vvEPA
United States
Environmental Protection
Agency
Health'Effects Research Laboratory EPA-600/8-80-038
Researqh Tn-ang|e park NC 2771 1 June 1 980
Research and Development
Manual
of Analytical Methods
for the Analysis
of Pesticides
in Humans and
Environmental Samples
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EPA-600/8-80-038
June 1980
ANALYSIS OF PESTICIDE RESIDUES IN
HUMAN AND ENVIRONMENTAL SAMPLES
A COMPILATION OF METHODS
SELECTED FOR USE IN
PESTICIDE MONITORING PROGRAMS
REVISIONS BY
THE ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS
JOSEPH SHERMA
MORTON BEROZA
CONTRACT NO. 68-02-2474
EDITED BY EDITORIAL PANEL
RANDALL R. WATTS R.G. LEWIS
. R.F. MOSEMAN
REVISED DECEMBER'1979 D.W. HODGSON
U.S. ENVIRONMENTAL PROTECTION AGENCY
HEALTH EFFECTS RESEARCH LABORATORY
ENVIRONMENTAL TOXICOLOGY DIVISION
RESEARCH TRIANGLE PARK, NORTH CAROLINA
h
Cilice. _.,
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U. S. Environmental Protection Agency, and approved for publication. Mention
of trade names or commercial products does not constitute endorsement or
recommendation for use.
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FOREWARD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory participates in the development
and revision of air quality criteria documents on pollutants for which
national ambient air quality standards exist or are proposed, provides the
data for registration of new pesticides or proposed suspension of those
already in use, conducts research on hazardous and toxic materials, and
is primarily responsible for providing the health basis for non-ionizing
radiation standards. Direct support to the regulatory function of the
Agency is provided in the form of expert testimony and preparation of
affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
This manual provides methodology useful in determining the extent of
environmental contamination and human exposure to pesticides and related
industrial chemicals. It has been compiled and produced in an effort to
promote general acceptance and adoption of uniform chemical methodology of
utmost reproducibility and accuracy and to ensure that analytical results
can be correlated and directly compared between laboratories. This
standardization of data collection will greatly increase our knowledge and
understanding of the extent of environmental contamination by pesticides.
F. 6. Hueter, Ph. D.
Director
Health Effects Research Laboratory
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ABSTRACT
This manual provides the pesticide chemist with methodology useful in
determining human exposure to pesticides and related industrial chemicals.
Methods are also presented for measuring the extent of environmental
contamination with these compounds. This manual has been compiled and
produced in an effort to promote general acceptance and adoption of uniform
chemical methodology of utmost reproducibility and accuracy and to ensure
that analytical results can be correlated and directly compared between
laboratories. Methods contained in this manual have generally been
developed and/or evaluated by this laboratory within the Environmental
Toxicology Division.
The analytical methodology compiled herein consists of both multi-
residue and specific residue procedures. Included also, are miscellaneous
topics treating a number of important activities such as the cleaning of
laboratory glassware, the preparation of analytical reference standards,
and the calibration and maintenance of the gas chromatograph. Several of
the methods have been subjected to collaborative studies and have thereby
been proved to produce acceptable inter!aboratory precision and accuracy.
These methods are designated by stars placed at the left of the title in
the TABLE OF CONTENTS. Other methods presented are thought to be accept-
able but have not been validated by formal interlaboratory collaboration.
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Revised 12/15/79
TABLE OF CONTENTS*
Section Subject
1. Introduction
2. Collection, preservation, and storage of samples
I. General comments
II. Sample containers
III. Storage of samples
IV. Sampling, general
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 - blood and adipose tissue
4. Gas-liquid chromatography
A. Electron capture detection
(1) Description of instrument and accessories
(2) Columns
(3) Detector
(4) Chromatography of sample
(5) Quantitation and interpretation
(6) Tables and figures
(7) Support-bonded Carbowax 20M columns
*Analytical methods designated by a star have been subjected to
interlaboratory study.
v
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Revised 12/15/79 Table of Contents
4. (Cont.)
B. Flame photometric detection
(1) Description of component modules
(2) Columns
(3) Detector
(4) Sample quantisation and interpretation
(5) Tables and figures
C. Hall electrolytic conductivity detector
(1) Description of instrument
(2) Columns
(3) Detector
(4) Sample quantisation and interpretation
(5) Retention data and chromatograms of carbamates
on Carbowax-modified supports
D. Nitrogen-phosphorus (N-P) detector
5. Chlorinated hydrocarbon pesticides and metabolites
A. In human tissues and excreta
(1) Adipose tissue
(a) Modification of Mills, Onley, Gaither
method for multiple chlorinated pesti-
cides and metabolites
(b) Hexachlorobenzene (HCB) and mirex, and
HCB confirmation
(2) Micro methods
(a) Liver, kidney, bone, marrow, adrenal,
gonads
(b) Brain and human milk
vi
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Revised 12/15/79 Table of Contents
5. (Cont.) (3) Blood
(a) Multiple chlorinated pesticides
(b) Pentachlorophenol
(4) Urine
(a) Pentachlorophenol and other chlorinated
phenols
(b) DDA in urine
(c) 2,4-D and 2,4,5-T
(5) Human blood and environmental samples
(a) Kepone
B. Cleanup by gel permeation chromatography (GPC)
6. Organophosphorus pesticides and metabolites
A. In human tissues and excreta
(1) General comments
(2) Urine
(a) Determination of metabolites or hydrolysis
products of organophoshorus pesticides
(b) Para-nitrophenol
(3) Blood
(a) Cholinesterase activity
7. Carbamate pesticides and metabolites
A. 1-Naphthol in urine
8. Sampling and analysis of air for pesticides
A. Sampling
B. Analysis
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Revised 12/15/79 Table of Contents
9. Analysis of nonpesticide pollutants: PCBs and TCDD
A. Introduction to the analysis of PCBs
B. Determination of PCBs in human milk
(1) Macro method
(2) Micro methods
C. Separation of PCBs from organochlorine pesticides
D. TLC semi-quantitative estimation of PCBs in adipose
tissue
E. Typical PCB chromatograms
F. Tables of relative retention and response ratios
of PCBs
G. Analysis of TCDD in milk, liver, fish, water, and
sediment
10. Analysis of water
A. Sampling and analysis of water for pesticides
B. Determination of some free herbicides in water
11. The analysis of soils, housedust, and sediment
A. Organochlorine insecticides in soils and housedust
B. Organochlorine and organophosphorusinsecticides in
bottom sediment
C. Carbamate pesticides in soil
12. Confirmatory procedures
A. Confirmation and determination of organochlorine
pesticides in human tissue and milk
B. Thin-layer chromatography
C. p-Values
vm
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Revised 12/15/79 Table of Contents
D. Derivatization techniques
(1) Microscale alkali treatment for confirmation
of sample cleanup
(2) Perchlorination of PCBs
E. Infrared spectroscopy
F. Polarography
13. High performance liquid chromatography
APPENDIX
Section Subject
I. Maintenance and repair of instruments
II. A. Analytical quality control
VI. Block diagram of tentative tissue, excreta and method
selection for abnormal pesticide exposure cases
VII. Pesticide analytical reference standards repository
VIII. Future manual revisions - very important
IX
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Revised 12/2/75 Section 1
Page 1
INTRODUCTION
The analytical methodology collected in this manual was primarily
intended for use by EPA Laboratories conducting analyses of pesticides in
various sectors of the environment and by laboratories under contract with
EPA to conduct community studies and the monitoring of concentrations of
pesticides in the human population.
One of the primary objectives of the Epidemiologic Studies and Monitor-
ing Laboratory program was 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 environ-
ment by pesticides and their metabolites. To accomplish this goal, data have
been continuously obtained on the levels of pesticides and their metabolites
in the human population and various elements of the environment. It is
important that uniform chemical methodology of utmost reproducibility and
accuracy be used by participating laboratories to ensure that analytical
results can be correlated and directly compared between laboratories.
A prime responsibility of the Environmental Toxicology Division is to
make new and improved 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 a primary facility to provide (1) high purity analytical reference
standards, (2) information on analytical quality control, (3) instrumental
troubleshooting and calibration, and further (4) to conduct research on
analytical methodology for the measurement of residues of pesticides and
other toxic residues in human and environmental media.
The analytical methodology compiled herein consists of both multi-
residue and specific residue procedures. Included also are miscellaneous
topics treating a number of important activities such as the cleaning of
laboratory glassware, the preparation of analytical reference standards, and
the calibration and maintenance of the gas chromatograph. Several of the
methods have been subjected to collaborative studies and have thereby been
proved to produce acceptable interlaboratory precision and accuracy. These
methods are designated by plus signs placed at the left of the title in the
TABLE OF CONTENTS. Other methods presented are thought to be acceptable but
have not been validated by formal interlaboratory collaboration.
A numbering system is used in this manual whereby each page bears a date
and numbers and/or letters designating the identity of the section and
subsection. Additions, deletions and revisions will be distributed to manual
holders as they are made available, with each such issuance bearing appropri-
ate section identification and revision date.
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Revised 12/2/75 Section 1
Page 2
The cooperation of scientists using this manual is solicited in helping
to improve and update the material. Suggestions and comments based on user's
experience will be welcomed. Such suggestions or requests for additional
copies of the manual should be directed to:
Director
Environmental Toxicology Division
EPA, Health Effects Research Laboratory
Research Triangle Park, NC 27711
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Revised 12/2/74 Section 2
Page 1
COLLECTION, PRESERVATION AND STORAGE OF SAMPLES
I. GENERAL COMMENTS:
In the procurement, storage, and transportation of samples intend-
ed for analysis for pesticide residues, the personnel involved should
be aware of some basic considerations to ensure delivery to the analyt-
ical chemist of samples that have not undergone degradation of any
pesticide present and that have not been contaminated with impurities
that might interfere with the analysis.
Although medically trained personnel may be inclined to consider
asepsis as the sole requirement, and, while aseptic handling may help
ensure freedom from unwanted contamination, there are other far more
important considerations. One example is the material of which the
sample container is made. Plastics are widely used in the container
industries but, although they take preference over glass for many pur-
poses, they should be rigidly avoided as containers for samples that
will be examined by gas chromatography. Minute traces of certain of
the components of plastics may play havoc in electron capture GLC.
Similarly, ferrous metal containers such as compression lid cans
or ointment tins which were used by pharmacies may contain trace
impurities that will cause interference in the analysis of GLC.
In general, glass, Teflon, and aluminum foil have been proved to
be the most suitable materials to come in direct contact with the
sample. Foil or Teflon is generally used as liner material for a
bottle or jar cap when the material in the normal cap may contribute
impurities. The containers listed in the next subsection are suggested
with the foregoing considerations in mind.
II. SAMPLE CONTAINERS:*
A. For tissues:
1. Wide-mouth bottles, glass, 2-1/16 in. high x 1-1/4 in. diam.,
approx. 1 oz. Owens-Illinois mold number AM-6764. Available
from many wholesale glass container distributors. These are
generally sold in lots of 1 to 10, 10 to 25, 25 to 50, 50 to
100 and over 100 gross with decreasing per-gross prices for
the larger quantities. No caps are included.
These containers are suitable for any autopsy sample not exceed-
ing about 25 grams.
*New containers should be cleaned as described in Section 3, A.
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Revised 6/77 Section 2
Page 2
2. The suggested screw caps for the above bottles are metal with
paper back foil liners, size 38-400, available in gross
quantities from glass container distributors.
B. For blood:
Glass vials in sizes of 45 x 15 mm, 5 ml and 60 x 17 mm, 9 ml.
These are available from Arthur H. Thomas Company, Philadelphia,
PA under catalog number 9802-G. Caps for these vials are
listed as catalog number 2849-A, sizes 13 and 15, respectively.
These are molded screw caps with cork back foil liners.
The size should be selected on the basis of the volume of sample
drawn and should not be less than 7 ml. Containers with rubber
caps should be avoided because of the possibility of contamin-
ation from impurities in the rubber. The same warning applies
to cork unless a layer of inert material such as foil or Teflon
is used on the side contacting the sample.
C. For water:
Water samples may be conveniently taken in glass bottles in which
organic solvents are supplied. For example, an emptied hexane
or acetone bottle makes an excellent water sample container.
The molded screw cap generally has a Teflon liner. If not, a
foil liner may be inserted. See Section 10, A for details.
D. For agricultural or environmental media:
Environmental or agricultural samples of 1-lb. or more may be
taken in pint, quart or 2-quart size Mason jars. One layer of
industrial gauge aluminum foil (0.001 in.) or two layers of
regular household grade foil should be used as cap liner.
Under no circumstances should the sample material be allowed
to come in contact with the paper liner material of the usual
metal screw caps.
III. STORAGE OF SAMPLES:
Tissue samples that are to be extracted within 24 hours may be
held at normal refrigerator temperature (+2° to +4°C). If extraction
is not to be carried out within this time, the samples should be deep
frozen at -12° to -18°C.
Blood samples that are to be separated for subsequent analysis of
the serum should be centrifuged as soon as possible after drawing. If
the serum is to be analyzed within a 3-day period, storage at +2° to
+4°C is suitable. If storage is to be for longer periods, it is
preferable to deep freeze at -12° to -18°C.
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Revised 12/2/74 Section 2
Page 3
Agricultural or environmental samples that are to be analyzed for
organophosphates should be placed in tight containers and stored in
deep freeze as soon as possible after sampling unless sample prepara-
tion is to be conducted within a few hours. Under no circumstances
should extraction be deferred longer than an overnight period, even
when the samples are frozen.
IV. SAMPLING, GENERAL:
A subsection on sampling guidelines is included in each method
section wherever feasible. In certain sections wherein the sampling
and storage may exert a profound influence over the quality of the
data obtained from the analysis, the subject is addressed in some
detail.
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Revised 12/15/79 Section 3, A
Page 1
MISCELLANEOUS INFORMATION
CLEANING OF LABORATORY GLASSWARE
In the pesticide laboratory involved in the analysis of samples contain-
ing 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 from contamination. Particular care must be
taken with glassware such as Kuderna-Danish flasks, evaporative concentrator
tubes, or any other glassware coming in contact with an extract that will be
evaporated to a lesser volume. The process of concentrating the pesticide in
this operation may similarly concentrate the contamination substance, result-
ing in extraneous chromatographic peaks that, in extreme cases, may complete-
ly overlap and mask the pesticide peak pattern.
Although chemists do not all agree on procedural details in the cleaning
of glassware, the majority are in agreement regarding the basic cleaning
steps. These are:
1. Removal of surface residuals immediately after use.
2. Hot soak to loosen and flotate most of soil.
3. Hot water rinse to flush away flotated soil.
4. Soak with deep penetrant or oxidizing agent to destroy traces
of organic soil.
5. Hot water rinse to flush away materials loosened 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
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Revised 12/2/74 Section 3, A
Page 2
serve to contaminate all other glassware placed therein. Many
instances of widespread laboratory contamination with a given pesti-
cide are traceable to the glassware washing sink.
2. The hot soak consists 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 some hard water scum resulting from the reaction
between calcium and magnesium salts with a fatty acid soap. This
hard water scum or curd would have an affinity particularly for the
chlorinated pesticides and, being almost wholly 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 dishwashing 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. Sparkleen, another powdered product, is
distributed by Fisher Scientific Company.
NOTE: Certain detergents, even in trace quantities, may
contain organics that will contribute significant
background contamination by electron capture
detection. For this reason any detergent selected
should be carefully checked to ensure freedom from
such contamination. The following procedure is
recommended:
Add 25 ml dist. water, previously checked for back-
ground contaminants, to a 250 ml sep funnel. Add
1 drop of the liquid detergent (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 ^SO^ to the hexane extract
and shake 1 minute. Transfer extract to a Kuderna-
Danish assembly fitted with a 10 ml evaporative concen-
trator tube containing one 3 mm glass bead. Reduce
extract volume to ca 3 ml in a hot water bath. Cool,
rinse down f joint and sides of tube with hexane,
diluting extract to exactly 5 ml. Stopper tube and
shake on Vortex mixer 1 minute. Chromatograph by
electron capture GLC and evaluate chromatogram for
contaminant peaks.
3. No comments required.
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Revised 12/2/74 Section 3, A
Page 3
4. The most common and highly effective oxidizing agent for removal of
traces of organic soils is the traditional chromic acid solution
made up of H2SOi,. and potassium or sodium dichromate. For maximum
efficiency, the soak solution should be hot (40° to 50°C). Safety
precautions must be rigidly observed in the handling of this solu-
tion. Prescribed safety gear should include safety goggles, rubber
gloves and apron. The bench area where this operation is conducted
should be covered with lead sheeting as spattering will disintegrate
the unprotected bench surface.
The potential hazards of using chromic sulfuric acid mixture are
great and have been well publicized. There are now commercially
available substitutes that possess the advantage of safety in
handling. These are biodegradable concentrates with a claimed
cleaning strength equal to the chromic 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 are further claimed to
remove radioactive traces and will not attach glass nor 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"
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 some contamination from
either the air or direct contact. To ensure against this, it is
good practice to flush the item immediately before use with some
of the same solvent that will be used in the analysis.
The drying and storage of the cleaned glassware is of critical impor-
tance to prevent the beneficial effects of the scrupulous cleaning from
being nullified. Pegboard drying is not recommended as contaminants may be
introduced to the interior of the cleaned vessels. Neoprene-coated metal
racks are suitable for such items as beakers, flasks, chromatographic tubes,
and any glassware then can be inverted and suspended to dry. Small articles
like stirring rods, glass stoppers and bottle caps can be wrapped in aluminum
foil and oven dried a short time if oven space is available. Under no cir-
cumstance 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.
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Revised 12/15/79 Section 3, A
Page 4
Pipet Hashing
The efficient washing of pipets offers some special problems. Hand
washing performed by soaking pipets in a pan or sink followed by rinsing
under running water is highly unsatisfactory, particulary as applied to
transfer or volumetric pipets. This procedure does not assure a complete
rinse of all surfaces inside the bulb. Therefore, an automatic or semi-
automatic washing system is strongly recommended. Self-contained equipment
for the entire operation, although available commercially, is quite
expensive.
The basic cleaning steps are the same as those listed earlier for
miscellaneous glassware, with the exception of the soap wash.
After use, all pipets should be rinsed with an appropriate solvent to
remove the bulk of residues remaining in the pipets. The pipets are cleaned
by immersion in a chromic acid cleaning solution. For this purpose, the
pipets should be in a standard (13.5 x 44 cm) nalgene pipet basket. The
entire assembly is submerged in chromic acid in a glass cylinder (16 x 39cm).
After 1-2 hours, the basket of pipets is withdrawn from the chromic acid
solution, allowed to drain about 1 minute, and then transferred to a stain-
less steel washer where rinse water (tap) is run through the washer at the
rate of ca.3 minutes per discharge for approximately 1 hour. If piped
distilled water is available, seven or eight discharges of this are run
through the system to remove all traces of metal contaminants left by the
tap water.
A final rinse with acetone, either from a wash bottle or from an over-
head syphon bottle, is then applied to each pipet. After draining, a
convenient and rapid method of drying is to wrap a bundle of pipets in
aluminum foil and place in a drying oven for at least 3 hours, or overnight.
NOTES: (a) Under no circumstances should plastic gloves be worn
by personnel during glassware cleaning or handling.
It has been determined beyond question that these
gloves can most effectively contaminate an entire
sinkful of glassware to such an extent that subsequent
solvent rinsing may not completely eliminate the
contaminants. This is a VERY IMPORTANT precaution.
(b) Drying racks of plastic or plastic-coated metal must
be avoided. The latter type of rack may be used,
however, after the plastic is scraped from the metal
prongs and the rack is cleaned thoroughly with a
suitable organic solvent.
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1/4/71
Section 3,A.
Page 5
FIGURE 1.
Bale handle of 1/8"
s.s. rod
Reinforced bands
of about 22 ga. s.s
PIPET BASKET
Perrine Primate Researcn branch
P.O. Box 490
Perrine, Florida 33157
Sidewalls may be of 1/8"
or 1/4" s.s. mesh or
perforated s.s. sheet.
Solder used to be
95/5
of 1/8" mesh
s.s. screen
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1/4/71
Section 3., A
Page 6
s.s. Pipet Basket
500 watt immersion
element
6" x 18" Pyrex
glass cylinder
Figure 2. Assembly of pipet washer showing pipet basket inside coiled
immersion heater, all contained in Pyrex jar.
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Revised 12/15/79 Section 3, B
Page 1
MISCELLANEOUS INFORMATION
PREPARATION, STORAGE, AND USE OF PESTICIDE ANALYTICAL STANDARDS
FOR GLC
EQUIPMENT, SOLVENTS. AND REAGENTS
1. Analytical balance capable of an accuracy of +_ 0.05 mg.
2. Spatula, stainless steel.
3. Pipets, Pasteur, disposable.
4. Flasks, volumetric, 25, 50, and 100 ml.
5. Bottles, prescription, % oz, 1 oz, or 2 oz, with plastic screw
caps. Available from any wholesale pharmacy supply firms.
With cap liners, Teflon, sizes 13, 15, 18, and 22 mm, Arthur H.
Thomas 2390-H22, H32, H42, and H62.
6. Vial, screw cap, size 1-6, Kontes # K-940100, with Microflex
valve # K-749100.
7. Serum bottle, 20-100 ml size, Wheaton # 223742 to # 223747,
with Teflon-faced septa # 224168 and seal # 224183.
8. Refrigerator, explosion proof, with freezer across top, capable
of maintaining + 4°C in refrigerator section and - 15°C in freezer.
NOTE: It is definitely preferable to have separate refrigerators
for chemicals and sample materials. However, if a labora-
tory is restricted to one refrigerator, sample materials
should be stored in air-tight glass containers to prevent
contamination by spillage or airborne vapors from
pesticides.
9. Primary pesticide standards. Available in approximately 50 mg
quantities to qualified laboratories from the reference standards
repository, ETD, HERL, U.S. EPA, Research Triangle Park, NC.
NOTE: The organophosphorus compounds are subject to a wide
variety of oxidation, rearrangement, and hydrolytic
reactions. These compounds should be stored in the
refrigerator in a large air-tight container (such as
a wide-mouth mayonnaise jar) or in a desiccator to
minimize moisture absorption and toxic vapor cross-
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Revised 12/15/79 Section 3, B
Page 2
contamination. ALL HANDLING OF THESE STANDARDS SHOULD
BE DONE WITH RUBBER GLOVES. SKIN CONTACT BY HIGHLY
CONCENTRATED MATERIALS CAN BE FATAL. Samples of organ-
ophosphates and metabolites should be equilibrated to
room temperature in a desiccator to avoid condensation
and possibility of long-term hydrolysis.
10. Sylon-C7 (Supelco) for silanization of empty glass gas chromato-
graphic columns and glass injection inserts.
11. Toluene, isooctane (2,2,4-trimethylpentane), ethyl acetate, or
hexane, pesticide quality, distilled in glass.
NOTES: 1. A 10 yl injection of each solvent (except ethyl
acetate) should result in a chromatogram with
zero background when examined by electron capture
GC with system sensitivity adjusted to concur with
the criteria outlined in Section 4,A,(4).
2. Isooctane or hexane are both suitable for standard
dilution. Isooctane, while more expensive, offers
the advantage of a 100°C boiling point and much
higher vapor pressure than hexane. The solvent is
much less likely to evaporate through long-term
leakage around the seal and during repeated bottle
openings.
3. Ethyl acetate is not recommended as a final solvent
for electron capture GC but may be necessary for
preparation of the first or concentrated solution
of polar materials.
II. INTEGRITY AND STABILITY OF STANDARDS:
1. Stability of the Solid or Liquid Primary Standard.
Standards that are not in solution are generally stable to
chemical decomposition, if kept refrigerated or frozen. Studies
done in the past have not shown significant chemical decomposition
for time periods in excess of one year. The generally used organo-
chlorines, organophospates, triazines, and carbamates are included
in this group. The organophosphate and carbamate standards are
subject to hydrolysis reactions. Storage of these compounds in a
refrigerated desiccator jar is recommended.
2. Stability of Standard Solutions.
Over the time period of one year, most compounds in hexane,
isooctane, or toluene solution are stable to chemical decomposition.
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Revised 12/15/79 Section 3, B
Page 3
Refrigeration of the standards when not in use is strongly
recommended.
Compounds in four classes have been studied. Organochlorine
and triazine compounds seem to be stable for long time periods.
Most organophosphates and carbamates are also stable. Disulfoton
is the only organophosphate that degraded chemically over the
period of one year, to the extent of 10% in three months.
The carbamate compounds CDEC and butyl ate have been found to
be unstable as stock solutions and as GC injection solutions.
Storage of these solutions for greater than one month is not
recommended.
3. Solvent Evaporation Problems.
Most of the problems with standard solution integrity were
found to be related to solvent evaporation and the resultant
solution concentration. The solvent, the storage temperature, and
the storage container are all factors that influence solvent
evaporation.
The rate of solvent evaporation from closed containers is
related to the vapor pressure of the solvent at the storage temp-
erature. Hexane evaporated 2.4 times faster than isooctane. The
vapor pressure of hexane is 3 times the vapor pressure of isooctane.
Although the relationship is not perfect, vapor pressure is a better
factor to relate to than the boiling point of the solvent. The
following is the order of the evaporation rates of the solvents
studied:
toluene < isooctane < benzene < methanol « hexane <
acetone < methylene chloride « diethyl ether « petroleum ether
For this reason toluene or isooctane is recommended as the
solvent for storage of standards. When solubility or reactivity
is a problem, the choice of a solvent should be based partially
on the necessary chemical properties and the vapor pressure of
the solvents. The use of solvents with high vapor pressures can
significantly shorten the shelf-life of standard solutions.
As expected, there is a dramatic difference between solvent
evaporation rates at ambient laboratory temperature and in the
refrigerator. The life of analytical standard solutions will be
lengthened considerably by refrigerated storage when not in use.
The choice of the storage container is a rather critical one.
Volumetric flasks are standard storage containers used by many
-------
Revised 12/15/79 Section 3, B
Page 4
laboratories. However, the standard taper stopper does not form
a good seal. The only way to reduce the effect of evaporation is
to store a large volume of the standard, thereby reducing the
relative solvent loss.
Better containers include prescription bottles with Teflon
seals in the cap; these are quite reliable and inexpensive. The
graduation mark on the side of the bottle or a piece of label tape
can be used to monitor the evaporation of the solvent. Once the
solvent evaporation is obvious, the standard is discarded. Two
other very good containers are the large volume screw cap vial sold
by Kontes and the serum bottle (with Teflon-faced cap) sold by
Wheaton. These two containers allow a minimum of solvent evapora-
tion when closed, and they are never opened in use. This reduces
the solvent loss dramatically. Once again, a mark or label on the
side of the container will serve as a graduation mark. Once the
solvent level in the bottle is significantly below the mark, the
standard should be discarded.
III. FORMULATION PROCEDURE:
1. Preparation of Concentrated Stock Standard Solutions.
Except for concentrates for special purposes, a concentration
of 200 yg/ml is suitable for the common chlorinated and organo-
phosphate pesticides. Ten milligrams of the primary standard,
corrected to a 100% purity basis, diluted to 50 ml will provide
this concentration (20 mg/100 ml).
Toluene is a suitable solvent for most of the primary stan-
dards. B-BHC dissolves readily in toluene, with stirring and a
slight application of heat from a hot water bath. For the tria-
zines, the use of ethyl acetate is necessary for the concentrated
stock solutions.
The concentrated standards of chlorinated compounds and
triazines should maintain uniform strength for a 12-month period
at - 10° to - 15°C. The organophosphate standards are less stable
than the organochlorines. It is recommended that the concentrated
stock solutions of phosphates and carbamates be held no longer
than 6 months at - 15°C.
NOTES: 1. Extreme care must be used in the formulation of this
standard. If an error is made here, all subsequent
dilutions for the life of the standard will be
inaccurate. Obviously, all quantisations of samples
will be similarly incorrect.
-------
Revised 12/15/79 Section 3, B
Page 5
2. Concentrated solutions of CDEC and butyl ate should
not be kept for longer than one month. Very rapid
decomposition of the compounds occurs under all
conditions.
2. Preparation of Standard Solutions of Intermediate Concentration.
These will be the standards from which the final working
mixtures will be prepared. Convenient intermediate concentrations
of a number of widely used compounds are given in Table 1.
The solvent for the intermediate standards must be of pesti-
cide quality. Hexane and isooctane are normally used. Isooctane
is preferred as discussed in Section 3,B,II.
The intermediate concentration standards of the chlorinated
compounds and triazines, if stored in the freezer at - 10° to
- 15°C, should be stable for a 12-month period.
The organophosphorus and carbamate intermediate standards
should be similarly stored in the freezer. The time limit of these
standards should not exceed 6 months.
3. Working Standard Mixtures.
A. Preparation and Storage.
Isooctane is favored as the solvent for the working
mixtures since the many repeated bottle openings greatly
increase the evaporation and subsequent concentration of
standards if a lower boiling point solvent is used.
The attached Table 2 is useful in rapid determination
of the aliquot volumes of the higher concentration solutions
required to result in given concentrations of the diluted
working standards.
The use of standard mixtures of varying concentrations is
a necessity for reliable quantisation of unknowns. The degree
of peak height variation between sample and standard ideally
should not exceed 10%, although variations up to 25% should
not result in appreciable error. A simple means of achieving
this is to have available working standard mixtures of three
concentrations. The suggested mixtures given in Table 3 have
proved very useful in the analysis of tissues. Those labora-
tories conducting analyses on environmental samples may wish
to make alterations in the compound content, but the multi-
concentration concept should be retained (Miscellaneous
Note 5).
-------
Revised 12/15/79 Section 3, B
Page 6
The selection of working standard containers and methods
of handling and storage are, to some extent, a matter of local
preference. Following are two procedures, both of which have
proved satisfactory.
1. After the working standard mixtures are diluted in
volumetric flasks, they are transferred to prescrip-
tion bottles with Teflon-lined screw caps, serum
vials, or vials with valve tops. These mixtures
should be stored in the refrigerator at all times
when not in actual use. The organochlorine and
organophosphorus working standards should be renewed
monthly. With this scheme, large volume glassware
should be used.
2. The working standard mixtures are transferred from
the volumetric flasks into several small volume
(up to 20 ml) containers. When not in use, the
standard solutions are kept in the deep freeze. When
needed, they are removed from the deep freeze and
used. Storage in the refrigerator when not in use
is recommended. When the project is completed or the
standard has evaporated, a new one should be obtained
from the deep freeze and put into use. This option
has the advantages of less frequent formulation of
working standards and reduced possibility of errors
arising from repeated opening of the working stan-
dard containers.
Working standards can be used for long periods of time
without chemical decomposition. Standards of carbaryl and
methiocarb do decompose when exposed to light. These stan-
dards should be replaced every 2 months. Disulfoton, CDEC,
and butyl ate decompose rapidly under all storage conditions.
Standards of these compounds must be replaced at least every
month.
All standards should be replaced when solvent evaporation
is obvious when compared to a reference line on the container.
B. Use of Working Standards.
At the start of each working day, after making certain
that column operating and instrumental parameters are properly
adjusted, it is good practice to make several consecutive
injections of standard mixtures to "prime" the column for that
day's work. When it has been determined that peak heights for
given compounds are constant, the first exploratory injection
of an unknown sample extract is made. From this, the
-------
Revised 12/15/79 Section 3, B
Page 7
chromatographer can now make a number of tentative peak
identifications by calculating relative retention values.
The peak height response of some of the compounds in the
sample extract may match, within reason, the peak heights
resulting from the prior working standard injections. In all
probability, certain other compound peaks will not match. The
operator will now-select from the three working standard con-
centrations the one that he estimates will produce matching
peak heights.
In some cases, it will be found that even the highest
concentration mixture will be insufficient to properly quan-
titate £,£'-DDE and £,£'-DDT. In this case, the sample
extract should be quantitatively diluted to a degree that is
calculated to produce peaks matching those of the working
standards. The pesticide concentrations in the mixtures in
Table 3 practically preclude any possibility of violating
the detector linearity range of the EC detector when volumes
of 5 to 6 yl are injected.
The range of the 63Ni detector is much more restricted
than that of the 3H detector in the DC mode. Each detector
must be checked for its linearity performance. Improved
performance from the 63Ni detector can be obtained in the
linearized or pulsed mode.
IV. MISCELLANEOUS NOTES:
1. In addition to the diluted working standard mixtures, each labora-
tory should maintain a standard of pure £,£'-DDT diluted to 60 pg/nl
(the highest concentration of the working mixture). This should be
chromatographed daily on each working column to provide current
information concerning on-column conversion (generally to p,p'-DDE
and/or £,£'-DDD). In case a breakdown peak greater than 3% of the
£,£'-DDT is noted, the silanized glass wool plug at the column
inlet and the Vykor glass injection insert should be changed. It
is most important that the glass injection insert also be silanized.
If, after an overnight period of normal operating temperature and
carrier gas flow, the situation has not improved, the column
should be discarded.
2. If a laboratory has occasion to analyze for endrin, a similar check
with an endrin analytical standard should be made weekly. The con-
centration should be ca.100 pg/yl. The manifestation of endrin
breakdown is a depression of peak height response in the main peak
accompanied by the formation of two additional peaks. One of these
is in the general area slightly later than £,£'-DDT. The other,
and largest, peak elutes extremely late, around the methoxychlor
-------
Revised 12/15/79 Section 3, B
Page 8
retention area on the OV-17/OV-210 column. If this is observed,
the silanized glass wool at the head of the column should be
replaced, as well as the glass injection insert.
3. In no case should any attempt be made to dilute standard concen-
trations to quantitate sample peaks of less than 10% full scale
recorder deflection. In view of the sensitivity of which the
MT-220 is capable, if all systems are functioning properly, there
should be no need to compare peaks with very small areas against
each other. The optimum range of peak heights for quantitation
lies between 20 and 70% full scale recorder deflection, provided,
of course, that the compound concentrations fall within the linear
range of the detector.
4. The importance of operating within the limits of the linearity
range of the detector cannot be over-emphasized. One means of
ensuring this is to operate at a relatively high sensitivity.
5. It is strongly preferable to use the same attenuation setting for
standard and sample. If, for any reason, it should appear
necessary to use different attenuations, the operator must carefully
consider detector linearity limitations and should have prechecked
the attenuator linearity. The use of multiconcentration standard
mixtures should minimize the need for peak height adjustment by
other means.
6. When a new working standard formulation is used for the first time,
the peak height response should be carefully compared with the
latest chromatograms of the previous mixture. This practice
enables the chromatographer to immediately detect any response
irregularity, thereby avoiding the use of an incorrect standard for
several weeks.
7. It is good practice to standardize injection volumes of standards
and sample extracts. A 5 yl injection provides a convenient
volume. If alternative volumes are used, they should be restricted
to the range of 3 to 8 yl, and each operator should make certain
that he can obtain linear response when injecting these volumes.
-------
Revised 12/15/79
Section 3, B
Page 9
TABLE 1. SUGGESTED CONCENTRATIONS OF THE INTERMEDIATE STANDARDS
OF SOME COMMON PESTICIDAL COMPOUNDS USED IN ELECTRON
CAPTURE GLC.
Organochlorine
a-BHC
3-BHC
Lindane
Heptachlor
Aldrin
Heptachlor Epoxide
£,£'-DDE
£,£'-DDE
Endosulfan
DDA (Methyl Ester)
Dieldrin
o,£'-DDD
Endrin
Perthane
£,£'-DDD
o.,£'-DDT
Oil an
Methoxychlor
Tetrad if on
Mi rex
Chlordane
Toxaphene
ng/yl
1
2
1
1
1
1
1
2
4
a
2
2
4
a.
4
4
10
10
20
10
10
a
Organophosphorus
Mevinphos
Phorate
Dimethoate
Diazinon
Methyl Pa rath ion
Ethyl Parathion
Malathion
Ethion
Carbophenothion
Azinphos Methyl
ng/yl
50
50
40
30
10
10
20
20
10
a.
aFinal working standard prepared directly from the 200 ng/yl
concentrate.
-------
Revised 12/15//9 Section 3, B
Page 10
TABLE 2. COMMONLY USED DILUTION VALUES. VALUE IN LEFT COLUMN IS THE AMOUNT (ML) OF CONCENTRATED
SOLUTION THAT MUST BE DILUTED TO 100 ML TO ARRIVE AT THE CONCENTRATION VALUE GIVEN IN
THE RIGHT COLUMN. VALUE AT HEAD OF EACH COLUMN IS THE CONCENTRATION OF THE STOCK
SOLUTION.
lug/iil
ml ng/pl
50
47.5
45
42.5
40
37.5
35
32.5
30
27.5
25
22.5
20
17.5
15
12.5
10
9.5
9
8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
500
475
450
425
400
375
350
325
300
275
250
225
200
175
150
125
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
200 ng/ul
ml ng/ul
50
47.5
45
42.5
40
37.5
35
32.5
30
27.5
25
22.5
20
17.5
15
12.5
10
9.5
9
8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
20 ng/ul
ml pg/pl
5
4.875
4.75
4.625
4.5
4.375
4.25
4.125
4
3.875
3.75
3.625
3.5
3.375
3.25
3.125
3
2.875
2.75
2.625
2.5
2.375
2.25
2.125
2
1.875
1.75
1.625
1.5
1.375
1.25
1.125
1
0.875
0.75
0.625
0.5
1,000
975
950
925
900
875
850
825
800
775
750
725
700
675
650
625
600
575
550
525
500
475
450
425
400
375
350
325
300
275
250
225
200
175
150
125
100
10 ng/ul
ml pg/ul
10
9
8
7
6
5
4.75
4.5
4.25
4
3.75
3.5
3.25
3
2.75
2.5
2.25
2
1.75
1.5
1.25
1
.95
.9
.85
.8
0.75
.7
.65
.6
.55
0.5
.45
.4
.35
.3
0.25
1,000
900
800
700
600
500
475
450
425
400
375
350
325
300
275
250
225
200
175
150
125
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
continued
-------
Revised 12/15/79
TABLE 2. (CONTINUED)
Section 3,
Page 11
4 nq/yl
ml pg/ul
20
18.75
17.5
16.25
15
13.75
12.5
11.25
10
9.375
8.75
8.125
7.5
6.875
6.25
5.625
5
4.375
3.75
3.125
2.5
2.375
2.25
2.125
2
1.875
1.75
1.625
1.5
1.375
1.25
1.125
1.
.875
.75
.625
0.5
0.25
0.125
800
750
700
650
600
550
500
450
400
375
350
325
300
275
250
225
200
175
150
125
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
10
5
2 ng/Ml
ml pg/iil
35
32.5
30
27.5
25
22.5
20
17.75
17.5
16.25
15
13.75
12.5
11.25
10
8.75
7.5
6.25
5
4.75
4.5
4.25
4
3.75
3.50
3.25
3
2.75
2.5
2.25
2
1.75
1.5
1.25
1
.75
0.5
700
650
600
550
500
450
400
375
350
325
300
275
250
225
200
175
150
125
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
1 ng/nl
ml pg/ul
50
45
40
37.5
35
32.5
30
27.5
25
22.5
20
17.5
15
12.5
10
9.5
9
8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.9
.8
.7
.6
.5
500
450
400
375
350
325
300
275
250
225
200
175
150
125
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
9
8
7
6
5
-------
Revised 12/15/79 Section 3,
Page 12
TABLE 3. SUGGESTED MIXTURES FOR QUANTITATION OF COMMON
CHLORINATED COMPOUNDS IN TISSUES.
SERIES
A. (6%)
B. (15&)
*C
D.
HCG
2$"-BHC
g-BHC
Aldrin
Oxychlordane
Heptachlor epoxide
t-Nonachlor
p,£'-DDE
o,£'-DDT
£,p'-DDD
£,£'-DDT
Mi rex (if suspected)
Aldrin
Dieldrin
Endrin (if suspected)
a-BHC
S-BHC
Heptachlor
Aldrin
p_,£'-DDE
Aroclor 1254
Aroclor 1260
Standard
1
2.5
5
7.5
5
7.5
7.5
7.5
40
15
20
25
40
5
10
12.5
5
5
5
5
10
125
125
concentration
2
5
10
15
10
15
15
15
80
30
40
50
80
10
20
50
10
10
10
10
20
250
250
in pg/yl
3
10
20
30
20
30
30
30
160
30
80
100
160
20
40
100
20
20
20
20
40
500
500
*This series contains only those compounds that are rarely found in tissues.
