eliminate both
electron capturing substances and non-volatile fatty substances. In many
instances thin layer chromatography or microcoulometric GLC cannot be
accomplished prior to alkali treatment. Electron capture responses to sulfur,
often a source of annoyance to the residue chemist, are also eliminated by
this treatment.
-------
11/1/72 Section 12,D,(1)
-7-
1. BRAND, K. and BUSSE-SUNDE1 ’W , A., Berichte, 75 B 1819 (1942).
2. SOLCMIAY, S. B., SCHECHThR, M. S., and JONES, H. A., Soap and Sanitary
Chemicals, 1946 Blue Book , 18th Ed., 215 (1946).
3. MILLS, P. A., J. Ass. Offic. Anal. Chem. 42 , 734-740 (1959).
4. KLEIN, A. K. and W LTfS, J. 0., J. Ass. Offic. Anal. Chem. 47 , 311-316
(1964).
5. MILLER, G. A. and WELLS, C. E, J. Ass. Offic. Anal. Chein., 52 , 548-553
(1969).
6. Pesticide Analytical Manual, Vol. I , Food and Drug Administration,
Washington, D. C., 2nd Ed., 1968; Revised July 1969, July 1970, and
April 1971.
7. KRAUSE, R. T., Private Communication, Food and Drug Administration,
Washington, B. C., May 1970.
8. GREVE, P. A. and WIT, S. H., J. Agr. Food Chem. 19 , 372-374 (1971).
-------
Revised 11/1/72 Section 13,A
Page 1
DETERMINATION OF MEII-JYL MERCURY IN FISH, BLOOD, BRAIN ANI) URINE
I. II’(rRODUCTI :
During the past year, mercury has been found to be a frequent con-
taininant of fish in North America. Much effort has been spent in im-
proving or developing methods for analyzing commodities for total mercury
content, usuaiiy by colorinietric (1) or atomic absorption procedures (2).
However, it has been shown (3) that most, if not all, mercury found in
fish is present as methyl mercury, a compound thought to have much greater
chronic toxicity than other forms of mercury. Therefore, appropriate
methodology, specific for methyl mercury, is desirable. (The form in
which mercury may be present in other contaminated commodities varies and
is not necessarily methyl mercury).
The most widely used method for methyl mercury analysis is that
described by West it5 (3,4,5,6). This method has been used successfully
for the analysis of fish, red blood cells, plasma and brain of hamsters
fed methyl mercury. The method described herein represents a modifica-
tion of the procedures described by West iti in her papers and private
communications.
REFERENCES :
1. “Official Methods of Analysis” 10th Ed., Association of
Official Agricultural Chemists, Washington, D. C., 1965,
pp. 375.
2. W. R. Hatch and W. L. Ott, Anal. Chem . 40, 2065 (1968).
3. G. Westtit , Acta. them. Scand . 21, 1790-1800 (1967).
4. G. West it5, Acta. them. Scand . 20, 2131-2137 (1966).
5. G. West itJ, Acta. Ghem. Scanã . 22, 2277-2280 (1968).
6. G. Westtitl, private communication.
7. Laboratory Information Bulletin, FDA, October, 1970
DETERMINATION OF MEI}IYL MERCURY IN FISH AND ANIMAL
TISSUES BY GAS LI( JID (i-IROMATOGRAPHY, Kamps, L., and
Malone, B.
-------
Revised 11/1/72 Section l3,A
-2-
II. PRINCIPLES :
In this procedure, methyl mercury chloride (CH 3 FIgC1) is formed by
addition of hydrochloric acid to honDgenized tissue, and the organic salt
is extracted into benzene. The methyl mercury is extracted from the
benzene solution by partitioning it with aqueous cysteine solution,
which layer is then acidified and the salt reextracted back into benzene.
The final benzene layer is analyzed by gas-liquid chromatography.
III. APPARARJS :
1. Gas chromatograph fitted with column 6’ x 4 n n i.d. of 5 1-IIEFF-1OB
on thromosorb W, H.P., 80/100 mesh and with in electron capture
detector.
2. Sorval homogenizer with 50-mi cups. Available from Ivan Sorval Co.,
Norwalk, Conn.
3. Centrifuge tubes, 35 ml, conical, Kimble #45201 or the equivalent.
4. Centrifuge bottles, 250 ml, Corning #1280, or the equivalent.
5. Centrifuge with adjustable speed up to 3,000 r.p.m. with carrier
heads appropriate for tubes and bottles listed above in 3 and 4.