-------
Revised 12/15/79 Section 3, C
Page 1
MISCELLANEOUS INFORMATION
GENERAL PURITY TESTS FOR SOLVENTS AND REAGENTS
In general, the solvents used in pesticide residues by GLC must be of
very high purity. If the laboratory intends to use the purchased solvents
without redistilling, materials bearing the manufacturer's designation of
"pesticide quality, distilled in glass" should be purchased. Even with this
designation, each lot must be checked for assurance of freedom from any
impurity that may have escaped the manufacturer's quality control. If drum
lots of technical or commercial grade solvents are bought, distillation
through an all-glass still is practically mandatory.
I. TEST FOR SUBSTANCES CAUSING INTERFERENCE IN ELECTRON CAPTURE GLC:
Electron capture GLC requires solvent that is free of substances
causing detector response at the electrometer attenuation normally used
in analytical work. Place 300 ml of the solvent in a specially cleaned
500 ml Kuderna-Danish concentrator fitted with a 3-ball Snyder column
and a 10 ml evaporative concentrator tube. Evaporate in a hot water
bath to 5 ml. Inject 5 yl of this concentrate into the gas chroma-
tograph and allow enough time for elution of any peak equaling the
retention time of the latest eluting compound of possible interest to
the laboratory. This would generally be the retention area of Guthion.
If no peaks elute at the retention sites of the compounds of interest,
and adjacently eluting peaks are not sufficiently large to create a
partial overlapping with pesticides, the indication is favorable for
the purity of the solvent. If any peak(s) of greater than 2% FSD elute
at the retention sites of one or more of the pesticides of interest,
the solvent would create problems in identification and quantisation
and would not be acceptable. The electrometer attenuation should be
that currently in use for sample analysis.
It may be possible to remove the contaminants by distillation
through an all-glass still. However, there is no certainty of this
because some organic materials may codistill with the solvent and
still be present in the distillate.
II. TEST FOR SUBSTANCES CAUSING PESTICIDE DEGRADATION:
Solvent impurities not detected by the above procedure may cause
degradation and loss of pesticides during analysis. Solvents should be
tested for suitability by carrying known amounts of both chlorinated
and organophosphate pesticides through the method in the absence of any
sample substrate. Solvents containing oxidants may cause noticeable
loss of organophosphate pesticides, especially carbonphenothion.
-------
Revised 6/77 Section 3, C
Page 2
III. REAGENTS:
A. Acetonitrile - Some lots of reagent grade acetonitrile are impure
and require redistillation. Vapors from impure CH3CN turn
litmus paper blue when moistened paper is held over mouth of
storage container.
B. Ethyl Ether - Must be free of peroxides. The test is outlined
in Section 5, A, (1) under REAGENTS.
NOTE: The ether from one manufacturer is sold in a metal can.
A polyethylene cap is provided for resealing the can
during use. It has been determined that contaminants
from the polyethylene cap can prove most troublesome,
particularly when the 200 ml of 15% ether fraction is
concentrated down to 1.0 ml in Method 5, A, (1).
C. Sodium Sulfate, Sodium Chloride and Glass Wool - These materials
used in the cleanup procedure, even of reagent quality, frequent-
ly cause interfering peaks. This is so prevalent that it is good
practice to Soxhlet extract with the solvent(s) to be used in the
method and dry in 130°C oven before use. Fifty extraction cycles
are usually sufficient to remove the impurities.
-------
Revised 12/15/79
Section 3, D
Page 1
MISCELLANEOUS INFORMATION
EVALUATION OF QUALITY OF FLORISIL
I. INTRODUCTION:
Florisil, PR grade, is available from a number of distributors or
from the Floridin Division of the Pennsylvania Glass Sand Company. It
is packed in various size units up to 20 Kilos.
The "PR" grade used for pesticide residue analysis is checked at
the producer's laboratory for activity characteristics to ensure
uniformity. However, these characteristics may vary slightly from
batch to batch, and, therefore, each new lot purchased should be
evaluated by the user to determine the elution and recovery character-
istics for the pesticides of interest in the user's laboratory.
If the material is purchased in fiber drums lined with poly-
ethylene, the evaluation sample may be drawn from the drum(s) in
accordance with the following guidelines: Immediately following
material should be transferred from the drum(s)
with foil-lined lids to avoid the possibility
the Florisil by trace quantities or organic
the evaluation, the
to glass containers
of contamination of
contaminants in the polyethylene drum liner.
II. SAMPLING:
A drum is sampled by taking six plugs from top to bottom with a
36 in. x 1 in. grain trier. The approximate plug pattern should be
as shown in the following sketch:
-------
Revised 12/15/79 Section 3, D
Page 2
The trier plugs from all drums are placed in a single container
and are mixed thoroughly. Three elution columns are prepared as
described in Section 5,A,(1), and the prepared columns are stored in
a 130°C oven at least overnight.
NOTE: If the normal procedure of the laboratory is to pack the
columns immediately before use, the prepacking of columns
for overnight activation may be avoided. In this case,
the flask of Florisil should be held in the 130°C oven at
least 16 hours before use.
III. STANDARD MIXTURES:
Prepare the following standard mixtures, using hexane or
isooctane as the solvent.
Compound
Hexachlorobenzene
Lindane
Heptachlor
Aldrin
Heptachlor epoxide
Dieldrin
Endrin
o.,£'-DDT
£,£' -ODD
j>,j>' -DDT
Mi rex
Diazinon
Methyl parathion
Malathion
Ethyl parathion
Carbophenothion
Florisil
20
20
20
40
60
40
100
80
50
80
100
2500
250
400
400
250
Standards pg/yl
6% 15% 50%
20
20
20
40
60
40
100
80
50
80
100
2500
250
400
400
250
The "Florisil" standard is used to elute through the Florisil columns. The
6%, 15%, and 50% standards are used as quantitation standards during gas
chromatographic analysis.
-------
Revised 12/15/79 Section 3, D
Page 3
IV. FLORISIL ELUTION:
All glassware used in this procedure should be meticulously
cleaned; chromic acid is recommended for thorough cleaning.
1. Remove the prepacked columns from the oven so they may cool before
use.
2. Read and record the percentage relative humidity in the room.
3. Place a beaker or flask under each column and prewet the packing
with 50 ml of petroleum ether.
NOTE: From this point on throughout the following elution
process, the solvent level should not be allowed to go
below the top of the Na2SOtt layer.
4. With 5 ml volumetric pipets, transfer 5 ml of the standard
mixture onto duplicate columns and 5 ml of hexane onto the third
column as a control.
5. Place 250 ml Kuderna-Danish assemblies with 10 ml graduated tubes
under each column and commence elution with 100 ml of diethyl
ether-petroleum ether (6:94 v/v) at a rate of 5 ml/minute.
Measure the 100 ml portion of elution solvent in a graduated
cylinder and apply to the column when the liquid level just
reaches the top of the Na2SOtt layer. At the instant the liquid
level of the first 100 ml of eluting solvent just reaches the
top of the Na2SOit layer, place a second 250 ml K-D assembly under
the column. Quickly add another 100 ml of eluting solvent and
let this solvent pass through the column.
6. Continue elution with 200 ml of diethyl ether-petroleum ether
(15:85 v/v) in two separate TOO ml portions. Collect the eluate
in two separate K-D assemblies identified as 200-300 ml and
300-400 ml.
7. Continue the elution with diethyl ether-petroleum ether (50:50
v/v), following the same procedure of collecting the 100 ml
increments that are designated as 400-500 ml and 500-600 ml.
8. Place the 18 K-D assemblies containing the 100 ml eluate incre-
ments on a 100°C steam bath and evaporate the contents to ca.
2-5 ml.
9. Evaporate remaining solvent under a nitrogen stream to 1 ml,
remove from the bath, and let cool. Dilute the samples contain-
ing pesticides with hexane to exactly 5 ml. Do not dilute the
control samples.
-------
Revised 12/15/79 Section 3, D
Page 4
10. Stopper the evaporative concentrator tubes and mix on a Vortex
mixer for 1 minute.
NOTE: Another way to evaluate the blank is to elute the column
with the full 200 ml volumes of the 6 and 15% solvents.
Then concentrate the eluate to 1.0 ml before injection.
V. GAS CHROMATOGRAPHY:
1. With a column of 1.5% OV-17/1.95% OV-210 installed in the
instrument, prime the column as described in Section 4,A and
equilibrate the instrument.
NOTE: Use only the column designated, on which the compounds
in the respective mixtures will overlap only minimally.
2. Make 5 yl injections of the fractions collected from each of the
three columns and the 6%, 15%, and 50% standards.
NOTE: In case of off-scale peaks or peaks of less than 10% FSD,
make appropriate attenuation adjustment for both standards
and eluates. For valid comparisons, measure both the
standards and the samples at the same attenuation.
VI. CALCULATIONS AND TABULATION:
1. Measure all peak heights from original standards and eluate
increments.
2. Based on peak heights measurement for each compound, calculate
the percentage of the compound appearing in each 100 ml increment
and in the original standard.
Example: Lindane, 0-100 eluate, peak ht. 30 mm
100-200 eluate, peak ht. 60 mm
Original standard 98 mm
?n
Percentages in the 0-100 eluate = 3Q " 6Q x 100 = 33%
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:
Recovery = 30 60 x 100 = 92%
-------
Revised 12/15/79 Section 3, D
Page 5
With the possible exception of aldrin, the recoveries of the
chlorinated compounds should fall in the range of 90 to 105%.
Aldrin may not exceed 80%. Some of the compounds may not yield
high recoveries. For example, trithion may yield no higher
than 40% recovery under certain conditions, as outlined in
Subsection VIII, Note 1.
4. Tabulation of results may be made on a form comparable to Table 1.
The decision of acceptance or rejection of each lot is based on a
consideration of the elution pattern and recovery efficiency of the
pesticides of interest in the program.
5. Evaluation of the control columns should also be taken into account
before the Florisil is accepted. There should be no peaks in the
chromatograms that would influence pesticide quantisation.
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 lines with polyethylene, which
may contribute unwanted contamination over a period of time.
Glass jars that have been found suitable for storage are available
from certain glass container distributors. A suitable jar bears
Owens-Illinois mold No. C-3122, with 100-400 finish, packed in cartons
of six jars. Metal screw caps with coated paper liners are used.
The jars may be washed by mechanical dishwashers, and then rinsed
with distilled water and acetone. After the jars are thoroughly dried,
the Florisil may be transferred with a 2 Ib aluminum sugar scoop
previously washed and acetone rinsed. The net content of each jar,
when filled within 1/2 inch of the rim with Florisil, is ca. 2 Ib.
A square of aluminum foil is crimped over the rim of the jar, and the
cap is screwed on tightly. Each jar is labeled with the lot number and
is now ready for storage.
VIII. NOTES:
1. Factors influencing the recovery efficiency, particularly of certain
organophosphorus compounds, include the presence of impurities in
the petroleum ether and the presence of peroxides in the diethyl
ether. This is discussed in more detail in the MISCELLANEOUS NOTES
of Section 5,A,(1).
2. The polarity of the elution solvents exerts a profound effect on
the selective elution of a number of compounds. The diethyl ether
must contain 2% v/v of ethanol to obtain compound elution patterns
comparable to those shown in Table 1. The following chart
-------
Revised 12/15/79
Section 3, D
Page 6
demonstrates the effects resulting from altering the amounts of
ethanol in the diethyl ether.
The effects of polarity variation of eluting solvent in Florisil
partitioning of 7 pesticides. Absolute diethyl ether mixed with
0, 2, and 4% absolute ethanol.
Elutioo Fraction"
Hept. Epoxide
D«ldrin
Endrin
Diozinon
Methyl Parothion
Ethyl Porolhion
Mold tt> ion
No Ethanol
I
100
n
87
100
100
16
m
13
100
64
'Eluting mixture!:
froct. ' - 6X Et,O in pet ether
Frocl.ll-15% - - - ••
Frocllll 5O% - - - -
2% Ethanol
I
100
n
100
100
100
100
100
Iff
100
Elulioo froetion"
Hepl Epo.ide
Dieldrm
Endrin
Diazinon
/Methyl Paraihior
Ethyl Porothion
Mololhion
If possible, the Florisil oven should be reserved only for
adsorbents and not used for general laboratory purposes. Any
spillage or introduction of organic materials inside the oven
may contaminate the Florisil (or other adsorbent materials)
and result in a profusion of contaminant peaks when the final
eluates are chromatographed.
In the assessment of extremely low concentrations of pesticides
in samples, it is not uncommon to concentrate the fraction
eluate(s) to as little as 1.0 ml rather than 5.0 ml. This may
pose a problem in background contamination not evident in the
5.0 ml concentrate. Scrupulous care must be taken in the
cleaning, storage, and handling of the glassware. When signif-
icant contaminant peaks are obtained with EC detection, the
operator is often inclined to fault the Florisil, which is a
possibility; however, it is far more common to find that the
actual problem is contaminated glassware.
-------
Revised 12/15/79
Section 3, D
Page 7
TABLE 1. ELUTION PATTERNS AND RECOVERY DATA FOR FLORISIL, LOT # 2854
BY METHOD SECTION 5,A,(1) (MANUAL OF ANALYTICAL METHODS)
FLORISIL COLUMN PREPACKED AND HELD IN 130°C OVEN AT LEAST 24 HOURS BEFORE USE
RELATIVE HUMIDITY IN LABORATORY 65 %
ELUTION INCREMENTS (ml)
Compound
a-BHC
3-BHC
Lindane
Heptachlor
Aldrin
Hept, Epox.
Dieldrin
Endrin
p,£'-DDE
p_,£'-DDT
jp_,£'-DDT
Ronnel
Methyl
Parathion
Malathion
Ethyl
Parathion
Diazinon
Trithion
6% Fraction
0 - 100 -
100 200
100
100
100
100
100
78 22
100
100
100
100
100
15% Fraction 50% Fraction
200 - 300 - 400 - 500 -
300 400 500 600 Recovery, %
97
95
96
91
100
105
85 15 96
89 11 99.6
97
99.6
90
93
47 53 1 03
100 99
78 22 96
100 83
43
Numerical values represent the percentage of each compound eluting
in the given fraction.
-------
-------
Revised 12/2/74
Section 3, E
Page 1
MISCELLANEOUS INFORMATION
LIMITS OF DETECTABILITY
The Analytical Chemistry Committee, comprised of representatives from
the Community Studies laboratories, the Perrine Chemistry Section and the
Division of Community Studies, Chamblee, Georgia, met in December 1969.
Among the topics discussed was that of the lower limits of detectability of
pesticidal compounds in human tissues.
The Committee recognized the necessity for
limits so that data from all laboratories would
manner.
the establishment of such
be reported in a comparable
The two tissues considered were blood and adipose tissue. The limit
recommendations were based upon data from quality control check samples,
recommendations from individual project chemists, and the experience of the
Committee members. The recommendations do not imply toxicological signifi-
cance, reflecting only the apparent analytical potential within the confines
of the currently prescribed methodology. It is entirely possible that
further studies may indicate the advisability of revising the limits. For
the present, the established limits are as follows:
Compound
a-BHC
Lindane
3-BHC
Aldrin
Heptachlor
Heptachlor Epoxide
^,J3'-DDE
J3,£'-DDE
Dieldrin
Endrin
0^2' -DDT
£,£'-DDD
£,£' -DDT
Cone, in ppb
Adipose Serum
10
10
20
10
10
10
20
10
10
20
20
20
20
1
1
1
1
1
1
1
1
1
2
2
2
2
-------
-------
Revised 12/15/79 Section 4, A, (1)
Page 1
GAS CHROMATOGRAPHY-ELECTRON CAPTURE
INSTRUMENT
In this section, operating instructions of a specific nature are intend-
ed to apply to the model Tracor 220 or 222 gas chromatograph manufactured by
Tracer, Inc., Austin, TX. This instrument may be equipped with a DC detector
containing 63Ni or 130 me 3H. However, many of the following guidelines are
broadly applicable to a wide range of chromatographic instruments.
I. FLOW SYSTEM:
The flow system consists of the entire system through which
nitrogen gas will flow, from the common point of entry at the exit of
the filter drier branching to (1) the purge line running through the
purge rotameter and flow controller thence through the detector, and
(2) the carrier flow line running through the rotameters, the flow
controllers, and the column, thence through the transfer line into the
detector.
It is essential that no leaks exist anywhere in the flow system.
Even a minute leak will result in erratic baselines with the 3H
detector. The 63Ni 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 MS-180 with the instrument
operating and observing recorder response. Spray short squirts close
to the connection. Dp_ not spray around the detector or injection port.
II. DETECTOR:
This subject is covered in detail later in Section 4, A, (3).
III. ELECTROMETER:
To ensure proper daily operation of the unit, set the attenuators
to the 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 be
obtained from the Electronics Shop at Research Triangle Park, NC.
Zero and bucking controls should operate "smoothly" and should not
cause erratic recorder response.
-------
Revised 11/1/72 Section 4, A, (1)
Page 2
Check the "maximum" polarizing voltage available. If at least
-130 v DC is not available on the rear panel, it is quite possible that
the power printed circuit board (PCB) is not functioning properly and
damage or noisy operation will result from continued use.
IV. TEMPERATURE PROGRAMMER:
The operator should be certain this unit is functioning properly.
When the unit is operating properly, the column over 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
temperature.
V. PYROMETER:
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 megohm
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-180 and wiping with dry cloth. To
prevent shorts, it is recommended 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 unused 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. MISCELLANEOUS:
A. Septums - There are a number of different types available, ranging
from the inexpensive plan black (or gray) silicone rubber to the
sophisticated "sandwich" type selling at a significantly higher
price.
-------
Revised 12/15/79 Section 4, A, (1)
Page 3
Excellent results have been reported using the blue silicone
rubber material marketed by Applied Science Laboratories as their
"W" series.
The 13 mm precut septums are available in lots of 100 under
catalog number W-13. The same material listed as "Type W" is
available in sheets of 12 in. x 12 in. About 400 13-ml septums
can be cut from this sheet with a No. 9 cork borer making the
price per hundred syptums about half that of the precut septums.
B. Column "0" Rings:
The conventional column "0" rings are of heat-resistant silicone
rubber, and must be used with brass ferrules. The "0" rings are
available in varying sizes from all suppliers of gas chromatog-
raphy accessories. The chromatographer may prefer to use Teflon
ferrules instead of brass. If these are used, no "0" rings are
required.
C. Prepurified nitrogen gas shall be used for the DC mode of opera-
tion. Argon containing 5% methane (P-5 Mix) is recommended for
linearized 63Ni detectors. This is piped to the instrument
through a filter drier of molecular sieve, 1/16" pellets, Linde
type 13X. Before the filter drier is charged with fresh molecular
sieve, the interior of the drier should be rinsed with acetone,
and the drier unit should be placed in a 130°C oven for at least
1 hour. The bronze frit should be rinsed with acetone and flamed.
After filling, the unit should be heated at 350°C for 4 hours with
a nitrogen flow of ca.90 ml/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.
NOTE: Argon-methane cannot be used for operation of the flame
photometric detector. Separate carrier gas systems must
be used on instruments equipped with linearized 63Ni
electron capture and flame photometric detectors.
-------
Revised 12/15/79 Section 4, A, (2)
Page 1
GAS CHROMATOGRAPHY-ELECTRON CAPTURE
COLUMNS
I. SPECIFICATIONS:
Column material shall be of borosilicate glass, 6 feet (1.8 m)
long, 1/4 in. (6 mm), o.d., 5/32 in. (4 mm) i.d. Because off-column
injection will be used, one side of the column shall be 1 in. longer
than the other. The Swagelok nut, ferrule and silicone "0" ring are
assembled as in Fig. 4. Complete column specifications for the
Tracer MT-220 gas chromatograph are given in Fig. 11.
II. COLUMN SELECTION:
There is a wide variety of column packing materials in the
marketplace, some of which are entirely suitable for use in pesticide
analysis, and otherswhich are of limited value. In general, the
columns selected as a "working pair" should be significantly different
in polarity and in their compound elution characteristics. One pair
that has proved very useful is given as A and C below. B provides
another alternative. The peak elution patterns for 13 chlorinated
pesticidal compounds on each of these columns are shown in Figures
1 through 3.
A. 1.5% OV-17/1.95% OV-210 - liquid phases premixed and coated on
silanized support, 80/100 mesh.
B. 4% SE-30/6% OV-210 - liquid phases premixed and coated on
silanized support, 80/100 mesh.
C. 5% OV-210 - coated on silanized support, 100/120 mesh.
III. PACKING THE COLUMN:
Make certain the column 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 Tracer Model 220 or 222, one column leg
should be 1 in. shorter than the other.
With a china marking pencil, place a mark on the long column leg
2 in. from the end. Place a similar mark 1-1/8 in. from the end of the
short leg.
Add the packing to the column through a small funnel, ca.6 in. at
a time, and bounce the column repeatedly on a semihard surface.
-------
Revised 12/15/79 Section 4, A, (2)
Page 2
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 because
the packing can be packed too densely, thus, introducing
the possibility of an excessive pressure drop when carrier
gas flow is started.
Pack silanized glass wool into both ends of the column just
tightly enough to prevent dislodging when carrier gas flow is started.
NOTE: 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 contam-
ination of the glass wool.
IV. COLUMN CONDITIONING:
The column is conditioned, or made ready for use, in two opera-
tions: (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-in. Swagelok to AN
adapter, part number 400-A-4ANF, connected to a 1/4-in. male union,
part number 400-6.
Before assembling, the bore of the union must be drilled out
with a 1/4-in. drill and burnished with a rat-tailed file so that
it will accept the 1/4-in. o.d. column glass.
with
sure
i ' _.-- __. ^ ______
The short column leg is attached to the above fitting, w
the end of the long leg venting inside the oven. The nut,
ferrule, and "0" ring are assembled as shown in Fig. 4. Make
the nut is tight, because the "0" ring will shrink during the
curing period, thus allowing carrier gas to escape.
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-in. Swagelok nut on a short piece of 6-mm glass
rod with ferrule and "0" ring.
-------
Revised 12/15/79 Section 4, A, (2)
Page 3
2. Silylating Treatment.
Treatment with a sil/lating compound such as Silyl 8 serves
to block active adsorption sites, particularly in a new column,
thereby somewhat improving efficiency and resolution character-
istics. 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-ml septum capped
bottles from the Pierce Chemical Company, P.O. Box 117, Rockford,
Illinois 61105.
At the end of the prescribed heat curing period, adjust the
oven tempset 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
injections of 25 yl each of Silyl 8, spacing the injections ca
1/2 hour apart. Allow at least three hours for the final injec-
tion to elute off the column before proceeding.
NOTES:
1. Syringe used for Silyl 8 injections should be used
for no other purpose, and should be flushed with
benzene immediately after use to avoid plugging of
the needle.
2. It is strongly advised that Silyl 8 be discarded
after one year and that fresh material be ordered;
observations in the Editors' laboratory have in-
dicated some troublesome side effects in electron
capture GLC arising from the use of old Silyl 8.
V. EVALUATION 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 injection 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 ensure 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 with a precalibrated dial face thermometer
-------
Revised 12/2/74 Section 4, A, (2)
Page 4
with the stem inserted through the oven door, or with a mercury ther-
mometer pushed down through an unused injection port. DO NOT RELY
WHOLLY ON THE INSTRUMENT PYROMETER.
Check the carrier gas flow rate using the sidearm buret device
sketched in Fig. 4 (a) attached to the purge exit of the detector.
DO NOT RELY ON THE INSTRUMENT ROTAMETER 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.
NOTE: 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 param-
eters 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 column influence may be roughly
determined by allowing several hours more for equilibration and
repeating the BGC profile. If an increase in BG current is obtained,
additional checks are made until no further increase is noted. A
typical BGC profile is shown in Fig. 5.
If the detector foil is new and the BG 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 run a
polarizing voltage/response curve as described in Subsection 4,A,(3)
OPTIMUM RESPONSE VOLTAGE. A polarizing voltage/response curve is
shown in Fig. 6.
-------
Revised 12/2/74
Section 4, A, (2)
Page 5
The operator should now be ready to chromatograph some standard
mixtures to evaluate the efficiency, resolution, compound stability
and response characteristics of the new column. A mixture that has
proved very useful in assessing performance is made up as follows,
the concentration of each compound stated in terms of picograms per
microliter:
a- BHC
3- BHC
Lindane
Heptachlor
Aldrin
10
40
10
10
20
Hept. Epoxide 30
£,£'-DDE 40
Dieldrin 50
Endrin 80
£,£'-DDD
£,£'-DDD
£,£' -DDT
£,£'-DDT
80
80
90
100
The mixture is made up in isooctane, and, if kept tightly
stoppered in the deep freeze, it should be usable for a year or more,
strictly for column evaluation purposes but not for quantisation.
Its value for column evaluation lies in the number of very closely
eluting peaks. The chromatograms in Figs. 1, 2, and 3 were obtained
from this mixture.
Several things about the new column can be learned from
chromatographing this mixture.
1
3.
The column efficiency can be determined from computation based
on the £,£'-DDT peak. The equation is given on next page.
If the computed efficiency is less than 2,700 theoretical plates,
and if the resolution between peaks is not comparable to that
shown by Figs. 1, 2 or 3, the indication is clear that something
has gone wrong in the preparation, conditioning and/or use of the
column, provided of course that high quality column packing was
used in preparation of the column.
Compute the relative retention value for £,£'-DDT and compare this
value to the values given in Table 2, a, b, or c. This should
enable the operator to determine his precise column temperature
and to relate this to the readout from pyrometer and outboard
thermometer.
Compute the absolute retention in minutes for £,£'-DDT from the
equation given below and compare with the value given on the
chromatogram furnished with the packing. If the value varies by
more than 2 minutes from the value stated in Table 2, it is
indicated that (1) one or both operating parameters are off,
-------
Revised 12/2/74
Section 4, A, (2)
Page 6
Peak A
(Aldrin)
Peak B
Injection Point
X
STT8
R>
R>
RRTn =
16.76
_x
25.4
A z
— x
(At l/4-in./min chart speed)
(At l/3-in./min chart speed)
(At l/2-in./min chart speed)
(At 2/3-in./min chart speed)
(At l-in./min chart speed)
Where N
= column efficiency in total theoretical plates.
Rx-|,x2, etc. = absolute retention, in minutes, for peak B.
= retention ratio, relative to aldrin, for peak B.
= measurements in millimeters.
RRT,,
,,
x,y,z
-------
Revised 12/2/74 Section 4, A, (2)
Page 7
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 table value
by more 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 6 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
some combination of two or more of these factors.
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, characteristics of efficiency absolute retention and
peak resolution do not compare reasonably well with the chromato-
grams and data furnished by Table I and Figures 1, 2 or 3, 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 to produce peak heights of 50 to 60% FSD.
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
breakdown peak(s) x 100 is the breakdown percentage.
-------
Revised 12/2/74 Section 4, A, (2)
Page 8
VI. MAINTENANCE AND USE OF COLUMN:
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 variables present in different laboratories.
Data from a column performance survey showed one laboratory using
the two working columns 3-1/2 months for an estimated number 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 deterioration.
In fact, the laboratory injecting fat predominantly was included
in the group showing superior overall column performance.
The Vykor glass injection insert used in off-column injections
serves as a trap to prevent a high percentage of dirty material
from befouling the front end of the column. If this insert is not
changed frequently, however, column performance characteristics
can be significantly altered. When a sufficient amount of residue
collects in this insert, lowered efficiency, compound breakdown,
peak tailing, and depressed 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 endrin 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 and lodges in the glass wool plug at the column inlet.
Indications from the survey mentioned above were that those lab-
oratories 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 column is idle overnight or over weekends, a low
carrier flow of ca 25 ml per minute through the column is advised.
Simultaneously, a purge flow of ca 25-30 ml through the detector
is also advised. If a column is out of the instrument longer than
2 or 3 days, reconditioning is advised wherein the column is not
connected to the detector, but is allowed to vent into the oven
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
-------
Revised 12/2/74 Section 4, A, (2)
Page 9
has been stable and first became erratic upon installation of a
new column, the probability of loose column connections is
indicated.
If any laboratory has trouble obtaining performance character-
istics equal to those indicated by the chromatograms and data
furnished in Table I and Figure 1, 2 or 3, 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.
VII. SOURCES OF COLUMN PACKINGS
The question is often raised concerning the advisability of a
laboratory making its own column packing or buying it precoated
from a commercial producer. If a laboratory staff member has
developed the expertise to make consistently high quality column
packing, this is the less expensive route. However, it should be
noted that few individuals possess this "knack". Coupled with
the science, there is a degree of art in the formulation of small
batch lots of quality column packing. Lacking this expertise,
the laboratory would be well advised to purchase precoated packing,
prescribing a set of quality specifications with the purchase
order. The specifications should include:
(1) A statement listing a group of pesticidal compounds
such as the list given on page 5 of this section along
with the required retention values, relative to aldrin,
at a given column temperature. This is of particular
importance for mixed liquid phase packing to ensure
the proper proportion of liquid phase components.
(2) A statement of minimum efficiency in terms of the total
theoretical plates in a 6-foot column as computed by
the method shown on page 6 of this section.
(3) A stated range of absolute retention, in minutes, for a
given compound such as p_,p_'-DDT when column is operated
at given parameters of temperature and carrier gas
velocity.
(4) A statement prescribing maximum decomposition limits
for such compounds as endrin and £,p_'-DDT under pre-
scribed operating parameters.
-------
Revised 12/2/74 Section 4, A, (2)
Page 10
VIII. MISCELLANEOUS 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 aldrin is reduced ca 25% when the off-column
is carried at 70 ml/min, 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 massive leak will be created. One easy means of
doing this is to slip swagelok fittings and an "0" ring on
the end of a short piece of 1/4 in. o.d. glass rod and install
in the unused outlet port.
3. Columns shorter than 6-ft. are generally suitable for chroma-
tography of specific, late eluting compounds as retention
time can be shortened for greater work output. However, for
multiresidue analysis on samples of unknown composition, the
shorter columns are not advised. Shorter columns are less
efficient and therefore yield much poorer peak resolution.
This can be an important factor in peak identification.
-------
Revised 12/15/79 Section 4, A, (3)
Page 1
GAS CHROMATOGRAPHY-ELECTRON CAPTURE
DETECTOR
Straight DC polarizing voltage should be supplied to the detector from
either an outboard power supply unit or a strip on the back of the electrom-
eter. Provided the column and all electronic circuits in the various
modules of the instrument are functioning properly, the degree of sensitiv-
ity in the electron capture mode relates 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. Subsequent periodic measurements should be made to
provide up-to-date information on the performance of the detector as influ-
enced by the condition of the foil or by any other effects such as column
bleed or contaminated carrier gas.
I. BACKGROUND SIGNAL PROFILE:
1. Zero recorder and electrometer in the normal manner.
2. With a well-seasoned column such as OV-17/QF-1 in the instrument,
set input attenuator on 1_0 and output attenuator on 256.
NOTE: The given attenuation values apply to electrometer Model
E2. If the dual channel, solid state unit is used, an
equivalent setting would be 102 x 128.
3. Set column and detector temp, and carrier flow rate to the levels
prescribed for the column in use. Apply ca 70 ml/minute of purge
gas.
4. Set OUTPUT POLARITY switch to the polarity opposite of that used
in normal operation.
5. Reduce polarizing voltage to zero using control on power supply
unit or in front of electrometer, and adjust bucking control of
electrometer to permit zeroing of pen on chart paper.
6. Set chart speed on 1/4 inch per minute, start chart drive, and
allow about 1/2 inch horizontal trace.
7. Advance polarizing voltage 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 produces no
further recorder deflection.
-------
Revised 12/2/74 Section 4, A, (3)
Page 2
Generally, a new detector or one with a new tritium foil should be
expected to produce a response of 60 to 80% full scale deflection. With
aging, as the response level approaches about 30% FSD, a replacement of
the foil is indicated. Figure 5 shows a background signal profile on a
detector in constant use for 2 months. At the time of original installa-
tion, the background signal profile produced 68% FSD.
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 quantisation of peaks. The
optimum response voltage is determined as follows:
1. Upon completion of the background signal profile, reset OUTPUT
POLARITY switch back to normal operating position and set
polarizing voltage control to the voltage that produced ca 60%
of the total BGC profile.
NOTE: If you are fairly certain that the optimum polarizing
voltage will fall in some fairly high range, i.e.,
20 to 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 aldrin standard in quantity known from current operation
to produce a peak about 1/2 full scale at the attenuation being
used.
NOTE: The volume injected must be carefully measured and
should not be less than 5 yl.
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.
-------
Revised 12/15/79 Section 4, A, (3)
Page 3
NOTE: Occasionally a new detector will require only around
7 volts, and it may be found that the 2.5-volt intervals
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.
7. Taking the exact peak height values, measured in millimeters,
plot a peak height vs voltage curve on linear graph paper
(Figure 6). Usually the optimum polarizing voltage is the next
voltage interval higher than the voltage producing the greatest
response, in other words, a point on the downslope of the curve.
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. The arrow in
Figure 6 indicates the voltage selected in this particular case.
III. DETECTOR LINEARITY:
In making chromatographic runs for quantisation, 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 or 102 x 8 (or 16) on the SS will preclude the
possibility of violating the linear range of the detector. If samples
are diluted so that quantifiable peaks are produced at these settings,
the large errors resulting from calculations based on nonlinear
response can be avoided.
The 63Ni detector operated in the DC mode is far more restrictive
in linearity characteristics than the 3H detector. The linearity curves
in Figure 6 illustrate the comparative linearity of 63Ni and 3H
detectors. Linearity curves should be run frequently and, most impor-
tant, on each new detector or on one subjected to overhaul.
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-------
Revised 12/15/79 Section 4, A, (3)
Page 6
IV. TRACOR LINEARIZED 63Ni ELECTRON CAPTURE DETECTOR:
The linearized EC detector is operated in the constant current
pulsed mode. As electron capturing compounds enter the detector, the
polarizing pulse frequency changes in order to keep the detector cell
current and standing current constant. The signal generated is
amplified and displayed on a strip chart.
Details of set-up, operation, theory of operation, GC parameters,
detector profiles, and circuit alignment can be found in the Operation
Manual 115314B supplied with the detector.
Advantages of the linearized EC detector include:
1. Wider linear range than the DC mode - a linear response of
+; 5% is obtainable with argon-methane (95:5 v/v) carrier gas
from 5 x 10~12 to 5 x 10~8 gram of lindane. In some cases,
operation to 2 x 10~7 gram of lindane within the above
linearity has been achieved.
2. Can be operated in a somewhat "dirty" (contaminated) condition
with less loss of performance.
3. Can tolerate more contaminants in the carrier gas.
4. Gives a generally narrower solvent front.
5. Has sensitivity comparable to the DC mode.
Disadvantages of the linearized EC detector include:
1. Argon-methane carrier gas is more expensive.
2. Nitrogen carrier gas can be substituted for argon-methane,
but the linear response range is reduced for higher
concentrations with the nitrogen carrier.
3. Linearity must be checked over each concentration range used
for actual samples.
4. Electronic alignments can be difficult.
-------
Revised 12/2/74 Section 4, A, (4)
Page 1
GAS CHROMATOGRAPHY-ELECTRON CAPTURE
CHROMATOGRAPHY 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 quantisation 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 excellent results
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; the concentration values are in nanograms per
micro!iter.
Lindane 0.5 Dieldrin 1.0
B-BHC 1.5 £,£'-DDT 1.5
Aldrin 0.5 £,£'-DDD 1.5
Hept. Epox. 1.0 £,£'-DDT 1.5
£,£'-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 injec-
tions.
In the early morning the priming may be conducted while the other daily
instruments 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 off the columns, the carrier flow should be carefully
adjusted for the working column using the bubble device shown in Figure 4(a).
The chromatograph should now be ready for the first working standard injec-
tion.
A sample extract concentration of 10 ml from a 5 gram sample contains
the tissue equivalent of 0.5 milligram per micro!iter. A 5 pi injection
of this extract (2.5 milligrams of sample) into an EC detector of average
sensitivity should easily produce quantifiable peaks at pesticide concentra-
tions 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 not more
-------
Revised 12/2/74 Section 4, A, (4)
Page 2
than 20 ml/min on this alternative column or, if preferred,
leave carrier flow at zero on a column of high thermal stability.
Set attenuation at an estimated appropriate sensitivity.
NOTE: The specified GLC instrument has a high sensitivity
potential provided that all modules are functioning
properly. It is important to take full advantage of
this potential by avoiding low sensitivity attenuation.
With a new detector foil, low sensitivity attenuation
may be necessary, but as the BGC 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 1%
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 1% full scale, some electronic
trouble shooting may be indicated.
2. If a column is used for which an RRT/Temp. table is available
(Tables 2, a, b, or c), the procedure for tentative peak identif-
ication in an unknown is relatively simple and requires far less
time than traditional "cut and try" methods. First it is
necessary to establish the prevalent true column temperature.
This is determined by chromatographing a standard mixture con-
taining aldrin and £,£'-DDT. Other compounds eluting earlier
than £,£'-DDT may be included, but their presence may be irrel-
evant for this mission. Calculate from the chromatogram the
RRTfl of £,£'-DDT, then by scanning horizontally across the column
opposite £,£'-DDT on the table, locate the RRTA value which most
closely matches the calculated value. The actual column tempera-
ture can now be obtained by reference to the top or bottom of
the table.
3. Inject 5 yl of the sample extract as a preliminary run to deter-
mine whether all peaks are on scale and are of quantifiable peak
height. If off-scale peaks are observed, make an estimated
dilution of a portion of the extract and reinject.
NOTE: Injections of volumes less than 5.0 yl should be avoided
in quantisation. The possibilities for error are greatly
enhanced by low volume injections.
-------
Revised 12/2/74 Section 4, A, (4)
Page 3
4. Calculate the RRT/\ values for all peaks appearing on the sample
chromatogram(s). By vertically scanning the appropriate temper-
ature column on the table, the calculated RRT^ values may be
compared with table values to obtain tentative peak identifica-
tions.
5. The information derived from Step 4 above should provide the
operator with sufficient intelligence re tentative compound
identities and estimated concentration ranges to facilitate
the selection of an appropriate working standard mixture for
precise quantisation. Subsequent injections of standards will
then be carried out bearing in mind that (1) peak heights
between sample and standard should vary not more than 25%,
(2) the concentration of all compounds must fall well within
the linear range of the detector, and (3) no peak of less than
10% FSD should be quantitated.
6. At this point the task of compound identification is incomplete,
and confirmation must be conducted on an alternative column of
completely different polarity (see Section 4,A,(2).
The chromatographer must be constantly aware that artifact peaks
may be obtained with one column which may have identical RRT^
values with certain pesticidal compounds; also that a number of
pesticidal compounds may have identical or near identical RRT/\
values on a given column. The last point must be carefully
considered in the selection of an alternative column that will
resolve such overlaps.
MISCELLANEOUS 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 mm 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.
-------
Revised 12/2/74 Section 4, A, (4)
Page 4
3. Electrometer attenuation should be adjusted to obtain a minimum
sensitivity level of a peak of 50% FSD resulting from the
injection of 100 picograms of aldrin.
4. Quantisation by referencing sample peaks against a standard curve
may be an acceptable practice provided that certain limitations
are carefully considered. It must be recognized that repetitive
injections of certain sample extracts may gradually depress
response characteristics of the GLC system. When this occurs,
a curve established from a standard or mixture of standards at
9 AM on a given day may be worthless by 11 AM on the same day.
This possibility must be monitored by interspersing standard
injections continually throughout the work day. In view of this
requirement, the construction of a curve becomes a superfluous
and unnecessary task as quantitative referencing can be made
against the interspersed standards.
5. At this point, detailed evaluations are made of all chromatograms.
If there is reason to suspect any peak identification or quanti-
tation, instrumental controls should be switched over to alterna-
tive column for further scrutiny. The isomers of BHC, o^jD'-DDE,
and Ojj}'-DDT frequently pose identification problems. If such
identification problems are present and cannot be confidently
resolved by any of the three prescribed columns, further confir-
matory work is required by electrolytic conductivity detection
and/or by TLC.
-------
Revised 12/2/74 Section 4, A, (5)
Page 1
GAS CHROMATOGRAPHY-ELECTRON CAPTURE
QUANTITATION AND INTERPRETATION
There are several methods for quantitating chromatographic peaks.
While we are not partial to any particular method, it is desirable in a
system of laboratories providing data to a central point that some degree
of uniformity be specified.
The preferred method of calculation is somewhat dependent on peak
shape. The major categories of peak shapes are: (1) Tall, narrow, and
symmetrical, generally illustrated by a £,jD'-DDT peak from a clean extract,
(2) Overlapping peaks where the overlap is estimated not to obscure the
peak height, (3) Unsymmetrical peaks such as are commonly encountered in
an uncleaned extract.
Broadly speaking, quantitation methods recommended for the various
types of peaks are:
I. PEAK HEIGHT:
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
(Figure 7).