6. Cylinder, grad., 100 ml.
7. Separatory funnels, 30, 60 and 250 ml with Teflon stopcock.
8. Pipets, transfer, 2,4,6 and 50 ml.
9. Pipets, measuring 1 and 2 ml, grad., in 0.01 ml.
IV. REAGENTS AN]) SOLVENTS :
1. Benzene, pesticide quality. Background contamination must be checked
by injecting 5 p1 of a 15 to 1 concentrate. No peaks should appear
in the area shortly after the solvent front.
2. Methyl mercuric chloride*. Available from K E K Laboratories, stock
number 23308. Stock Standards of:
a. 1 mg/mi in benzene.
-------
Revised 11/1/72 Section 13,A
-3-
b. 1 mg/mi in ethanol-water (1:1).
Dilute stock solutions a. and b. as needed for recovety trials
and GLC working standards.
NOTE : The stability of U -I HgC1 dilute standard solu-
tions is questionab’e. Stock solutions, 1 mg/mi
in benzene or in (1:1) alcohol/FL,O should be
stable indefinitely. It is sugg sted that
dilute solutions in H O be made fresh every 2 or
3 days and dilute soli tions in benzene every
2 months.
*S of the mercury standards will be available from the
Perrine Repository.
3. Ethyl mercuric acetate. Available from Ventron, P.O. Box 159,
Beverly, Mass. 01915, stock #87301.
Prepare benzene solution of 1 mg/mi.
4. Phenyl mercuric acetate. Available from K K Laboratories, stock
#17412.
Prepare benzene solution of 1 mg/mi.
5. Methyl mercuric iodide. Available from K K Laboratories, stock
#2508.
Prepare benzene solution of 1 mg/mi.
6. Column conditioning mixture: Combine equal portions of foregoing
items 2a, 3, 4 and 5 and set aside for column conditioning.
7. Cysteine hydrochloride-nx)nohydrate. Aldrich Chemical Co., stock
#C 12, 180 - 0.
8. Sodium acetate .3H 2 0, Mallinckrodt #7368 or the equivalent.
9. Sodium chloride, reag. grade, fine crystal.
10. Sodium sulfate, reag. grade, anhydrous.
NOTE : Contaminant peaks are sometimes observed from
this material. Each batch should be checked by
injecting a benzene extract into the gas chroma-
to graph.
-------
Revised 11/1/72 Section l3,A
-4-
11. Cysteine Solution - Dissolve 1.00 g cysteine hydrochloride, 0.775 g
sodium acetate. (3 H 2 0) and 12.5 g anhydrous Na 2 SO in distilled
H 2 0 and dilute to 100 ml. Prepare fresh every 3 da s.
12. Mercuric Chloride Solution - Dissolve 5.0 g HgC1.) and 17.0 ml conc.
HC1 in distilled H 0 and dilute to 100 ml. Shak 4 times (3 mi-n.
each) with 50-mi p rtions of benzene to remove impurities.
13. Hydrochloric acid, conc., reagent grade.
14. 6.0 N Hydrochloric acid - Prepare by diluting 185 ml conc. HC1 to
1 liter with dist. water.
NOTE : It has been found that reagent grade HC1 may
occasionally be contaminated with materials
that interfere with the chromatographic deter-
mination of CH 3 HgC1. It is suggested that the
acid be triple washed with equal volumes of
pesticide quality benzene in a sep. funnel.
15. Column packing - 5% HIEFF-lOB (phenyldiethanolanuine Succinate).
Available from Applied Science Laboratories, Stock #09120. Coat on
80/100 mesh Chromosorb W, H.P. After the 6-ft. column is packed, conditi
conditioning should be carried out as follows: Place in condition-
ing oven in the manner described in Section 4,A,(2), raise the
temperature to 210 C and apply a carrier flow of ca 25 mi/mm.
Maintain conditioning for 4 days. Ixiring this period, the organic
mercury mixture (Subsection IV,6) is injected into the column in
three sessions during each day, each injection session consisting
of three 10 id injections spaced one or two minutes apart. There-
fore, at the end of the 4-day period, the column should have
received a total of 36 injections of the mixture (see MISC. NOTE 1).
V. PROCELUBE :
All glassware used in the procedure shall, after washing in the
usual manner, be rinsed successively with NHAOH, distilled water and
ethanol (see MISC. NOTE 2). The following pi ocedure, unless otherwise
noted, is intended for fish containing 0.1 ppm or more of CI-1 3 1-Ig;
-------
Revised 11/1/72 Section 13,A
-5-
Extraction and Partitioning
A. Fish, Blood Brain
1. Weigh 10 grains of sample into 50-mi homogenization cup. Measure
55 ml of dist. water in a 100-mi grad. cylinder and add a suffi-
cient amount to just cover sample. Homogenize at medium speed
for 3 minutes.
2. Transfer sample to a 250-mi sep. funnel rinsing with the water
remaining in the cylinder. Add 14 ml of conc. HC1, 10 grams
of NaG and mix.
NOTE : In the analysis of red blood cells, plasma or
brain, add at this point 2 ml of the uripurity-
free UgCl 2 solution (Subsection 1 1 1,12).
3. Add 70 ml. of benzene, stopper funnel and shake vigorously for
5 minutes (a reciprocating mechanical shaker is preferable if
available ).
4. Transfer sample to a 250-mi centrifuge bottle, cover with foil
and centrifuge at 2000 r.p.m. for 10 minutes.
NOTE : The initial 10 -minute centrifuging may not
adequately separate the aqueous-organic phases.
The solid material at the organic-aqueous inter-
face rust be quite compact to allow easy removal
of 50 ml benzene. If recentrifugation is
necessary, stir the sample vigorously with a
glass rod before centrifuging. Should the ben-
zene layer appear cloudy, centrifuge for an
additional 10 minutes.
S. With a vol. pipet, carefully transfer 50 ml of the benzene layer
into a 60-mi sep. funnel. Take care not to disturb the solids
collected at the benzene-water interface.
6. Add 6.0 ml of the cysteine solution to the 60-mi sep. funnel,
stopper and shake vigorously for 2 minutes.
NGI’E : To increase the limit of detectability, the
volume of cysteine solution should be reduced
to 1.2 ml, applied with a 2-ml Mohr pipet.
Further modifications in this connection are
given in NC11’E form below.
-------
B vised 11/1/72 Section 13,A
-6-
7. Allow to stand 10 minutes, then drain aqueous layer emulsion
into a 35-mi centrifuge tube. Centrifuge at 3000 r.p.m. for
20 minutes. If emulsions persist, stir vigorously with a glass
rod and repeat centrifuging as necessary.
8. With a volumetric pipet, transfer 2 ml of the cysteine solution
from the centrifuge tube to a 30-mi sep. funnel. Add 1.2 ml of
6N HC1 and 4.0 ml benzene, stopper and shake vigorously for 2
minutes.
NOTE : For lower detectability levels, transfer 0.80 ml
(or as unich as possible to remove and measure)
of the cysteine solution, then reduce the addi-
tion volumes of 6N HC1 ar 1 d benzene to 0.6 and
2.0 ml, respectively.
B. Procedure for Urine
1. Pipet 15 ml of urine into a 60-mi separatory funnel. Add conc.
HC1 until a pH of 1 is obtained, and mix slowly to avoid loss
of sample due to foaming.
2. Mci 30 ml of benzene and shake vigorously for 5 minutes (a re-
ciprocating mechanical shaker is preferable if available).
3. Transfer sample to a 100-nil centrifuge bottle, cover and
centrifuge at 2000 r.p.m. for 10 minutes.
4. Volumetrically transfer 25 ml of the benzene layer to a 60-mi
sep. funnel.
5. Add 5 ml of cysteine solution tc the 60-mi sep. funnel, stopper
and shake vigorously for 2 minutes.
6. Dram aqueous layer emulsion into a 35-mi centrifuge tube.
Centrifuge at 3000 r.p.m. for 10 mm. If emulsions persist,
stir with a glass rod and repeat centrifugation as necessary.
7. Carefully transfer 4 ml of the cysteine solution to a 30-mi
sep. funnel. Add 1.2 ml of 6N HC1 and 5 ml of benzene, stopper
and shake vigorously for 2 minutes.
8. Allow to stand for 10 minutes, discard aqueous layer and dry
benzene by adding a pinch of anhydrous Na 2 SO 4 prior to GLC.
-------
Revised 11/1/72 Section 13,A
-7-
Gas Chromatography
Set column temperature at 170°C and adjust carrier flow to 120 ml
per minute. Set attenuator to obtain a peak height of 60-80% f.s.d.
resulting from the injection of 1.6 ng of methyl mercuric chloride in
benzene. If column is properly prepared and conditioned, the peak
should be of reasonable symmetry with minimal tailing as illustrated in
Figure 1. Make a sufficient number of repetitive standard injections
to assure response reproducibility before making sample injections.
It has been observed that all methyl mercury salts elute at the same
retention time. These include the Cl, Br and I salts of methyl mercury,
as well as Panogen (methyl mercury dicyandiaiuide).
VI. CALCULATIONS :
If 50 ml of benzene and 2.0 ml of cysteine are recovered during the
procedure for 10 g samples, the amotzit of sample per ml of final benzene
solution is 0.595 g. This calculation is as follows:
g/ml=Ax x F where
A = of sample analyzed.