II. PEAK HEIGHT X WIDTH AT HALF HEIGHT:
A. Separated, symmetrical, and fairly wide peaks (Figure 8).
III. TRIANGULATION OR INTEGRATION:
A. Separated unsymmetrical 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
EC 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.
-------
Revised 12/15/79 Section 4, A, (5)
Page 2
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 inter-
pretation is one requiring careful study of the data and the applica-
tion of sound judgment. The presence of chromatographic peaks which
precisely match the absolute and relative retention values of those of
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 same as a-BHC and/or £,p>DDE. Confirmation by ancillary
techniques has never supported the electron capture detector indica-
tions, however. In one instance methyl parathion was reported in a
blood sample. Had the individual conducting the interpretation
exercised sound judgment, it should have been immediately apparent that
the presence of the parent compound of parathion 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 electron
capture 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, some
further confirmation is required. This may be done by (1) using an
alternative column and electron capture detection, (2) applying elec-
trolytic conductivity detection, (3) thin-layer chromatography, (4)
chemical derivatization, (5) gas chromatography-mass spectrometry, or
(6) high performance column liquid chromatography.
-------
Revised 6/77 Section 4, A, (6)
Page 1
TABLES AND FIGURES
Tables and figures in this section will assist the analyst in column
selection and operation by providing retention data on compounds for tenta-
tive identification of unknown peaks in a multiresidue analysis (see Sub-
section 4,A,(4).
Figures 1 through 3d are typical chromatograms of a 13-compound mixture,
each column operated at a temperature and carrier gas flowrate providing
maximum efficiency with reasonable retention times. Since the parameters may
differ widely, comparisons of retention parameters on different chromatograms
should not be made. For example, retention times in Figures 3a and 3d are
not directly comparable because chart speeds were 0.5 and 0.25 inch per
minute, respectively.
Because the elution pattern of compounds with DEGS differs from the
patterns from most other columns used in pesticide analysis, the DEGS column
(Figure 3a) often proves useful in resolving problems relating to peak
identification. Field reports, however, warn of bleed problems with DEGS and
suggest that the DEGS column not be used for a "working" column since it is
likely to foul the detector. For the brief periods the column is used for
confirmations, bleed effects are not apt to be troublesome.
Stationary phases that are chemically similar and give similar elution
patterns are listed in the following table. For example, the elution pattern
for a given pesticide mixture for 5% DC-200 will be very similar to that from
5% SE-30 or OV-101.
-------
Revised 6/77 Section 4, A, (6)
Page 2
Stationary Phases Commonly Used in Pesticide Analysis
No.
Designation Chemical Name Similar Phases
1. DC-200 Methyl silicone OV-1, OV-101, SE-30,
SP-2100, DC-11, SF-96
2. QF-1 Trifluoropropyl methyl silicone OV-210, SP-2401
3. SE-30 (See No. 1)
4. SE-52 Methyl silicone, 10% phenyl OV-3
substituted
5. SF-96 (See No. 1)
6. XE-60 Cyanoethyl methyl silicone OV-225
7. OV-17 Methyl silicone, 50% phenyl SP-2250
substituted
8. OV-7 Methyl silicone, 20% phenyl
substituted
9. OV-210 (See No. 2)
10. DEGS Diethyleneglycol succinate none
-------
Revised 12/2/75 Section 4. A, (6)
Page 3
TABLE 1. CONDITIONING, OPERATION PARAMETERS AND PERFORMANCE EXPECTATIONS FOR 6-FT. X 1/4-IN. O.D. COLUMNS OF
PRECOATEO PACKINGS, 3t DEGS INCLUDED SOLELY AS A CONFIRMATORY COLUMN, NOT FOR ROUTINE USE.
Parameters
Liquid phase(s)
Solid Support
Heat Curing Temp °C
Time Hours
Operating Temp °C
Detector Temp °C (tritium)
Carrier Flow ml/minute
Elution Time for p_,p'-DDT
Approx. (minute)
1.52: OV-17
-""^TTgW OV-210
Silicone OV-17
Silicone DC QF-1
(FS1265)
Chromosorb W, H.P.
or Gas-Chrom Q
100/120 mesh
245
48 (minimum)
200
205
50-70
16-20
45! SE-30
^""M OV-210
Silicone SE-30
Silicone DC QF-1
(FS1265)
Chromosorb W, H.P.
or Gas-Chrom Q
80/100 mesh
245
72 (minimum)
200
205
70-90
16-20
5% OV-210
OV-210
Tri f 1 uoromethyl propyl
Silicone
Chromosorb W, H.P.
or Gas-Chrom 0
100/120 mesh
245
48 (minimum)
180
205
45-60
16-20
n DEGS
DEGS
Stabilized
Diethylene
Glycol
Succinate
(Analabs C4)
Gas-Chrom P
80/100 mesh
235
20 (exact)
195
205
70-90
16-20
Expected Minimum
Efficiency (Total theor.
plates in 6-ft. column
basis p,£/-DDT)
3000
3000
3000
2800
-------
Revised 6/77
Table 2(a)
Section 4, A, (6)
1.5%OV-17/1.95% OV-210
Column
170
|
0.25
0.32
*0.34
0.38
0.44
0.42
0.48
0.54
0.56
0.54
0.67
0.65
0.66
0.76
0.82
0.82
0.94
1.00
1.17
1.17
1.49
1.41
1.49
1.71
1.70
1.82
2.07
1.92
2.02
2.14
2.32
2.15
2.20
2.75
2.97
2.80
3.34
3.26
3.47
3.98
4.65
4.45
5.57
6.1
6.4
7.7
10.7
13.1
12.4
16.9
22.1
1
170
174
1
0.25
0.32
0.34
0.38
0.45
0.42
0.48
0.54
0.56
0.54
0.67
0.65
0.67
0.76
0.82
0.82
0.94
1.00
1.16
1.16
1.48
1.40
1.49
1.69
1.69
1.80
2.04
1.91
2.00
2.12
2.28
2.13
2.18
2.72
2.93
2.77
3.29
3.23
3.43
3.94
4.57
4.39
S.43
5.97
6.2
7.6
10.5
12.7
12.1
16.5
21.5
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
26
32
35
38
45
43
49
54
56
55
66
65
67
76
82
0.82
0
1
1
1
1
1
1
1
1
1
2
1
1
2
2
2
2
2
2
2
3
3
3
3
4
4
5
5
6
7
10
12
11
16
20
93
00
15
16
45
39
47
67
68
78
01
89
99
09
25
11
16
68
88
75
25
19
40
88
49
34
39
85
1
5
3
4
8
1
9
174
1
0.26
0.32
0.35
0.38
0.45
0.43
0.49
0.54
0.56
0.55
0.66
0.65
0.67
0.76
0.82
0.82
0.92
1.00
1.14
1.15
1.43
1.38
1.47
1.66
1.67
1.76
1.E8
1.88
1.97
2.07
2.22
2.09
2J5
2.64
2.84
2.72
3.20
3.16
3.36
3.83
4.41
4.28
5.29
5.73
5.99
7.3
10.1
12.0
11.6
15.7
20.3
I
178
|
0.26
0.32
0.36
0.39
0.45
0.44
0.50
0.55
0.56
0.56
0.66
0.66
0.67
0.75
0.81
0.82
0.92
1.00
1.13
1.14
1.41
1.36
1.46
1.64
1.66
1.74
1.95
1.85
1.95
2.05
2.19
2.07
2.13
2.61
2.79
2.69
3.15
3.13
3.33
3.77
4.33
4.23
5.20
5.61
5.88
7.3
9.9
11.6
11.3
15.3
19.6
1
178
1
0.27
0.32
0.36
0.39
0.45
0.44
0.50
0.55
0.56
0.56
0.66
0.66
0.67
0.75
0.81
0.32
0.91
1.00
1.11
1.14
1.40
1.35
1.45
1.62
1.65
1.72
1.92
1.85
1.93
2.03
2.16
2.05
2.11
2.58
2.75
2.67
3.11
3.09
3.29
3.71
4.26
4.17
5.11
5.49
5.76
7.1
9.7
11.2
11.0
14.9
19.0
1
182
|
0.27
0.32
0.36
0.39
0.45
0.44
0.50
0.55
0.56
0.56
0.66
0.66
0.67
0.75
0.81
0.82
0.90
1.00
1.10
1.13
1.38
1.34
1.44
1.60
1.64
1.70
1.89
1.83
1.92
2.01
2.13
2.03
2.10
2.54
2.71
2.64
3.06
3.06
3.26
3.66
4.18
4.11
5.01
5.36
5.64
7.0
9.5
10.8
10.7
14.5
18.4
1
182
Temperature , °C. I
186 190 194 198 f
I, *
1 1 1
0.27 0.28 0.28 0.28 0.29 0.29 0.29 0.30 0.30
0.32 0.32 0.32 0.33 0.33 0.33 0.33 0.33 0.33
0.37 0.37 0.38 0.38 0.38 0.39 0.39 0.40 0.40
0.39 0.39 0.40 0.40 0.40 0.40 0.41 0.41 0.41
0.46 0.46 0,46 0.46 0.46 0.46 0.47 0.47 0.47
0.45 0.45 0.45 0.46 0.46 0.47 0.47 0.48 0.48
0.51 0.51 0.52 0.52 0.52 0.53 0.53 0.54 0.54
0.55 0.55 0.55 0.55 0.55 0.56 0.56 0.56 0.56
0.56 0.56 0.56 0.56 0.56 0.56 0.56 0.56 0.56
0.57 0.57 0.58 0.58 0.58 0.59 0.59 0.60 0.60
0.65 0.65 0.65 0.65 0.65 0.65 0.54 0.64 0.64
0.66 0.66 0.67 0.67 0.67 0.67 0.67 0.68 0.68
0.68 0.68 0.68 0.68 0.68 0.68 0.69 0.69 0.69
0.75 0.75 0.74 0.74 0.74 0.74 0.73 0.73 0.73
0.81 0.81 0.81 0.81 0.80 0.80 0.80 0.80 0.80
0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82 0.82
0.90 0.89 0.88 0.88 0.87 0.37 0.36 0.85 0.85
1.00 1.00 1.00 1.00 1.00 1.00 l.CO 1.00 1.00
1.09 1.08 1.07 1.06 1.05 1.03 1.02 1.01 1.00
1.12 1.12 1.11 1.10 1.09 1.0° 1.08 1.07 1.07
1.36 1.34 1.32 1.31 1.29 1.27 1.25 1.23 1.22
1.33 1.32 1.31 1.30 1.29 1.23 1.27 1.26 1.25
1.44 1.43 1.42 1.42 1.41 1.40 1.39 1.39 1.38
1.59 1.57 1.55 1.53 1.52 1.50 1.48 1.47 1.45
1.63 1.62 1.61 1.59 1.58 1.57 1.56 1.55 1.54
1.68 1.66 1.64 1.62 1.60 1.58 1.56 1.54 1.52
1.87 1.34 1.81 1.78 1.75 '1.72 1.69 1.66 1.63
1.81 1.80 1.78 1.77 1.75 1.74 1.72 1.71 1.69
1.90 1.88 1.86 1.85 1.83 1.82 1.79 1.78 1.75
1.98 1.96 1.94 1.92 1.30 1.88 1.86 1.84 1.82
2.09 2.06 :.03 2.00 1.97 1.93 1.90 1.87 1.84
2.01 1.99 1.97 1.96 1.94 1.92 1.90 1.88 1.86
2.08 2.06 2.05 2.03 2.01 2.00 1.98 1.37 1.95
2.51 2.47 2.43 2.40 2.37 2.33 2.30 2.27 2.23
2.66 2.62 2.57 2.53 2.49 2.44 2.40 2.35 2.31
2.61 2.59 2.56 2.53 2.51 2.48 2.45 2.43 2.40
3.01 2.97 2.92 2.88 2.83 2.77 2.74 2.59 2.65
3.03 3.00 2.96 2.93 2.90 2.37 Z.S3 2.80 2.77
3.22 3.18 3.15 3.12 3.08 3.04 3.01 2.97 2.93
3.60 3.54 3.48 3.43 3.38 3.32 3.27 3.21 3.16
4.10 4.02 3.94 3.87 3.79 3.71 3.64 3.61 3.48
4.05 3.99 3.94 3.88 3.82 3.76 3.71 3.65 3.59
4.92 4.83 4.74 4.64 4.55 4.46 4.36 4.27 4.18
5.24 5.12 5.00 4.88 4.76 4.64 4.52 4.40 4.28
5.52 5.40 5.28 5.16 5.04 4.92 4.30 4.68 4.E6
6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1
9.3 9.1 8.9 8.7 8.5 8.3 8,1. 7.9 7.7
10.4 10.0 9.7 9.3 8.9 8.5 8.1 7.7 7.3
10.4 10.1 9.8 9.5 9.3 9.0 8.7 8.4 8.1
14.1 13.7 13.3 12.9 12.5 12.1 11.7 11.3 10.9
17.7 17.1 16.5 15.8 15.2 14.6 14.0 13.4 12.7
1 1 | 1 | 1
186 190 194 198
202
0.30
0.33
0.40
0.41
0.47
0.48
0.54
0.56
0.56
0.60
0.64
0.68
0.69
0.73
0.80
0.82
0.84
1.00
0.99
1.06
1.20
1.24
1.37
1.43
1.53
1.50
1.60
1.68
1.74
1.79
1.81
1.84
1.93
2.20
2.27
2.37
2.60
2.74
2.90
3.10
3.40
3.54
4.09
4.16
4.44
6.0
7.5
7.0
7.8
10.5
12.1
202
Page 4
204
|
0.31
0.33
0.41
0.41
0.47
0.49
0.55
0.56
0.56
0.61
0.64
0.68
0.69
0.73
0.80
0.82
0.83
1.00
0.98
1.05
1.18
1.23
1.36
1.41
1.52
1.48
1.57
1.66
1.73
1.77
1.78
1.82
1.91
2.17
2.22
2.35
2.56
2.70
2.87
3.0*
3.32
3.48
4.00
4.04
4.32
5.85
7.3
6.6
7.5
10.2
11.5
1
204
Compound
•Dimethyl Phthalate
Mevlnphos
Tecnazene
Dlethyl Phthalate
2.4-n(ME)
Hexachlorobenzene
C.-BHC
COEC
2,4-D(IPE)
Chlordene
Olazlnon
PCNB
Llndane
2.4,5-T(ME)
e-BHC
Heptachlor
2,4,5-T(IPE)
Aldrin (REFERENCE)
Dlmethoate
Ronnel
01 butyl Phthalate
1-Hydroxychlordene
Oxychlordane
M. Parathlon
Heptachlor Epoxi'de
DCPA
Malathion
Chlordane, Gamma
rroKs-Nonachlor
o,p'-DDE
£. Parathlon
Chlordane, Alpha
Endosulfan I
p,p'-DDE
*DDA(ME)
Dieldrln
o,p'-DOD
Chlordecone
Endrin
o,p'-DDT
p,p'-DDD
Endosulfan II
n,p'-DDT
Tthlon
Carbophenothlon
M1rex
Endrin Ketone "153"
Dioctyl Phthalate
Methoxychlor
Tetradi fon
Dlphenyl Phthalate
Retention ratios, relative to aldrin, of 49 compounds at temperatures from
170 to 2C4*C> support of Gas Chrom Q, 100/120 mesh; electron capture detector;
tritium source, parallel plats; all absolute retentions measured from injection
point. Arrow indicated optimum column operating temperature vith carrier flow
at 60 ml per minute.
-------
Revised 6/77
170 174
I
6.25
0.27
0.34
0.39
0.39
0.39
0.42
0.44
0.54
0.54
0.54
0.57
0.60
0.59
0.66
0.80
0.89
0.96
1.01
1.00
1.04
1.41
1.43
1.49
1.53
1.64
1.70
1.67
1.65
1.84
1.86
2.09
1.99
2.16
2.27
2.34
2.43
3.02
2.76
3.22
2.97
3.19
4.08
4.04
4.08
6.7
6.1
7.3
11.0
12.2
11.6
1
170
0.25
0.28
0.34
0.39
0.39
0.40
0.43
0.44
0.54
0.54
0.54
0.57
0.60
0.59
0.66
0.80
0.89
0.95
1.00
1.00
1.04
1.39
1.42
1.48
1.53
1.63
1.68
1.66'
1.64
1.83
1.85
2.07
1.98
2.14
2.25
2.31
2.41
2.97
2.73
3.17
2.94
3.16
4.02
3.98
4.02
6.5
6.0
7.2
10.8
11.9
11.3
1
0.26
0.28
0.34
0.39
0.39
0.40
0.43
0.45
0.54
0.55
0.55
0.57
0.60
0.60
0.66
0.81
0.88
0.94
1.00
1.00
1.04
1.37
1.42
1.47
1.52
1.61
1.66
1.65
1.63
1.82
1.33
2.05
1.97
2.11
2.22
2.29
2.39
2.93
2.71
3.13
2.91
3.13
3.96
3.92
3.98
6.4
5.96
7.1
10.5
11.6
11.1
174
1
0.26
0.28
0.35
0.39
0.40
0.41
0.44
0.45
0.54
0.55
0.55
0.58
0.60
0.60
0.66
0.81
0.88
0.94
0.99
1.00
.03
.36
.41
.46
.51
1.60
1.64
1.63
1.62
1.80
1.82
2.02
1.95
2.09
2.19
2.27
2.37
2.88
2.69
3.08
2.89
3.10
3.89
3.86
3.90
6.3
5.87
7.0
10.2
11.3
10.8
i
178
|
0.26
0.29
0.35
0.40
0.40
0.41
0.44
0.46
0.54
0.55
0.56
0.58
0.59
0.60
0.65
0.81
0.87
0.93
0.98
1.00
1.03
1.34
1.40
1.45
1.50
1.59
1.63
1.62
1.61
1.79
1.81
2.00
1.94
2.07
2.16
2.24
2.35
2.84
2.67
3.04
2.86
3.07
3.81
3.80
3.83
6.1
5.78
6.9
9.9
11.0
10.6
1
178
Table 2(b)
4%SE-30/6%OV-210
Column Temperature , °C. 1
182 186 190 194 198 *
1
0.27
0.29
0.36
0.40
0.40
0.41
0.44
0.46
0.55
0.56
0.56
0.58
0.59
0.61
0.65
0.81
0.87
0.93
0.98
1.00
1.03
1.33
1.40
1.44
1.50
1.57
1.61
1.60
1.61
1.78
1.80
1.98
1.93
2.05
2.13
2.22
2.33
2.80
2.64
2.98
2.83
3.04
3.76
3.73
3.78
5.98
5.68
6.8
9.6
10.7
10.3
1
I
0.27
0.29
0.36
0.40
0.41
0.42
0.45
0.46
0.55
0.56
0.56
0.59
0.59
0.61
0.65
0.81
0.87
0.92
0.97
1.00
1.03
1.3?
1.39
1.43
1.49
1.56
1.59
1.59
1.60
1.77
1.78
1.96
1.91
2.02
2.10
2.19
2.31
2.76
2.62
2.94
2.80
3.00
3.68
3.67
3.72
5.84
5.60
6.7
9.3
10.4
10.1
1
182
1
0.27
0.30
0.36
0.40
0.41
0.42
0.45
0.47
0.55
0.57
0.57
0.59
0.59
0.61
0.65
0.82
0.86
0.91
0.96
1.00
1.03
1.30
1.39
1.42
1.48
1.54
1.57
1.57
1.59
1.76
1.77
1.94
1.90
2.00
2.07
2.17
2.29
2.73
2.60
2.90
2.73
2.97
3.60
3.61
3.56
5.70'
5.52
6.5
9.0
10.1
9.8
— 1
0.28
0.30
0.37
0.40
0.42
0.43
0.46
0.47
0.55
0.57
0.57
0.59
0.59
0.62
0.65
0.82
0.86
0.91
0.96
1.00
1.03
1.28
1.38
1.41
1.48
1.53
1.55
1.56
1.58
1.75
1.76
1.91
1.89
1.98
2.04
2.15
2.27
2.68
2.58
2.86
2.75
2.94
3.53
3.54
3.59
5.57
5.43
6.4
8.7
9.8
9.6
1
186
,1,1,
0.28 0.23 0.29 0.29 0.29 0.29 0.30
0.30 0.30 0.31 0.31 0.31 0.32 0.32
0.37 0.38 0.38 0.38 0.39 0.39 0.40
0.41 0.41 0.41 0.41 0.41 0.42 0.42
0.42 0.42 0.43 0.43 0.43 0.44 0.44
0.43 0.43 0.44 0.44 0.45 0.45 0.45
0.46 0.47 0.47 0.48 0.48 0.48 0.49
0.48 0.48 0.48 0.49 0.49 0.50 0.50
0.55 0.55 0.55 0.55 0.55 0.55 0.55
0.57 0.58 0.58 0.59 0.59 0.59 0.60
0.58 0.58 0.58 0.59 0.59 0.60 0.60
0.59 0.60 0.60 0.60 0.60 0.61 0.61
0.59 0.59 0.58 0.58 0.58 0.58 0.58
0.62 0.62 0.63 0.63 0.63 0.64 0.64
0.65 0.65 0.65 0.64 0.64 0.64 0.64
0.82 0.82 0.82 0.83 0.83 0.63 0.83
0.85 0.85 0.85 0.84 0.84 0.83 0.83
0.90 0.89 0.89 0.88 0.87 0.87 0.86
0.95 0.94 0.94 0.93 0.92 0.92 0.91
1.00 1.00 1.00 1.00 1.00 1.00 1.00
1.03 1.03 1.03 1.02 1.02 1.02 1.02
1.27 1.25 1.24 1.22 1.21 1.19 1.18
1.37 1.37 1.36 1.36 1.35 1.34 1.34
1.40 1.39 1.38 1.37 1.36 1.35 1.34
1.47 1.46 1.46 1.45 1.44 1.44 1.43
1.52 1.51 1.49 1.48 1.47 1.45 1.44
1.53 1.51 1.49 1.48 1.46 1.44 1.42
1.55 1.53 1.52 1.50 1.49 1.47 1.46
1.57 1.56 1.55 1.54 1.54 1.53 1.52
1.74 1.73 1.71 1.70 1.69 1.68 1.67
1.74 1.73 1.72 1.71 1.69 1.68 1.67
1.89 1.87 1.85 1.83 1.80 1.78 1.76
1.87 1.86 1.85 1.83 1.82 l.SO 1.79
1.95 1.93 1.91 1.89 1.86 1.84 1.82
2.01 1.98 1.96 1.93 1.90 1.S7 1.84
2.12 2.10 2.07 2.05 2.03 2.00 1.98
2.25 2.22 2.20 2.18 2.16 2.14 2.12
2.64 2.50 2.56 2.52 2.47 2.43 2.39
2.55 2.53 2.51 2.49 2.46 2.44 2.42
2.82 2.77 2.73 2.68 2.64 2.59 2.55
2.72 2.69 2.67 2.64 2.61 2.59 2.56
2.91 2.88 2.85 2.81 2.78 2.75 2.72
3.47 3.40 3.32 3.27 3.20 3.13 3.05
3.48 3.43 3.36 3.30 3.24 3.18 3.12
3.52 3.47 3.40 3.34 3.28 3.22 3.16
5.42 5.29 5.16 5.01 4.88 4.73 4.60
5.33 5.24 5.15 5.06 4.97 4.88 4.79
6.3 6.2 6.1 5.98 5.84 5.75 5.64
8.4 8.1 7.8 7.5 7.2 6.9 6.6
9.5 9.2 8.9 8.6 8.3 8.0 7.7
9.3 9.0 8.8 8.5 8.3 8.0 7.8
| , 1 1 i 1
190 194 198
202
1
0.30
0.32
0.40
0.42
0.44
0.46
0.49
0.50
0.55
0.60
0.60
0.61
0.58
0.64
0.64
0.83
0.83
0.85
0.90
1.00
1.02
1.16
1.33
1.33
1.42
1.43
1.40
1.45
1.52
1.66
1.66
1.74
1.78
1.80
1.81
1.96
2.10
2.35
2.40
2.51
2.53
2.69
2.98
3.05
3.10
4.46
4.70
5.53
6.4
7.4
7.5
2C2
Section 4,A,(6)
Page 5
204
1
0.30
0.33
0.40
0.42
0.45
0.46
0.50
0.51
0.55
0.61
0.61
0.61
0.58
0.65
0.64
0.83
0.82
0.85
0.90
1.00
1.02
1.14
1.33
1.32
1.42
1.41
1.38
1.43 '
1.50
1.65
1.64
1.72
1.76
1.77
1.78
1.93
2.03
2.31
2.37
2.46
2.50
2.66
2.90
2.98
3.03
4.32
4.62
5.42
6.1
7.1
7.3
1
204
Compound
Dimethyl Phthalate
Mevinphos
Tecnazene
01 ethyl Phthalate
2.4-D(ME)
Hexachlorobenzene
o-BHC
CDEC
2.4-DUPE)
Lindane
Chlordene
S-BHC
Dlazlnon
PCNB
2,4,5-T(ME)
Heptachlor
2,4,5-T(IP£)
Dlmethoate
Ronnel
Aldrin (REFERENCE)
l-Hydroxych1ordene
Dibutyl Phthalate
Oxychlordane
M. Parathlon
Heptachlor Epoxfde
D C P A
Malathlon
o,p'-DOE
Chlordane, Gaima
Chlordane, Alpha
rrone-Nonachlor
E. Parathlon
Endosulfan I
p,p'-DDE
"DOA(ME)
o.p'-DOO
Uie'ldrln
0,p'-DDT
Endrin
p,p'-DDO
Chlordecone
Endosulfan II
Ethlon
p,p'-DDT
CarbophenotMon
Methoxycblor
M1rex
Endrin Ketone"153"
Dlocty! Phthalate
Dlphenyl Phthalate
Tetradlfon
Retention ratios, relative to aldrin, of 49 compounds at temperatures from
170 to 204°Cs support of Gas Chrom C, 80/100 mesh; electron capture detector;
tritium source, parallel plate; all absolute retentions measured from injection
point. Arrow indicated optimum column operating temperature with carrier tlow
at 70 ml per minute.
-------
Revised 6/7
170
1
0.43
0.51
0.52
0.58
0.58
0.65
0.69
0.73
0.75
0.78
0.35
0.83
0.36
0.93
1.00
1.41
1.44
1.59
1.66
1.38
1.88
1.96
2.05
2.25
2.21
2.21
2.54
2.60
2.69
2.83
2.97
3.00
2.95
3.08
3.71
4.01
4.45
4.15
4.38
4.78
5.28
5.90
7.3
13.5
12. 9
20.0
21.0
170
I
0.43
0.51
0.53
0.59
0.58
0.66
0.69
0.73
0.75
0.79
0.85
0.33
0.86
0.93
1.00
1.39
1.43
1.58
1.64
1.37
1.87
1.94
2.03
2.21
2.18
2.19
2.52
2.58
2.65
2.79
2.92
2.95
2.91
3.05
3.66
3.94
4.31
4.09
4.31
4.70
5.17
5.77
7.1
13.1
12.5
19.4
20.4
7
174
0.44
0.51
0.53
0.59
0.59
0.66
0.69
0.72
0.75
0.79
0.85
0.34
0.87
0.92
1.00
1.38
1.42
1.57
1.53
1.35
1.85
1.92
2.02
2.17
2.16
2.16
2.49
2.55
2.61
2.76
2.86
2.89
2.87
3.01
3.51
3.88
4.17
4.03
4.23
4.63
5.06
5.63
6.9
12.7
12.3
13.9
19.7
1
174
1
0.45
0.51
0.54
0.60
0.60
0.66
0.69
0.72
0.74
0.79
0.84
0.84
0.87
0.92
1.00
1.37
1.41.
1.56
1.61
1.83
1.83
1.91
2.00
2.13
2.13
2.13
2.46
2.53
2.57
2.72
2.31
2.84
2.82
2.98
3.56
3.81
4.04
3.93
4.16
4.55
4.95
5.50
6.7
12.3
12.0
13.3
19.1
i
173
1
0.45
0.52
0.54
0.60
0.61
0.67
0.69
0.72
0.74
0.79
0.34
0.84
0.87
0.92
1.00
1.35
1.40
1.55
1.60
1.81
1.82
1.89
1.98
2.10
2.10
2.11
2.44
2.50
2.55
2.69
2.75
2.79
2.73
2.94
3.51
3.74
3.90
3.92
4.08
4.48
4.84
5.36
6.55
11.9
11.3
17.3
13.5
1
173
t
i
0.46
0.52
0.55
0.61
0.62
0.67
0.69
0.72
0.74
0.79
0.84
0.34
0.87
0.92
l.CO
1.34
1.39
1.54
1.58
1.80
1.80
1.87
1.95
2.06
2.09
2.09
2.41
2.43
2.49
2.66
2.70
2.73
2.74
2.91
3.46
3.67
3.76
3.86
4.01
4.40
4.73
5.23
6.4
11.4
11.5
17.3
17.8
i
Table 2(c)
5% OV-210
Column Temperature , °C.
182 135 190 194
0.46
0.52
0.55
0.61
0.62
0.67
0.69
0.72
0.74
0.30
0.34
0.34
0.87
0.92
1.00
1.33
1.38
1.53
1.56
1.78
1.78
1.85
1.94
2.02
2.04
2.05
2.38
2.46
2.45
2.62
2.65
2.68
2.70
2.37
3.41
3.60
3.62
3.80
3.93
4.33
4.62
5.09
6.2
n.o
11.2
16.7
17.2
1
132
i 1
0.47 0.48
0.53 0.53
0.55 0.56
0.62 0.62
0.63 0.64
0.57 0.68
0.69 0.69
0.71 0.71
0.73 0.73
0.30 0.80
0.84 0.83
0.34 0.34
0.37 0.87
0.92 0.92
1.00 1.00
1.32 1.30
1.37 1.36
1.52 1.51
1.55 1.53
1.76 1.74
1.77 1.75
1.84 1.83
1.92 1.90
1.99 1.95
2.01 1.98
2.02 2.00
2.35 2.33
2.43 2.41
2.41 2.37
2.59-2,55
2.59 2.54
2.63 2.58
2.66 2.61
2.34 2.80
3.36 3.31
3.53 3.46
3.49 3.35
3.74 3.69
3.85 3.78
4.25 4.18
4.51 4.40
4.96 4.82
6.0 5.84
10. S 10.2
10.9 10.5
15.2 15.6
16.5 15.9
1 I
186
i
0.48
0.53
0.55
0.63
0.65
0.63
0.68
0.71
0.73
0.80
0.83
0.84
0.87
0.92
1.00
1.29
1.36
1.50
1.52
1.73
1.73
1.80
1.89
1.91
1.95
1.97
2.30
2.38
2.33
2.52
2.43
2.52
2.57
2.77
3.26
3.39
3.21
3.63
3.70
4.11
4.28
4.69
5.66
9.7
10.3
15.1
15.2
1
0.49
0.53
0.57
0.63
0.66
0.68
0.58
0.71
0.72
0.80
0.33
0.84
0.87
0.92
1.00
1.28
1.35
1.49
1.50
1.71
1.72
1.79
1.87
1.87
1.92
1.94
2.27
2.36
2.29
2.49
2.43
2.47
2.53
2.73
3.21
3.32
3.08
3.57
3.63
4.03
4.17
4.55
5.49
9.3
10.1
14.6
14.6
190
i
0.49
0.54
0.57
0.64
0.66
0.69
0.68
0.71
0.72
0.81
0.83
0.84
0.87
0.92
1.00
1.26
1.34
1.48
1.43
1.69
1.70
1.77
1.85
1.84
1.89
1.92
2.25
2.34
2.25
2.45
2.38
2.42
2.49
2.70
3.12
3.25
2.94
3.51
3.55
3.96
4.06
4.42
5.31
8.9
9.8
14.0
13.9
1
0.50
0.54
0.58
0.64
0.57
0.69
0.68
0.70
0.72
0.81
0.83
0.85
0.88
0.91
1.00
1.25
1.33
1.47
1.47
1.68
1.63
1.75
1.83
1.80
1.87
1.89
2.22
2.31
2.21
2.42
2.32
2.36
2.45
2.66
3.11
3.19
2.80
3.45
3.47
3.88
3.95
4.28
5.13
8.5
9.5
13.5
13.3
194
0.51
0.54
0.53
0.65
0.68
0.69
0.68
0.70
0.71
0.81
0.82
0.85
0.88
0.91
1.00
1.24
1.32
1.45
1.45
1.65
1.67
1.74
1.81
1.76
1.84
1.86
2.19
2.29
2.17
2.38
2.27
2.31
2.40
2.63
3.06
3.12
2.67
3.40
3.40
3.81
3.84
4.15
4.95
8.0
9.2
13.0
12.6
1
193
0.51
0.54
0.59
0.65
0.69
0.70
0.68
0.70
0.71
0.81
0.82
0.35
0.88
0.91
1.00
1.22
1.31
1.44
1.44
1.64
1.65
1.72
1.79
1.72
1.81
1.84
2.17
2.26
2.13
2.35
2.21
2.26
2.36
2.59
3.01
3.05
2.53
3.34
3.32
3.73
3.73
4.01
4.77
7.5
8.9
12.4
12.0
198
202
I
0.52 0
0.55 0
0.59 0
0.66 0
0.70 0
0.70 0
0.68 0
0.70 0
0.71 0
0.81 0
0.32 0
0.85 0
0.33 0
0.91 0
1.00 1
1.21 1
1.30 1
1.43 1
1.42 1
1.62 1
1.63 1
1.70 1
1.77 1
1.69 1
1.78 1
1.81 1
2.14 2
2.24 2
2.09 2.
2.32 2
2.16 2.
2.21 2
2.32 2
2.56 2
2.96 2
2.98 2
2,39 2
3.28 3
3.25 3
3.66 3
3.62 3
3.39 3.
4.60 4.
7.2 6.
8.6 8
11.9 11
11.4 10.
1
52
55
60
67
70
70
68
70
71
82
82
85
88
91
00
20
29
42
40
61
62
68
75
65
75
78
11
22
Q5
28
11
15
23
52
91
91
25
22
17
58
51
74
4?
4
3
7
202
Section 4, A, (6)
Page 6
204
0.53
0.55
0.60
0.67
0.71
0.70
0.68
0.59
0.70
0.82
0.82
0.35
0.83
0.91
1.00
1.19
1.28
1.41
1.39
1.59
1.60
1.67
1.74
1.61
1.72
1.75
2.08
2.19
2.01
2.25
2.05
2.10
2.24
2.49
2.86
2.84
2.12
3.16
3.09
3.50
3.40
3.61
4 24
6.4
8.1
10.8
10.1
I
204
Cornoound
Hexachlorobenzene
Dimethyl Phthalate
Tecnazene
Chlordene
a-BHC
CDEC
Mevinphos
Dlethyl Phthalate
Diazinon
Lindane
2,4-0(IPE)
PCNB
Heptachlor
S-BHC
Aldrin (REFERENCE)
Ronnel
1 -Hydroxychl orde"p
Oxychlordane
o,p'-DOE
rrono-Nonachlor
Chlordane, Garma
Heotachlor Epoxide
Chlordane, Alpha
Dibutyl Fhthalate
Oimethoate
p.p'-DOE
Endosulfan I
o,p'-DOD
DCPA
Chlordecore
o,p'-OOT
MaTathion
M. Parathion
Dieldrin
Endrin
p,p'-DDO
E. Parathion
Ml rex
p,p'-DDT
Endosulfan II
Carbophenothion
Ethion
Methoxychlor
Dloctyl Phthalate
Endrin Ketone "153'
Tetradifon
Diphenyl Phthalate
Retention ratios, relative to aldrin, of 47 compounds at temperatures from
HO to 204"C; suDDort of gas Chrom q, 30/100 r.esh; electron capture detector;
Mi source; all absolute retentions measured from injection point. Arrow
Indicated optimum coiurm operating temperature with carrier flow at 50 ml
per min.
-------
Revised 6/77
Table 2(d)
Section 4,A,(6)
Page 7
10% DC-200
Column Temperature, °C
170
174
178
182
186
190
194
198
202 204
J I L
0.16
~0.27
034
0.35
0.35
0.38
0.39
041
052
0.43
048
0.38
0.60
0.46
0.78
0.87
0.71
0.52
045
1.00
0.82
1.01
068
1.29
0 77
1.10
1 29
1 65
1.43
1.03
1.64
2.09
1.81
1.21
1 30
1.99
260
2.13
2.90
2.22
3.07
2.72
2.29
3.04
3.72
3.42
3.23
6.9
6.1
4.07
6.4
0.16
0 28
0.34
0 35
0.35
0.38
0.39
0.41
052
043
0 48
0.38
0 60
0.45
0.78
0.67
0 70
052
0 45
1.00
0 81
1.01
0.66
1 29
078
1 09
1.28
1 64
1 42
1.02
1 63
2.07
1.79
1 21
1.29
1.98
2.56
2.11
2.86
2.20
3.03
2.69
2.27
3.00
3.67
3.37
3.19
6.8
6.0
4.02
6.3
6.3
0.16 0.16
~0.28 0.28
0.35 0 35
0.36 0.36
0.36 0.36
0 38 0.39
0 39 0 39
0.41 041
0.52 0.52
0 44 0.44
0.48 0.48
0 39 0.39
0.60 0 60
0 46 0.46
078 078
067 067
0 70 0 70
0 52 0 52
045
1 00
081
1 01
0.68
1 28
0 78
1.09
1 27
0 45
1.00
081
1 Ot
0.68
1 23
0 78
1.08
1 27
1.62 1 61
1 41 1.40
1 02 101
1.62 1.61
2 05 2.02
1.77 1.75
1 21 1 20
1.29 1.28
1 96 1.95
2 53 2 49
2.08 206
2 83 2.79
2.19 2.17
3.00 2.96
2 66 2 62
2 25 2.24
2.95 2.91
3.62 3.57
3.32 3.28
3.15 3.11
6.7 6.6
5 89 5.77
3.98 3.89
6.2 6.1
6.2 6.0
0.17
0.29
035
037
037
039
0.40
042
052
0.45
0 48
040
0 60
0.47
0 79
067
0 70
0.52
045
1 00
0.81
1.00
069
1.27
0.78
1 08
1.26
1 59
1 40
1 00
1 61
2.00
1.73
1 20
1 27
1.93
2.45
2 04
2.76
2 16
2.93
2.59
2.22
286
3.51
3.21
3.06
6.5
5.66
3.83
5.97
5.92
0 17
0.29
0.36
0 37
037
0 40
040
0.42
052
0.45
049
040
0.61
0 47
0 79
0 66
0.70
052
0.45
1 00
080
1.00
069
1.27
0 79
1.07
1 25
1 56
1 39
1 00
1.60
1 98
1 71
1 19
1.27
1.92
2.41
2 01
2 72
2.14
290
256
2.20
2.81
3.46
3.16
3.02
8.4
5.54
3.77
S.85
5.81
0.17 0 17
0.29 0 30
0.36 037
0 38 0.38
037 0.38
0.40 0 40
0.40 040
042 0.42
0 52 0.52
0 46 0.46
0 49 0 49
0.41 041
0.61 0.61
0 47 0 48
0 79 0.79
0 66 0 66
070 070
052 052
0 45 0 45
1 00 1 00
080 080
1 00 1 00
0.69 0 69
1 26 1 26
0 79 0 79
1 06 1 06
1.24 1.24
1 57 1 55
138 1.37
0.99 0 99
1 59 1 58
1 96 1 94
1 69
1 19
1 26
1.90
2.38 2 34
1 99 1 97
2 68 2 65
213 211
2 86 2.83
2 52 2 49
2.18 2.17
2.77 2.72
3.40 3 35
3.11 3.06
2.98 1.93
6.3 S.2
5.42 5.30
3.70 3.64
S 73 6.62
5.69 5.58
0.18 0.18
0.30 0.30
0.37 038
039 0.39
0.38 0.39
041 0 41
041 0 41
0.43 043
0.52 0.52
0.47 0.47
0.49 0.49
0.42 0.42
0.61 0.61
0 48 0 49
060 080
066 066
070 0 70
052 OS2
045 046
1 00 1 00
080 080
1 00 1 00
069 069
•1 25 1 24
0 79 0 80
1 05 1 05
1 67
1 18
1.26
1.89
1 23
1.54
1 36
0.98
1.56
1 92
1 65
1.18
1.25
1.87
I 22
1 53
1 36
0 98
1 57
1 90
1 63
1.17
I 24
1.86
2.30 2.27
1 94 1.92
2.61 2.58
2.09 2 08
2.79 2 76
2.4S 2 42
2.15 2.13
268 263
3.29 3.24
3.01 2.96
2.89 2.65
6.1 60
5.13 5.06
3.58 3 52
5.50 538
5.46 5 34
0.18
0.30
038
0.40
0.39
041
0.41
043
0.52
0.48
0.49
043
061
0 49
0 80
0 66
0 70
0 52
0 46
1 00
0 79
1 00
069
1 24
0.80
1 04
1 22
1 51
1 35
097
1 56
1 88
1 61
1.17
1 24
1 84
2.23
1 90
2 54
2.06
2.72
2.39
2.11
2.58
3.18
2.90
2.80
5 90
4.93
3.45
5.27
5.23
0 18
0.31
" 6.38"
0.40
039
042
041
0.43
0.52
0.48
049
043
061
0 49
0 80
0 66
069
0 52
0 46
1.00
0 79
0 99
069
1 23
080
1 03
1 21
1 50
1 34
0.96
1 !.5
1 86
1.60
1 16
1 23
1.83
2 19
1.87
2 50
2 05
2.69
2.35
2.10
2 54
3 13
2.85
2.76
5.80
4.82
3.39
5 15
5.12
0.18 0/19
0.31 oTsi
djY~OJ»
"6.41" 0~.41
0.40 0.40
0.42 0.42
0.41 0.42
0.43 0.44
0.52 O.S2
0.49 0.49
0.50 0.50
0.44 0 44
0.61 061
0.50 0 50
080 0.81
0.66 0 65
069 069
0 53 0 53
0 46 0 46
1.00
0 79
0 99
0 69
1 00
0 79
0 99
069
1 23
0 80
1 03
1 20
1 48
1 33
1 22
0 81
1.02
1 20
1 47
1 33
0.96 0 95
1 54 1.54
1 63 191
1 58 1 66
1.16 1 15
1 23 1.22
1 81 1 80
2.15 2 11
1 85 1 83
2 47 2.43
2.03 2 01
266 262
2.32 229
2.08 2 06
2.49 2.44
3.08 3.02
2.80 2.75
2 72 2 67
5.69 5.59
4.69 4.58
3 33 3.27
5.03 4.92
5.00 4.88
JJ.19
032
6.40
042
0.41
043
0.42
0.44
052
0.50
0.50
045
061
051
081
0 65
0 69
053
0 46
1 00
0 78
0 99
0 69
1 22
0 81
1 02
1.19
1 46
1 32
095
1 53
1.79
1 54
1 15
1.22
1.79
2 08
1.80
2.40
2.00
2 59
2 25
2.05
2.40
2.97
2.70
2.63
548
4 45
3.20
4 80
4.77
J0.19 0.19
0.32 0.32
0.40 040
0.42 6.42
0.41 0.41
0.43 0 43
0 42 0.42
0.44 0.44
0.52 0.52
0 50 0 50
0 50 0 50
0 45 0 46
0.61 061
0.51 051
081 081
0 65 0 65
0.69 0 69
0 53 0 53
0.46 0 46
1 00 1 00
0 78 0 78
0 99 0 99
069 069
1 21 1.20
081 081
1.01 1 01
1 18 I 17
1 44 1 43
1 31 1 30
0.94 0 93
1 52 1.51
1 77 1.75
1 52 1 50
1 14 1.14
1.21 1.20
1.77 1.76
2.04 2.00
1.78 1.76
2.36 2.32
1.98 1.97
2.55 252
2.22 2.19
2 03 2.01
2.35 231
2.92 2 86
2 65 2.60
2.58 2.54
5.38 5.28
4.33 4.22
3.14 3.08
468 4.57
4.66 4 54
0.20
0.33
0.41
"0~43
0.42
0 44
042
044
0.52
0.51
0.50
0.46
061
0 52
082
0 65
069
053
046
1 00
078
0.98
0 69
1 20
082
1 00
1 17
1 41
1.30
0 93
1.50
1 73
1.48
1.13
1.20
1.74
1 97
1.73
2.29
1.95
2.49
2.15
2 00
2 io
2.81
2.55
2.50
5.17
4.10
302
4 45
4 42
I
I
T
170
174
178
182
190
194
202
Mevinphot
2,4-OIME)
Phorate
a BHC
CDEC
2,4-DOPE)
Simazina
Atrazine
Diazinon
Lindane
2,4.5-T(ME)
/3BHC
2,4D(BE)I
S-BHC
Heptachlor
2.4.5-TIIPE)
2.4-OIBEMI
Dichtone
Dimethoate
Aldrin (REFERENCE)
Ronnel
1-Hydroxychlordene
M. Parathion
Heptachlor Epoxide
Malathion
D C P A
Dyrene
o.p'-DDE
Chlorbensid*
E. Parathion
findotulfon I
p,p'-DOE
DDAIMEI
Captan
Folpet
DieWrin
Perthana
o.p'-DDO
o,p'-DDT
Endrin
Chlordecon.
p.p'-DDO
Endoiulfon H
Ethion
p,p'-DOT
Corbophvnothion
Dilan I
Mtrax
Metboxychlcr
Dilan II
T.I rod if on
Azinpnpsmvrhyl
204
Retention ratios, relative to aldrin, of 48 pesticides on a column of 10% DC-200 at temperatures from
170 to 204°C; support of Chromosorb W.H.P., 80/100 mesh; electron capture detector,
tritium souroa. parallel plate; all absolute retentions measured from injection point. Arrow indicates
optimum column operating temperature with carrier flow at 120 ml per minute.
-------
Revised 6/77 - Section 4,A, (6)
Table 2(e)
5%DC-200/7.5%QF-1
Column Temperature, °C.
170
I
029
0.40
043
0.43
045
0.54
0.54
0.55
0.59
OSS
067
O.S7
0.83
0.65
080
0.90
098
0.84
0.95
100
102
1.27
1.54
1.54
1 73
1.70
154
166
1.62
214
2.00
2.15
228
2.35
224
2.40
2.42
2.37
2.91
2.81
2.96
3.22
3.22
4.10
4.0V
4.12
6.3
5.98
6.6
75
116
12.5
1
0.29
0.40
0.44
0.43
0.45
0.54
0.54
0.55
0.59
0.55
0.67
058
0.83
0.65
080
089
0.97
084
0.94
1 00
1 01
1.27
1 53
1.53
1.71
1 68
1.53
1.65
1 61
2.11
199
213
2.25
2.33
2 23
2.38
2.39
2.35
2.87
2.79
2.93
3.18
3.19
4.04
4.00
4.08
8.2
5.91
6.5
7.3
11.4
12.2
1
174
0.29
0.40
044
044
045
054
054
055
059
056
0.67
058
083
0.66
080
089
096
084
094
100
1 01
125
151
1 53
1 69
1 67
1 51
1.63
1.60
2.09
197
2.11
2.22
231
221
2.37
2.36
233
2.84
276
2.91
3.14
3.16
3.98
3.93
4.00
8.1
5.83
6.4
7.2
11.2
12.0
1
0.29
040
0.44
0.44
045
054
054
0.55
0.59
056
0.67
058
082
066
0.80
089
095
084
093
1 00
1 01
1 24
1 51
1 52
167
165
1 50
1 62
1 59
207
1 96
209
2.19
228
219
23S
233
230
281
2.74
2.89
3.10
3.13
3.91
3J»
3.94
5.91
5.78
6.2
7.0
10.9
11.7
1
178
1
030
040
044
044
046
0.54
054
0.56
0.58
056
066
0.58
082
066
0.81
088
095
084
093
1 00
1 00
1 23
149
1 51
1 65
1 63
1 49
1 61
158
205
1 95
2.07
2.16
2.28
2 17
2.33
2.30
228
278
272
2.88
3.06
311
385
3.79
3.88
5.87
5.68
8.1
«.9
10.7
11.4
1
1
030
0.40
044
045
046
0.54
054
056
0.58
0.57
0.66
058
082
0.66
081
088
094
084
093
1 00
1.00
1 22
1.49
1 51
163
162
l'48
159
1 57
2.03
1 94
205
2 13
2.23
2.15
232
2.27
2.25
2.75
2.69
284
302
307
3.79
3.72
3.82
5.76
5.61
5.97
8.8
10.4
11.2
I
182
1
0.30
041
0.44
0.45
046
055
054
0.56
0.58
0.57
0.66
0.58
081
067
081
087
093
0.84
092
1 00
1 00
1 21
1 47
1 50
1 61
1 60
1 47
158
1 56
201
1 93
203
210
221
2 13
230
2.24
2.23
272
267
2.81
298
3.06
3.73
3.6S
3.78
S.6S
5.53
5.83
6.6
102
10.9
I
0.30
041
045
045
047
0.55
054
056
058
057
066
059
081
067
081
087
092
084
092
1 00
099
1 20
1 46
1 49
159
1 59
1 46
1 57
1 55
1 99
1 91
2.01
207
2.19
2 12
229
2.21
221
269
265
279
2.94
3.02
3.66
3.58
3.70
5.S3
5.46
5.71
8.5
10.0
106
1
186
030
041
045
046
047
055
054
056
058
058
0.66
0.59
081
067
081
086
0.91
085
091
1 00
0 99
1 19
1 45
1 49
157
1 57
1 45
1.55
1 54
197
1 90
1.99
204
2 17
2 10
2.27
2.18
2 18
265
2.62
2.76
290
299
3.60
3.51
3.63
5.42
5.38
5.58
8.4
97
10.4
1
I
030
0.41
045
048
0.47
0.55
0.54
0.56
0.58
058
066
059
080
067
0.81
086
091
085
091
1 00
099
1 18
1 44
1 48
155
1 55
1 44
154
1 53
t 95
189
t 97
201
214
2 06
225
2 15
2.16
262
260
274
286
2.96
3.54
3.44
3.57
5.31
5.30
5.43
8.2
9.5
10.1
1
190
1
0.30
0.41
0.45
046
048
055
0.54
056
058
0.58
066
059
080
068
081
085
090
085
091
1 00
098
1 17
1 43
1 53
1 54
143
1 53
t 52
1 93
t 88
1 95
1 99
212
2 O6
224
2.12
2.13
2.58
258
271
282
2.93
347
337
3.50
520
5.22
530
6.1
9.2
9.9
|
030
0.41
0.45
04;
0.48
055
0.54
056
0.57
0.59
066
059
0.80
068
081
085
089
085
090
1 00
098
1 16
1 42
1 51
1 52
1 41
1 51
1 51
1.91
1 87
1 93
1.96
209
2O4
222
2.08
2.11
256
255
269
2.78
290
341
3.30
3.44
5.10
5.15
5.18
594
90
9.6
~l
194
I
0.31
0.42
0.46
0.47
0.48
0.55
0.54
0.57
057
059
065
060
079
068
082
084
089
085
090
1 00
098
1 15
1 41
1 49
1 51
1 40
1 50
1 50
1 88
1 86
1 90
193
2.07
203
221
2 05
208
253
253
266
274
2 87
3.35
323
338
500
5.08
5.03
580
8.8
93
0.31
0.42
0.46
047
0.48
0.55
054
057
057
0.59
065
060
079
069
082
084
088
085
089
1 00
096
1 14
1 40
1 47
1 49
1 39
1 49
1 49
186
1 85
1 88
1 90
2.05
201
2 19
2.02
206
243
251
264
270
284
329
3.16
332
488
5.00
4.88
567
85
9.1
198
0.31
0.42
046
0.48
049
0.55
054
057
057
060
0.65
0.60
078
069
082
083
087
085
089
100
096
1 13
139
1 45
1 48
1.38
1 47
1.48
184
183
1 86
1 87
2.02
1 99
2.18
1 99
203
2.46
2.48
2.61
266
281
3.22
309
3.26
478
4.92
4.78
5.52
8.3
8.8
¥
|
0.31
042
0.48
048
049
0.55
054
057
0.57
060
0.65
0.60
0.78
069
082
0.83
086
085
089
1.00
096
1.12
1.38
143
1 46
1.37
1.46
1.47
182
1 82
1.S4
184
2.00
1.97
2.16
196
2.01
2.43
2.48
259
2.62
2.78
3.16
3.02
3.20
463
4.85
464
5.40
8.0
85
1 '
202
0.31
0.42
0.46
0.48
0.49
055
054
057
057
060
0.65
060
078
069
082
082
0.85
085
0.88
1 00
096
1 11
137
1 41
1 44
1.36
145
1 46
180
1 81
182
181
198
1.95
2.15
193
1.99
2.40
2.44
257
258
275
3 10
2.96
3.14
4.56
473
4.50
5.26
78
8.3
204
|
031
0.42
0.46
0.49
050
0.55
054
0.57
0.57
0.61
065
060
077
070
032
082
085
085
088
1 00
094
1 10
1 36
1 39
1 43
1 35
1 43
1 45
1 78
1 80
1.80
1 78
1.96
1 93
2 13
1.90
1 96
2.37
2.41
2.54
2.54
272
3.03
2.88
3.08
446
4.70
4.33
5.11
7.8
8.0
I
Mfvinphoi
2,4-DIME)
Phorale
a -BMC
CDEC
2,4-OIIPE)
Simazine
Atrazine
Diazinon
Lindane
2.4,S-T(ME)
/9BHC
2,4~D(BEH
5-BHC
Heptachlor
2,4,5-TIIPE)
2,4-O(BEIII
Dichtone
Oimethoate
Aldrm (REFERENCE)
Ronnel
1 Hydroxychlordene
M. Parathion
Hepuchlor Epoxide
Malathion
D CPA
Dyrens
o,p'-DDE
Chlorhenside
E. Parathion
Endosulfon I
n.p'-OOE
DDA(ME)
Captan
Folpet
Dieldnn
Perthane
o,p'-DDD
o,p'-DDT
Endrin
Chlord«cone>
p.p'-DDD
Endesulfon H
Ethion
P.P'-DOT
Corboprnnothion
Dilan 1
Mirex
Methoxychlor
Dilan II
Tetrodifon
Axlnphosmethyl
170 "4 178 182 186 190 194 198 202 204
Retention ratios, relative to aldrin, of 43 pesticides on a column of 5% DC-200/7.5%QF-1 at temperatures
from 170 to 204°C; support of Chromosorb W.H.P., 80/100 mesh; electron capture detector, tritium source,
parallel plate; all absolute retentions measured from injection point. Arrow indicates optimum column
operating Umperatura with carrier flow at 120 ml per minuta.
-------
Revised 6/77 Section 4,A,(6)
Page 9
Table 2(£)
1.6%OV-17/6.4%OV-210
Column Temperature, °C.
170
1M 178 182 '86 1*0 1W 193 202
I | | | J L I I I I I I I I I 1
0. 36
0.48
0. 51
0.49
0.56
0.62
0. 72
0. 70
0.71
0. 69
0. 84
0.84
0.96
0.97
0. 83
1.02
1.13
1.14
1.30
1.00
1.24
1.43
2.02
1.81
2.37
2.06
2.14
2.04
2.18
2.80
2. 33
2.67
3.07
3. 53
3. 51
2.94
3.24
3.26
3.81
3. 68
3. 18
4.63
4.60
6.2
5.46
6.35
2.1
7.0
10.9
10.5
17.5
24.5
n
n
0
0.
n.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
1.
1.
1.
1.
1.
2.
1.
2.
2.
2.
2.
2.
2.
2.
2.
3.
3.
3.
2.
3.
3.
3.
3.
3.
4.
4.
6.
5.
6.
8.
6.
10.
10.
17.
23.
17
48
SI
50
56
62
72
70
70
69
84
84
96
97
83
02
12
14
29
23
42
01
79
33
04
12
02
16
76
31
64
02
54
46
92
18
20
75
64
15
55
54
0
36
2
9
9
6
3
2
8
0
0
0
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
1.
1.
1.
1.
1.
1.
1.
J.
2.
2.
2.
2.
2.
2.
2.
2.
3.
3.
2.
3.
3.
3.
3.
3.
4.
4.
5.
5.
6.
8.
6.
10.
10.
16.
23.
17
48
SI
SO
56
62
71
70
70
69
83
84
95
97
83
01
12
14
29
22
41
99
78
30
02
09
00
14
73
29
60
97
49
42
89
12
16
70
59
12
46
48
92
26
1
7
8
4
0
a
2
0.37
0.48
0.51
0.51
0.56
0.62
0.71
0.70
0.69
0. 69
0.83
0.84
0.95
0.97
0.83
1.01
1.11
1.14
1.28
1.22
1.41
1.98
1.77
2.26
1.99
2.07
1.96
2.11
2.69
2.27
2.57
2.92
3.44
3.37
2.86
3.07
3.10
3.64
3.54
3.09
4.38
4.42
5.79
5.16
5.96
8.5
6.7
10.2
9.3
16.4
22. 5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
I
1
2
1
2
1
2
2
2
2
2
3
3
2
3
3
3
3
3
4
4
5
5
5
8
6
10
9
16
21
37
49
52
52
57
62
71
70
69
70
82
84
94
97
83
00
10
14
28
21
40
96
76
22
97
04
95
09
66
25
53
87
40
33
83
01
05
58
50
06
30
35
67
06
83
3
6
0
6
0
9
0
0
0
0
.37
.49
.52
.52
0.57
0
0
0
0
0
0
0
0
.62
.71
.70
.69
.70
.82
.84
.93
0.97
0
1
1
1
1
1
1
1
1
2
1
2
1
2
2
2
2
2
3
3
2
2
3
3
3
3
4
4
5
4
5
8
6
9
9
15
21
.33
.00
.09
.13
.27
.20
. 40
.95
.75
.18
. 95
.02
.i3
.07
.62
.23
.49
.82
.35
.28
.80
. 95
.00
.52
.45
.03
.21
. 30
. 54
. 96
.69
.2
.5
.7
.4
. 7
.3
0.
0.
0.
3V
49
52
0.52
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
1.
1.
1.
1.
1.
1.
2.
1.
2.
1.
2.
2.
2.
2.
2.
3.
3.
2.
2.
2.
3.
3.
3.
4 .
4 .
5.
4.
5.
8.
6.
9 ,
9.
15.
20.
57
62
71
70
68
70
32
34
93
97
33
99
08
13
26
20
39
93
73
15
93
00
91
04
59
21
46
78
30
24
77
90
96
46
40
00
13
24
42
86
56
0
4
4
1
3
7
0.33
0.49
S.52
0. .33
0.57
0.62
0.71
0.70
0.68
0.70
0.81
0. 84
0.92
0.97
0.83
0.99
1.07
1. 13
1.26
1.19
1.38
1.92
1.72
2.11
1.91
1.97
1.89
2.02
2.55
2.19
2.42
2.73
3.26
3.20
2.74
2.84
2.90
3.40
3.36
2.97
4.05
4.17
5.29
4.76
5.43
7.8
6.3
9.2
8.9
14.9
20.0
0.38 0
0.50 0
0.52 0
0.53 0
0.58 0
0.62 0
0.71 0
0.70 0
0.67 0
0.70 0
0.81 0
0.34 0
0.91 0
0.97 0
0.83 0
0.98 0
1.06 1
1.13 1
1.25 1
1.18 1
1.38 1
1.90 1
1.71 1
2.07 2
1.38 1
1.95 1
1.87 1
2.00 1
2.52 2
2.18 2
2.39 2
2.63 2
3.21 3
3.15 3
2.71 2
2.78 1
2.86 2
3.35 3
3.32 3
2.94 2
3.36 3
4.12 4
5.16 5
4.66 4
5.30 5
7.6 7
6.2 6
9.0 3
3.7 8
14.5 14
19.4 18
38 0
SO 0
53 b
j4 0
58 0
62 0
71 0
70 0
1,7 0
70 0
00 0
84 0
91 0
97 0
83 0
98 0
05 1
13 1
25 1
17 1
37 1
88 1
-0 1
04 2
36 1
92 1
85 1
97 1
48 2
16 2
35 2
63 2
17 3
10 3
68 2
72 2
80 2
28 3
27 3
91 2
88 3
05 4
04 4
56 «
16 5
4 7
1 5
8 8
4 8
1 13
8 18
38
50
53
54
58
62
70
70
66
70
80
84
90
97
83
98
04
13
24
17
36
87
69
00
84
90
83
95
44
14
32
58
12
06
65
66
76
23
22
88
80
00
92
46
03
2
97
5
2
8
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
3
3
2
2
2
3
3
2
3
3
4
4
4
7
5
6
9
13
17
38 0
50 0
S3 0
55 0
58 0
02 0
70 0
70 0
(6 0
7C- 0
80 0
84 0
90 0
97 0
83 0
97 0
04 1
13 1
23 1
16 1
36 1
85 1
68 1
?6 1
32 1
87 1
80 1
93 1
41 2
12 2
28 2
54 2
07 3
02 2
62 2
bl 2
71 2
17 3
18 3
85 2
72 3
3t 3
79 4
36 4
90 4
0 6
87 5
3 8
0 7
4 13
6 17
39 0
50 0
S3 0
b5 0
59 0
62 0
70 0
70 0
65 0
71 0
79 0
34 0
89 0
97 0
S3 0
96 0
03 1
12 1
23 1
15 1
35 1
84 1
66 1
431
80 1
85 1
78 1
90 1
37 2
10 2
24 2
43 2
03 2
97 2
60 2
55 2
66 2
11 3
13 3
82 2
63 3
63 3
67 4
26 4
77 4
8 6
77 5
0 7
8 7
0 12
0 16
39 0
SI 0
54 0
56 0
59 0
62 0
70 0
70 0
65 0
71 0
79 0
£4 0
88 0
97 0
83 0
96 0
02 1
12 1
22 1
15 1
34 1
82 1
65 1
83 1
77 1
83 1
76 1
88 1
34 2
08 2
21 2
44 2
98 2
93 2
57 2
492
61 2
05 3
09 3
80 2
55 3
82 3
54 4
16 4
63 4
7 6
67 5
8 7
5 7
6 12
3 15
39 0
51 0
54 0
57 0
59 0
62 0
70 0
70 0
64 0
71 0
78 0
64 0
88 0
97 0
83 0
96 0
01 1
12 1
22 1
14 1
34 1
81 1
64 1
86 1
75 1
SO 1
74 1
86 1
30 2
07 2
17 2
39 2
94 2
88 2
54 2
44 2
56 2
00 2
04 2
77 2
46 3
75 3
42 4
06 3
50 4
5 6
57 5
6 7
3 7
3 11
7 15
39 0
51 0
54 0
57 0
59 0
62 0
70 0
70 0
64 0
71 0
78 0
34 0
37 0
97 0
83 0
95 0
CO 0
12 1
211
13 1
33 1
79 1
63 1
82 1
73 1
78 1
72 1
84 1
27 2
05 2
14 2
34 2
89 2
34 2
51 2
38 2
512
94 2
99 2
74 2
38 3
t>9 3
29 4
96 3
37 4
3 6
48 5
3 7
1 6
9 11
1 14
39 0
51 0
54 0
53 0
59 0
62 0
70 0
70 0
64 0
71 0
78 0
84 0
86 0
97 0
83 0
95 0
J9 0
12 1
211
13 1
33 1
78 1
62 1
78 1
71 1
76 1
70 1
81 1
23 2
03 2
10 2
29 2
84 2
79 2
48 2
32 2
46 2
88 2
95 2
•"1 2
30 3
153 3
17 4
86 3
24 4
1 5
38 5
1 6
8 6
5 11
5 lj
39 Me/inphos
51 2,U-D(ME)
i4 Phorate
58 a-BHC
60 CDEC
62 2,k-D(IPE)
70 Slmazlne
70 Atrazine
63 Diazincn
71 Lindane
77 2,I»,5-T(ME)
84 S-.-HC
86 2,1-D(BE)I v
97 S-^HC
83 Heptachlor
94 2,ll,5-T(IPE)
Jl> 2,li-D(BE):i
12 Dlchlone
20 Dimethoate
"*" Aldrin (BEFEBEHCE)
32 Fonnel
32 1-Hydroxychlordene
76 M. Pirathion
60 Heptachlor Epoxide
74 Maiathion
69 DCPA
?3 Ityrcne
68 o ,p ' -DDE
79 Chlorbenside
20 E. Parathijr.
ul Endosulfan I
06 p,p'-DDE
24 DDA(ME)
80 Captan
75 Folpet
45 Dieldrin
26 Perthane
40 o,p'-DDD
82 o,p'-DDT
90 Endrin
68 Chluraecone
22 p,p'-DDD
57 Endosulfan II
04 Ethion
76 p,p'-DDT
10 Carbophenothion
90 Dilan I
28 Mirex
B Met^oxychlor
6 Dilan II
1 Tetradifon
8 Azinphosmethyl
—I—I—I—j—,—I I I I I I I I I
174 178 182 186 '*° . '*« 1*8 205
Retention ratios, relative to aldrin, of 49 pesticides on a column
of 1.6IOV-17/6.4JOV-210 at- temperatures from 170 to 204°C; support
'of Chromosorb M.H.P., SO/100 mesh; electron capture detector,
tritium source, parallel plate; all absolute retentions measured
from injection point. Arrow Indicates optimutr column operating
temperature with carrier flow at 70 ml per minute.
-------
Revised 11/1/72
Section U,A,(6)
Page 10
1.5%OV-17/1.95V. QF-1
T
Figure 1
4%SE-30/6a/.OV-'>in
§
Figure 2
5% OV-21Q
Figure 3
-------
P£
Ol2-/\u xr 9//1-AO V9T
q£
LL
(9) 'V 'fr uoi:pa$
-------
Revised 11/1/72
Section 4, A, (6)
Page 12
Fig. 4 — Column to Port Assembly-Exploded View
Silicon* O-Ring
Bock F»rrul»
Nut
0 - Ring Retainer
Glass Col umn
Fig.4(a)— Bubble Flowmeter
Bur.t, 50 ml
Poly.th. Tubing,1/8 o.d.
Tygon Tubing , l/8"i.d.—
Rubbor Bulb , 30 ml —
' Snoop"
-------
Revised 12/2/74
Section 4, A, (6)
Page 13
Figure 5. Standing current profile from % detector in constant
use 60 days. Instrument Tracer MT-220; electrometer
attenuation 10 x 2-56, detector temp. 200°C., column
3% OV-1, column temp. 180°C., carrier gas nitrogen,
flow rate 45 ml/min, purge flow 30 ml/min
30v. 35 v.
*
25v.
20 v.
15 v.
Optimum polarizing
voltage (see Fig. 6)
2 10 v.
5v.
-------
Revised 12/2/74
Section 4, A, (6)
Page 14
Figure 6. Voltage/Response curve for 50 pg of aldrin from •%
detector in continual use 60 days. Instrument Tracer MT-220;
electrometer attenuation 10 x 32; column 3% OV-1, column temp.
180°C., detector temp. 200°C., nitrogen carrier flow 60 ml/min
7.5v.
-------
Revised 12/15/79
Section 4, A, (6)
Page 15
Fig. 7
Peak Height = CD
1
Fig. 8
Peak Area = CD x AB
Half Height
Fig. 9 Triangulation
Peak Area =--L(FG)(JH)
-------
Revised 12/2/74
Fig. 10.
Copied from Pesticide Analytical Manual, Vol.
Drug Administration.
Section 4, A, (6)
Page IB
1, U.S. Food 8
Fig. 2—Baseline construction for some typical gas chromatographic peaks.
a, symmetrical separated flat baseline; b and c, overlapping flat baseline;
d, separated (pen does not return to baseline between peaks); e. separated
sloping baseline; f, separated (pen goes below baseline between peaks);
g, a- andy-BHC sloping baseline; h,a-./J-, and 7-BHC sloping baseline;
i, chlordane flat baseline; j. heptachlor and heptachlor epoxide super-
imposed on chlordane; k, chair-shaped peaks, unsyir.metrical peak; 1,
p,p'-DDT superimposed on toxaphene.
-------
Revised 12/2/74 Section 4, A, (6)
Page 17
Figure 11. Six-foot gas chromatographic column. Borosilicate glass, 1/4" o.d.,
5/32" i.d. - Corning No. 237300 or equivalent. Tubing o.d. to be
tested for assurance it will accomodate 1/4" Swagelok nut.
(If butt-jointed, the butt to be on one side, not at U-bend.)
-------
Revised 12/15/79 Section 4, A, (7)
Page 1
SUPPORT-BONDED CARBOWAX 20M COLUMNS
I. INTRODUCTION:
Highly inert column packings have been prepared by chemically
bonding Carbowax 20M to different GC supports. The Carbowax is coated
on acid washed support, and, after heat conditioning, the nonbonded
phase is removed by solvent extraction. A thin layer of liquid phase
remains bonded to the support surface. Packings prepared in this way
have been used for the GC of chlorinated pesticides without further
treatment, and for heat-labile nitrogen-containing and other polar
pesticides after being coated with liquid phases such as OV-101 or
OV-210. The columns have been used with electron capture (Section 12,
A), Hall electrolytic conductivity (Section 4,C), and nitrogen-
phosphorus thermionic (Section 4,D) GC detectors for the separation and
analysis of pesticides.
The preparation of columns described in this section differs from
vapor phase deposition of Carbowax 20M (Section 4,B,(2), IV,2) used to
make columns more suitable for the GC of organophosphorus pesticides.
This earlier form of support bonding produces only temporary and less
complete deactivation and had to be repeated periodically.
REFERENCES:
1. Synthesis and Chromatographic Applications of Bonded, Monomolecular
Polymer Films on Silicic Supports, Aue, W. A., Hastings, C. R.,
and Kapila, S., Anal. Chem., 45_, 725 (1973) and J. Chromatogr.,
77_, 299 (1973).
2. Laboratory Preparation and Applications of Modified Carbowax 20M
Bonded Supports to the Gas Chromatography of Pesticides,
Winter!in, W. L., and Moseman, R. F., J. Chromatogr., 153, 409
(1978).
3. Rapid Procedure for Preparation of Support Bonded Carbowax 20M
Gas Chromatographic Column Packing, Moseman, R. F., J. Chromatogr.,
166, 397 (1978).
-------
Revised 12/15/79 Section 4, A, (7)
Page 2
II. PREPARATION OF SUPPORTS:
Commercial support-bonded Carbowax 20M column packings are costly
and have proved to be variable among batches and suppliers. The
following laboratory procedure for preparing the packing is much more
rapid than earlier methods and produces a highly deactivated, low-bleed
material. It is possible for one person to prepare support-bonded and
coated column packings for use in less than four days.
1. Wash the commercial support (e.g., Chromosorb W or G) by slurrying
with hot 6 N HC1 in a 350 ml coarse-frit Buchner funnel. Draw
off the acid with vacuum produced by a water aspirator. Repeat
the washing until all traces of yellow color are removed; no more
than 3 or 4 washings are usually required.
NOTE: Acid treatment of supports, whether acid washed by the
manufacturer or not, greatly improves their chromatographic
performance, particularly for many pesticides that are
difficult to chromatograph.
2. Wash the support in the funnel with several portions of distilled
water to remove excess acid.
3. Oven dry the support at 100°C overnight and then coat with 3-5%
Carbowax 20M, using rotary vacuum (EPA Pesticide Analytical QC
Manual, Section 4G) or vacuum filtration (Technical Bulletin
No. 2A, 1967, Applied Science Laboratories, Inc.; see Section
4,A,(4).
4. Carry out the heat treatment (support bonding) process in a 100 ml
volumetric pi pet as follows:
a. Pack the portion of the pipet below the bulb with uncoated
support held in place with glass wool plugs. Fill the
remainder of the pipet with coated support.
NOTE: The lower bed of support prevents back diffusion
of oxygen into the coated support.
b. Connect the pipet to the inlet of a Tracer MT-222 or
equivalent conventional gas chromatograph, using Swagelok
fittings drilled to the proper size and a ferrule fabricated
with PTFE to obtain a gas-tight seal between the pipet and
fitting (Figure 1).
c. Sweep nitrogen through the column packing at a flow rate
of 60 ml/minute for at least 2 hours at room temperature.
-------
Revised 12/15/79 Section 4, A, (7)
Page 3
d. Program the temperature of the GC oven to 270°C at
l°C/minute and hold for 16 hours.
e. Cool the oven to room temperature while maintaining the
nitrogen flow.
f. Remove the pipet and empty the contents into a 350 ml
coarse-frit Buchner funnel.
5. To remove nonbonded Carbowax 20M, slurry the support with the
solvent used for coating in Step 3 and draw off the solvent with
vacuum into a filter flask.
6. Repeat this process four or five times until two successive
washes yield no yellow color.
7. Transfer the packing material to a sheet of aluminum foil and
air dry in a fume hood.
8. If the support is to be used without coating, pack into a GC
column (Section 4,A,(2),III) and condition for at least 16
hours at 230°C (Section 4,A,(2),IV).
NOTES: 1. Be sure to purge oxygen from the column during
the conditioning process before increasing the
oven temperature.
2. An occasional reconditioning of columns at 230°-
240°C for overnight periods can be beneficial in
restoring performance of columns used for analysis
of lipid extracts.
3. A new Carbowax 20M column can exhibit a sharp
increase in response after injection of a number
of lipid-containing extracts. It is probably that
the improved column performance is due to coverage
of residual active sites on the support.
9. If the support is to be coated with a liquid phase, use any
standard coating method, e.g., vacuum filtration (Step 3 above).
If fines appear to be a problem, pass the packing through the
proper mesh-size screen.
10. If properly prepared and used, columns can be stable for at least
several months. Exclusion of oxygen during operation at elevated
temperatures is an important factor.
-------
Revised 12/15/79 Section 4, A, (7)
Page 4
III. APPLICATIONS. CHROMATOGRAMS. AND DATA:
Coating the support-bonded material with OV-101 provides a column
packing that allows chromatography of many polar or heat labile
compounds such as intact carbamate pesticides, chlorinated anilines,
and metabolites of triazine herbicides, usually without derivatization.
Figure 2 shows the gas chromatogram for a nine-component mixture of
pesticides of different classes on a 3% OV-101 column coated on
Chromosorb W support-bonded Carbowax 20M (A) and unbonded Gas-Chrom Q
support (B). All nine compounds chromatographed well on the support-
bonded packing, while the carbamate pesticides propoxur and carbaryl
did not chromatograph on the conventional column. The elution order
of eicosane (C2o)> atrazine, and pentachloronitrobenzene (PCNB) can be
seen to vary on the two columns, presumably due to the presence of the
small amount of Carbowax. The carbamate pesticides carbofuran,
aminocarb, and mexacarbate also chromatographed well on the support-
bonded Carbowax 20M packing.
Figure 3 illustrates the separation of two N-dealkyl metabolites
of triazine herbicides that could not be chromatographed on convention-
al column packings. Figure 4 demonstrates the GC of several chlorin-
ated anilines that ordinarily require derivatization prior to
chromatography on a methyl silicone liquid phase. The chromatograms
shown in Figures 1-4 were all obtained using a packed 1.8 m x 4 mm i.d.
glass column and a flameless nitrogen-phosphorus detector.
Twenty-one pesticides varying greatly in polarity and chromato-
graphic behavior on conventional silicone-coated columns were evaluated
on modified Carbowax 20M supports with and without OV-210 coating.
Many of the chosen compounds are thermally unstable, yield unfavorable
separations, and/or give peaks characterized by tailing or broadening.
Supports were packed in 1.8 m x 2 mm i.d. U-shaped glass columns, and
compounds were detected with a 3H or 63Ni electron capture detector.
Tables 1 and 2 compare relative retention values and chromato-
graphic efficiency as indicated by peak shape for the 21 pesticides
on each of five Carbowax 20M modified supports both with and without
OV-210 coating. It was found that Gas-Chrom P and Q bonded with
Carbowax 20M gave, in general, the most desirable chromatographic
behavior. In addition, coating the modified supports with OV-210
generally altered the relative retention values and improved the
chromatographic behavior and separations of the pesticides. Figure 5
depicts a typical improvement in peak shape due to coating a modified
support with OV-210.
As expected, considerable improvement in chromatographic behavior
was also obtained for Carbowax 20M deactivated supports coated with
OV-210, compared to nontreated supports coated with OV-210 (Figure 6).
-------
Revised 12/15/7 Section 4, A, (7)
Page 5
Carbowax 20M modified supports were also found to offer significant
advantages over columns treated with traditional silylating reagents
(Section 4,A,(2),IV,2).
All of the data and figures in this section emphasize the
importance of a highly deactivated support when attempting the gas
chromatography of many polar and labile pesticides of current
importance to the analyst.
-------
TABLE 1. PEAK SHAPE AND RELATIVE RETENTION ON CARBOWAX 20M MODIEIED SUPPORTS COATFD WITH OV-210
Peak shape is defined by numbers: 1 = sharp peak with little or no tailing; 3 = broad but symmetrical
with little or no tailing; 4 = moderate tailing; 5 = severe tailing; N.P. = no peak. RRT = relative
retention time (Parathion - 1.00). All column temperatures were held constant at 200.
Pesticide
~\Q% OV-210 on
Chromosorb P
Phosphamidon
Mevinphos
Methamidophos
Diazinon
Lindane
Disulfoton
Atrazine
Simazine
Benefin
"Iri flural in
Aldrin
Dioxathion
Chlorpyrifos
Monocrotophos
Methyl Parathion
Parathion
Chlorpyrifos oxyqen
analogue
p,p' -DDT
Paraoxon
TEPP
Azinphos-methyl
Peak
shape
3
3
-
3
3
3
3
3
-
-
3
3
3
-
3
3
-
3
N.P.
-
-
RRT
0.10
0.15
-
0.20
0.23
0.24
0.29
0.33
-
-
0.27
0.28
0.43
-
0.79
1.00
-
1.18
N.P.
-
-
10% OV-210 on
Chromosorb G
Peak
shape
1
1
3
1
1
1
1
1
1
1
1
3
1
3
1
1
3
3
1
3
1
RRT
0.10,0.12
0.15
0.15
0.23
0.24
0.25
0.30
0.34
0.25
0.25
0.28
0.30
0.45
0.25,0.84
0.80
1.00
0.86
1.26
1.30
1.47
7.36
5% OV-210 on
Chromosorb W
Peak
shape
1
1
5
1
1
1
1
1
1
1
1
1
1
4
1
1
1
1
1
1
RRT
0.08,0.10
0.17
0.17
0.20
0.23
0.25
0.26
0.28
0.28
0.29
0.28
0.29
0.44
0.76
0.78
1.00
1.06
1.35
1.40
5.74
5? OV-210 on
Gas-Chrom P
Peak
shape
1
1
3
1
1
1
1
1
1
1
1
1
1
3
1
1
1
1
1
1
1
RRT
0.12,0.14
0.17
0.15
0.21
0.25
0.26
0.28
0.29
0.29
0.29
0.29
0.31
0.46
0.71
0.80
1.00
0.23,1.00
1.15
1.34
1.41
6.06
5% OV-210 on
Gas-Chrom Q
Peak
shape
4
1
4
1
1
1
1
1
1
1
1
1
1
4
1
1
1
1
1
3
RRT
0.12,0.
0.17
0.15,0.
0.15
0.26
0.27
0.30
0.32
0.28
0.28
0.29
0.32
0.46
0.26,0.
0.80
1.00
0.62,1.
1.33
1.39
7.10
15
26
74
17
'Very poor response with many small peaks and a large hump.
•
-------
TABLE 2. PEAK SHAPE AND RELATIVE RETENTION ON UNCOATED CARBOWAX 20M MODIFIED SUPPORTS
Peak shape is defined by numbers: 1 = sharp peak with little or no tailing; 2 = sharp but tailing;
3 = broad but symmetrical with little or no tailing; 4 = moderate tailing; 5 = severe tailing;
6 = peak poorly distinguished; N.P. = no peak. RRT = relative retention time (Parathion = 1.00).
All column temperatures were held constant at 175° with the exception of Chromosorb P which had a
column temperature of 220°.
in
•»»
^4
10
Pesticide
Phosphamidon
Mevinphos
Methamidophos
Diazinon
Lindane
Disulfoton
Atrazine
Simazine
Benefin
Trifluralin
Aldrin
Dioxathion
Chlorpyrifos
Monocrotophos
Methyl Parathion
Parathion
Chlorpyrifos oxygen
analogue
2.,2.'-DDT
Paraoxon
TEPP
Azinphos-methyl
Chromosorb P
Peak
shape
4
1
4
3
1
1
4
4
1
1
1
1
1
4
1
1
6
3
N.P.
1
N.P.