B = ml benzene added (normally 70 ml).
C = ml benzene recovered (normally 50 ml).
D = ml cysteine solution added (normally 6.0 ml).
E = ml cysteine solution recovered (normally 2 ml).
F = ml benzene added for final partition (normally 4.0 ml).
This calculation may be applied to all variations of the CI-L HgC1
method. It is readily apparent that sample size affects the limXt of
detectability; however, the analysis of samples larger than 10 g is not
recommended. Results in terms of ppm Q-I 3 HgCl can be converted to ppm Hg
by multiplying by a conversion factor of 0.80.
VII. MISCELLANE(XJS NOTES :
1. Obtaining and maintaining acceptable gas chromatography of methyl
mercury is difficult. Chromatography has been tried on the follow-
-------
Revised 11/1/72 Section l3,A
-8-
ing liquid phases: 10% Carbowax 20 in, 20% Carbowax 20 m, 15% DEGS
(stabilized), 5% HIEFF- 1OB [ Phenyldiethanolamine Succinate (PDEAS)].
Acceptable chromatography was not obtained on a 15% DEGS column. To
date 5% HIEFF-10B has produced the best chromatography. Proper
pre-conditioning of the column is very critical, Provided that
column is properly conditioned, it should be possible to obtain a
peak of at least 50% f.s.d. resulting from the injection of 0.150
nanograms of C}f HgCl. If the sensitivity should fall short of this,
further conditi6ning with repeated inj ections of the concentrated
mixture may be indicated.
2. Glassware such as pipets which come in contact with fairly con-
certtrated solutions of (]-( 3 HgCl should be rinsed with NI-1 4 C*1, distilled
1120, and C 7 HCOH, but it is probably not necessary to rinse the rest
of the gla sc are with anything but distilled f f20.
3. Benzene solution of Ct-I 3 HgC1 cannot be concentrated without signifi-
cant loss of CH HgCl. Limit of detectability is therefore restricted,
as discussed abthre in Subsection VI.
4. The analysis should not be stopped overnight at any point except when
the sample is in benzene solution. Prolonged contact with cysteine
solution results in low recoveries.
-------
1/L /71
—9—
Section L,A
Figure 1. Typical chromatograni of 1.6 ng ‘ ( H HgC1 cn 5% }lT ’FF—10B
at 170°C. and carrier flow of 2 ini 1 minute.
— PC Lk Ht’ gut
I
-------
Revised 11/1/72 Section 13,B
-3-
5. Masterfiex Pump: With electronic speed control. Any peristaltic
pimIp capable of delivering 1 liter of air per minute may be used.
6. Flowmeter: Capable of measuring an air flow of 1 liter per minute.
7. Aeration Tubing: A straight glass frit having a coarse porosity.
Tygon tubing is used for passage of the ir.ercury vapor from the sini le
bottle to the absorption cell and return.
8. Drying Tube: 6” x 3/4” diaii ter tube containing 20 grams of magne-
sium perchiorate. The apparatus is assembled as shown in Figure 1.
NOTE : In place of the magnesium perchlorate drying tube
small reading lamp with 60W bulb may be used to pre-
vent condensation of moisture inside the cell. The
lamp is positioned to shine on the absorption cell
ma ntaining the air temperature in the cell about
10 C above ambient.
9. Bottles, B.O.D., 300 ml, with glass stoppers. Fishers #2-926, or
the equivalent.
10. Pipets, transfer - 1, 2 and 5 ml.
11. Pipets, measuring - 1,2 and 5 ml.
IV. REAGENTS :
1. Sulfuric acid, conc., reagent grade.
2. Sulfuric acid, 1.0 N. Dilute 28.0 ml of conc. }-1 2 S0 4 to 1000 ml with
distilled water.
3. Sulfuric acid, 0.5 N. Dilute 14.0 ml of conc. l I2SO4 to 1000 ml with
distilled water.
4. Nitric acid, conc., reagent grade, of zero or low Hg content.
NOTE : If a high reagent blank is obtained, it may be
necessary to distill the HNO 3 .
5. Staimous sulphate, reagent or purified grade. Add 25 g of the
reagent to 250 ml of 0.5 NI L,SOA. This mixture is a suspension
and requires continuous sti riñg during use.
NOTE : Stannous chloride or hydroxylanu.ne hydroch]oride may
be substituted for the stannous sulphate.
-------
Revised 11/1/72 Section 13,B
-4-
6. Sodium chloride - hydroxylanane sulfate solution. Dissolve 12 g
of each compound in distilled water and dilute to 100 ml.