RRT
0.13
0.21
0.17
0.24
0.27
0.28
0.32
0.33
0.29
0.29
0.32
0.32
0.46
0.78
0.82
1.00
-
1.11
-
2.36
-
Chromosorb G
Peak
shape
1
4
3
3
3
3
3
1
1
3
3
3
4
3
3
6
3
3
3
3
RRT
0.05,0.07
0.12
0.32
0.31
0.26
0.28
0.61
0.72
0.15
0.14
0.25
0.35
0.59
0.16,1.48
0.84
1.00
_
2.32
1.06
2.90
12.06
Chromosorb W
Peak
shape
1
1
4
1
1
1
4
4
1
1
1
4
1
5
1
1
6
3
4
1
3
RRT
0.08
0.16
0.62
0.37
0.24
0.31
0.62
0.70
0.18
0.18
0.26
0.36
0.60
2.22
0.72
1.00
_
0.82,1.99
1.35
3.17
10.17
Gas-Chrom P
Peak
shape
1
1
3
1
1
1
1
1
1
1
1
3
1
2
1
1
1
1
1
1
1
RRT
0.52,0.78
0.14
0.28
0.29
0.31
0.29
0.65
0.82
0.17
0.17
0.26
0.37
0.55
1.49
0.90
1 .00
0.78
2.39
1.04
2.51
13.00
Gas-Chrom Q
Peak
shape
1
1
4
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
RRT
0.11,0.85
0.14
0.18
0.35
0.31
0.30
0.65
0.86
0.18
0.18
0.28
0.38
0.56
1.46
0.90
1 .00
0.72,1.84
2.34
1.06
2.58
'Peaks on a solvent front; could not distinguish peak shape.
-------
Revised 12/15/79
Section 4, A, (7)
Page 8
PREPARATION OF SUPPORT-BONDED CARBOWAX 20M GC PACKING
GC INLET
'k inch SWAGELOK N.UT
AND FERRULES
FEMALE AOAPTEn
l/«mch TUBE TO 11 ,nrh PIPE
SV\AG£LOKNO 501 A 2F
MALE CONNECTOR
5/16 .nth TUCE TO I 8 ,"h PIPE
SWAGE LOK NO 500 1 Z
100 ml VOLUMETRIC P'PET
FIGURE 1. Swagelok adapter for heat treating in a GC oven.
1 T
I I T r
a
r
5
6 8
MINUTES
10
12
FIGURE 2. (A) Gas chromatogram of m'neTComponent mixture on 3% OV^-101 coated
on 80-100 mesh Chromosorb W support-bonded Carbowax 20M. (B) Same mixture on
3% OV-1 coated on 80-100 mesh Gas-Chrom Q. Column temperature, 185°; helium
flow-rate, 57 ml/min. Perkin-Elmer N-P detector, nitrogen mode.
-------
Revised 12/15/79
Section 4,A, (7)
Page 9
CM,
t
5 10
MINUTES
6 e
MINUTES
10
12
14
Figure 3.
Gas chromatogram of N-dealkyl
metabolites of triazine herbi-
cides with same conditions as
in Fig. 2A.
Figure 4.
Gas chromatogram of chlorinated anilines
on 2% OV-101 coated on 80-100 mesh
Chromosorb W support-bonded Carbowax 20M.
Column temperature Drogrartmed from
100 to 160 C at 8^/minute; 4 minute
initial hold.
-------
Revised 12/15/79
Section 4,A, (7)
Page 10
B
It Z> 21 22 nun
24 I I 10 12 14
Fig. 5. Chroitatogranis of monocrotophos on Carbowax 20M modified Chromosorb W
support before (A) and after (B) coating with 5% OV-210. Column
temperature 175 C in (A) and 200°C in (B).
B
Fig. 6. Chromatograms of disulfoton on (A) nontreated Chromosorb P coated
with 10% OV-210 and (B) Carbowax 20M modified Chromosorb coated
with 10% OV-210. The column temperature was 200 C in both cases.
-------
Revised 12/15/7S
TECHNICAL
BULLETIN >o. 2A
Section «,A, (7)
Page 11
Applied Science
Laboratories, Inc.
PO. Box 440 / State College, Pennsylvania 16801
Preparation of Coated Packings
Use of the HI-EFF0 FLUIDIZER
This bulletin describes the Drocedures used in making coated
packings by means of the HI-EFF- Flu,dizer* Although the
procedures described in this bulletin may at first glance appear
complicated, with a little study and oract.ce packings of the
highest quality can be easily and quickly made (1)
tains a 6 mm thermowell to permit measurement of gas temp-
erature An interchangeable hose bio and a compression fitting
are 'ncluded with the unit, permitting the use of either metal
CONTENTS
Section
I. Description
II. Cleaning
III. Fluidization Gases
IV. Precautions
V. General Operation
VI. Temperature Calibration
VII. Filtration-Fluidization Procedure
VIII. Slurry-Fluidization Procedure
IX. Teflon Coating Procedure
X. Other Drying Techniques
Page
1
1
2
2
2
2
2-3
4
4
4
«US. Patent 3,513.562
n ) Kruppa, R l= , R S Heniy, and 0 L Smeaa, improved Gas Chroma-
tograohv Packings with Flutdized Drying, Anal Chem 39, 35! ' 1967)
I. DESCRIPTION OF THE FLUIDIZER
If a gas is passed upward through a porous plate covered
with a finely divided solid, the particles are suspended and in-
timately mixed in the gas stream. The suspended particles ex-
hibit fluid characteristics and the process is called fluidization
This process provides an ideal method for drying pack.rgs in-
tended for gas chromatography Fluidization drying prevents
clumping of the packing and insures uniform coating of tne
support with stationary phase. The basic parts of the HI-EFF
Fluidizer are shown and labeled ,n the exploded view Base
section, cap, and barrel are made of chrome-plated brass The
screen cap prevents the packing from blowing out of the barrel
if the gas rate is initially too nigh The barrel screws into the
base section and clamps the porous plate in position The base
is designed with a spiral insert (silver soldered in place! which
provides good heat transfer characteristics for preheating the
gas when the Fluidizer is set on a hot plate The base also Con-
or rubber tubing to connect the Fluidizer to a gas cylinder
The unit is already assembled when received but can be
stripped down to its basic oarts simoly by unscrewing the bar-
rel from the base, exposing the porous bronze plate. The plate
rests on a flange and drops out when the base is inverted
II. CLEANING
The Fluidizer can be cleaned simply by blowing out residual
packing with compressed gas. but for thorough cleaning the
unit should be disassembled and the barrel, cap, and porous
plate soaked in a suitable solvent Clean the base section with
the solvent used for the phase if a solution of phase has
inadvertently been poured into the unit without gas flow
WARNING If this happens when the base is heated, the base
can become plugged and the porous plate ruined
-------
Revised 12/15/79
III. FLUIDIZATION GASES
Nitrogen is recommended for the fluidization gas because it
is inert and inexpensive, although compressed air can be used
for phases which are not sensitive to oxidation A HYDRO-
PURGE® or similar molecular sieve trap can be inserted in the
gas ime between the cylinder and Fluidizer, if desired A cylin-
der reducer valve for the 0-50 psig range is recommended, al-
though a 0-250 psig valve may be used if great care is taken
when turning on the fluidizing gas.
IV. PRECAUTIONS
This unit may be used to prepare almost every type of pack-
ing used for gas chromatography columns except those contain-
ing highly corrosive materials such as silver nitrate, strong acids
iH,P04. H;SOa, etc), strong bases (NaOH, KOH, etc.) or
corrosive organics.
There is no need to operate this unit above 150°C fits tem-
perature limit) Discoloration of the chrome plating on the base
can be expected from prolonged usage or excessive high temp-
erature exposure but this will in no way interfere with the op-
eration of the unit 1 he barrel should be replaced if it becomes
badly discolored, although this is unlikely to happen if the
temperature limit is observed
V. GENERAL OPERATION
A small amount (50 g) of GAS-CHROM S is supplied with
the HI-EFF Fluidizer To become familiar with the operation
of the unit it is suggested that the user practice with this sup-
port by carrying the procedure through the filtration stage
ISection VII) with sclvr it alone (a stationary phase is not
necessary in this case) A maximum of 50 g of packing can be
made with one filling of the Fluidizer. Heat is supplied to the
packing by preheating the fluidizmg gas This is easily accom-
plished by setting the Fluidizer on a hot plate (any type avail-
able in the laboratory .s satisfactory) Heat from the hot plate
is transferred to the gas as it flows through a long spiral path
in the base of the Fluidizer and emerges below the porous
plate Gas temperature can be measured by inserting a labora-
tory thermometer in the well in the base. The heat supply
should be controlled by operating the hot plate through a lab-
oratory Vanac iSee Section VI) In this way the gas tempera-
ture below the porous plate can be maintained within a 5°C
range.
In general, good packings can be produced with gas temper-
atures in the range of 40 to 100°C A good rule of thumb is to
operate with the gas temperature jus* below the boiling point
of the solvent being removed With solid stationary phases oest
results appear to be obtained with gas temperatures below the
melting point of the phase The Fluidizer will operate correctly
with gas pressures of 1 to 2 psig.
VI. TEMPERATURE CALIBRATION
Although the gas temperature can be measured while drying
is in progress, we recommend that a simple temperature versus
Vanac voltage calibration plot be made initially with the user's
hot plate. This allows the voltage to be preset when a packing
is to be dried.
Section 4 . A, •!}
Page 12
To carry out the calibration, place anout 25 grams of dry
support las supplied) in the Fluidizer, place the Fluidizer on
the hot plate, and apply a low heat (about 20 volts Vanac set-
ting). After 10 minutes gradually start the gas flow and set a
rate which will result in gentle ftuidization of the support (See
Notes 1 and 2 tn section VII) Take successive temperature
readings at 5 minute intervals until tjvo consecutive readings
agree within 5 degrees. Record this temperature and voltage
Raise the voltage in several increments until a gas temperature
of 100°C is reached, record the temperature for each voltage
setting, and make a plot of gas temperature versus Vanac vol-
tage
Use of this plot without measuring temperatures while dry-
ingshould be satisfactory However, if desired, the gas tempera-
ture can be measured during operation and a firver control ot
temperature obtained by small ad|ust
-------
Revised 12/15/79
StepS. Put the cap in place and leave the unit alone for 2
minutes. After 2 minutes the packing should begin to
fluidize. At this point
-------
Revised 12/15/79
Section 4,A, (7)
After filtration
F = 1 1 5 mi
S = 6a - F = 150 - 115 = 35 ml
100Sb
a ^ Sb
= 100
J35UO 13)
25 + (35)10 13)
i 5 4% coating
VIII. SLURRY-FLUIDIZATION PROCEDURE
The quality of packings prepared by this technique can
equal that of packings prepared by the Fiitration-Fiuidization
technique described >n the previous section However, great
attention to derail during the orehmmdry drying steo is essen-
tial to achieve the best possioie oackmg
Step 1 Seiect the desired number of grams of packing to oe
made, eg 50 0 g {see examole at end of secfoni
Vlultiply this number LV the percent onase coat de-
sired, expressed js a dec Ta' . e 0 03 tor 3%) Care-
fullv A'eigh out m a beaker the amount of phase calcu-
lated Subtract this weight from the grams of packing
desired to get the weight of support Weigh out the
support in a separate beaker Dissolve the phase in a
jo I u me of 3olvent equai 'm ml/ to 3 5 times the weight
of pack'ng desired, then aod the supoort siowiy to the
solution iwith constant stirring) to form a slurry It
mav be necessary to add additional solvent to com-
plete the slurry The tinal slurry snould have the con-
sistency of thick cream Transfer the Blurry to a fiat
dish and dry siowiy under an infrared lamp or on a
warm not plate
Step 2. When the transfer of the slurry to the dish is com-
plete, excess solution can readily oe seen Solvent from
the excess solution must be evaporated during this
step, but the packing must still be wet when trans-
ferred to the Fluidizer While it ts drying the packing
must be stirred constantly and gently to maintain uni-
formity The packing is ready for the Flutdizer when
it looks like wet sand or cottage cheese No solution
should be evident, but the packing should still be quite
wet and sticky The wet packing is now transferred to
the preheated Fluidizer The transfer should be made
with some gas flowing through the Fluidizer If the
packing is too wet when it is placed in the Fluidizer
the porous disc may be plugged by the excess solution
An inexperienced operator will tend to transfer the
packing either too soon or too iate during the prelim-
inary drymgstep. Step 2 is the critical step m this pro-
cedure
Step 3. Final drying by fluidization is now accomplished as
described m Steps 4, 5, and 6 of the Filtration Fluidi-
zation procedure (Section VII) The Slurry Ftuidiza-
tion procedure is not recommended for Teflon sup-
ports A sample calculation for this procedure is given
below
Packing desired - 50 grams co
Weight of SE 30 - 0 10 x 50 0 - 5 0 g
Weight of support = 50 - 5 = 45 0 g
Dissolve the SE-3Q >n 50 0 times 3 5 or approximately
175 ml or chloroform For all slurry coatings the
exact amount of phase and support s calculated as
above
IX. TEFLON1- COATING PROCEDURE
Excellent uniform coated packings may be prepaied from
Teflon supports by the Filtration-Fluidizadon procedure The
method is the same 35 'hat described tn Section VII but the
Ftuidizer is not heated Consequent^ Step 2 of Section VII is
omitted The damp Teflon oackmg is transferred from the fun-
nel to the Fluidizer (n 3 manner identical to that described
earlier For regular GC packings Immediately after transferring
the damp Teflon packing to the Fiuidtzer the rluidizmg gas is
turned on Solvent evaporation jvill coo! the pluidizer. Teflon,
and phase to oelow the transition temperature of Teflon M 9" C)
This cooling will permit flu:dization of the material without
undue aggregation of the particles (lump formation > After 5
minutes the Teflon pacKmg should oe fluichzmq if not, a vig-
orous shake of the F'uidizer will start the orocess When the
odor of solvent >s ^o longer evident (usually after 20 to 25 min-
utes) the packing ,s finished and can be poured into a ptastic
bottle for storage Do not attempt to scrape out the material
which adheres to the wails or cap of the Fluidizer, oecause it is
unevenly coated and aggregated Use caution when cleaning the
unit after working with Tetlon because of 'ts tendency to stick
to the unit at room temperature.
We suggest that you use highly volatile solvents for Teflon
coating Such solvents as methylene chloride, acetone, ether,
and chloroform seem to work best
For more information about working with Teflon as a sup-
port see Kirkland J J , Anal Chem 35, 2003 (1963) and Ap-
plied Science's GAS-CHROM Mewsletter 8, No 3 (July 1967)
Reprints of both articles are available from Applied Science
Teflon1^' is a registered trademark of the E.I Dupont Company
HI-EFF - is a registered trademark of Applied Science Laboratories, Inc.
X. OTHER DRYING TECHNIQUES
!f the Fluidizer is not to be used as the drying method,
the basic procedures outlined m [his bulletin can still be fol-
lowed The other drvmg methods are well described in these
references
Horning, E C , Moscatelli, E A., Sweety, C.C , Chem. and Ind
(London), 751(1959)
Zuoyk. WJ, Conner, AZ, Anal Chem 36, 912(1960)
Wotiz, H H , Chattorai, SC, Anaf Chem 36, U67M964)
Purnel!, Howard, "Gas Chromatography," Wiley, New York,
1962. p 240
-------
Revised 12/2/74 Section 4, B, (1)
Page 1
GAS CHROMATOGRAPHY-FLAME PHOTOMETRIC
INSTRUMENT
I. INTRODUCTION:
Some of the instructions in this section apply specifically to
the Mel par flame photometric unit marketed by Tracer, Inc. Other
guidelines are broadly applicable, irrespective of the make of the
gas chromatograph.
It should be borne in mind that the selection of a proper combi-
nation of operating parameters is critical for operation at maximum
sensitivity and minimum noise; therefore, the following guidelines
should be carefully considered for the achievement of these
objectives.
II. FLOW SYSTEM:
The reader is referred to page 1 of Section 4,A,(1). 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.D. requires Hydrogen,
Oxygen and possibly, air to operate. Leaks in these systems can be
hazardous from the explosion standpoint.
III. DETECTOR:
This subject is covered in detail later in Section 4,B,(3).
IV. ELECTROMETER:
See Section 4,A,(1), III. The electrometer for the F.P.D., must
deliver at least 750 VDC, and should be capable of delivering at
least 1 x 10~6 amperes bucking current.
V. TEMPERATURE PROGRAMMER:
See Section 4,A,(1), IV.
VI- PYROMETER:
See Section 4,A,(1), V.
-------
Revised 12/2/74 Section 4, B, (1)
Page 2
VII. MISCELLANEOUS:
See Section 4,A,(1), VI.
-------
Revised 12/2/74
Section 4, B, (2)
Page 1
GAS CHROMATOGRAPHY-FLAME PHOTOMETRIC
COLUMNS
I. SPECIFICATIONS:
The specifications given in Section 4,A,(2), page 1 should be
reviewed.
II. COLUMN SELECTION:
A. 4% SE-30/6% OV-210 - liquid phases premixed and coated on
silanized support, 80/100 mesh.
B. 5% or 10% OV-210 - coated on silanized support, 100/120 mesh.
See Section 4,A,(2), II.
III. PACKING THE COLUMN:
See Section 4,A,(2) for details.
IV. COLUMN CONDITIONING:
1. Heat condition a 6-foot column of 4% SE-30/6% OV-210 (QF-1)
according to Section 4,A,(2), IV,1.
2. It has been determined that a Carbowax deposition treatment will
significantly enhance the F.P.D. response of GLC columns com-
prised of Chromosorb W, H.P. as the support. The treatment has
not appeared to produce any difference in columns of Gas-Chrom Q
support. The Carbowax treatment outlined differs slightly from
the method reported by Ives and Guiffrid&i/ in that the 10%
Carbowax is packed directly into the front end of the column in
the published procedure. In the following procedure, the 10%
Carbowax is contained in a short piece of extension tube, and
attached to the front end of the column, thus leaving the front
end portion of the GLC column undisturbed.
a. Place a small wad (ca 1/2 in.) silanized glass wool in one
end of a 3 in. length of 1/4 in. 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 (Figure 1).
_i/ Gas-Liquid Chromatographic Column Preparation for Adsorptive
Compounds, Ives and Guiffrida, JAOAC, 53, 5, 1970, 973-977.
-------
Revised 12/2/74 Section 4, B, (2)
Page 2
b. Place Swagelok nut, ferrule and "o" 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.
c. Attach other end of tube to the column inlet port in the
oven, tightening Swagelok nut as much as possible. Place
1/4 in. nut on inlet end of the 6-ft. GLC column (pre-
viously heat conditioned) and insert into the male union
until it touches bottom end of 3 in. tube, then slack off
very slightly to prevent glass ends from grinding together
when nut is tightened (Figure 2).
d. 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 period. A Little
Jack works nicely. Bring oven heat up to 230° to 235°C and
apply a carrier gas flow of 20 ml/min. Hold for a 17-hour
period.
NOTES:
1. The combined parameters of temperature, time and
carrier flow are critical in the assurance of
uniformity of vapor phase deposition as affecting
ultimate retention characteristics.
2. Special materials needed include:
(a) A 3-in. length of borosilicate glass tubing,
1/4 in. o.d. x 5/32 in. i.d. (or 6 mm x 4 mm).
(b) Silanized glass wool.
(c) 10% Carbowax 20M on a silanized support,
10/100 mesh.
(d) Swagelok male union #400-6, 1/4 in. This
must be drilled out to accommodate the
1/4 in. o.d. tubing.
3. DO NOT USE A SILYLATED COLUMN WITH F.P.D. The
Silyl-8 will bleed into the F.P.D. and fog the
heat shield excessively.
4. See Section 4,A,(7) for a more permanent and
deactivating Carbowax treatment.
-------
Revised 12/15/79 Section 4, B, (2)
Page 3
V. EVALUATION OF COLUMN:
See Section 4,A,(2), V for general evaluation guidelines.
After overnight equilibration, recheck the oven temperature
and carrier gas flow rate. For optimum performance, it is advisable
at this point to adjust all operating parameters.
A. See the F.P.D. operation manual for a schematic of the recom^
mended gas flow pattern to the detector. Typical approximate
gas flows are as follows:
Nitrogen carrier 70-80 ml/minute
Hydrogen flow 50-100 ml/minute
Oxygen content of air flow 0.2-0.4 of the hydrogen
flow
Total air flow 1.5 times the hydrogen
flow
Example:
Hydrogen flow = 60 ml/minute
60 ml x 0.3 =18 ml/minute oxygen needed
18 ml v 0.20 (% 02 in air) = 90 ml/minute
air required
NOTES:
1. If the F.P.D. and F.I.D. are in operation
simultaneously, the oxygen-to-hydrogen ratio
should be closer to 0.4, which will result in
decreased sensitivity for the F.P.D. response.
2. Higher gas flows will increase background
noise.
-------
Revised 12/15/79 Section 4, B, (2)
Page 4
B. Suggested approximate operating temperatures are as follows:
Column 200°C
Injection block 225°C
Detector 175-225°C
Transfer line 235°C
NOTES:
1. Do not heat the column until the detector has reached
operating temperature.
2. The F.P.D. will operate within the range of 120-250°C,
but do not heat above 250°C or damage to the plastic
photomultiplier tube housing may result.
C. When flow and temperature parameters have been adjusted to
produce optimum signal-to-noise ratio, baseline noise should
not exceed 2.5% FSD, and an injection of 2.5 nanograms of
parathion should result in a peak of at least 50% FSD.
The following mixture should produce approximately equal peak
heights of at least 10% FSD.
Compound nc[
Ethyl Parathion 0.50
Methyl Parathion 0.38
Diazinon 0.17
Ronnel 0.25
Malathion 0.51
Trithion 1.22
Ethion 0.58
NOTES:
1. 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.
2. With some organophosphorus compounds, it will be
necessary to make several consecutive injections with
fairly high concentrations of the compound to achieve
adequate and reproducible response. This is an
important consideration if quantisation is to be
conducted.
-------
Revised 12/15/79 Section 4, B, (2)
Page 5
VI. MAINTENANCE AND USE OF COLUMN:
See Section 4,A,(2), VI.
The effects of the vapor phase deposition from Carbowax appear
to persist at least three months with a slow decrease in response
becoming evident, depending on the particular column and the amount
and type of use.
Each operator should monitor the response characteristics in
relationship to the column just after treatment.
NOTES:
1. Response will sometimes decrease rapidly for several days
after treatment, then stabilize.
2. Retreatment of columns appears to rejuvenate the response
but may result in shifts of some of the RRT-P values.
See Table 1, Section 4,B,(5). Retreatment is not advised,
therefore, because the data contained in Tables 2, 3, and
4 would become unusable.
-------
Revised 12/15/79 Section 4, B, (3)
Page 1
GAS CHROMATOGRAPHY-FLAME PHOTOMETRIC
DETECTOR
I. OPERATING PARAMETERS:
DC voltage should be supplied to the detector from either an
outboard power supply or from a strip on the back of the electro-
meter. Provided the column and all electronic circuits in the
various modules of the instrument are functioning properly, the
degree of sensitivity in the flame photometric mode relates to
four factors: (1) condition of the photomultiplier (P.M.) tube;
(2) voltage applied to P.M. tube; (3) flow rates of hydrogen,
oxygen and air; and (4) condition of interior of detector.
II. OPTIMUM RESPONSE VOLTAGE:
In order to determine the optimum response voltage for the
P.M. tube, a variable power supply is necessary which allows the
voltage to be increased with little increase in electronic noise.
Increasing the voltage from the electrometer will increase the
electronic noise inordinately.
1. Optimize all temperature and flow parameters as
described in Section 4,B,(2).
2. With flows at optimum, set power supply at 750 V DC.
Inject enough ethyl parathion to give 35 to 60, FSD.
3. Reset voltage to 850 V. Inject the same amount of
ethyl parathion as before.
4. Repeat in 100 V increments until the signal-to-noise
ratio reaches maximum and starts to decrease (.Figure 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.
-------
Revised 12/15/79
III. DETECTOR LINEARITY:
IV.
Section 4, B, (3)
Page 2
The FPD has a broad range of linearity for phosphorus.
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 attenuation will depend
on the sensitivity of the particular system used. It is best to
operate at the minimum detection level and dilute the sample when
necessary, however.
In the sulfur mode, the response is proportional to the square
of the sulfur concentration. The detector is offered with a square
root function circuit that linearizes the detector output, a neces-
sity when electronic integration and automation in the sulfur mode
are desired {-Figure 1).
PHOSPHORUS MODE:
When the detector is fitted with a 526 nm optical filter between
the flame and the photomultiplier, it is highly selective for
phosphorus, but large amounts of sulfur will give a response in
this mode.
SULFUR MODE:
When the detector is fitted with a 394 nm filter, it becomes
selective for sulfur. Sensitivity for sulfur is usually an order
of magnitude less than for phosphorus.
500
ETHION
TRITHION
500
PARATHION
METHYL
PARATHION
TRiTHION
10 30 50
FPD Response vs. Concentration
(Sultur mode wnnout square foot function )
10 30 50 70
FPO Response vs Concentration
(Sulfur mode witn square root (unction )
Fig. 1. Comparison of concentration vs response plots for FPD with and
without sulfur-mode linearizer. Conditions: column, 183 cm x 4 mm
glass,containing 3% OV-1 on high performance Chromosorb W; temperature,
200 C; carrier, nitrogen at 60 ml/minute.
-------
Revised 12/2/74 Section 4, B, (4)
Page 1
GAS CHROMATOGRAPHY-FLAME PHOTOMETRIC
SAMPLE QUANTITATION AND INTERPRETATION
I. See Section 4,A,(4).
The priming mixture below is given in nanograms 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 sample injections.
II. Peak Height:
See Section 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. Retentions, relative to parathion (RRT ), on a given column
matched with a standard or matched against the RRT values
given in Tables 2, 3 or 4.
-------
Revised 12/2/74 Section 4, B, (4)
Page 2
2. Suspect compound in the correct Florisil elution.
3. Response on a selective detector.
4. Sulfur to phosphorus ratio matched against a standard.
-------
Revised 12/2/74 Section 4, B, (5)
Page 1
TABLE I. RETENTION AND RESPONSE RATIOS, RELATIVE TO ETHYL PARATHION
ON COLUMN OF 4% SE-30/6% QF-1
COMPOUND
TEPP
Dichlorvos
Demeton-Thiono
Naled
Phorate
Sulfotepp
Diazinon
Demeton-thiolo
Dioxathion
Disulfoton
Diazi non-oxygen analog
Dimethoate
Monocrotophos
Ronnel
Ronnel -oxygen analog
Chlorpyrifos
Fenthion
Methyl Parathion
Malathion
Methyl Parathion-oxygen analog
Malathion-oxygen analog
Fenitrothion
Ethyl Pasiatklon (ti&fieAe.nce.}
Phosphamidon
Ethyl Parathion-oxygen analog
Merphos
DEF
Carbophenothion-oxygen analog
Ethion
Carbophenothion
Phenkapton
Fensulfothion
Imidan
EPN
Azinphos methyl
Coumaphos
RRT-P-1/
0.08
.10
.22
.28
.28
.28
.35
.37
.38
.40
.42
.49
.54
.57
.58
.68
.72
.75
.81
.83
.85
.85
I. 00
1.02
1.10
1.23
1.25
1.78
1.83
1.90
3.04
3.16
3.91
3.95
6.03
11.84
RPH-P-3/
5.0
5.0
2.0
0.02
4.0
5.2
2.5
2.0
0.5
3.8
1.0
0.50
0.08
1.42
0.25
1.4
1.6
0.71
0.71
0.10
0.063
0.80
1.00
0.16
0.50
0.35
0.80
0.12
0.71
0.36
0.20
0.03
0.02
0.134
0.044
0.20
_s/ Retention ratio, relative to ethyl parathion. Retention measurements
from injection point.
2/ Peak height ratio, relative to ethyl parathion.
-------
12/2/74 Section 4,B,(5)
Table 2 Pa9e 2
4%SE-30/6% OV-210
Column Temperature, °C.
170
0.06
0.04
0.14
0.15
0.16
0.19
0.20
0.21
0.23
0.23
0.27
0.30
0.32
0.30
0.38
0.40
0.46
0.48
0,51
P,55
0.60
0.68
0.57
0.62
0.67
0.72
0.81
0.86
0.94
0.90
0.93
0.91
0.85
0.96
1.00
1.02
1.02
1,11
1,18
1,19
1,1?
1,25
1.23
1,37
1,87
1,8?
1,89
3,18
3.96
4.65
4.66
5,67
7.4S
7,53
17,4
1
0.06
0.04
0.14
0.15
0.16
0.20
0.21
0.22
0.23
0.24
0.27
0.30
0.32
0.31
0.38
0.40
0.46
0.48
0.51
0.55
0.60
0.59
0.58
0.62
0.67
0.72
0.81
0.86
0.94
0.90
0.93
0.91
0.84
0.96
1.00
1.02
1.02
1.11
1,18
1.18
1.17
1.24
1,22
1.36
1.85
1,88
1.88
3.15
3.9?
4,60
4,61
5,«
7.38
7.42
_UJ!_
174
0.06
0.04
0.14
0.15
0.16
0.20
0.21
0.22
0.23
0.24
0.27
0.30
0.32
0.31
0.38
0.40
0.46
0.48
0.51
0.55
0.60
0.59
0.58
0.62
0.67
0.72
0.80
0.85
0.93
0.90
0.93
0.91
0.84
0.96
1.00
1.02
1.02
1.10
1.17
1.18
1.17
1.24
1.22
1.36
1,84
1.87
1.87
3.1?
3-8.7
4.55
4,55
5.59
7.27
7.3?
16,7
1
1
0.06 0
0.04 0
0.14 0
0.16 0
0.17 0
0.20 0
0.21 0
0.22 0
8
1
06 0.07
05 0.05
15 0.15
16 0.16
17 0.18
21 0.21
22 0.22
23 0.23
0.24 0.24 0.24
0.24 0
0.28 0.
0.31 0.
0.33 0.
0.32 0.
0.38 0.
0.41 0.
0.46 0.
0.48 0.
0.51 0.
0.56 0.
0.60 0.
0.59 0.
0.58 0.
0.62 0.
0.68 0.
0.73 0.
0.80 0.
0.85 0.
0.93 0.
0.90 0.
0.93 0.
0.91 0.
0.84 0.
0.96 0.
1.00 1.
1.01 1.
1.02 1.
1.10 1.
1.17 1.
1.17 1.
1.17 1.
1.23 1.
1.22 1.
25 0.25
28 0.28
31 0.31
33 0.33
32 0.33
38 0.38
41 0.41
47 0.47
49 0.49
52 0.52
56 0.56
60 0.60
59 0.59
59 0.59
63 0.63
68 0.68
73 0.73
80 0.80
85 0.85
93 0.92
90 0.90
92 0.92
91 0.90
63 0.83
96 0.97
00 1 .00
01 1.01
02 1.02
10 1.10
17 1.16
17 1.17
17 1.16
23 1.22
22 1.22
1.35 1.35 1.34
1 .83 1 .
1.86 1.
1.86 1.
82 1.81
85 1.84
85 1.84
3.09 3.06 3.03
3.83 3.
4.50 4.
4.51 4.
5.55 5.
7.17 7.
7.21 7.
16.3 16
79 3.75
45 4.40
46 4.41
51 5.47
06 6.96
10 6.99
0 15.6
T
0.07
0.05
0.15
0.17
P. 18
0.21
0.23
0.23
0.25
0.25
0.26
0.32
0.33
0.33
0.38
0.42
0.47
0.49
0.52
0.56
0.60
0.60
0.59
0.63
0.68
0.73
0.80
0.84
0.92
0.90
0.92
0.90
0.83
0.97
1.00
1.01
1.03
1.10
1.16
1.16
1.16
1.22
1.21
1.33
1.80
1.83
1.83
3.00
3.70
4.35
4.36
5.42
6.85
6.89
15.3
1
0.07
0.05
0.15
0.17
0.16
0.21
0.23
0.24
0.25
0.26
0.29
0.32
0.34
0.33
0.38
0.42
0.47
0.50
0.52
0.57
0.60
0.60
0.59
0.63
0.68
0.74
0.79
0.84
0.91
0.90
0.92
0.90
0.82
0.97
1.00
1.00
1.03
1.10
1.15
1.16
1.16
1.21
1.21
1.33
1.78
1.82
1.82
2.98
3.66
4.31
4.31
5.38
6.75
6.78
15.0
T
0.07
0.06
0.16
0.17
0,19
0.22
0.23
0.24
0.25
0.26
0.29
0.32
0.34
0.34
0.39
0.42
0.47
0.50
0.53
0.57
0.60
0.60
0.60
0.64
0.68
0.74
0.79
0.84
0.91
0.90
0.92
0.90
0.82
0.97
1.00
1.00
1.03
1.10
1.15
1.15
1.15
1 21
1.21
1.32
1.77
1.80
1.80
2.95
3.62
4.26
4.26
5.34
b.64
6.67
14.6
1
1
0.08 0
0.06 0
0.16 0
0.18 0
0.19 0
0.22 0
0.24 0
0.24 0
0.25 0
0.26 C
0.29 0
0.33 0
0.34 0
0.34 0
0.39 0.
0.43 0.
0.48 0.
0.50 0.
0.53 0.
0.57 0
0.60 0
0.60 0.
O
1
08 0.08
06 0.06
16 0.17
18 0.19
19 0.20
22 0.23
24 0.24
25 0.25
26 0.26
27 0 27
30 0.30
33 0.33
35 0.35
35 0.35
39 0.39
43 0.43
48 0.48
50 0.51
53 0.54
53 0.58
60 0.61
60 0.61
0.60 0.60 0 61
0.64 0.
0.69 0.
0.74 0.
0.79 0.
0.84 0.
0.91 0.
0.90 0.
0.91 0
0.90 0
0.83 0
0.97 0
1.00 1
1.00 1
1.03 1
1.10 1
1.15 1
1.15 1.
1.15 1
1.20 1
1.21 1
1.32 1
1.76 1
64 0.64
69 0.69
75 0.75
79 0.79
85 0.85
90 0.90
90 0.90
91 0 91
90 0.90
84 0.86
97 0.97
00 1.00
00 1.00
03 1 .03
09 1.09
14 1.14
14 1.14
15 1.14
20 1.19
20 1.20
31 1.31
75 1.74
1.79 U78 1.77
1.79 1
2.92 2
3.58 3
4.21 4
4.21 4
5.30 5
6.54 6
6.57 6
14.3 13
78 1.77
89 2.86
53 3.49
16 4.11
16 4.11
26 5.21
43 6.33
46 6.35
9 13.6
I1
n
0
0
0
n
n
0
n
n
n
0
n
n
n
n
n
n
n
n
n
n
0
0
0
0.
0
n
n
0
n
0
0
n
n
l
i
i
i
i
l
i
i
i
i
i
i
i
4
08
06
17
19
?0
?1
?4
?•;
?f
?7
in
14
IS
15
IP
44
48
SI
54
58
61
fil
61
64
69
75
79
85
IW
9(1
91
91
87
98
on
ni
m
m
11
14
14
19
?n
30
73
76
76
2.83
3
4
4
5
6
6
13
44
06
06
17
??
24
2
1
0.08
0.06
0.17
0.19
0.21
0.23
0.25
0.26
0.27
0.28
0.30
0.34
0.35
0.36
0.39
0 44
0.49
0.51
0.54
0.58
0.61
0.61
0.61
0.64
0.69
0 75
0.78
0.85
0.89
0.90
0.90
0.91
0 88
0.98
1.00
1 .01
1.03
1 .09
1 13
1 13
1.14
1.18
1 .20
1.30
1.72
1.75
1 .75
2.80
3.41
4.01
4.00
5.13
6.12
6.14
12.9
T
0.09
0.07
0.17
0.20
0.21
0.23
0.25
0.26
0.27
0.28
0.31
0.34
0.36
0.36
0.39
0.44
0.49
0.51
0.54
0.59
0.61
0.62
0.61
0.64
0.70
0.76
0.78
0.86
0.88
0.90
0.90
0.91
0 89
0.98
1.00
1.01
1.03
1.09
1 13
1 13
1 13
1.18
1.19
1.29
1.70
1.74
1.74
2.77
3.36
3.96
3.95
5.09
6.01
6.03
12.6
T
1
0.09
0.07
0.18
0.20
0.21
0.24
0.25
0.27
0 27
0 28
0.31
0.35
0.36
0.37
0.40
0.45
0.49
0.5?
0.55
0.59
0 61
0 62
0.62
0.65
0.70
0.76
0.78
0.86
0.88
0.90
0.90
0.91
n.90
0.98
i.on
1.01
1.03
1.09
1.12
1.12
1.13
1.17
1.19
1.29
1.69
1.73
1.73
2.74
3.32
3.91
3.90
5.05
5.91
5.92
12.2
T
0.09
0.07
0.18
0.20
0.22
0.24
0.26
0.27
0.27
0.28
0.31
0.35
0.36
0.37
0.40
0.45
0.49
0.52
0.55
0.59
n.ei
0 62
0.62
0.65
0.70
0.76
0.78
0.86
0.88
0.90
0.90
0 91
0 92
0.98
1 .00
1 01
1 03
1.09
1 12
1.12
1 13
1.17
1.19
1.28
1 68
1 72
1.72
2 72
3.28
3.87
3.85
5.01
5.80
5.81
11.9
204
1
0.09
0.07
0 18
0 21
0.23
0.24
0.26
0.27
0.28
0.29
0.31
0 35
0 37
0.37
0.40
0.45
0.49
0.52
0 55
0 59
0.6!
0 62
0.62
0.65
0 70
0.76
0.76
0 86
0 87
0.90
0.90
0 91
0 93
0.98
1.00
1.01
1.03
1.08
1.11
1.12
1.12
1.16
1.19
1.28
1.67
1 71
1.71
2.69
3.23
3.82
3.80
4.96
5.70
5 71
11.5
Dlchlorvos
TEPP
Mevlnphos
Demeton Thiono
TMonazin
Ethoprop
Phorate
Sulfotepp
Naled
Oxydemeton Methyl
Dlazinon
Dioxathlon
Demeton Thiolo
Disulfoton
Diazoxon
Dichlofenthion
Oimethoate
Ronnel
Cyancx
Ronnoxon
Monocrotophos
Chlorpyrlfos
Zytron
Fenthion
Malaoxon
Methyl Parathion
Malathion
Fern trothion
Bromophos
Methyl Paraoxon
Phenthoate
Bromophos Ethyl
Schradan
Dicapthon
E. ParathionllMnniJ
Amldithion
lodofenphos
Cruf ornate
DEF
Phosphamidon
Folex
Ethyl Paraoxon
Methidathion
Tetrachlorvinphos
Ethion
Carbophenoxon
Carbophenothion
Phenkapton
Fensulfothion
Imidan
EPN
Famphur
Azlnphos Ethyl
Azlnphos Methyl
Coumaphos
Retention ratios, relative to parathlon, of 54 organophosphorous pesticides on a column of
4» SE-30/6J OV-210 «t temperatures from 170 to 204-C; support of Gas Chrom-Q, 80/100 mesh-,
flame photometric detector, 5260 A'fllter; all absolute retentions measured from Injection
point. Arrow Indicates optimum operating temperature with carrier flow set at 75 ml per minute.
-------
12/2/74
Table 3
Section 4,B,(5J
Page 3
10 °/o OV-210
Column Temperature , "C.
T
0.04
6.66
6.12
O*1
0.14
6.15
6.16
017
B"1B
6.19
6.64
6.??
6.25
0.29
0.29
0.33
6.34
0.46
0.43
0.4?
0.48
6.4)
0.51
0.53
0.59
6.67
6.75
0.69
0.77
6.75
0.77
6 74
6.81
6.84
1.62
0.9S
.02
.00
1.64
.03
.13
1.22
1.27
1.38
1.42
1.43
T.54
2.69
4.26
4.37
4.65
6.98
(.66
7.61
19.1
170
1
0.04
6.66
6.12
6.14
6.14
0.15
6.16
0.17
0.18
6.19
6.04
6.22
6.25
0.29
0.29
6.33
6.34
6.40
0.43
0.47
0.48
0.47
0.51
6.53
0.59
6.67
0.75
0.69
0.77
0.75
6 77
6.74
6.81
0.84
1.61
6.95
.02
.06
.03
.03
.13
.22
.27
.37
1.41
.43
1.63
2.08
4.21
4.32
4.59
6.83
6.76
(.90
18.7
1
174
1
0.04
6.66
6.12
0.15
6.14
0.15
6.16
0.18
0.18
0.26
6.64
6.23
6.25
O.J9
0.29
0.33
6.34
6.41
0.43
6.4?
0.48
0.48
6.61
6.54
6.59
6.66
0.75
0.69
0.77
0.75
6.77
6.75
0.81
0.84
1.01
0 95
.62
.60
.03
.U3
.13
.21
.26
.36
.40
.42
.62
2.06
4.16
4.27
4.54
6.7?
6.66
6.79
18.3
,74
i
0.04
6.66
6.12
'fl.15
6.15
0.16
0.17
0.18
0.19
0.26
6.64
6.23
0.26
0.29
0.30
0.33
6.35
6.41
6.44
0 47
0.48
0.48
0.52
6.54
6.59
6.66
0.74
6.76
0.77
0.75
0.77
0.75
0.81
0.84
1.66
0.95
.61
.60
.03
.UJ
.13
.20
.26
.36
.39
.41
.62
2.04
4.11
4.22
4.48
6.66
6.56
6.69
17.9
1
r
0.04
6.07
0.13
0.15
6.15
0.16
6.17
6,18
0.19
0.21
0.05'
6.23
6.26
0.29
0.30
0.34
6.35
6.41
6.44
0.47
0.49
0.48
0.52
0.54
0.60
0.66
6.74
6.76
6.77
6.74
6.77
0 75
6.86
6.85
6.99
6.95
1.61
1.60
1.03
.UJ
.13
1.20
1.25
1.35
1.38
.46
.61
2.02
4.06
4.18
4.43
6.55
6.47
6.58
17.5
178
1
0.05
6.67
0.13
0.15
6.15
6.U
6.17
0,18
0.19
0.21
0.05
0.24
0.26
0.29
0.30
0.34
0.35
0.41
0.44
6.47
0.49
0.49
6.52
0 55
0 60
0.66
6.73
6.70
6.77
0.74
0.77
6.76
0.86
0.85
6.99
6.95
1.00
1.00
1.62
.02
1.13
1.19
1.25
1 34
1.37
1.39
1.66
2.00
4.01
4.13
4.37
6.44
6.37
6.47
17.1
1
182
0.05
0.07
6.13
0.16
6.16
0.17
0.18
0.19
0.20
0.21
0.05
6.24
0.27
0.30
0.31
0.34
0.36
0.41
6.45
6.48
0.49
0.49
6.53
O.E5
6.60
6.66
0.73
0.70
0.76
0.74
0.77
0.76
0.86
0.85
0.98
0.95
1.06
1.00
1.02
I.IK
1.13
1.19
1.24
1.33
1.37
1.38
1.59
1.98
3.97
4.08
4.32
6.33
6.27
6.36
16.7
1
1
0.05
0.07
0.13
6.16
0.16
0.17
0.18
0.19
0.20
0.22
0.05
0.24
0.27
0.30
0.31
0.34
0.36
0.42
0.45
0 48
0.49
0.49
6.53
0.56
0.60
0.65
0.73
0.70
0.76
0.74
0 77
0 76
0.80
0.85
6.97
6 95
1.00
1.00
1.62
.02
1.13
1.18
1.24
.32
1.36
.37
1.58
1.96
3.92
4.03
4.26
6.22
6.17
6.25
16.3
1
T
0.05
,0.07
0.13
0.16
0.16
0.18
0.18
0.19
0.20
0.22
0.05
0.25
0.28
0.30
0.3Z
0.34
0.37
0.42
0.45
0.48
0.49
0.50
6.53
0.56
0.60
0.65
0.72
0.76
0.76
0.74
6.77
0.76
0.80
0.85
6.96
6.95
0.99
.00
.62
.02
.13
.16
.24
.31
.35
.37
.57
.94
3.87
3.98
4.21
6.11
6.08
6.14
15.9
I
190 194 If
1 1 1 1
0.05 0.05 0.05 0.06 0.06 0.
T.O 0.68 0.08 0.08 0.08 67
0.13 0.13 0.13 0.14 0.14 0.
0.16 O.Y? (T.T7 ~67T7 0.17 B7
0.16 0.17 0.17 0.17 0.17 0.
0.18 0.19 0.19 0.19, 0.20 0.
0.19 0.19 0.19 0.20 0.20 0.
0.19 0.20 0.20 0.20 0.20 0.
0.20 0.21 0.21 0.21 0.22 0.
0.23 0.23 0.24 0.24 0.24 0.
0.06 0.06 0.06 0.06 0.06 0.
0.25 0.26 0.26 0.26 0.27 0.
0.28 0.28 0.29 0.29 0.29 0.
0.30 0.30 0.31 0.31 0.31 0.
0.32 0.32 0.33 0.33 0 33 0.
0.35 0.35 0.35 0.35 0.35 0.
0.37 0.37 0.38 0.38 0 38 0.
0.42 0.42 0.42 0.43 0.43 0.
0 46 0.46 0.46 0.46 C.47 0
0.48 0.48 0.49 0.49 0.49 0.
0 50 0.50 0.50 0.50 0.50 0.
0.50 0.51 0.51 0.51 0.52 0
6.53 0.54 0.54 0.54 0.54 0
0.56 0.57 0.57 0.57 0.58 0
0.60 0.60 0.60 0.61 0 61 0
0.65 0.65 0.65 0.64 0 64 0
0.72 6.71 0.71 0.71 0 70 0
0 70 6 70 6.71 0.71 0.71 0
0.76 0.76 0 75 0.75 0.75 0
6.74 0.74 0.75 0.75 0.75 0
0.77 0.77 6.77 0 77 0.77 0
0.76 6.77 6.77 6.77 6 77 6
0.80 0.8C 0.80 0.81 0 81 0
0.85 6.85 0.86 0.86 0 86 0
0.96 0.95 0.94 0 94 0.93 0
6.95 0 96 0.96 0.96 0.96 0
0.99 0.99 0.98 0.98 0.98 0
.00 .00 1.00 1.00 .00 1
01 .01 1.01 1.01 .00 1
.13 .13 1.12 1.12 .12 1
.17 .17 1 16 1 16 .15 1
.23 .23 1 22 1.22 .21 1
.31 .30 1.29 1.28 .27 1
.34 4.33 .32 1.32 .31 1
.36 .35 1.34 1.33 .32 1
.56 .55 1.54 1.53 .53 1
.92 .90 .88 1.86 .84 1
3.82 3.77 3.72 3.68 3.63 3
3.93 3.88 3.83 3.78 3.73 3
4.15 4.10 4.04 3.99 3.94 3
6.00 5.89 5.78 5.67 5.56 5
5.98 5.88 5.79 5.69 5.59 5
6.03 5.92 5.81 5.70 5.59 5
15.5 15.1 14.7 14.3 13.9 13
1 | . | 1
190 194 U
B '
1
06 0,06
6J^.09
14 0.14
Yfi tt.Te
18 0.18
20 0.20
21 0.21
21 0.21
22 0.22
25 0.25
07 0.07
27 0.27
30 0.30
31 0.31
3"4 0.34
36 0 . 36
39 0.39
43 0.43
47 0.47
49 0.49
51 0.51
52 0.52
55 0.55
58 0.58
61 0.61
64 0.64
76 6.70
71 0.71
75 0.75
75 0.75
77 0.77
78 0.78
81 0.81
86 0.86
92 0.92
96 0.96
97 0.97
00 .00
00 1.00
12 1.12
15 1.14
21 1.20
26 1.26
30 1.29
31 1.31
52 1.51
82 1.80
58 3.53
68 3.63
88 3.86
45 5.34
49 5.40
48 5.37
5 13.1
1
8
T
0.06
0.09
0.14
0.18
0.18
0.20
0.21
0.21
0.22
0.25
0.07
0.28
0.30
0.32
0.34
0.36
0.39
0.43
0.47
0.50
0.51
0.53
0.55
0.59
0.61
0.64
0.69
0.71
0.75
0.75
0.77
0.78
0.81
0.86
0.91
0.96
0.97
1.00
1.00
1.12
1.13
1.20
1.25
1.28
1.30
1.50
1.79
3.48
3.58
3.77
5.24
5.30
5.26
12.7
1
204
1
0.06
0.09
0.14
0.19
0.19
0.21
0.22
0.21
0.23
0.26
0.07
0.28
0.31
0.32
0.35
0.36
0.40
0.43
0 47
0.50
0.51
0.53
0.55
0.59
0.61
0.63
0.69
0.71
0 74
0.75
0.77
0.79
0.81
0 86
0.90
0 96
0.96
1.00
0.99
1 12
1.12
1.19
1.24
1.27
1.29
1.49
1.77
3.43
3.53
3.72
5.1 3
6.26
5.15
12.3
204
TEPP
Dlchlorvos
Demeton Thlono
Mevtnphos
Thlonazln
Phorate
Ethoprop
Dlazinon
Sulfotepp
Naled
Oxydemeton Methyl
Disulfoton
Dioxathlon
Demeton thlolo
Dichlofenthion
Dlazoxon
Ronnel
Chlorpyrlfos
Zytron
Bromophos
Fenthlon
Dimethoate
Ronnoxon
Cyanox
Bromophos ethyl
Monocrotophcs
Malathion
lodofenphos
OFF
Phenthoate
Folex
Methyl parathion
Schradan
Feni trothlon
Kalaoxon
Dicapthon
Crufomate
Parathion (R«f»r«n
-------
12/2/74 Section 4,B,(5)
Table 4 Pa9e 4
1.5%OV-17/1.95% OV-210
Column Temperature , * C. i
170
0.04
0.06
0.12
0.16
0.19
0.20
0.23
0 24
0.26
0.32
0.31
0 34
0 36
0.37
0.38
0.44
0.51
0.53
0.55
0.57
0.78
0.66
0.74
0.74
0.79
0.93
0.92
0.85
0.90
0.90
1.02
1.25
1.00
1.06
1.05
1.12
1.23
1.40
1.36
1.51
1.51
1.57
1.72
1.74
2.69
2.88
2.99
4.65
5.57
6.07
6.63
7.95
10.9
14.4
22.2
170
1
0.04
0.06
0.13
0.16
0.19
0.20
0.23
0.25
0.27
0.32
0.31
0.34
0 36
0 38
0.39
0.44
0.51
0.53
0.55
0.57
0.77
0.66
0.74
0.74
0.79
0.93
0.92
0.85
0.90
0.90
1.01
1.23
1.00
1.06
1.05
1.12
1.22
1.39
1.35
1 50
1.50
1.57
1.71
1.73
2.67
2.85
2.97
4.60
5.50
5.99
6.53
7.84
10.7
14.1
21.8
1
174
0.05
0.07
0.13
0.17
0.19
0.20
0.24
0.25
0.27
0.33
0.32
0.34
0.37
0.38
0.39
0 45
0.51
0.53
0.56
0 57
0 76
0.67
0 74
0.74
0.79
0.92
0.92
0 86
0 90
0.90
1.01
1.21
1.00
1.06
1.05
1.12
1.22
1.38
1.35
1.49
1.49
1.56
1.70
1.73
2.64
2.82
2.94
4.56
5.4?
5.90
6.44
7.72
10.6
13.9
21.3
174
1
0.05
0.07
0.14
0.17
0.20
0.21
0.24
0.25
0 28
0.33
0.32
0.35
0.37
0.39
0.39
0.45
0.52
0.54
0 56
0.58
0.76
0.67
0 74
0.75
0 79
0.92
0.92
0.86
0.90
0.90
1.00
1.19
1.00
1.06
1.05
1.11
1.22
1.38
1.34
1.49
1.49
1.56
1.69
1.72
2.62
2.79
2.92
4.51
5.35
5.82
6.34
7.61
10.4
13.6
20.9
'
178
0.05
0.07
0.14
0.17
0.20
0.21
0.25
0.25
0.28
0.33
0.33
0.35
0.38
0.39
0.40
0.45
0.52
0.54
0.57
0 58
0 75
0.67
0.75
0.75
C 79
0.91
0.91
0.86
0.91
0.91
1.00
1.18
1.00
1.05
1.06
1.11
1.21
1.37
1.34
1.48
1.48
1.56
1.68
1.72
2.59
2.76
2.89
4.46
5.27
5.74
6.25
7.50
10.3
13.3
20.4
178
1
0.05
0.08
0.15
0.18
0.21
0.22
0.25
0.26
0.28
0.33
0.33
0 35
0.38
0.39
0.40
0.46
0.53
0.54
0.57
0.59
0.74
0.67
0.75
0.75
0.79
0.91
0.91
0.86
0 91
0.91
l.OC
1.16
1.00
1.05
1.06
1.11
1.20
1.36
1.33
1.47
1.47
1.55
1.67
1.71
2.57
2.73
2.87
4.42
5.20
5.65
6.16
7.38
10.1
13.0
20.0
'
T
0.06
0.08
0.15
0.18
0.21
0.22
0.25
0.26
0.29
0.34
0.34
0.36
0.38
0.40
0.40
0.46
0.53
0.55
0.57
0.59
0.73
0.68
0.75
0.76
0.79
0.91
0.91
0.87
0.91
0.91
0 99
1.14
1.00
1.05
1.06
l.ll
1.20
1.35
1.33
1.46
1.46
1.54
1.66
1.71
2.54
2.70
2.84
4.37
5.12
5.57
6.06
7.27
10.0
12.8
19.5
1
1
0.06
0.08
0.16
0.18
0.21
0.22
0.26
0.27
0.29
0.34
0.34
0.36
0.39
0.40
0.41
0.46
0.53
0.55
0.58
0.59
0.73
0.68
0.75
0.76
0.80
0.90
0.91
0.87
0.91
0.91
0.99
1.13
1.00
1.05
1.06
1.11
1.19
1.34
1.32
1.45
1.45
1.54
1.65
1.70
2.52
2.67
2.82
4.32
5.05
5.49
5.97
7.15
9.8
12.5
19.1
1
T
0.06
0.09
0.16
0.19
0.22
0.23
0.26
0.27
0.30
0.34
0.35
0.36
0.39
0.41
0.41
0.47
0.54
0 55
0.58
0.60
0.72
0.68
0.7^
0.77
0.80
0.93
0.91
0.87
0.91
0.91
0.98
1.11
1.00
1.05
1.06
1.11
1.19
1.33
1.32
1.44
1.44
1.53
1.65
1.70
2.49
2.64
2.79
4 27
4.97
5.40
5.88
7.04
9.7
12.2
18.6
i
1
0.06
0.09
0.17
0.19
0.22
0.23
0.27
0.27
0.30
0.34
0.35
0.37
0.40
0.41
0.41
0.47
0.54
0.56
0.59
0.60
0.72
0.68
0 75
0.77
0.80
0.89
0.90
0.87
0.91
0 91
0.98
1.09
1.00
1.05
1.06
1.10
1.19
1.32
1.31
1.43
1.44
1.53
1.64
1.69
2.46
2.61
2.77
4.23
4.90
5.32
5.78
6.93
9.5
11.9
18.2
1
T
0.07
0.09
0.17
P.19
0.23
0.24
0.27
0.28
0.30
0.35
0.36
0.37
0.40
0.41
0.42
0.47
0.55
0.56
0.59
0.61
0.71
0.69
0.75
0.77
0.80
0.89
0.90
0.88
0.91
0.91
0.98
1.07
1.00
1.05
1.05
1.10
1.19
1.32
1.31
1 43
1.43
1.53
1.63
1.69
2.45
t.58
2.75
4.18
4.82
5.24
5.69
6.81
9.3
11.7
17.2
i
1
0.07
0.10
0.18
0.20
0.23
0.24
0.27
0.28
0.31
0.35
0.36
0.37
0.40
0.42
0.42
0.48
0.55
0.56
0.59
0.61
0.70
0.69
0.75
0.78
0.80
0.89
0.9C
0.63
0.91
0.91
0.97
1.06
1.00
1.05
1.05
1.10
1.19
1 31
1 30
1 42
1.42
1.52
1.62
1.6E
2.42
2.55
2.72
4.13
4.75
5.15
5.59
6.70
9.1
11.4
17.3
1
T
0.07
0.10
0.18
0.20
0.23
0.24
0.28
0.28
0.31
0.35
0.36
0.37
0 41
0.42
0.42
0.48
0.55
0.57
0.60
0.61
0.69
0.69
C.76
0.78
0.80
0.88
0.90
0.88
0 90
0.92
0 97
1.04
1.00
1.04
1.05
1.10
1.20
1.30
1 30
1.41
1.41
1 52
1.61
1.68
2.40
2.52
2.70
4.08
4.67
5.07
5.50
6.58
8.9
11.1
16.8
1
194
1
0.07
0.10
0.19
0.20
0.24
0.25
0.28
0.29
0.32
0.35
0.37
0.38
0.41
0.43
0.4'
0.48
0.56
0.57
0.60
0.62
0.68
0.69
0.76
0.78
0.80
0.88
0.90
0.88
0.90
0.92
0 96
1.02
1.00
1.04
1.05
1.10
1.20
1.29
1.29
1.40
1.41
1.51
1.60
1.67
2.37
2.49
2.67
4.04
4.60
4.99
5.41
6.50
8.7
10.9
16.4
1
T
0.08
0.11
0.19
0.21
0.24
0.25
0.29
0.29
0.32
0.36
0.37
0.38
0.42
0.43
0.43
0.49
0.56
0.57
0.61
0 62
0.68
0.70
0.76
0.79
0.81
0.87
0.89
0.89
0.90
0.92
0.96
1.00
1.00
1.04
1.04
1.09
1.20
1.28
1.29
1.39
1.40
1.51
1.59
1.67
2.35
2.46
2.66
3.99
4.52
4.90
5.31
6.36
8.5
10.6
15.9
i
198
T
i
0.08
0.11
0.20
0.21
0.25
0.26
0.29
0 29
0.32
0.36
0.38
0.38
0.42
0.43
0.43
0 49
0.57
0 58
0.61
0.63
0.67
0.70
0.76
0.79
0.81
0.87
0.89
0.89
0.90
0.92
0.96
0.99
1.00
1.04
1.04
1.09
1.20
1.27
1.28
1 38
1.39
1.50
1.58
1.66
2.32
2.43
2.62
3.94
4.45
4.82
5.22
6.24
8.3
10.3
15.5
1
T
0.08
0.11
0 ?1
0.21
0.25
0.26
0.29
0.29
0.33
0.36
0.38
0.39
0.42
0.44
0.44
0.49
0.57
0.5S
0.61
0.63
0 66
0.70
0.76
0.79
0.81
0.87
0.89
0.89
0.90
0.92
0.95
0.97
1.00
1.04
1.04
1.0?
1.20
1.26
1 ?7
1 17
1.38
1.50
1.57
1.65
2.30
2.40
2.60
3.89
4.38
4.74
5,1?
6,13
8.1
10.1
15.0
202
204 ,
|
0.09 TEPP
0.12 Dichlorvos
0 21 Mevinphos
0.22 Demeton thiono
0.25 Thionazin
0.26 Ethoprop
0.30 Phorate
0.33 Oxydemeton methyl
0.37 Diazinon
0.39 Naled
0.39 Demeton thiclo
0 43 Disulfoton
0.44 Oioxathion
0.44 Diazoxon
0.50 Dichlofenthion
0 57 Cyanox
0.58 Dimethoate
0.62 Ronnel
0.63 Ronnoxon
0 65 Monocrotophos
C 71 Zytron
0 76 Chlorpynfos
0 80 Methyl Parathion
0.81 Methyl Paraoxon
0 86 Malaoxon
0.89 Malathidn
0.9C Broroophos
0.90 Fenthion
0.92 Femtrothion
O.S5 Schradon
1 On Paratnion(R«f»f»ne«)
} C4 Ethyl Paraoxon
1 03 Dicapthon
1 09 Bromophos Ethyl
1 .20 Anndithion
1.25 Crufomate
1.27 Phenthoate
1.36 Folex
1 37 DEF
1 .49 lodofenphos
1 56 Tetrachlorvlnphos
1.65 Methidathion
2.27 Carbophenoxon
2.37 Ethion
2.57 Carbophenoth 1 on
3.85 Fensulfothion
4.30 Phenkapton
4.65 Famphur
5.03 EPN
6.01 I mi dan
7.9 Azinphos Methyl
9.8 Azlnphos ethyl
14.6 Coimaphos
I
204
Retention ratios, relative to ethyl pt ilon, of 54 organophosphorous pesticides on a column of
1.51 OV-17/1.951 OV-210 at temperature;, from 170 to 204-C; column support of Sas Chrom-Q, 100/120
mesh; flame photometric detector, 5260 A« filter; all absolute retentions measured from Injection
point. Arrow Indicates optimum column operating temperature vrtth carrier flow at 70 ml per minute.
-------
11/1/72 Section 4,B,(5)
Page 5
FIGURE 1
Carbowax Tube Section
_^___ lO°o Carbewax
s£53IZIZL_ZZIZ^ZIZIIZZI)
' Glass Wool
FIGURE 2
Cutaway Viev/ of Column with Carbowax Assembly
-------
11/1/72
FIGURE 3
Section 4, B, (5)
Page 6
Chromatcgroms of a mixture of 7 organophosphorous pesticides
on an untreated column of 4% SE-30/6% GF-1 (Fig.1),and on the
same column treated with Carbowax(Fig.2)
Column:4% SE-30/6% QF-l;amps,full scale 0.8 xlO -8 ;voltage 850 v.
OPERATING PARAMETERS
TEMP., C.
Column
Inlet
Detector
Transf.line
Vent
200
225
195
235
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-------
11/1/72
Section 4, B, (5)
Page 7
FIGURE 4. Calculations of Signal/Noise Ratio
|03X id, (i.e. Xio-sfln/°S FULL.
-------
-------
Revised 12/15/79 Section 4, C, (1)
Page 1
GAS CHROMATOGRAPHY - HALL ELECTROLYTIC
CONDUCTIVITY DETECTOR
INSTRUMENT
I. INTRODUCTION:
The Hall electrolytic conductivity detector (HECD) can be
operated in the reductive mode selective for nitrogen- or halogen-
containing compounds or in the oxidative mode selective for sulfur
detection. Most applications to date have been for organochlorine
and organonitrogen compounds. The selectivity of the detector allows
its use for obtaining confirmatory evidence for residues tentatively
identified by electron capture GC, while the sensitivity is adequate
for quantisations at sub-ppm levels.
Effluent from the gas chromatograph is pyrolyzed in a quartz
combustion tube in the presence of specified gases and sometimes
catalysts. During pyrolysis, specific elements present in the
organic pesticides form soluble electrolytes that are then combined
with deionized liquid in a gas-liquid contactor. The electrical
conductivity of the liquid is continuously measured. Only those
combustion products that are readily soluble and ionized in the
liquid change its electrical conductivity and produce a response
on the recorder.
Most of the material in this section applies specifically
to the Tracer Model 700 Hall detector connected to a conventional
gas chromatograph such as the Tracer MT 222, or equivalent. The
Model 700 monitors the electrical conductivity of the liquid
utilizing an AC bridge circuit and auxiliary recorder. The newer
Model 700A detector is similar in principle but is engineered
exclusively for use with the Tracer Model 560 digital processor
controlled gas chromatograph. The 700A features more precise
flow regulation, a microreactor furnace, lower dead volume, improved
scrubbers, automatic solvent venting, and a new differential con-
ductivity cell design combined with a pulse cell excitation system
for improved detection specificity and sensitivity and baseline
stability.
-------
Revised 12/15/79 Section 4, C, (1)
Page 2
II. FLOW SYSTEM:
See page 1 of Section 4,A,(1). Hydrogen is the combustion gas
for both chlorine and nitrogen detection in the reductive mode. Air
is the reactor gas for sulfur compounds in the oxidative mode.
Helium only (preferably ultra pure) is the recommended carrier
gas for operation in the nitrogen mode. Nitrogen carrier in this
mode is unsatisfactory because a small percentage of nitrogen is
converted to ammonia causing a high background and low sensitivity.
Helium may also be used in the chloride (reductive) mode, but best
results are obtained only with the ultra pure grade. Nitrogen or
helium may be used as carrier gas in the sulfur mode. In all cases,
metal diaphragm pressure regulators should be used with helium to
prevent contamination of the carrier gas with air. A Go-Getter
gas purifier (General Electric, Schenectady, NY, distributed by
Alltech Associates, Inc., Arlington Heights, IL) can be used to
ensure removal of impurities from helium. Hydrogen must be free
of oxygen to be suitable. Electrolytic generators constructed with
a palladium diffusion membrane have been successfully used with
the HECD.
The HECD is extremely sensitive to carrier gas leaks, with
generally are manifested as wandering baseline and noise.
III. DETECTOR:
See Section 4,C,(3). For connection of the detector to the
chromatograph and operation of the system, see Sections II and III
of the Model 700 Operation and Service Manual 115008A.
IV. ELECTROMETER:
See Section 4,A,(1), III.
The HECD does not require as high a voltage as the electron
capture and FPD detectors. The power supply for the HECD shares
a printed circuit board with the AC bridge. The function of the
power supply is to provide + 15 V regulated.
V. TEMPERATURE PROGRAMMER:
See Section 4,A,(1), IV.
VI. PYROMETER:
See Section 4,A,(1), V.
-------
Revised 12/15/79 Section 4, C, (1)
Page 3
VII. MISCELLANEOUS:
See Section 4,A,(1), VI.
A heated transfer line carries the GC column effluent to the
combustion tube, and this line plays a critical role in successful
operation of the detector. Care must be taken to ensure that all
transfer line areas are sufficiently hot to prevent analyte loss
and/or tailing.
Use of septa coated with polyimide (Pursep-P, L. C. Co., Inc.,
Schaumburg, IL) has been reported to reduce background noise during
HECD operation (FDA PAM, Section 315.42, 6).
-------
-------
Revised 12/15/79 Section 4, C, (2)
Page 1
GAS CHROMATOGRAPHY - HALL ELECTROLYTIC
CONDUCTIVITY DETECTOR
COLUMNS
I. SPECIFICATIONS:
See Section 4,A,(2), page 1.
II. COLUMN SELECTION:
Although normal pesticide column packings (Section 4,A,(2))
have most often been used with the HECD, it is best to select very
stable columns because acid products resulting from the bleed of
halogenated liquid phases, such as OV-210, XE-60, or QF-1, can
produce inordinately high noise levels in the Cl-mode and scrubber
depletion in the N-mode. For this reason, surface-bonded Carbowax
20M columns (Section 4,A,(7)), uncoated or coated with a low loading
of a stable liquid phase, are especially recommended for analyses
with the HECD (Section 12,A). Columns containing 3% OV-1, 3% and
5% OV-101, 3% STAP, 5% Carbowax 20M, and wall-coated OV-101
(capillary column) have also been successfully used.
The use of 1.8 m (6 ft.) x 2 mm i.d. GC columns rather than
4 mm i.d. has been recommended in the FDA PAM (Section 315.41 (3)).
The smaller column requires only 30-40 ml/minute carrier gas flow
to produce the same chromatogram from the larger column with
100-120 ml/minute flow, leading to increased detector sensitivity
and reduced system back pressure. The smaller column may not,
however, tolerate a large number of relatively "dirty" samples.
III. PACKING THE COLUMN:
See Section 4,A,(2), III.
IV. COLUMN CONDITIONING:
See Sections 4,A,(2), IV and 4,A,(7), II.
V. COLUMN EVALUATION:
See Section 4,A,(2), V for general evaluation guidelines.
Figures 1 and 2 show typical sensitivities attainable for
pesticides with the nitrogen and chloride modes of the Model 700
-------
Revised 12/15/79 Section 4, C, (2)
Page 2
HECD using the following operating conditions:
Column 1.8 m (6 ft.) x 4 mm (k in.), 3% OV-1
Column oven 200°C
Inlet 200°C
Transfer line 275°C
Helium carrier 50 ml/minute
Hydrogen reaction gas 50 ml/minute
Electrolyte n-propanol-deionized water (1:1 v/v)
or 100% methanol for the chlorine mode
Electrolyte flow 0.8 ml/minute
Furnace temperature 850°C
Background level <]% FSD at 8 x 10
Noise 5% peak to peak at 1 x 10
Chart speed 0.5 in./minute
Figure 3 shows the sensitivity for simazine with a 5% OV-101
column with the nitrogen mode Model 700 HECD. The operating
conditions are listed on the figures.
The chromatograms shown in the figures in Section 4,A,(7)
indicate sensitivities and retention times to be expected with the
Model 700 HECD and Carbowax 20M columns.
Figures 4, 5, and 6 present chromatograms for pesticides in
the halide, nitrogen, and sulfur modes, respectively, with the
Model 700 HECD detector. The columns were 3% OV-101 for chlorinated
and phosphorus pesticides and 3% STAP for triazines. In general,
1-2 orders of magnitude better sensitivity can be expected for the
Model 700 A HECD compared to the Model 700.
If columns are properly prepared and conditioned and operating
conditions are optimized, the analyst should be able to reproduce
or improve the sensitivities and chromatograms illustrated in these
sections for pesticide standards.
-------
Revised 12/15/79 Section 4, C, (2)
Page 3
Relative retention times and responses for numerous pesticides
on 5% or 10% DC-200 columns with the nitrogen-mode HECD are listed
in the FDA RAM, Table 335-A. A chromatogram of 2.5-5 ng of seven
OC1 insecticides on a 5% OV-101 column with the HECD in the halogen
mode is given in the FDA RAM, Figure 335-B.
VI. MAINTENANCE AND USE OF COLUMNS:
See Sections 4,A,(2), VI and 4,A,(7), II.
-------
Revised 12/15/79
Section 4,C,(2)
Page 4
50-
40—
30 —
20 — -..
-*•) I MIN )•*-
10 --; --.~ rfc—T
0—
Fig. 1. Typical chrcmatogram Hall
electrolytic conductivity
detector in the nitrogen
mode.
80 —
70 —
60-
50 —
40 —
30 —
20-
10 —
0 '
I MIN.
•_ ATTEN 2 x 10
F-ig. 2. Typical chrcmatogram Hall
electrolytic conductivity
detector in the chloride mode.
-------
Revised 12/15/79
Section 4,C,(2)
Page 5
0"-
1 Solvent
2 Simazine
(50pg)
Conditions
Detector: Model 700 Hall Detector
Mode: Nitrogen with Ni Catalyst
Reaction Gas. H2, 40 cc/min
Furnace Temp. 800° C
Solvent: 1-4IPA/water
Column: 6' Glass; 5% OV-101 on Gas
Chrom Q
Sensitivity: 10 x 1
Col. Temp: 200° C
Fig. 3. Chrcmatography of simazine.
700A Halide Mode
3% OV-101 Column @200°C
Attenuation 10x4
Pesticides
Fig. 4. Chronatogram of chlorinated pesticides.
-------
Revised 12/15/79
Section 4,C,(2)
Page 6
CONDITIONS
GC: TRACOR MODEL 560
Column: 3% STAR on 80-100 CWHP,
6' x 2mm ID Glass
Column temp.. 195°C
Carrier: 30 ml/mm, He
Injector temp.. 210°C
Detector: 700A HECD
Detector temp.. 225"C
Reactor temp.: 800"C
Reaction gas: 80 ml/mm, H2
COMPOUNDS
1 Propazine
2 Atrazme
3 Simazine
4 Prometryne
> 4 ng each,
4 8
Time, minutes
Fig. 5- Chromatogram of nitrogen herbicides.
CONDITIONS
GC: TRACOR MODEL 560
Column. 3% OV-101 on 80-100 CWHP
6' x 2mm ID Glass
Column temp.: 190°C
Carrier. 25 ml/mm. He
Injector temp.: 210°C
Detector: 700A HECD
Detector temp.. 225°C
Reactor temp.- 850°C
Reaction gas: 100 ml/mm, air
Compounds
1 Thimet = 1.8 ng
2 Malathion
3 Parathion
4 Captan ) 3.6 ng each
5 Ethion
6 Tnthion
4 8 12
Time, minutes
16
Fig. 6. Chromatogram of sulfur pesticides.
-------
Revised 12/15/79 Section 4, C, (3)
Page 1
GAS CHPOMATOGRAPHY - HALL ELECTROLYTIC
CONDUCTIVITY DETECTOR
DETECTOR
I. OPERATING PARAMETERS:
See Section 4, C, (2), V for a list of typical but not critical
operating conditions for the HECD. The column oven temperature will,
of course, affect elution time in the usual way. The inlet and trans-
fer line temperatures may be decreased by as much as 50°C with little
effect. The helium carrier flow rate can be increased or decreased
5-10% without major effect (except for the expected change in elution
time). The hydrogen reaction gas flow rate can be set anywhere in the
range of 40-50 ml/minute; above this rate, response may decrease. The
concentration of ji-propanol will not drastically affect results within
the range of 40-60%. The peak height is inversely related (but probably
not linearly) to the electrolyte flow into the gas liquid contactor.
However, at flow rates less than 0.3-0.4 ml/minute, background noise
becomes appreciable. The conversions in the furnace proceed well at
temperatures of 820-850°C. Above this temperature, response increases
for some compounds and decreases for others. Certain classes of
compounds may be selectively screened for at a given temperature.
For example, at 600°C, reduced response is noted for chlorinated pesti-
cides, but PCBs elicit no response at all. Therefore, theoretically
the pesticides could be quantitated at this temperature, and the PCBs
could be determined by difference after determining total chlorine
response at 830°C.
II. MODES OF DETECTION AND SELECTIVITY:
The basic components of the electrolytic conductivity detector are
displayed in Figure 1. The chromatographed sample is converted to the
monitored species by oxidative or reductive pyrolysis in the high
temperature furnace. The reaction products are swept into a gas-
liquid contactor where they are mixed with a conductivity solvent. The
liquid phase is separated from insoluable gases in a gas-liquid separator
and then passed through a conductivity cell (Figure 2).
The Hall detector, which is a miniaturized version of the original
Coulson electrolytic conductivity detector, possesses significantly
higher sensitivity. This is the result of modifications in cell design
and geometry leading to decreased detector dead volume, and improved
electronic measuring of electrolytic conductivity. The selectivity
-------
Revised 12/15/79 Section 4, C, (3)
Page 2
of the HECD in the N-, C1-, and S-modes vs hydrocarbons is equal to 10
or greater in all cases.
Nitrogen Detection - Organic nitrogen-containing pesticides are
converted to ammonia at 800-900°C with a nickel wire catalyst and
hydrogen reaction gas according to the following equation:
R_CN Nl> Hz > NH3+ Lower Alkanes
The increase in conductivity due to the formation of ammonium hydroxide
is the basis of response.
Selectivity to nitrogen compounds is based on the conversion of po-
tentially interfering substances to reaction products that either
produce little electrolytic conductivity or can be easily removed
with a post-pyrolysis scrubber prior to the cell. Specifically,
pesticides containing halogens and sulfur are converted to HX and
H2S (and/or H2SOs, H2SCK), respectively, all of which are removed
by inserting a nitrogen mode scrubber tube containing, e.g., strontium
hydroxide on Fiberfrax or quartz wool, inside the exit end of the
combustion tube. Compounds containing oxygen are converted to water
(no response). Lower alkanes (primarily CHi+) are also produced in
all cases, but these have low solubilities in the conductivity solvent
and are not ionized when dissolved (no response). Thus, ammonia is
the only product from common organic compounds that gives a response,
thereby explaining the high specificity to nitrogen compounds.
The selectivity due to the scrubber is illustrated by Figure 3.
Chromatogram A shows the separation of two representative nitrogen-
containing compounds, atrazine and caffeine, at the 10 ng level, along
with four chlorinated insecticides at the 100 ng level. Note that the
atrazine and caffeine peaks (numbered 1 and 3) are nearly obscured by
the chlorinated compounds. Chromatogram B shows the same mixture run
under identical conditions except that the strontium hydroxide scrubber
was inserted in the furnace tube to remove the interfacing halogen
peaks. For the maximum selectivity, the scrubber tube must be main-
tained in efficient condition.
In practice, sample extracts with microgram quantities of sulfon-
ated compounds will cause significant detector response. Sample im-
purities containing nitrogen can also be present in sample extracts
and will cause unwanted response.
Chlorine Detection - In the reductive mode, ammonia will not form
from nitrogen-containing compounds unless the nickel catalyst is present.
Therefore, chlorinated pesticides can be selectively detected as HC1
-------
Revised 12/15/79 Section 4, C, (3)
Page 3
using an empty pyrolysis tube and hydrogen reactor gas. As in the
nitrogen mode, the products H2S and CH^ will cause little or no response.
Any oxygen in the system, introduced either as a carrier or com-
bustion gas impurity or from the sample itslef (e.g., a sulfoxide or
sulfone group), will form acid complexes that cause considerable
response. Small amounts of solvents containing oxygen, such as ethyl
acetate, can render the detector inoperable. Selectivity to chlorine,
therefore, depends on the high purity of the carrier and combustion
gases and solvents, efficiency of oxygen traps in the gas lines, and
cleanup of sample matrices to remove impurities containing groups such
as S-0. Bleed of GC liquid phases containing halogenated compounds
can also cause interferences.
Adequate cleanup of many samples for determination with the HECD
in the nitrogen and chloride modes can be obtained by use of the modi-
fied MOG procedure described in Section 5,A,(1). Additional cleanup
of fatty samples, if required, can be achieved by gel permeation
chromatography (Sections 12,A and 5,B).
Sulfur Detection - When used in the oxidative mode, the eluting
compounds are combined with air in an empty pyrolysis tube, forming
different reaction products. Chlorine-containing compounds again
produce HX, which is removed by a silver nitrate scrubber. Nitrogen-
containing compounds form N2 or N02 in very low yield (no response).
Hydrocarbons produce CO, C02, and H20 (no response). Sulfur groups
produce S02 and S03, which dissolve in the electrolyte to yield ionized
compounds providing the selective response of the detector.
Almost no use has been made of the sulfur mode of the HECD for
practical residue analyses. The reportedly lower selectivity and
sensitivity compared to the chloride and nitrogen modes will apparently
limit the potential of the sulfur mode for pesticide determinations.
III. BACKGROUND SIGNAL CHECK:
1. Set attenuator to infinity (INF) position and zero recorder.
2. Set the attenuator to 8, the conductivity range to 10, and the
zero suppression switch to off.
3. Normally, with a well stabilized system, the background should be
less than 10% of full scale. A background significantly higher
than this may result in negative peaks. High background levels
are normally caused by column bleed, contamination of the reaction
or carrier gas, leaks or contamination elsewhere in the system, or
incompletely conditioned furnace tubes. In the latter event, the
background level will gradually decrease over a period of time and
-------
Revised 12/15/79
Section 4, C, (3)
Page 4
4.
will normally reach acceptable to excellent levels in a day or
so (background less than 5% and noise less than %% at 10 x 8).
When the instrument is first set up, typical background and noise
levels may be as high as 60% and 3%, respectively, at an
attenuation of 10 x 8.
Activate zero suppression switch and adjust coarse zero and fine
zero controls to suppress background signal to zero.
IV. SENSITIVITY:
See the chromatograms in Section 4,C,(2) for typical sensitivities
obtained for the HECD in various modes with different GC columns. In
general, the HECD is less sensitive than electron capture or N-P
detectors, due, in part, to the relatively large dead volume of the
detector.
Chlorine Detection - the 2 mm i.d. furnace tube is recommeneded
over the 4 mm tube for chlorine detection since better chromatography
with less peak broadening results from its use. Best sensitivity is
obtained when the conductivity solvent is neutral or slightly acid.
Under ideal conditions, a response of h FSD to 1 ng of heptachlor
epoxide can be obtained. For reliable, noise-free operation, h FSD
for 5-10 ng heptachlor epoxide should readily be obtainable (FDA
PAN, Section 315.32 (1)).
Nitrogen Detection - The 4 mm i.d. furnace tube is preferable
because contact time between the sample and the Ni catalyst is in-
creased and conversion to ammonia is enhanced. A larger amount of
scrubber can also be placed in the larger tube. Conductivity solvent
should be neutral or slightly basic for optimum sensitivity. Negative
peaks can occur if pH is less than 7. As little as 0.1-0.5 ng of
nitrogen (corresponding to ca. 1.5-7.5 ng carbaryl or ca. 0.3-1.5 ng
atrazine) can produce % FSD response during optimized operation,
although 5-10 times more nitrogen may be required for the same re-
sponse during routine operation (FDA PAM, Section 315.32 (2)).
The Tracer company reports the following sensitivity specifi-
cations for the HECD:
Model 700
below 0.01 ng N, CL, or S
with noise <1% at 1 x 10
attenuation
Model 700A
halogen - 5 x 10 g Cl/second
12
nitrogen - 2-4 x 10" g N/second
sulfur - 1-2 x 10" 2 g S/second
-------
Revised 12/15/79 Section 4, C, (3)
Page 5
A rapid decrease in response can result from injection of lipid
extracts that have not been subject to rigorous cleanup.
V. LINEARITY:
Linearity of response is reported by the manufacturer to be 10
for Cl (Fig. 3), 104 for N, and >104 for S. The FPD is not linear
for sulfur unless electronically compensated. Linearity at low con-
centrations depends on the cleanliness of the system and the correct
pH of the conductivity solvent as determined by the condition of the
ion exchange resin. The upper end of the linear range for nitrogen
is determined by the amount and condition of the nickel catalyst as
well as its position in the furnace (FDA PAM, Section 315.33).