7. Potassium permariganate, cryst., reag. grade. Dissolve 5 g in 100 ml
of distilled water for a 5% w/v solution.
8. Potassium persulfate, reagent grade. Dissolve 5 g in 100 ml of
distilled water for a 5% w/v solution.
9. Mercuric chloride, reag. grade, cryst., A.C.S.
a. Stock mercury solution - dissolve 0.1354 grams of HgCl 2 in
lOOmlofl.0NH 2 50 4 . lml=lmgHg.
b. Working mercury solution - Make two successive 1/100 dilutions
• of the stock solution to obtain a final working standard con-
taining 0.1 pg per ml.
NOTE : The stock solution is relatively stable and
nay be held, tightly stoppered, for periods
UP to a month. The working solution should
be prepared weekly.
V. PROCEDURE :
Calibration
1. Transfer 1, 2 and S ml aliquots of the working Hg solution to 300-mi
BUD bottles arid add distilled water to each bottle for a total
volume of 100 ml.
M IrE : In additional BUD bottle, add 100 ml of distilled
water and carry through all successive steps as
a reagent blank.
2. Stopper and shake bottles vigorously for cinplete mixing.
3. Add S ml of conc. H 2 SOA, mix by swirling and then 2.5 ml of conc.
HNO 3 to each bottle and, without stoppering, mix gently by swirling.
NOTE : Loss of Hg may occur at elevated temperatures.
However, with the specified volumes of acids, the
temperature should rise only 13 C and no Hg
losses have been observed under these conditions.
-------
Revised 11/1/72 Appendix II,A
Page 1
ANALYTICAL ( 1ALITY CC TROL
INTERLABORATORY
I. BACKG1 )UND :
The term “quality control” may connote to some a system of control-
ling the quality of a manufactured product. Truly, the phrase is widely
applied in this role, but it also can be aptly applied to a “level of
performance”. It is in the latter sense that the term is applied in the
chemical analysis for pesticide residues. A program of quality control
is a means of assuring the output of reliable and valid analytical data.
There are basically two general types of analytical quality control,
INTERLABORAIDRY and INTRALABORATORY . The former term indicates the in-
volvement of two or more laboratories, providing a mathematical basis
for the confidence placed in the results of sample analyses and an in-
sight into analytical areas needing attention. An intralaboratory pro-
grain provides some systematic guidelines within a laboratory to assist
in the prevention of a sizable amount of inaccurate analytical data.
II. ThE INTERLABORAIORY PROGRAM :
The prime objectives of this program are:
A. To provide a measure of the precision or reproducibility of methods
of analysis.
B. To measure the precision and accuracy of analytical results between
laboratories.
C. To identify weak methodology.
D. To detect training needs.
E. To upgrade the overall quality of laboratory performance.
The Perrine Technical Services Section functions as the coordinating
unit for an interlaboratory quality control program which has been oper-
ating since 1966. The program takes in all the Coninunity Pesticide
Studies and National Monitoring Laboratories, operating under contract
with the United States government to conduct analytical chemical monitor-
ing for pesticidal residues in man and in the environment.
The quality control program includes the following areas of activity:
A. An interlaboratory check sample program.
-------
Revised 11/1/72 Appendix II,A
-2-
B. The operation of an electronic facility for the repair and cali-
bration of laboratory instruments.
C. A repository to provide analytical grade pesticide standards.
D. Providing uniform analytical methods.
E. Furnishing materials of uniform standard quality such as precoated
GLC column packings and cleanup adsorbent.
F. Providing abbreviated, informal on-the-job training for specific
requirements.
G. Assisting with problems related to analytical methodology by phone,
mail or on-site consultations.
The services provided by above items B, B and F are confined to
laboratories within the U. S. Environmental Protection Agency and those
under contract with E.P.A.
The pesticide analytical standards repository currently stocks about
400 standards which are available to federal, state, and municipal labora-
tories, U. S. universities and international agencies such as World Health
Organization. The standards are listed in a separate manual available on
request to qualified recipients.
Currently, 35 laboratories participate in the interlaboratory check
sample program (Item A above). Sample materials are selected which
relate to the routine work of the majority of the participants. Sub-
strates which have been used include fat, blood, brain, liver, standard
pesticide mixtures in solvent, and simulated air samples (ethylene glycol
spiked with pesticides at concentration levels found in actual air samples).
After selection of the substrate, a bulk sample is thoroughly homo-
genized and transferred to the vials in which the samples will be mailed
to participants. One of the Perrine chem.ists with experience in the
appropriate analytical method then runs a series of four or five analyses,
in duplicate, using the GLC detection mode(s) which will be suggested
to the participants. Confirmatory techniques such as TLC, p-values,
derivatizations are applied as required for assurance of compound identi-
fications. All data are examined to establish potential reproducibility
of analysis from vial to vial and to determine approximate quantitative
values.
1 hen samples are mailed, a covering letter is sent to all partici-
pants providing any special instructions for handling the sample.
-------
Revised 11/1/72 Appendix II,A
-3-
Generally, the tine allocated for analyzing and reporting results
approximates that which would be considered normal for processing a routine
sample. The participants are requested to report their results on forms
supplied. The supplemental data requested includes information on sample
size, extent of sample extract concentration, all instrumental operating
parameters, identity of GLC columns used, and numerical data upon which
quantitative calculations are based. The participants are requested to
include with their report all pertinent chromatogTalns, fully identified
so that material on chromatog-rams may be easily related to numerical
data on report forms.
When the report from each laboratory is received at Perrine, the
quantitative results are entered on a S114 IARY OR RESULTS sheet. If any
result appears to be completely erroneous, the laboratory is contacted at
once and requested to review their work and advise us promptly if they
see fit to change their reported value. There have been occasional
instances of a misplaced decimal point or failure to apply a divisor or
multiplier dilution factor, or simply a transcription error. When all
laboratory results have been recorded, the statistical workup is made,
computing the mean standard deviation and relative standard deviation
for each pesticide reported by the majority of the laboratories. A
relative performance or ranking chart is then made up establishing a
numerical ranking level for each laboratory. This is based on (1) extent
of deviation of each reported pesticide from the calculated mean (in the
case of spiked sample, the formulation value is the base of reference),
(2) false identification of compounds not present in the sample, and
(3) failure to identify compounds known to be present.
Upon completion of the performance calculations, the data sheets and
chromatograzns of the laboratories demenstrating relatively poor per-
formance are subjected to detailed examination to try and determine the
potential factors responsible for the relatively inferior performance. A
detailed critique is then written, each such laboratory is contacted by
phone to immediately apprise them of their performance problem, and to
make such suggestions as may be appropriate so that corrections can be
initiated at once.
The reports from all the rest of the laboratories are then scanned
for any irregularities that might lead to future problems. A general
letter is drafted, a copy of which is mailed to all laboratories. In
this letter, mutual analytical difficulties experienced by several lab-
oratories are discussed, and suggestions are offered which appear to apply
broadly. Included with the letter to all laboratories are (1) a copy of
the SiMVIARY OF RESULTS with each laboratory identified by code number,
(2) a copy of the RELATflIE PERFORMANCE TABLE, and (3) a private critique
to each laboratory where indicated.
-------
Revised 11/1/72
Appendix II,A
-4-
Front information obtained front the interlaboratory check sample
program, a number of actions have been taken towards standardization.
For example, bulk lots of Florisil are purchased on specification
directly from the manufacturer for distribution in smaller units to
the laboratories; similarly, bulk lots of GLC column packing are pur-
chased on very rigid specifications; standard analytical methods have
been distributed and are updated frequently.
The overall success of the quality control efforts is well illus-
trated in the following tables. Of greatest significance is the con-
tinuing improvement in interlaboratories precision over a four-year
period.
Tables showing progression of interlaboratory precision front 1968
thru 1972:
FAT ANALYSIS
Interlab Check
Sample Number
Number of
Labs
Number of
Compounds
Mean Rel. *
Std. Dev. %
Year
9
1968
21
7
38
11
1969
19
7
24
21
1972
16
7
19
BLOOD SERUM ANALYSIS
1971
1972
20
6
10
.1968
16
22
1969
17
6
1970
36
22
22
29
14
21
14
17
17
14
* Formerly cafled “Coefficient of Variation”
-------
Revised 11/1/72 Appendix ii:, B
Page 1
ANALYTICAL QIJALI1Y CONTR()L
INTRALABOPATORY *
There are a n nber of factors in every laboratory of which any
one or ccgnbination, if riot carefully considered, can lead to inaccurate
analytical data. This discussion is intended to nighliglit a nunber
of these factors and to suggest a program for the avoidance of these
pitfalls. There is no intent to dwell on any SL,r.tf C points of
methodology in the preparative or detenninative areas of analysis as
these points are covered in detail in the appropriate section of this
manual. Some of the more important broad factors ii,clude:
1. Sensitivity and accuracy of analytical balances.
2. Purity of reagents such as solvents, disti!lcd water,
Na 2 SO 4 , NaC1, compressed air or nitrogen, et’ .