VI. EQUIPMENT AND REAGENTS:
1. A potential source of interference is unsuspected reactions in
the furnace caused by contamination. For this reason, it is
recommended that Vespel (DuPont), graphite, or glass filled
Teflon ferrules be used throughout the system, together with
the most stable column materials available. Silicone 0-rings
may be used in place of ferrules for lower temperature work,
but a certain amount of bleed will be encountered until they
become well conditioned.
NOTE: Vespel ferrules sometimes stick in the fitting after
high temperature operation and cannot be removed
easily by pulling or twisting the quartz furance
tube. The best procedure is to back off the Swagelok
nut completely and grasp the exposed portion of the
ferrule with tweezers as close to the metal fitting
as possible and twist slightly until it is dislodged.
To avoid this problem, glass filled Teflon ferrules
(Tracer Part No. 76458-0012) can be used with no
sacrifice in performance up to 275°C.
2. For convenience, the type "T" solvent vent of the Model 700 can
be replaced with a four port high temperature valve (e.g., Model
CV-4-HTA, Valco Instruments Co., Houston, TX). This valve vents
solvent while still maintaining gas flow through the detector
from the column in use. In addition, effluent from either of
two different GC columns can be readily delivered to the pyrolysis
furnace.
To operate, the valve should be opened immediately before
a sample injection, left open for a time period sufficient to
vent all the solvent, and closed before the first sample peak
begins to elute. For best results, it is important that the
venting time be repeated, as closely as possible, for each
-------
Revised 12/15/79 Section 4, C, (3)
Page 6
successive injection. Under normal operating conditions, 30
seconds vent time is usually adequate for most solvents. If
10 microliters or more is injected, more time may be required.
3. The needle valve solvent flow controller to the gas-liquid con-
tactor can be replaced with a more exact 10-turn calibrated
control (e.g., stainless steel Nupro fine metering valve with
vernier, catalog No. M-ZMA) for more stability and reproducibility.
4. Chlorine and nitrogen are both detected in neutral electrolyte
solution. Neutrality (pH 7) is maintained by the mixed bed resin
supplied by Tracer for removing ions from the circulating
electrolyte. Detection of chlorine is optimum in slightly
acidic solution, achieved by using a 1:4 mixture of anion
(Duolite APA-366) and mixed bed (Duolite ARM-381) resins
(FDA RAM, Section 315.42 (2)). Ions exchange resin should
be extracted in a Soxhlet apparatus with water and the alcohol
to be used with it prior to use.
5. A 50% (v/v) mixture of n-propanol in deionized water is
recommended as the conductivity solvent. Methanol, isopropanol,
and ethanol have also been used as the alcohol. Reducing the
percentage of alcohol increases the sensitivity, especially in
the nitrogen mode. Cell wall wetting may also be impaired,
however, and flow irregularities may result in increased noise
and poorer reproducibility. Twenty to thirty percent alcohol
in water is usually the lowest practical choice for the best
signal to noise ratio in the nitrogen mode. Methanol (100%)
has been successfully used in the chlorine mode.
6. The quartz combustion tube is h in. (0.6 cm) o.d. (4 mm or 2 mm
i.d.) x approximately lh in. (19 cm) long. Separate tubes should
be conditioned and maintained for nitrogen and chloride modes, re-
spectively. Do not interchange the tubes once they have been
used. A tube used in the chloride mode should be first heated
to a dull red over its entire surface with a propane/oxygen
torch to burn off surface impurities. This pretreatment, which
has to be done only once, shortens the time required to reach
maximum response and reduces peak tailing. In the nitrogen mode,
the tube should not be treated as above.
7. The nickel catalyst is approximately 100 in. (254 cm) of 8 mil
wire wrapped in a bundle 1-3/8 (3.5 cm) in. long. The catalyst
should be inserted in the tube after the tube is installed and
with reaction gas (hk) turned on. The catalyst must be centered
in the hot zone of the furnace. Normally, some response for
10 ng of atrazine should be obtained in just a few minutes after
initial setup of standard operation conditions. However, the time
required to reach maximum sensitivity may vary from one hour to
-------
Revised 12/15/79 Section 4, C, (3)
Page 7
overnight depending on the condition of the catalyst. If the
system is not used for long periods of time, it is advisable to
have only reaction gas flowing over the catalyst. If the catalyst
should slip so that a portion of it is outside the hot zone
(expecially downstream), some of the converted sample could be
adsorbed by contact with the metal. For this reason, it is wise
to spread the ends of the wire bundle so it fits snugly in the
furnace tube and to avoid bumping the instrument. Conversion
of nitrogen to ammonia is increased by greater nickel surface
area, but the amount of nickel wire is limited by the size of
the combustion tube.
8. When changing modes of operation, also change or clean the
1/16 in. (0.16 cm) o.d. Teflon tubing between the combustion
tube and the cell to prevent salt formation and sample loss
in the line. In addition, install the proper ion exchange tube.
9. The Tracer Standard Sample contains atrazine (10 ng/yl) and
aldrin (5 ng/yl) in methanol. In the nitrogen mode with a
strontium hydroxide scrubber, only one peak for atrazine is
observed (Fig. 1, Section 4,C,(2)). In the chloride mode,
a peak representing the single chlorine in atrazine and a
larger peak for aldrin should be obtained (Fig. 2, Section
4,C,(2).
10. The strontium hydroxide scrubber must be used in the nitrogen
mode to eliminate any interference from acid gases. Insert a
plug of the scrubber approximately % in. (1.3 cm) long, such
that the outside edge of the plug is % in. (0.6 cm) inside the
furnace. To maintain stability, routinely replace the scrubber
after 50-100 analyses.
11. Choice of solvent for sample and reference materials can be
critical with the HECD, because several common solvents have
adverse effects on the detector. Hydrocarbon solvents are
considered preferable. When operated in the nitrogen mode,
the detector responds to acetonitrile, even at low levels and
even though the majority of the solvent is vented. The
injection should be vented for at least two minutes if there
is acetonitrile in the solution. Methylene chloride as the
injection solvent depletes the strontium hydroxide scrubber
needed for nitrogen mode operation and makes the detector
inoperative. A vent time of 3 minutes is needed when using
methylene chloride. Ethyl acetate often contains trace amounts
of acetic acid, which also depletes the scrubber if this solvent
is injected without venting. A minimum vent time of 2 minutes
is recommended. Chlorine mode operation precludes the use of
halogenated solvents in the HECD. See also Subsection II above
-------
Revised 12/15/79 Section 4, C, (3)
Page 8
for a description of the problems encountered when using solvents
containing oxygen.
VII. MAINTENANCE AND TROUBLESHOOTING:
Consult the Tracer Operation and Service Manual, the FDA RAM
Section 315.6, and the EPA Pesticide AQC Manual, Section 5,G,b.
Some further operating characteristics and maintenance instructions
by Bayer are described in Section 5,G,b of the EPA Pesticide AQC
Manual. The greater time and attention required for maintenance
of the HECD compared to the EC and FPD is a disadvantage of the
detector.
Problems encountered in the use of the HECD can usually be
recognized prior to significant deterioration in performance and
can often be solved simply and immediately if the analyst is
familiar with the chemistry of the detector. Common problems
in nitrogen detection include poor linearity and peak shape. Poor
linearity is usually caused by neutralization of the NJKCH. This
is due to insufficient basicity of the conductivity solvent and/or
exhausted scrubber. Neutralization problems are readily recognized
by a sharp dip in the baseline just prior to the peak, followed by
a negative dip after the peak that gradually increases to the base-
line (Fig. 5). The peak may be totally negative if the solvent is
acidic or the quantity of nitrogen compound is very small. Peak
tailing is usually due to a contaminated scrubber, contaminated
transfer line from the furnace to the cell, deactivated catalyst,
or the presence of acidic reaction products that are not removed
by the scrubber. The presence of acidic reaction products is also
normally indicated by negative peaks. Negative peaks are prevented
by using a properly packed ion exchange tube, high purity gases, and
an efficient scrubber. Other solutions are obvious and include re-
placing the catalyst, ion exchange resin, scrubber, and transfer
line as required. A "dirty" combustion tube can cause peak tailing
and loss of sensitivity.
-------
Revised 12/15/79
Section 4,C,(3)
9
Reaction
Gas
Solvent
Reservoir
Pump
1
ChroiSoVngl. Fu"""=e ~
Gas -Liquid
Contactor
Gas -Liquid Conductivity Conductivity
Separator Cell Meter
Fig. 1. Block diagram of the electrolytic conductivity detector.
KO.Sin-
Fig. 2. Microelectrolytic conductivity detector cell assembly. A,
gas-liquid contactor; B, Teflon solvent delivery tube; C,
Teflon reaction products delivery tube; D, stainless steel
detector block; E, solvent vent; F, Teflon insulator sleeve;
G, gas-liquid exit tube and center electrode.
-------
Revised 12/15/79
Section 4,C,(3)
Page 10
Sample Peak
S
1
2
3
4
5
6
Identity
Solvent
Atrazine dOng)
Lindane OOOng)
Caffeine (10 ng)
Heptachlor (100ng)
Aldnn(IOOng)
Dieldrm (100ng)
Conditions
Column: 6 x V Glass
packed with 3% OV-1 on
Chromasofb WHP
80/100 mesh
Col Temp- 200s C
Carrier: H2at SOcc.'mm
Chromatograph- Model
550
Detector: Hall Model 700
Mode- Nitrogen
Catalyst. Nickel wire
Furnace Temp 850° C
Sensitivity: 10x4
A. Scrubber OUT
B. Scrubber IN
Fig. 3. Hall detector selectivity.
-------
RELATIVE RESPONSE
Ul
-o CO
cu m
sa o
!£
-•3
n
U)
-------
Revised 12/15/79
Section 4,C,(3)
Page 12
Fig. 5. Peak shapes obtained for nitrogen-containing compounds:
A, normal peak; B, peak obtained with an insufficiently
basic conductivity solvent; C, peak obtained with an
acidic conductivity solvent.
-------
Revised 12/15/79 Section 4, C, (4)
Page 1
GAS CHROMATOGRAPHY - HALL ELECTROLYTIC
CONDUCTIVITY DETECTOR
SAMPLE QUANTITATION AND INTERPRETATION
Methods of quantitation with the HECD are similar to those
used with the electron capture (Section 4,A,(4)) and flame photo-
metric (Section 4,B,(4)) detectors.
As with other element selective detectors such as the FPD
(Section 4,B,(4), V), interpretation is greatly simplified with
the HECD compared to the electron capture detector. Figures
1 and 2 compare this selectivity to the N-P detector (Section 4D)
and the sulfur mode of the FPD.
Figure 1 shows that the HECD does not respond to levels of
hydrocarbons (e.g., 100 ug of octadecane) that elicit significant
response with the N-P detector. The HECD is specific for nitrogen;
the response depends solely on the nitrogen content of the molecule
detected. It is even possible to tune the HECD to only certain
types of nitrogen compounds.
Figure 2 compares the response of the HECD in the sulfur
mode to that of the FPD. The detectors display similar selectivities
for the quantities of material shown, but the selectivity of the
FPD would be reduced by a factor of three if the response were
linearized. Linearization of the output of the FPD also enhances
peak tailing. The output of the HECD is linear and suffers none
of these disadvantages.
-------
O
IB
o.
RESPONSE
o
O
CD
m
CD
to
-Azobenzene(4ng)
Octadecone (40,000ng)
o
OD
•Azobenzene (4ng)
-Octadecane (40,000ng)
m
o
o
o
m
H
m
o
o
T3
O
m
rn
o
o
CD
O
-Atrazine
(4ng)
•Atrazine
(4ng)
RESPONSE
RESPONSE
I"
;3
a.
•d
13
O
o
—i—
Ul
O
o
o
-H
o
-nr—
O Q-
Ul
O
z
-^
O
o
I
m
o
o
0
3-
(t')'D't'
-------
Revised 12/15/79
Section 4, C, (5)
Page 1
RETENTION DATA AND CHROMATOGRAMS OF CARBAMATE PESTICIDES ON
CARBOWAX 20M-MODIFIED SUPPORTS WITH DETECTION
BY THE HALL ELECTROLYTIC CONDUCTIVITY DETECTOR
REFERENCE
Hall, R. C., and Harris, D. E., J. Chromatogr. 169. 245 (1979).
CONDITIONS:
Tracor Model 560 gas chromatograph
Tracor Model 700 HECD
Instrument
Detector
Electrolyte
Transfer line temperature
Furnace temperature
Hydrogen reaction gas flow
Columns
Support
Liquid phase coating
Column packing
Column conditioning
Helium carrier gas flow
15% isopropanol in water,
0.5 ml/minute
200°C
720°C
80 ml/minute
Glass, 6 ft. (.1.8 m) x 2 mm i.d.,
silanized with Supelco Sylon-CT,
and ends plugged with silanized
glass wool
Commercial bonded Carbowax 20M,
designated Ultra-Bond
(RFR, Hope, RI)
By evaporation technique, using
rotary evaporator operated at
20 rpm for solvent removal
By use of slight vacuum and gentle
tapping with plastic rod
At 190°C for 24 hours with normal
carrier gas flow
25 ml/minute
-------
Revised 12/15/79
Retention Indices for Carbamate Pesticides
Relative to Carbofuran on Ultra-Bond and Coated Ultra-Bond
Column temperature is 170°
Section 4, C, (5)
Paae 2
Compound*
EPIC
Butylate
Pebulate
Vernolate
Propham
Diallate
Meobal
CDEC
Pyramat
Trillate
Propoxur
2,3,5-Landrin
Cnlororopham
Bux
Terbutol
3,4,5-Landrin
Benthiocarb
Aminocarb
Mexacarbate
Carbofuran
SWEP
Dimetilan
Methiocarb
Carbaryl
Purity***
99.5
99.5
99.0
99.0
100.0
99.0
99.0
99.5
98.0
99.5
98/99
98.0
99.5
98.0
98.0
98.0
98.0
98.0
99.0
99.5
98.0
98.0
99.0
99.5
Ultra
Bond
—
--
—
0.19***
0.20***
0.33
0.34
0.35
0.53
0.55
0.60
0.61
0.78
0.82
0.85
0.85
0.93
0.98
1.00
1.36
1.37
2.10
2.75
2%
OV-101
0.20
0.25
0.25
0.28
0.31
0.67
0.59
0.66
0.62
1.01
0.55
0.69
0.66
1.04
1.47
0.94
1.80
1.07
1.32
1.00
1.19
1.79
2.25
2.48
n
OV-17
0.08
0.09
0.12
0.12
0.19
0.31
0.42
0.40
0.43
0.48
0.48
0.51
0.45
0.72
0.91
0.78
1.26
0.89
0.98
1.00
0.97
1.93
2.13
2.41
1 *
Carbowax
20M
0.07
0.07
0.09
0.08
0.22
0.21
0.52
0.30
0.29
0.26
0.53
0.58
0.59
0.71
0.66
0.85
0.32
0.95
0.94
1.00
1.47
1.38
2.28
3.10
T?
OV-210
__
__
—
0.32
0.56
0.40
0.39
0.39
0.63
0.65
0.56
0.75
0.82
0.88
0.75
1.02
1.02
1.00
1.19
1.36
1.96
2.81
0.5% OV-210 +
0.65% OV-17
__
--
._
0.22
0.28
0.50
0.37
0.36
0.38
0.52
0.58
0.55
0.71
0.78
0.85
1.02
0.94
0.96
1.00
1.23
1.64
2.20
2.82
*Compounds are listed by common names.
**Standards came from the EPA Protection Agency, Health Effects Research Laboratory,
Environmental Toxicoloqy Division, Research Triangle Park, NC 27711, USA
***Co1umn temperature is 150°C.
Compound
EPIC
Butylate
Vernolate
Pebulate
Retention Parameters for the More Highly Volatile Carbamate
Pesticides on Ultra-Bond
tR = retention time; t'R = retention relative to butylate
Purity
99.5
99.5
99.0
99.0
Temperature
90°
tR(min)
3.27
4.72
5.29
6.00
0.69
1.00
1.12
1.27
100°
tR(mi
00
75
06
3.44
0.73
1.00
1.11
1.25
120'
(mm)
1.09
1.25
1.42
1.55
0.87
1.00
1.14
1.24
-------
Revised 12/15/79
Section
Page 3
o
TIME,min
Fig. 1 Chromatograms of carbamate pesticides separated on a 3% OV-101 on Ultra-Bond column
operated at 170'. Sensitivity: 10 < 8 Compounds in order of eluUon are: (A) butylate.CDEC carbo-
furan, Dimetilan and methiocarb; (B) EPTC, chlorpropham, triallale, SWEP and lerbutol. Sample
quantity: 10 ng each.
10
0-
12
TIME, mm
24
f-ig. 2. Chromatogram of carbamate pesticides separated on a I % Carbowax 20M column operated
at 170'. Sensitivity 10 x 8. Compounds in order of elution are EPTC, chlorpropham, triallale,
SWEP and terbmol. Sample quantity: 10 ng each.
-------
Revised 12/15/79
Section
Page 4
10]
4 8 12
TIME, mm
16
Fig. 3. Chromatogram of carbamate pesticides separated on a 0.65°;; OV-17 I 0.5 /„ OV-210 on
Ultra-Bond column operated at 170'. Sensitivity: 10 x 8. Compounds in order of clution are:
Propham. diallatc, triallate, mcobal, 3,4,5-Landnn, carboftiran, mcxacarbatc, SWEP, Dimctilan,
methiocarb and carbaryl. Sample quantity 10 ng each with the exception of diallale at 5 ng.
08
08
0
Fig. 4. Chromatogram of carbamate pesticides separated on a 065% OV-17 I 05",, OV-210 on
Ultra-Bond column. (A) Temperature-programmed from 115-175" at lOVmm. Sensitivity 10 > 8.
Compounds in order of elution: propham, diallatc, triallate, mcobal, 3,4,5-Landrm, carbofuran,
mexacarbate, SWEP, Dimctilan, methiocarb and carbaryl. Sample quantity. 10 ng except for diallale
at 5 ng. (B) Temperature-programmed from I 10-185" at 10'/mm. Sensitivity: 10 > 8. Compounds in
order of elution are- EPTC, butylate, vernolaic, pebulate, propham, diallatc,, triallate, mcobal, 3,4,5-
Landrm, carbofuran, mexacarbate, SWEP, Dimetilan, methiocarb and carbaryl. Sample quantity
same as in A.
-------
Revised 12/15/79 Section 4, C, (5)
Page 5
SUMMARY OF RESULTS:
A wide variety of carbamate pesticides can be chromatographed as
the intact compounds, using Carbowax 20M-modified supports with or
without additional liquid phase coatings. It is important that moderate
column temperatures (<185°C) and relatively short analysis times be used.
Collection of chemical-ionization mass spectra (isobutane reaction gas)
of the parent carbamates of all compounds injected proved that among the
24 pesticides that could be readily chromatographed, carbaryl was the
only compound that exhibited significant degradation (% 50%). In all
other cases, the results of the CI-MS study indicated that the compounds
detected by the electrolytic conductivity detector were the intact carba-
mates. The retention time of carbaryl was considerably longer than that of
the other pesticides, which probably contributed to its degradation.
Eleven carbamate pesticides can be separated on an OV-210/OV-17 mixed phase
column under isothermal conditions. A total of 15 carbamates can be
separated with baseline resolution on the same column with temperature
programming.
-------
-------
Revised 12/15/79 Section 4, D
Page 1
GAS CHROMATOGRAPHY - NITROGEN -
PHOSPHORUS (N-P) DETECTOR
I. INTRODUCTION:
Alkali flame ionization detectors (AFID) for selective
analysis of phosphorus- and nitrogen-containing pesticides have
been described and discussed in Section 313 of the FDA PAM and
Section 5,E of the EPA Pesticide AQC Manual. The flame in the
AFID serves the dual purposes of volatilizing the alkali salt
source and ionizing the sample. Because the amount of hydrogen
determines the flame temperature and, therefore, the extent of
the above processes, small changes in hydrogen flow rate affect
the detector response considerably. The flame temperature is
also influenced by the flow rates of carrier gas and air. In
actual practice, it is difficult to maintain gas flows within
required tolerances for stable operation at high detector
sensitivities.
The difficulty of maintaining very accurate gas (especially
hydrogen) flow rates and the problems associated with the con-
tinually changing salt surface have resulted in decreased interest
by pesticide analysts in the AFID in favor of "flameless" alkali
sensitized detectors. These detectors, which have been recently
sold under the name "nitrogen-phosphorus (or N-P) detector"
because of their selective response to these elements, offer an
order of magnitude improvement in sensitivity and selectivity.
However, the low cost and ease of conversion of common flame
ionization detectors to AFID operation makes this detector still
attractive for analyses not requiring the higher stability of the
flameless detector.
For general information on the gas chromatograph, GC columns,
and sample quantisation and interpretation, consult Section 4,A.
REFERENCES:
1. Rapid Procedure for Preparation of Support Bonded Carbowax
20M Gas Chromatographic Column Packing, Moseman, R. F.,
J. Chromatogr., 166, 397-402 (1978).
2. Ionization Detector for GC with Switchable Selectivity for
Carbon, Nitrogen, and Phosphorus, Kolb, B., Auer, M., and
Pospisil, P., J. Chromatogr., 134, 65 (1977).
-------
Revised 12/15/79 Section 4, D
Page 2
3. Analytical Performance of a Novel Nitrogen-Sensitive Detector
and its Applications with Glass Open Tubular Columns, Hartigan,
M. J., Purcell, J. E., Novotny, M., McConnell, M. L., and Lee,
M. L., J. Chromatogr., 99, 339 (1974).
4. Study of the Nitrogen Response Mode of the Thermionic Rubidium
Silicate Detector, Lubkowitz, J. A., Glajch, J. L., Semonian,
B. P., and Rogers, L. B., J. Chromatogr., 133. 37 (1977).
II. DESCRIPTION OF N-P DETECTORS:
Figure 1 shows a schematic diagram of the Perkin-Elmer N-P
detector used by R. F. Moseman of the EPA (reference 1 above)
with support bonded Carbowax 20M columns (Section 4,A,(7)).
This detector and those available from Varian, Hewlett-Packard,
and Tracer have similarities in the position of the alkali source
above the detector jet, use of cylindrical collector electrodes,
application of a negative potential to the source, and use of air
and hydrogen only in quantities necessary to produce a low temper-
ature plasma (rather than a flame) surrounding the source. The four
commercial detectors are, however, distinct in geometry, alkali
source, and electrical technique used for heating the source. The
Perkin-Elmer detector uses a rubidium glass (silicate) bead source,
the Hewlett-Packard uses an unspecified alkali salt contained in
a ceramic reservoir, the Varian uses a proprietary alkali-ceramic
bead, and the Tracer uses a mixture of alkali salts in a silica gel
matrix. The sources have a relatively short bead life of about
three months.
The N-P detector is similar in basic design to the AFID and
can be considered as a modified AFID with an electrically heated
source that is operated with reduced hydrogen and air flows. The
plasma generated around the electrically heated beam is responsible
for ionizing the sample. Initial installation and positioning of
the bead is critical for optimum response. The bead temperature
is easily set by a control knob. Thus, the system becomes more
stable and has a wider linear range. Figure 1 shows a schematic
diagram of the Perkin Elmer nitrogen-phosphorus detector. When
this detector is used in the "phosphorus only" mode, the bead
heating circuit is switched off, and the flame is ignited.
-------
Revised 12/15/79 Section 4, D
Page 3
III. MECHANISM OF SELECTIVITY:
As in the case of the AFID, the mechanism of response of the
N-P detector is not fully understood. However, a brief probable
explanation of the mechanism of selective detection of the most
widely studied Perkin-Elmer detector follows:
N-P Mode - (Sensitivity to nitrogen and phosphorus.) The N-P
mode uses a negatively polarized jet and low hydrogen flow rate.
Because of the lack of a hot flame, organic compounds do not burn
completely. Rather, a partial pyrolysis takes place, producing
intermediate stable CN radicals from nitrogen-containing organic
compounds. The radicals take on an electron from the alkali,
resulting in a symmetrical cyanide ion and a positive alkali ion.
The alkali ion is recaptured by the bead, while the cyanide ion
migrates to the collector electrode and liberates an electron.
Collection of the electrons creates the specific response. A
similar mechanism has been formulated for phosphorus, except that
PO and/or POp are assumed to be the intermediate radicals. It
should be emphasized that there is no mode of the detector se-
lective for nitrogen only; there is strong response to phosphorus
plus nitrogen in the N-P mode.
P-Mode - (Sensitive to phosphorus only.) A hot flame exists
because of an increased hydrogen flow rate, and the jet of the
detector is gounded. The organic compounds are fully burned,
and the electrons produced by the normal combustion process are
conducted to ground. The combustion products of phosphorus react
with the alkali on the surface of the bead and produce ions that
are captured by the collector electrode, thus producing the
response. Nitrogen compounds give a reduced response in this mode.
IV. RESPONSE CHARACTERISTICS:
Compared with the flame ionization detector, the N-P detector
is about 50 times more sensitive for nitrogen and 500 times more
sensitive for phosphorus. The sensitivity for three pesticides is
illustrated in Figure 2. All of the compounds contain phosphorus,
and diazinon also contains nitrogen. The sensitivity of detection
calculated from the chromatogram for malathion is 6 x 10"11* g/second,
or, calculated for P, 6 x 10~15 g/second. Nanogram to picogram
quantities of most nitrogen compounds can be routinely determined,
and selectivity is high if no phosphorus-containing compounds are
co-injected. Sensitivity of the detector depends on detector
background. The background is affected by bead heating current,
gas flow rates, and condition of the bead. Operation of the
detector at a fixed background current requires only occasional
adjustment of the detector and has resulted in very uniform response
-------
Revised 12/15/79 Section 4, D
Page 4
with time. The hydrogen, air, and carrier (nitrogen or helium) flow
rates should be optimized for maximum signal to noise ratio for
the pesticide(s) of interest.
Figure 3 shows the wide linear range for the pesticide
malathion. Selectivity of the N-P detector relative to organic
molecules containing N or P atoms depends somewhat on the analytical
conditions and the type of molecule. However, it has always been
found to be better than 1:5000. This is important not only for
positive identification of residues but also to eliminate the inter-
ference of the large solvent peak that might overlap early peaks
in trace analysis when a nonselective detector is used. Figure 1
in Section 4,C,(4) illustrates the selectivity of the N-P detector.
V. GLC COLUMNS:
The N-P detector is not usable with columns of liquid phases
containing halogen, phosphorus, or nitrogen (OV-210, XE-60,
stabilized DEGS). Columns that have been used successfully include
support bonded Carbowax 20M (Section 4,A,(7)), 3% OV-1, 8% Apiezon L,
5% Carbowax 20M, and 3% OV-17.
Figure 4 shows the separation and detection of pg levels of
three triazine herbicides. Figure 5 compares detector response
to nitrogen and hydrocarbon. The chromatogram demonstrates a
nitrogen-.carbon selectivity of 3 x 105.
VI. INSTALLATION, OPERATION. MAINTENANCE:
Consult the operations manual of the particular detector to
be used. An advantage of the N-P detector is a relatively low
degree of required maintenance.
-------
Revised 12/15/79
Section 4,D
Page 5
VENT
COLLECTOR
ELECTRODE
RUBIDIUM
BEAD
FLAME JET
AIR
JET
POLARIZING
LEAD
HYDROGEN
L—?
COLUMN
EFFLUENT
- NP-
MODE
MODE
Fig. 1. Left: schematic diagram of the Perkin-Elmer nitrogen-phosphorus detector.
Right: the two possible modes of operation. Parts with negative polarity
are indicated in a light shading, and parts with positive polarity in black
Hatched area represents insulation. The electrical heating of the rubidium
glass bead is not indicated.
Column: 6 ft. x 0.08 in. ID glass, containing
3% SE-30 on Chromosorb W HP 80 /100 mesh.
Column temperature: 210'C.
Sample volume: 1 /j.\.
OIAZINON (10.3% P; 9.2% N) 1 x 10~l°g
MALATHION (9.4% P) 1 x 10-«>g
ETHION (16.1% P) 1.3 x 10-'°g
2 « 6 «
—-MINUTES
Fig. 2. Chrcnatogram of pesticides.
-------
Revised 12/15/79
Section 4,D
Page 6
V »-•
KS"
„-»
"
'MALATHION
SAMPLE WEIGHT, 4
Fig. 3. Linearity plot of the
nitrogen-phosphorus
detector for malathion.
COLUMN 6lt i 2mm 10 gloss
containing 15% Coroowax 20M
on 80/100 mesh Gos-Chrom Q
COLUMN TEMPERATURE 200°C
ATTENUATION I « 8
2 4
TIME (mmufesl
Fig. 4. Chroitiatogram of 200 pg each of
atrazine, simazine, propazine,
and prometryne.
Fig. 5. Selectivity of the N-P detector. A: 5 ng each of CYJ>
C-ior C,n, and Con normal hydrocarbons; B: 200 pg of
j_o j_y ^u
atrazine and 100 pg of methyl parathion. Attenuation,
10 x 4.
-------
Revised 12/2/74 Section 5, A, (1), (a)
Page 1
MODIFICATION OF MILLS, ONLEY, GAITHER METHOD FOR THE
"V" DETERMINATION OF MULTIPLE ORGANOCHLORINE PESTICIDES
AND METABOLITES IN HUMAN OR ANIMAL ADIPOSE TISSUE
I. INTRODUCTION:
This procedure combines some of the extraction features of the
de Faubert Maunder et al method and the Florisil partitioning and
cleanup basics of the Mills et al procedure. The modified procedure
has been collaboratively studied over a period of years and has been
found to yield interlaboratory relative standard deviation values of
15 percent or better for the chlorinated pesticidal compounds most
commonly found in the fat of humans and animals.
REFERENCES:
1. de Faubert Maunder, M. J., Egan, H., Godly, E. W., Hammond,
E. W., Roburn J., and Thompson, J. The Analyst, 89: 168,
1964.
2. Mills, P. A., Onley, J. H., and Gaither, R. A., J.A.O.A.C.
46, 186-191, 1963.
II. PRINCIPLE:
A 5 g. sample is dry macerated with sand and Na2SOit and the fat
is isolated by repetitive extractions with pet. ether. Pesticide
residues are extracted from the fat with acetonitrile and then parti-
tioned back into pet. ether by aqueous dilution of the acetonitrile
extract. Pet. ether extract is concentrated to 5 ml by Kuderna-Danish
evaporation and transferred to a Florisil column for successive
elutions with 6% and 15% ethyl ether/pet, ether. The respective
eluates are both concentrated to suitable volumes in K-D evaporators
and the final extracts are examined by electron capture gas-liquid
chromatography.
III. EQUIPMENT:
1. Gas chromatograph equipped for electron capture detection.
Specific GLC columns and recommended operating parameters are
given in Section 4,A.
*This method, with appropriate modifications, may be used for the
analysis of other tissues if original sample size is adequate.
-------
Revised 12/2/74 Section 5, A, (1), (a)
Page 2
2. Aluminum foil, household type.
3. Beakers, 250 ml, stainless steel or heavy duty glass.
4. Beakers, 250 ml, Griffin low form.
5. Stirring rods, glass 10 mm diameter.
6. Water bath with temperature adjustment of 90-100°C.
7. Filter paper - Whatman No. 1, 15 cm diameter.
8. Funnels, glass, ca 60 ml diameter.
9. Separatory funnels - 125 ml and 1 liter, Kimble 29048-F, or equiv.
10. Chromatographic columns - 25 mm o.d. x 300 mm long, with Teflon
stopcocks, without fritted glass plates, Kontes 420530, Size 241.
11. Filter tubes, 150 x 24 mm, such as Corning #9480.
12. Erlenmeyer flasks - 500 ml capacity.
13. Kuderna-Danish concentrator fitted with grad. evaporative con-
centrator tube. Available from the Kontes Glass Company, such
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 in., stock #K-662750
d. Concentrator tubes, 10 ml, size 1025, stock #K-570050
14. Modified micro-Snyder columns, 19/22, Kontes K-569251.
15. Glass beads, 3 mm plain, Fisher #11-312 or equivalent.
16. Glass wool - Corning #3950 or equivalent.
IV. REAGENTS:
1. Petroleum ether - Pesticide Quality, redistilled in glass,
b.p. 30° - 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
-------
Revised 12/2/74 Section 5, A, (1), (a)
Page 3
stabilizer, and it is therefore unnecessary to add ethanol unless
peroxides are found and removed.
NOTE: To determine the absence of peroxides in the ether, add
1 ml of freshly prepared 10% Kl 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
the 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 either 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.
NOTE: Neither of the eluting mixtures should be held longer
24 hours after mixing.
5. Florisil, 60/100 mesh, PR grade, to be stored at 130°C until
used.
NOTES: 1. In a high humidity room, the column may pick up
enough moisture during packing to influence the elution
pattern. To ensure uniformity of the Florisil fraction-
ation, it is recommended 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 RTP, NC laboratory on order, 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 laboratories may differ some-
what from that in RTP, NC. Each new batch should be
tested by the procedure described in Section 3,D for
assurance that the operator can obtain recoveries and
compound elution patterns comparable to the data given
on the accompanying table.
-------
Revised 12/2/74 Section 5, A, (1), (a)
Page 4
6. Acetonitrile, reagent grade, saturated with pet. ether.
NOTE: Occasional lots of CHsCN are impure and require redistil-
lation. Generally, vapors from impure acetonitrile 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 trans-
ferring 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 ml beaker and evapor-
ate down to ca 5 ml. Inject 5 yl 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 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.
NOTE: See Note 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.
11. Hexane, redistilled, pesticide quality.
V. SAMPLING:
The majority of human adipose tissue samples are taken during
autopsy and the chemist has little or no control over the sampling
process. Wherever possible, it should be recommended to the autopsy
physician that the sample be placed in a glass container with Teflon
or foil-lined screw cap. Plastic bags should be avoided as traces
of impurities such as phthalates may contaminate the sample and result
in many spurious chromatographic peaks when the final sample is exam-
ined by electron capture GLC.
-------
Revised 12/2/74 Section 5, A, (1), (a)
Page 5
VI. SAMPLE PREPARATION & EXTRACTION:
1. On a cupped sheet of lightweight aluminum foil, weigh 5 grains of
the previously minced fat. Transfer entire cup to a 250 ml
stainless steel or heavy duty glass beaker.
2. Add ca 10 grams of clean, sharp sand, ca 10 grams of anhydrous
Na2S04 and 1.0 ml of hexane solution containing 200 nanograms
of aldrin.
NOTE: The aldrin is added here for the dual purpose of (1) pro-
viding a built-in retention marker for direct peak
identification on all chromatograms 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 Na2SOtf to give a uniform, dry granular mass.
4. Add 50 ml of pet. ether and warm carefully on a water bath with
continuous stirring until solvent boils gently.
NOTE: Some laboratories have reported satisfactory recoveries
resulting from the use of hexane instead of pet. ether
as the extracting solvent. In all probability, hexane
would function as a satisfactory substitute but the
modification has not been subjected to collaborative
study, and therefore no supporting data is available
to validate this hypothesis.
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. Extract the contents of the first beaker with two more 50-ml
portions of pet. ether as described in Steps 4 and 5.
7. Transfer insoluble material to the filter paper and ringe 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 removed and allow to cool to room temperature in a dessicator.
9. Weigh beaker and record for calculation of percent fat in the
sample.
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Revised 12/2/74 Section 5, A, (1), (a)
Page 6
10. Accurately weigh between 2.8 and 3.0 grams of the fat obtained
in Step 9 into a 125-ml separator. Add 12 ml of pet. ether
previously saturated with acetonitrile.
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 amount of acetonitrile used in the
partitioning step should be increased to 40 ml.
VII. LIQUID-LIQUID PARTITIONING:
1. Add 30 ml 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 ml of a 2% solution of Nad
and 100 ml of pet. ether.
3. Similarly extract the pet. ether layer in the 125-ml separator
three more times with 30-ml 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-ml portions 2% NaCl and discard the
aqueous washings.
8. Prepare a 2-inch column of anhydrous, granular N32S04 in a 150 x
24 mm filter tube and position over a 500-ml K-D evaporator
fitted with a 10-ml grad. concentrator tube containing one glass
bead. Dry the pet. ether by filtering through this column.
Rinse the separator twice with 10-ml portions of pet. ether and
finally rinse down sides of the filter tube with 10 ml pet. ether.
9. Attach a 3-ball Snyder 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.
-------
Revised 12/2/74 Section 5, A, (1), (a)
Page 7
10. Concentrate the extract to ca 5 ml, 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 5 ml under a gentle stream
of nitrogen at room temperature.
VIII. FLORISIL FRACTIONATION:
1. Prepare a chromatographic column containing 4 inches (after
settling) of activated Florisil topped with 1/2 inch of anhydrous,
granular Na2SOi,.. A small wad of glass wool, preextracted with
pet. ether, is placed at the bottom of the column to retain the
Florisil.
NOTES: 1. If the oven is of sufficient size, the columns may
be prepacked and stored in the oven, withdrawing columns
a few minutes before use.
2. The amount of Florisil needed for proper elution
should be determined for each lot of Florisil.
2. Place a 500-ml Erlenmeyer flask under the column and prewet the
packing with pet. ether (40-50 ml, or a sufficient volume to
completely cover the Na2SOtt layer).
NOTE: From this point and through the elution process, the
solvent level should never be allowed to go below the
top of the Na2SOi+ layer. If air is introduced, channeling
may occur, making for an inefficient column.
3. Using a 5-ml Mohr or a long disposable pipet, immediately trans-
fer 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-ml portions 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 Kuderna-Danish evaporative assemblies complete with
10 ml graduated evaporative concentrator tubes. Place one glass
bead in each concentrator tube.
6. Replace the 500-ml Erlenmeyer flask under each column with a
500-ml Kuderna-Danish assembly and commence elution with 200 ml
of 6% diethyl ether in pet. ether (Fraction I). The elution rate
should be 5 ml per minute. When the last of the eluting solvent
reaches the top of the Na2SOit layer, place a second 500-ml
-------
Revised 12/2/74 Section 5, A, (1), (a)
Page 8
Kuderna-Danish assembly under the column and continue elution
with 200 ml of 15% diethyl ether in pet. ether (Fraction II).
7. To the second fraction only, add 1.0 ml of hexane containing 200
nanograms of aldrin, place both Kuderna-Danish evaporator assem-
blies in a water bath and concentrate extract until ca 5 ml
remain in the tube.
8. Remove assemblies from bath and cool to ambient temperature.
9. Disconnect collection tube from Kuderna-Danish flask and care-
fully rinse joint with a little hexane.
10. Attach modified micro-Snyder column to collect tubes, place tubes
back in water bath and concentrate extracts to 1 ml. If prefer-
red, 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).
12. Should it prove necessary to conduct further cleanup on the 15%
fraction, transfer 10 grams MgO-Celite mixture to a chromato-
graphic 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
ensure quantitative recoveries.
IX. ASSESSMENT OF EXTRACT CONCENTRATION:
1. Inject 5 yl 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 and one or more off-scale peaks in the 6% fraction.
-------
Revised 12/2/74 Section 5, A, (1), (a)
Page 9
2. If off-scale peaks are obtained in either fraction it will be
necessary to dilute volumetrically with hexane to obtain a
concentration that will permit quantisation of those peaks from
a 5 yl injection.
NOTE: A 5-ml dilution of a 3.0 gram sample containing .01 ppm
of a given pesticide will yield 30 picograms of the
pesticide per 5-microliter injection. Provided the
detector is operating at average sensitivity, it should
be possible to obtain quantifiable peaks of most compounds
likely to be present at this concentration.
X. MISCELLANEOUS NOTES:
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 must be
pretested to ensure 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 to
100%. Each laboratory should conduct its own recovery studies
to make certain of its 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
appropriate 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 to 100 ml of saturated NaCl
solution and all of the aqueous layer is discarded. The ether
is then transferred to a 5 flask containing a large excess of
anhydrous sodium sulfate 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 column into a third K-D evaporator assembly, concen-
trating the eluate as described for the 6% and 15% eluates.
-------
Revised 12/2/74 Section 5, A, (1), (a)
Page 10
6. Table 1 gives the elution pattern for a number of common pesti-
cides. On occasion it may be observed that a portion of a given
compound may elute into a different fraction than the one given.
For example, some operators have difficulty eluting all the
dieldrin in the 15% fraction. This is generally caused by 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% (v/v) ethanol.
Ether without the ethanol or with too much would expectedly
result in an altered elution pattern.