3. System of cleaning glassware.
4. Accuracy of analytical standard solutions.
5. Condition of gas chromatograph and accessoites: recorder,
electrometer, detector, carrier gas system. coli.nuris and
heating components- tempsets, variac , prog rauuners.
6. Deviations fi-nm analytical methods ad.pteJ foi use in program.
7. Demonstrated capability of personnel to obtain satisfactory
recoveries.
8. Training and experience of personnel.
A. ANALYTICAL BALANCES :
In the pesticide laboratory, the most critical i eig1itngs will be
those of the primary analytical standards. Obviously, if the balance
is not sufficiently sensitive and precise, the acLuracy cf the final
standard solutions will be open to question. The balance snould be
capable of sensitivity of 0.05 mg and should be checked no less than
yearly by a qualified balance specialist.
B. PURITY OF F EAGENTS, SOLVENTS AND DISTILLED WATER :
This is an area of extreme importance, particularly in those
laboratories working with substrates having residues in the parts per
billion range. Reagents or solvents from certain iianufacturers are
often found to yield excessive background contamination whereas those
from other manufacturers yield minijual background. In general, pesticide
*SoIp.e of the procedural instructions written specifically for a given
network of laboratories.
-------
1/4/71 Appendix Ii, B
-2-
quality solvents, distilled in glass, will give the least trouble.
However, each lot from source should be checked before use,
evaporating a portion by as great a concentration factor as would ever
be employed in any method for which the solvent is used. See Section 3, C.
Any reagent or material used in a preparative method is a potential
source of contamination which can result in extraneous chromatograpiic
peaks. It is good practice and requires little tine and cffort to
soxhiet extract such materials as sodnm sulfate, glass beads, NaC1, glass
wool, etc., t ing as the extracting solvent(s) that or those used
in the method.
Reagent blanks must be run constantly for each of the analytical
procedures, with final extracts being reduced to the same concentration
level noimally used for the sample material.
Distilled water can be troublesome, particularly in a procedure
where a large volim e is used. Such a procedure is the Mills, Onley,
Gaither cleanup method for adipose tissue where 700 ml of water is
used in the acetonitrile partitioning step. The final extract
concentration may be as little as 5 ml so that a potential contaminant
concentration factor of 140 x must be considered. Since the source of
extraneous peaks in laboratory water is organic in nature, distillation,
even though an all-glass still, is not necessarily sufficient cleanup.
Co-distillation of organic material with water is a well documented
phenomenon. The use of an activated charcoal filter just before the
distillation procedure has been found to significantly improve toe water
quality. If deionization of the water is desirable, the cnarcoal filter
should be installed between deionizer and the distillation equipment for
trapping the organic materials eluting from the polystyrene matrix of
the deionizing column before the water enters the still.
C. CLEANING OF GLASSWARE :
There are a few areas of analytical chemistry where tnis operation
is any more critical than in the residue laboratory. This job is
frequently done by lower grade personnel with little or no tecnnical
training. Therefore, the responsibility falls on the chemist to maintain
close control over cleaning procedures to be certain that the glassware
will be entirely free of contamination. In general, the cleaning operation
should include (1) soaking and washing in high temperature bath of
synthetic detergent in water, (2) rinsing with tap water, (3) rmsing with
distilled water, and (4) rinsing with acetone. In the cleaning of
glassware to be used in sample concentrations, (such as evaporative
concentrator tubes) the tap water rinse should be followed by a soak in
hot chromic acid cleaning solution. This step insures removal of any
traces of organic material. This soak is followed by thorough rinsing wit fl
tap and distilled water, and finally with acetone and hexane.
-------
Revised 11/1/72 Appendix II,B
-7-
H. TRAINING, EXPERIENCE AND RESPONSIBILITIES OF PERSONNEL :
A number of the routine functions of the residue laboratory can be
carried out by a technician who has been given careful on-the-job train-
ing by the chemist. This relieves the chemist to devote his efforts to
the more complex problems of instrumentation and interpretation.
In the small laboratory of three to six people, the responsibility
rests with each chemist to sufficiently acquaint himself with his analy-
tical instruments to perform routine adjustments and preventive mainten-
ance. Such duties as module replacements, replumbing and simple servic-
ing should be considered as the normal responsibility of the chemist.
The more complex electronic servicing is generally the function of the
electronics specialist.
The laboratory administration must recognize the need and importance’
of continuing training for the residue chemist. There are few areas of
analytical chemistry more complex or challenging than that of pesticide
residue analysis of biological or environmental media. Any skilled analyst
appreciates all too well that the pathways are liberally strewn with
booby traps. In the administration of the interlaboratory check sample
program over the years, many instances have been observed where the analyst
was obviously not sufficiently trained to recognize certain pitfalls. A
few large laboratories may have sufficient personnel to conduct in-house
training programs. However, the average smaller laboratory of three to
five people cannot divert personnel to conduct the type of intensive
training required. Furthermore, technological advances in instrumentation
dictate the need for even the most skilled chemists to take refresher
courses from time to time.
I. SIJMNI&TION :
The type of program discussed above requires an expenditure in man
hours and/or money. Total work output is somewhat reduced. The question
should rightfully be asked “Is it worth the effort?” This may be answered
in terms of the type of pesticide laboratory involved. In a laboratory
primarily concerned with law enforcement, the analytical chemist may be
called at any time as an expert witness to defend in court the accuracy
and precision of his work on a given sample. Revelation of the lack of
an active program of analytical quality assurance provides the opposition
with valuable aninunition for discrediting the laboratory’s case.
Pesticide laboratories concerned with the develop nt of analytical
-------
Revised 11/1/72 Appendix II,B
-8-
data of a survey nature have a pressing responsibility to insure quality
analytical performance. Generally, such laboratories are part of a net-
work feeding data to a central point for processing. Erroneous data from
one or n re of the component laboratories of the network can lead to
false conclusions concerning the pesticide profile of the sample material
in the areas assigned to those errant laboratories. For example, let us
assume that a laboratory assigned to the analysis of general population
blood in a given area is consistently, but erroneously, finding higher
pesticide levels than is being reported by laboratories in other areas.
Plotted on a national scale, it would then appear that the people of this
area contain higher levels of pesticides than the rest of the national
population.
We think it appropriate to reiterate here the importance of periodic
checking of pesticide profiles in substrates by confirmatory techniques.
The reporting of pesticides not actually present is just as misleading as
failure to identify pesticides which are present.
A properly functioning intralaboratory quality control program greatly
reduces the possibility of any laboratory straying into a pattern of con-
tinuing error since the periodic recovery checks serve to flash a warning.
The lack of such a program inspires little confidence in the routine data
from any laboratory. When the analytical work program of a laboratory is
planned, definite consideration imist be given to the allowance of suff i-
cient time to conduct a quality assurance program. A low total output of
reliable analytical data is at least useful. An impressively high volume
of uncontrolled data, however, is worse than none at all because of the
distorted and misleading profile that could result.
-------
1 / 4/71
Suspected CH1.
Pestic. Exposure
Suspected PCP
Exposure
Suspected 2,4-D
or 2,4,5-T
Exposure
TENTATIVE TISSUE, EXCRETA NJD METhOD SELLCTION FOR
ABNogv PESTICIDE EXPOSURE CASES; BLOCK DIAGRAM
LIVE DONORS
BLOOD URINE
5,A,(3),(a) 5,A,(4),(b)
4ML 1OML
Suspected o.c. .j
Pestic.Exposure
Suspected
Carbaryl Exposure
Suspected cl-IL.
Pestic. Exposure
Suspected Hg
Exposure
URINE
6,A,(2), (a)
20 ML
URINE
5,A, (4), (b)
10 ML
Suspected PCP Exposure —
Suspected 2,4-D or
2,4,5-T Exposure _____
Suspected OGP Pest-
icide Exposure
URINE 7
6,A,(4),(a)
L 5ML
AUTOPSY SAMPLES
ADIPOSE TISSUE
5,A, (1)
5 GRAMS
SAME AS FOR LIVE DONORS
6,A, (3), (a)
3 i’LL
BLOOL)
5,A, (3), (a)
100 GRALv!S
OR
‘MOD I FT LATIUI
Suspected Carbaryl
xposure -
Saniple size given represents a maximum, number and letter designations refer to
method number as listed in the TABLE OF CONTENTS.
Appendix VT,
Page 1
TISSUE BIOPSY
5,A, (2), (a)
0.5 (.R.AMS
URINE
5,A, (4), (c)
Suspected Hg Exposure
BLOOD-TISSUE
13, A
10 GRkIS
URINE
7,A
ML
S
-------
11/1/72 Appendix VII
PESTICIDE ANALYTICAL STANDARDS
The Perrine Prunate Laboratory operates a repository for pesticide
reference standards. The current standards index lists approximately 400
pesticidal compounds which are available in limited quantity to governmental,
university, and commerc al laboratories holding government contracts for
pesticide residue analyses.
The complete standards index, recently revised, is available to qualified
recipients and a copy may be obtained by writing to:
Technical Services Section
Perrine Primate Lahora tory
P. 0. Box 490
Perrine, Florida 33157
------- |