7. If this method is used for the detection and quantisation of
organophosphorous compounds, some 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 recov-
eries. 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.
8. If the presence of HCB is suspected in the sample, the analyst
would be well advised to apply the confirmatory procedure
described in Section %,A,(ll),(b) since recoveries by the method
described in this section (5,A,(1),(a), are expectedly poor.
If HCB is detected in a significant number of routine samples,
a modification in the extraction stage (Subsection VI,10) would
prepare for the confirmation contingency and save some time.
In Step 10 weigh 3.4 grams of fat and transfer to a 13 ml grad.,
conical centrifuge tube. Add pet. ether to bring the volume to
the 10 ml mark. Stopper securely and mix on a rotary mixer 30
minutes at ca 50 rpm. Quantitatively transfer 2 ml of the
extract to a small vial, seal and set aside under refrigeration
for possible use in confirmation. Transfer the remaining 8 ml
of extract to a 125 ml separator, rinsing tube with two 2 ml
portions of hexane. Proceed with Subsection VII.
-------
Revised 12/15/79 Section 5, A, (1), (a)
Page 11
TABLE 1. A COMPILATION OF FLORISIL ELUTION PATTERNS AND
RECOVERY DATA OF PESTICIDES
INTRODUCTION:
The data contained in the following table were copies from "Analytical
Behavior Data for Chemicals Determined Using AOAC Multiresidue Methodology
for Pesticide Residues in Foods," McMahon, B., and Burke, J. A., J. Assoc.
Off. Anal. Chem., 61, 640 (1978). Reproduction here is intended to provide
the reader with the elution characteristics and recovery potential of many
pesticides and industrial chemicals in addition to those that are normally
found in adipose tissue.
The elution behavior and recovery data for many of these compounds were
obtained from fatty foods (FDA PAM, Sections 211.1/231.1(6); Official Methods
of Analysis of the AOAC, 12th ed., (1975), Sections 29.001, 29.002, 29.005,
29.008-29.010, 29.012, 29.014, 29.015, 29.018; and Changes in Methods, J.
Assoc. Off. Anal. Chem., 59., 471 (1976), Sections 29.B01-29.B06), but because
of the similarity of the extraction and Florisil partitioning steps used in
analyzing adipose tissue, it would be expected that results would be very
similar or identical in the analysis of human or animal fat.
Circumstances under which the data were obtained varied widely.
Different data have been validated by many analysts or by only one, with
or without sample present, through complete methods or through individual
procedures of a method. Much of the data has been proven valid during a
number of years of routine use of the methodology in many laboratories.
When complete data on the behavior of a compound are unavailable, the
available data are given and the missing information is indicated.
Available information is presented on elution of compounds from the
Florisil column with additional eluants in cases where the 6% and 15% ethyl
ether-petroleum ether eluants were insufficient.
CODE:
C: Complete (>80%) recovery; may apply to the complete method or to only
the Florisil column elution by the specific eluant(s) shown.
P. Partial (>80%) recovery; may apply to the complete method or to only
the Florisil column elution by the specific eluant(s) shown. Approx-
imate percent recovery expected is given in parentheses, when known.
V. Variable recoveries or inconsistent elution patterns.
-------
Revised 12/15/79 Section 5, A, (1), (a)
Page 12
NR. Not recovered; may apply to the complete method or to only the Florisil
column elution by the specific eluant(s) shown.
ND. No data; indicates compound has not been tested through complete
procedure.
FLORISIL ELUTION NOTATIONS:
1. Percentages in this column refer to percent ethyl ether in petroleum
ether eluants in 200 ml portions in which the compounds eluted. Unless
otherwise indicated, percentages above 15% were used in addition to the
usual 6% and 15% eluants.
2. Appearance of C, P, or NR plus the appropriate eluant(s) indicates that
the information was obtained during testing of Florisil elution only.
3. Appearance of appropriate eluant alone indicates that the information
was obtained during testing of the entire method.
(continued)
-------
Revised 12/15/79
Compound
TABLE 1. (continued)
Acarol
Acetyl tributyl citrate
Acetyl triethyl citrate
Acetyl tris (2-ethyl hexyl)citrate
Alachlor (Lasso)
Aldrin
Allidochlor (Randox)
Anilazine (Dyrene)
Aramite
Aroclor 1016
Aroclor 1221
Aroclor 1242
Aroclor 1248
Aroclor 1254
Aroclor 1260
Aroclor 1262
Aroclor 4465
Aspon
Atrazine
Azinphos-ethyl (Ethyl Guthion)
Azinphos-methyl (Guthion)
Benfluralin (benefin)
Bensulide (Prefar)
Benzoylprop-ethyl (Suffix)
a-BHC
S-BHC
(J-BHC (lindane)
S-BHC
Binapacryl
Bis(2-ethoxyethyl)phthalate
Bis(2-methoxyethyl)phthalate
Bis(trich1oromethyl)disulfide
Bis(3,3,5-trimethylcyc1ohexyl)
phthalate
Bomyl
Bromacil
Bromophos
Bromophos-ethyl
Bulan
Butoxy ethyl ester 2,4-D
Butoxy ethyl ester 2,4,5-T
Butyl benzyl phthalate
jl-Butyl ester' 2,4-D
n^Butyl ester 2,4,5-T
Butyl isodecyl phthalate
Butyl octyl phthalate
Butyl phthalyl butyl glyocate
Captafol (Difolatan)
Captan
Captan epoxide
Method
Recovery
C
P
P
ND
ND
C
ND
P
NR
C
r
C
C
C
C
C
ND
ND
ND
C
C
ND
C
C
C
C
P(65)
ND
ND
ND
ND
ND
ND
ND
ND
P(75)
P
P
P(70)
P(10)
P
C
ND
P
ND
ND
ND
Section 5, A, (1), (a)
Page 13
Florisil
Elution
C, 15 + 50%
50%
50%
50%
NR, 6, 15?
6%
NR, 6, 15?
15?
P, 15%
6%
6%
6%
6%
6%
6%
6%
6%
6?
C, 50%
P, 50%
NR, 6, 15?
C, 6%
C, 50%
NR, 6, 15, 50%
6%
6?
6, 15% V
P(75-90), 15%
ND
ND
6%
15%
ND
NR, 6, 15, 50%
6%
6%
15%
15%
15%
C, 15 + 50%
15%
15%
15 + 50%
15 + 50%
50%
P(64), 50%
50% V
NR, 6, 15%
(continued)
-------
Revised 12/15/79
Compound
TABLE 1. (continued)
Method
Recovery
Section 5, A, (1), (a)
Page 14
Florisil
Elution
Carbophenothion (Trithion) P(60)
Carbophenothion oxygen analog ND
CDEC (Vegadex) " C
Cereclor"s-45 (chlorinated
paraffin) C
Cereclor S-52 (chlorinated
paraffin) C
Chloroenside C
Chlordane (technical) C
Chlordane (cis) C
Chlordane (trans) C
Chlordecone (Kepone) P
Chlorfenvinphos NO
a-Chlorfenvinphos NR
Chlornidine (Torpedo) P(70)
Chlorobenzilate P(75)
Chloroneb ND
Chloropropylate C
Chlorowax 40 (chlorinated
paraffin) C
Chlorowax 500C (chlorinated
paraffin) C
Chlorowax 70 (chlorinated
paraffin) C
Chlorothalonil (Daconil 2787) NR
Chlorpham (CIPS) C
Chlorpyrifos (Dursban) C(74-83)
Chlorpyrifos (DursDan) oxygen
analog ND
Chlorthion C
Clorafin 40 (chlorinated paraffin) P
Clorafin 50 (chlorinated paraffin) C
Coumaphos (Co-Ral) ND
CP-40 (chlorinated paraffin) C
Cresyl diphenyl phosphate C
Crotoxyphos (Cioidrin) iND
Crufomate (Ruelene) ND
Cumylphenyl diphenyl phosphate C
Cyanazine (Bladex) ND
Cypromid ND
Dacthal P
o_,p_'-DDE C
p_,p_'-DDE C
o,£'-ODT C
p_,p_'-DDT C
DEF P(60)
Demeton (Systox) ND
Diablo 700X (chlorinated paraffin) C
Dialifor P(50)
ND
6%
6%
6%
6%
6%
6'o
6%
P, 15, 50% V
ND
ND
C, 155
C, 15 + 50%
6%
C, 15 + 50%
6%
NR, 6, 15, 50%
15%
ND
6+15%
6 + 15%
NR, 6, 15
6 + 15%
50%
ND
ND
50%
ND
NR, 6, 15%
15?^
6%
6%
6%
5%
C, 15 + 50%
ND
6%
C, 15%
30%
(continued)
-------
Revised 12/15/79
Section 5,
Page 15
TABLE 1. (continued)
A, (1), (a)
Compound
Diallyl phthalate
Diazinon
Dibutoxyethyl phthalate
Di-n-butyl phthalate
Dicapthon
Dichlobenil (Casoron)
Dichlofenthion (Nemacide)
Dichlone
p_-Dichlorobenzene
ni-Dichlorobenzene
p_,£' -Dichlorobenzophenone
p_,p_' -Dichlorobenzophenone
Dichlorvos (DOVP)
Oicloran (Sotran)
Dicofol (Kelthane)
Dicrotophos (Bidrin)
Dicyclohexyl phthalate
Di-£-decyl phthalate
Dieldrin
Diethyl phthalate
Di-2-ethylhexyl isophthalate
Di-2-ethylhexyl phthalate
Diisobutyl phthalate
Diisodecyl phthalate
Diisohexyl phthalate
Diisononyl phthalate
Diisooctyl phthalate
Diiscpropyl phthalate
Dilan
Dimethoate
Dimethoate oxygen analog
Dimethyl isophtnalate
Dimethyl phthalate
Di(methylisobutylcarbinyl)
phthalate
Di(1-methylheptyl)adipate
Dinocap (Karathane)
Dinonyl phthalate
Di-£-octyl phthalate
Dioxathion (Delnav)
Dipentyl phthalate
Oiphenamid
Diphenyl phthalate
Dipropyl phthalate
Disulfoton (Di-Syston)
Disulfoton oxygen analog
(demeton-S)
Disulfoton sulfone
Method
Recovery
ND
C
C
C
C
C(80)
P(>70)
NR
C
C
C
C
NR
P(50)
P
ND
C
ND
C
P
C
C
ND
C
ND
ND
C
ND
P(65)
ND
ND
ND
ND
ND
C
P(60)
ND
C
ND
ND
ND
ND
ND
ND
ND
ND
Florisil
Elution
15 •*- 50%
15%
15
C, 15
C, 15
C j \ D 10
C, 6%
NR, 6, 15,
6%
6%
15%
15%
NR, 5, 15, 50%
P, 15%, C, 15 •
6, 15% V
ND
50%
50%
50%
+ 50%
+ 50% V
50%
20%
15
15
15%
15 + 50%
15%
C, 15 +• 50%
C(ca 80), 15 + 503;
15 + 50%
C, 15 + 50%
15%
C, 15 + 50%
15 + 50%
15%
ND
ND
NR
P, 6, 15, 50%
15%
5 Of
7o
P(75), 1
15 + 50%
C, 15 +
ND
15%
ND
15 + 50%
15 + 50%
P(25-40)
ND
ND
5,"
50%
, 6%
(continued)
-------
Revised 12/15/79
TABLE 1. (continued)
Section 5, A, (1), (a)
Paqe 16
Compound
Oiuron
Endosulfan I (Thiodan I)
Endosulfan II (Thiodan II)
Endosulfan sulfate
Endrin
Endrin alcohol
Endrin aldehyde
Endrin ketone (Delta Keto 153)
EPM
Epoxyhexachloronorbornene
EPTC (Eptam)
Ethton
Ethoorop (MOCAP)
2-Ethylhexyl diohenyl phosphate
Ethyl hexyl este- 2,4-D
Ethyl phthalyl ethyl glycolate
Famphur
Fenitrothion (Sumithion)
Fensulfothion (Oasanit)
Fensulfothion oxygen analog
Fensulfothion sulfone
Fenthion
Folpet (Phaltan)
Fonofos (Dyfonate)
Genite 923
Halowax 1001 (chlorinated
naphthalene)
Halowax 1013
Halowax 1014
Halowax 1031
Halowax 1051
Halowax 1099
Halowax 2U1
Hatcol 149 (mixed alkyl
phthalates)
Hatcol 190
Heptachlor
Heptachlor epoxide
Heptachloronorbornene
Hexachlorobenzene
Hexachloro-1,3-tutadiene
Hexachlorocyclopentadiene
Hexachloroethane
Hexachloronorbornadiene
Hexachlorophene
Hydroxy chloroneb
Isobenzan (Telodrin)
Isobutyl ester 2,4-D
Method
Recovery
ND
C
C
C
C
C
C
C
r
ND
ND
P(45)
C
C
MR
ND
C
ND
ND
ND
ND
P(50)
C
C
C
C
C
C
C
C
C
ND
ND
C
C
C
P(60)
P
ND
C
C
ND
ND
C
r
Florisil
Elution
NR,
15%
15 -
50%
15%
C,
C,
25%
15%
6%
p
6%
50%
50%
15%
NR,
ND
15%
ND
ND
ND
6, 1
C, 1
6%
C, 1
6, 15, 50%
50%
15 + 50°;!
15 + 50%
(following 6% only)
151!
6, 15, 50^;
5 + 50% V
C, 6%
6%
6, 15%
C, 6%
6%
15 + 50%
15 + 50%
6%
6%
ND
6%
6%
6%
ND
ND
NR, 6, 15, 50%
ND
6%
15%
(continued)
-------
Revised 12/15/79
Compound
TABLE 1. (continued)
Isodecyl isooctyl phthalate
Isodrin
Isooctyl ester 2,4-D
Isooctyl ester 2,4,5-T
Isopropyl biohenyl
Isopropyl ester 2,4-D
Isopropyl ester 2,4,5-T
Korax (Lanstan)
Leptophos (Phosvel)
Ma lath ion
Malathion oxygen analog
Merphos
Methidathion (Supracide)
2,p_' -Methoxychlor
p_,£' -Methoxychlor
Methyl phthaiyl ethyl glycolate
Methyl Trithion
Mevinphos (Phosdrin)
Mi rex
Mi rex, 2,8-dihydro-(photoproduct)
Mi rex, 10,10-dihydro-(photoproduct)
Mi rex, S-monohydro-(photcproduct)
Mi rex, 10-monohydro-(photoproduct)
MO
Monobutyl phthalate
Monocrotophos (Azodrin)
Monuron
Ma led
Neburon
Nitrofen (TOK)
Nonylphenyl diphenyl phosphate
Octachlor epoxide (oxychlordane)
Octachloro-dibenzo-£-dioxin
Octachlorostyrene
Ovex (chlorfenson)
Oxadiazon
Parathion
Parathion-methyl (methyl parathion)
Parathion-methyl oxygen analog
Parathion oxygen analog
Paroil 1400V (chlorinated paraffin)
Paroil 1500V
Pentachloraniline
Pentachlorobenzene
Pentachlorobenzoitrile
Pentachlorophenyl methyl sulfide
Method
Recovery
ND
C
P(75)
P(65)
C
ND
C
C
ND
C
P(50)
C
ND
MR
C(depends on
Florisil)
ND
P(70)
ND
ND
ND
C
ND
ND
ND
ND
ND
C
C
C
ND
ND
C
P(75)
C
C
ND
ND
C
C
C
C
P(60)
C
Section 5, A, (1), (a)
Page 17
Florisil
Elution
15 + 50%
6%
15%
15;i
5%
15%
15%
NR, 6, 15%
C 6%
15, 50% V
ND
6, 15, 50% V
507,
NR, 6, 15, 50%
6% V
ND
6%
6%
6%
6 + 15% V
15%
ND
NR, 6, 15, 50%
ND
NR, 6, 15, 30%
C, 15%
SO*,
6%
NR, 6, 15%
6%
15%
C, 15%
15%
15%
ND
ND
6%
5%
6%
C, 6%
15%
C, 6%
(continued)
-------
Revised 12/15/79
TABLE 1. (continued)
Perthane
Perthane olefin
Phenkapton
Phorate (Thimet)
Phorate oxygen analog sulfone
Phosalone
Phosmet (Imidan)
Phosphamidon
Phostex
Photodieldrin A
Planavin
Prolan
Prometryn
Propachlor (Ramrod)
Prooanil (Stam F-34)
Propazine
PX-316 (mixed alkyl phthalates)
Qinnotozene (PCNB)
Ronnel (fenchlorphos)
Ronnel oxygen analog
Schradan (OMPA)
SD 7438
Simazine
Strobane
Sulfotepp
Sulphenone
T-146 (mixed n-alcohol phthalates)
T-147
T-148
o_,£'-TDE
R,£'-TDE
£,£'-TDE olefin
Tecnazene (TCNB)
Terbacil
Terbufos (Counter)
Terbuthylazine
2,3,4,5-Tetrachloroanisidine
2,3,4,6-Tetrachloroanisidine
2,3,5,6-Tetrachloroanisidine
2,3,4,5-Tetrachloroanisole
2,3,4,6-Tetrachloroanisole
2,3,5,6-Tetrachloroanisole
1,2,3,4-Tetrachlorobenzene
1,2,3,5-Tetrachlorobenzene
2,3,4,5-Tetrachlorobenzene
2,3,7,8-Tetrachlorodibenzo-p_-dioxin
2,3,4,5-Tetrachloronitroanisole
Method
Recovery
C
C
ND
P(80)
ND
C
ND
ND
ND
C
P(70)
P(25)
P(70)
ND
ND
NR
ND
C
C
ND
ND
C
ND
P(65)
ND
ND
ND
ND
C
C
C
C
ND
ND
ND
ND
ND
ND
ND
ND
ND
C
C
C
P(70)
ND
Section 5, A, (i;
Page 18
Florisil
Elution
6%
6%
6%
6%
ND
C, 50%
NO
ND
6%
15%, final trace, 50%
P(50-80), 50%
15%
P(67), 50%
NR, 6, ]5%
NR, 6, 15*.
C(80-94), 15 + 50% V
15 +50%
6%
6%
ND
ND
C, 15%
C, 50%
5 + 15
50%
V
50%
50%
50%
15
15
15
6%
6%
6%
5%
NR, 5, 15%
6%
15 + 50%
6%
50/
,1
6%
6%
5%
6%
ND
ND
ND
p, 6, 15% V
(continued)
-------
Revised 12/15/79 Section 5, A, (1), (a)
Paqe 19
TABLE 1. (continued)
Method Florisil
Compound Recovery Elution
2,3,4,6-Tetrachloronitroanisole ND 6%
2,3,5,6-Tetrachloronitroanisole ND 6%
Tetrachlorvinphos (Gardona) ND ND
Tetradifon (Tedion) C 15%
Tetraiodoethylene P(65) 6%
Tetrasul C C, 6%
Thionazin (Zinoohos) NR C(80), 15 + 50% V
Toxaphene (camphechlor) C 6%
Tri(2-butoxyethyl)phosphate NR NR
Tributyl citrate NR ND
Trichlorobenzenes P(60) C, 6%
Tricresyl phosphate C 50%
Triethyl citrate NR ND
Tri(2-ethylhexy1)phosphate P 50%
Trifluralin C 6%
Triphenyl phosphate C 50%
Tris(l-bromo-3-chloroisopropyl)
phosphate P 50%
Tris(8-chloroethyl)phosphate NR NR
Tri's(2,3-dibromopropyl )phosphate NR NR
Tris(l,3-dichloroisopropyl)
phosphate P 50%
Tris(dichloropropyl)phosphate P(V) 50%
Tris(isopropy1pheny1)phosphate C 50%
vernolate (Vernam) ND P, 15%
Zyt'ron C 6%
-------
-------
Revised 12/15/79 Section 5, A, (1), (b)
Page 1
ANALYSIS OF ADIPOSE TISSUE
DETERMINATION OF HEXACHLOROBENZENE AND
MIREX WITH CONFIRMATION OF HEXACHLOROBENZENE
I. INTRODUCTION:
The detection and quantitation of hexachlorobenzene (HCB) in
adipose or other fatty tissues have posed problems to the analyst
for two basic reasons: (1) in chromatography with electron capture
detection, the retention characteristics of the HCB peak are quite
similar to the alpha isomer of BHC (hexochlorocyclohexane) on a
number of GC columns; (2) because of the unfavorable partition ratio
of HCG (and mirex) in the acetonitrile/petroleum ether partition
cleanup system, low recoveries are obtained using the multiresidue
method outlined in Section 5,A,(l),(a). The procedure below offers
improved recoveries and an HCB confirmatory derivative analysis.
The confirmation scheme is especially useful for HCB because HCB
elutes very early from the GC columns commonly used for pesticide
residue analysis. Although the procedure was developed specifically
for determination of HCB residues, it has proven useful for determina-
tion of mirex residues in adipose tissue.
REFERENCES:
1. Rapid Determination and Confirmation of Low Levels of Hexa-
chlorobenzene in Adipose Tissue, Crist, H. L., Moseman,
R. F., and Noneman, J. W., Bull. Environ. Contam. Toxicol.
11, 273-280 (1975).
2. Collaborative Study of an Improved Method for Hexachloroben-
zene and Mirex and Hexachlorobenzene Confirmation in
Adipose Tissue, Watts, R. R., Hodgson, D. W., Crist, H. L.,
and Moseman, R. F., J. Assoc. Off. Anal. Chem., submitted
for publication. (Official First Action status has been
granted by the AOAC.)
II. PRINCIPLE:
An adipose tissue sample is dissolved in hexane and applied
directly to a Florisil column. The HCB and mirex residues are eluted
with hexane and determined by direct EC-GC of the concentrated eluate.
HCG residues are then confirmed by EC-GC analysis of a disubstituted
ether derivative (bis-isopropoxytetrachlorobenzene; BITB) formed by
reaction with 2-propanol. Mirex residues do not survive this reaction.
-------
Revised 12/15/79 Section 5, A, (1), (b)
Page 2
III. APPARATUS:
1. Gas chromatograph fitted with 3H or 63Ni electron capture
detector and 1.8 m x 4 mm i.d. columns of 1.5% OV-17/1.95%
OV-210 and 5% OV-210 on 80-100 mesh support. Operating param-
eters: column temperature, 200°C (OV-17/OV-210), 180°C (OV-210);
detector (tritium) 210°C, (nickel) 300°C; inlet block 220°C;
transfer line 220°C; carrier gas flow 60 ml/min.
2. Columns, glass, Chromaflex, size 241, 300 x 25 mm o.d., Kontes
No. K-420530.
3. Kuderna-Danish concentrator assembly, Kontes No. K-570000,
fitted with 25 ml graduated evaporative concentrator tube
(K-570050), size 2525, 19/22).
4. Micro-Snyder column, Kontes No. K-569250.
5. Disposable pipets.
6. Compressed, gaseous nitrogen equipped with regulator valve for
pressure reduction to approximately 5 lb/in2.
7. Water bath, with temperature range of 50-100°C.
8. Glass wool, pre-extracted in a Soxhlet apparatus with hexane.
9. Vortex mini-mixer.
IV. REAGENTS AND SOLVENTS:
1. Hexachlorobenzene and mirex analytical reference standards,
available from the Quality Assurance Section, U.S. EPA, ETD,
HERL, MD-69, Reserach Triangle Park, NC 27711.
2. Pyridine, Burdick and Jackson, or equivalent, suitable for liquid
or gas chromatography. Test the reagent for intereferences by
using the derivatization procedure.
3. Potassium hydroxide, reagent grade; 10% solution in 2-propanol.
4. Sodium sulfate, anhydrous, granular. Soxhlet extract with hexane
and oven dry at 130°C.
5. Sodium sulfate, 2% aqueous solution, prepared from pre-extracted
reagent.
6. Florisil, PR grade, the Floridin Company, Berkeley Springs, WV.
Prepare Florisil column by packing Chromaflex column with 100 mm
-------
Revised 12/15/79 Section 5, A, (1), (b)
Page 3
adsorbent and 12 mm Na SO on top [see Section 5,A,(l),(a), III,
IV, and VIII]. Hold in 130°C + 2°C oven for at least 16 hours
prior to use. Remove stopcocks before placing columns in oven.
Prewash with 50 ml hexane just before use.
7. Keeper solution, 1% paraffin oil in hexane.
8. Hexane, 2-propanol, pesticide quality, or equivalent.
V. PROCEDURE:
1. Accurately weigh 0.5 g of rendered or extracted fat in a 13 ml
centrifuge tube.
2. Dissolve the fat in ca 0.5 ml of hexane and quantitatively trans-
fer to a Florisil column prewashed with 50 ml of hexane. Rinse
the sample tube with tow 0.5 ml portions of hexane and add each
to the column.
3. Allow the column to drain until the solvent level is just at the
top of the Na^O^.
4. Rinse the column insides above the adsorbent bed with 2-3 ml of
hexane.
5. Elute with 200 ml of hexane at a flow rate of 5 ml/minute.
NOTE: The elution characteristics of each lot of Florisil
should be tested for both compounds, and the elution
volume should be adjusted if necessary.
6. Collect the eluent in the Kuderna-Danish assembly containing a
3 mm glass bead or carborundum chip in the 25 ml concentrator
tube.
7. Immerse the concentrator tube in a boiling water or steam bath
to about 1/3 of its depth and concentrate the extract to ca
10 ml.
8. Remove the K-D assembly from the bath, cool, and carefully
remove the concentrator tube, rinsing the joint with ca 3 ml
of hexane.
9. Place the tube under a nitrogen stream and reduce the extract
volume to ca 3 ml. Do Not Allow to go to Dryness!!!
-------
Revised 12/15/79 Section 5, A, (1), (b)
Page 4
10. Rinse the sidewalls of the tube with hexane and adjust the
volume to 5 ml. Stopper and Vortex mix one minute.
NOTES:
1. On the basis of the gas chromatographic analysis below,
it may prove necessary to further dilute or concentrate
the extract.
2. In addition to HCB and mirex, the 200 ml hexane fraction
may contain heptachlor, aldrin, £_,p_'-DDE, p_,p_'-DDT, and
PCBs. No attempt should be made to quantitate any com-
pounds other than HCB that may appear in this eluate,
as the elution may be incomplete and, therefore, give low
recoveries.
VI. GAS CHROMATOGRAPHY:
Determine the amount of HCB and mirex in the sample by injecting
3-8 yl amounts of standards and samples into an OV-17/OV-210 GC
column with the parameters stated in Subsection 111,1. Alternatively,
quantitate mirex on an OV-210 column. The RRT,, of HCB, the HCB
derivative, and mirex are in the following table.:
RRTA of Compounds (Conditions in Subsection 111,1)
5% OV-210 1.5% OV-17/1.95% OV-210
HCB
HCB derivative
Mirex
0.46
N.D.1
3.78
0.48
0.86
6.1
lHot determined.
Adjust sample volumes as required to produce major peak
responses, so that no peak less than 20% f.s.d. is quantitated.
Peak heights of standards and samples should not vary by more than
25%, and concentrations must fall within the linear range of the
detector. It is best to work at the same attenuation setting for
samples and standards.
-------
Revised 12/15/79 Section 5, A, (1), (b)
Page 5
VII. CONFIRMATION OF HCB BY DERIVATIZATION:
1. Add 5 drops of paraffin oil keeper solution to the sample
(step 10, Subsection V) and place under a gentle nitrogen
stream in a warm water bath. Continue evaporation until
0.1-0.2 ml of hexane remains.
NOTE: At least three concentrations of the HCB standard
should be derivatized along with samples. Choose
concentrations that bracket the concentration of
the HCB present in the sample as determined by the
initial GC analysis. The responses of the HCB
standard derivatives should be linear. Add 2-3
drops of paraffin oil keeper solution before evap-
orating to 0.1-0.2 ml.
2. Add 0.2 ml of pyridine and 0.5 ml of 10% KOH in 2-propanol,
attach a modified micro-Snyder column to the concentrator
tube, and place in a boiling water bath for exactly 45
minutes.
3. Remove the tube, cool under tap water, and add 10 ml of
2% ^SOtt solution and exactly 2 ml of hexane. Stopper
and mix vigorously for one minute.
NOTE: The 2 ml of hexane should be delivered with a
volumetric pi pet, because this volume is used
in calculating the amount of HCB residue.
4. After the phases have completely separated, inject 3-8 yl of
the hexane extract (upper layer) into the gas chromatograph
fitted and adjusted as described in Subsection 111,1. An
additional quantitative volume of hexane may be added, as
estimated, to bring the BITB peak on scale. After such a
dilution, the tube must be stoppered, again shaken, and
time allowed for layer separation before sampling for GC.
If the neight of the BITB peak is less than 10% f.s.d.,
some further concentration is required by evaporation under
a nitrogen stream. Quantisation is obtained by comparison
of results to the reference standards of HCB carried through
the derivatization procedure along with the unknown. Mirex
is not recovered in the derivatization procedure.
NOTE: Using the prescribed column temperature of 200°C, the
RRTA of the BITB peak on the OV-17/OV-210 GC column
shoOld be 0.86 (see the Table in Section VI).
-------
Revised 12/15/79 Section 5, A, (1), (b)
Page 6
VIII. RECOVERY RESULTS:
1. Studies by the authors of the original work (Reference 1)
over a concentration range of 0.01 to 1.0 ppm have indica-
ted recoveries from 86 to 107% when a 100 ml (rather than
200 ml) Florisil eluate is quantitated as specified in the
earlier version of this method (see Table 1).
2. Tables 2-4 present a summary of the collaborative results
(Watts et_ aJL , Reference 2) for the unknown standard solutions
(8 ng/ml of HCB and 96 ng/ml of mirex), fat blank, spiked
samples of HCB before and after derivatization, and spiked
samples of mirex. Each value reported for fortified fat
represents an average of the three repetitive determinations
with the standard deviation of the three values directly
underneath. Using these figures, the percent intralaboratory
coefficient of variation (CV) for each spiking level and
the average intralaboratory CV value are calculated and
reported on the same horizontal line of the table. The
inter!aboratory results are given at the bottom of each
column. Results marked with a 1 superscript have been
determined to be "outlier" values as calculated by the Fit-
ness Test method described in the EPA Quality Control Manual,
Chapter 2, Section K,e.
The standard solution average result of 7.95 ng/ml for the
direct HCB standard analysis represents a relative accuracy
of 99.4%. The GCB inter!aboratory average recoveries from
Table 2 of 89.6, 87.4, and 92.6% (after blank value subtraction)
for the 20.0, 33.3, and 50.0 ppb fortified samples represent
good efficiencies over the range tested. The respective inter-
laboratory CV values ranged from an excellent 6.8% to a good 9.96%.
The results in Table 3 for the HCB confirmatory analysis by
formation of the BITB derivative show three HCB interlaboratory
average percent recoveries of 79.8, 78.8, and 76.9, respectively,
for the 20.0, 33.3, and 50.0 ppb fortifications when corrected
for the fat blank of 2.25 ppb. The respective interlaboratory
CV values of 15.7, 18.6, and 19.0% demonstrate acceptable precision,
although understandably not nearly as good as direct HCB deter-
minations.
The Table 4 results for mirex indicate no difficulty with
the unknown standard solution or the three fortification levels.
The standard solution mean of 97.5 ng/ml represents an accuracy
of 101.6% relative to the 96 ng/ml actual value. The three mirex
average results, when corrected for the average blank of 27.5 ppb,
yielded interlaboratory percent recovery values of 89.0, 90.2, and
-------
Revised 12/15/79 Section 5, A, (1), (b)
Page 7
92.3, respectively, for the 150, 300, and 500 ppb fortification
levels. The respective interlaboratory CV values of 7.6, 16.5,
and 18.1% represent excellent to acceptable precision values.
IX. MISCELLANEOUS NOTES:
1. Collaborator comments on the HCB derivative scheme indicated
some unresolved problems with formation of only the disub-
stituted (BITB) derivative. Significant quantities of the
monosubstituted derivative were often formed that prevented
accurate quantitations. Collaborator No. 2 did not report
derivative results for this reason, and large intralaboratory
CV values in Table 3 also indicate the same problem. The
derivative scheme is, therefore, recommended as qualitative
and semi-quantitative confirmation of HCB.
2. The analyst may find that the hexane eluate will yield a
clearly delineated peak of the precise RRTn value for HCB.
In this case, there may be little need for the derivatization
step. However, for more reliable confirmation, the deriva-
tization step is recommended. Early eluting compounds such
as isomers of hexachlorocyclohexane (BHC) and heptachlor,
which could present interfering peaks, are altered and do not
interfere after derivatization. Aldrin, dieldrin, endrin,
p_,£'-DDE, and PCBs are not altered, but their normal elution
characteristics pose no interference problems.
3. If the analyst has been alerted to the possible presence of
HCB in the sample during a routine analysis by method 5,A,(1),(a),
it is feasible to provide ahead of time for this contingency
during the extraction step. See Miscellaneous Notes,
Subsection 8, of Section 5,A,(l),(a).
-------
Revised 12/15/79 Section 5, A, (1), (b)
Page 8
TABLE 1. RECOVERY OF HCB FROM FORTIFIED CHICKEN FAT
BY DIRECT ELUTION WITH 100 ML OF HEXANE1
Fat, mg
403
494
477
474
583
482
530
497
446
555
528
525
HCB
Added, ng
4
8
16
25
50
48
150
160
250
350
500
1000
HCB
Recovered, ng
4.1
7.0
14.8
24.8
51.5
51.2
143
160
254
301
477
1003
HCB
Cone, ug/g
0.010
0.016
0.034
0.053
0.086
0.100
0.283
0.322
0.561
0.631
0.947
1.900
Recovery
%
102
88
92
99
103
107
95
100
102
86
95
100
Mean 97.4%
Range 86-107%
Standard Deviation ±6.3%
iCrist et al. (Reference 1)
-------
Revised 12/15/79
TABLE ?.. HCB COLLABORATIVE RESULTS (WATTS ET AL.. REFERENCE 2)
Section 5, A, (1), (b)
Page 9
Fat Fortification (ppb)
Lab
1
2
3
4
5
6
7
8
9
10
11
1?
Unknown
Std. (8jjg/Ml)
^
8.0
100.0
7.44
93.0
7.75
96.9
12. 51
156.3
8.0
100.0
7.5
93.8
7.28
91.0
7.7
96.3
9.3
116.3
7.91
98.9
9.0
112.5
7.6
95.0
Fat
Blank
(ppb)
2.80
2.95
2.05
2.00
2.20
1.45
5.58
2.70
7.01
3.02
3.70
0
20.0
33.3
50.0
Average (ppb)
Standard Deviation
22.0
1.00
21.2
0.29
20.7
2.01
22.7
5.85
19.60
0.40
18.6
2.11
17.3
4.62
21.7
?.08
28. 81
10.83
17.8
4.48
22.1
2.46
21.6
1.15
31.3
3.06
35.9
8.72
32.3
2.17
32.3
4.04
31.0
2.65
30.3
1.15
29.7
1.03
31.7
2.52
40. 71
5.14
27.9
1.82
34.0
3.14
32.8
1.51
50.3
7.77
51.9
10.22
49.6
1.42
50.0
22.72
51.5
3.32
38.9
0.81
42.4
2.31
46.3
3.21
55.5
13.18
34. 81
5.20
47.7
0.55
53.8
1.12
20.0
33.3
50.0
Intralab Coefficient
of Variation (%)
4.5
1.4
9.7
25.8
2.0
11.3
26.7
9.6
37.6
25.2
11.1
5.3
9.8
24.3
6.7
12.5
8.5
3.8
3.5
7.9
12.6
6.5
9.2
4.6
15.4
19.7
2.9
45.4
6.4
2.1
5.4
6.9
23.7
14.9
1.2
2.1
Average Intralab
Coefficient of
Variation (%)
9.9
15.1
6.4
27.9
5.6
5.7
11.9
8.1
24.6
15.5
7.2
4.0
Mean 7.95 2.58 20.5 31.7 18.9
Mean (%) 99.4 89. 6? 87.4? 92. 62
Std. Oev. 0.64 1.86 2.14 4.87
Coef.Var.(%) 8.05 9.1 6.8 9.96
'Outlier
2Mean Corrected for Blank
-------
Revised 12/15/79
Section 5, A, (1), (b
Page 10
TABLE 3. HCB DERIVATIVE COLLABORATIVE RESULTS (WATTS ET AL., REFERENCE 2)
Fat Fortification (ppb
Lab
1
2
3
4
5
6
7
8
9
10
11
12
Fat
Blank
(ppb)
3.0
2.0
--
4.0
--
7.541
0
0
2.86
3.9
--
20.0
ft
Stan
19.3
1.15
--
15.8
6.9
—
21.3
2.31
17.7
2.2
22.1
5.7
17.0
6.6
4.31
3.9
14.7
2.0
21.2
2.5
14.9
1.2
33.3
50.0
verage (ppb)
dard Deviation
26.3
5.51
—
20.0
16.2
--
34.0
6.2-1
26.6
2.5
33.1
3.9
30.7
11.9
7.01
2.8
23.3
4.4
36.1
1.7
26.7
1.3
37.0
9.54
--
38.7
15.6
--
53.0
5.2
35.7
1.9
44.8
5.5
35.7
13.3
9.31
5.2
31.3
6.2
52.8
1.1
37.7
4.8
20.0
33.3
50.0
Intralab Coefficient
of Variation (%)
6.0
43.7
10.8
12.4
25.8
38.8
90.7
13.6
11.8
8.1
21.0
81 .0
18.4
9.4
11.8
38.8
40.0
18.9
4.7
4.9
25.8
40.3
9.8
5.3
12.3
37.3
55.9
19.8
2.1
12.7
Average Intralab
Coefficient of
Variation (%)
17.6
55.0
13.0
9.0
16.6
38.3
62.2
17.4
6.2
8.6
Mean 2.25 18.2 28.5 40-7
Mean (%) 79.8'- 78.8? 76.9?
Std. Oev. 2.86 5.30 7.74
Coeff.Var.(%) 15.7 18.6 19.0
T0utliers
?Mean Corrected for Blank
-------
Revised 12/15/79
TABLE 4. MIREX COLLABORATIVE RESULTS (WATTS ET AL., REFERENCE 2)
Section 5, A, (1), (b)
Page 11
Fat fortification (ppb)
Lab
1
2
3
4
5
6
7
8
9
10
11
12
Unk.
Std. (96 pg/wl)
%
89.8
93.5
96.2
100.2
92.0
95.8
103.0
107.3
96.0
100.0
97.6
101.7
101
105.2
100.0
104.2
99.2
103.3
101
105.2
97.2
101.3
84. 71
88.2
150
300
500
Fat
Blank Average (£pb)
(ppb) Standard Deviation
10.0
32.3
15.7
20.0
8.0
._
6.26
55.0
86.3
13.fi
..
148
5.29
166
5.10
177
14.8
160
23.8
156
1.53
1251
6.03
145
10.8
182
16.2
169
17
151
45.5
149
3.06
164
10.1
287
9.54
317
61.0
341
21.6
212
30.2
290
16.6
209
11.5
298
15.6
346
14.0
377
1.27
286
15.7
297
3.61
315
4.6
490
54.8
523
66.0
568
20.0
349
131
507
9.50
313
27.2
504
46.5
555
33.5
582
9R.6
409
77.7
485
2.52
578
26.0
150
300
500
Intralab Coefficient
oF Variation (%)
3.6
3.1
8.4
14.9
1.0
4.8
7.4
8.9
10.1
30.1
2.1
6.2
3.3
19.2
6.3
14.2
5.7
5.5
5.2
4.0
0.34
5.5
1.2
1.5
11.2
12.6
3.5
37.5
1.9
8.7
9.2
6.0
16.9
19.0
0.52
4.5
Average Intralab
Coefficient of
Variation (%)
6.0
11.6
6.1
22.2
2.9
6.3
7.3
6.3
9.1
18.2
1.3
4.1
Mean 97.5 27.5 161 298 489
Mean (%) 101.6 89. 07- 90. 22 92. 3J
Std. Dev. 3.97 12.2 49.3 88.3
Coef. Var.(%) 4.1 7.6 16.5 18.1
'Outlier
2Mean Corrected for Blank
-------
-------
Revised 12/2/74
Section 5, A, (2), (a), & (b)
Page 1
II.
MICRO METHOD FOR THE DETERMINATION OF CHLORINATED
PESTICIDES IN HUMAN OR ANIMAL TISSUE AND HUMAN MILK
INTRODUCTION:
The size of many tissue samples is so minimal that the method
described in Section 5,A,(1) is unsuitable. This procedure,
requiring only 0.5 grams, is suitable for most biopsy samples and
for wildlife (small animal or bird) tissues.
REFERENCE:
Presentation at Fall meeting, ACS, Chicago, IL,
1967 MICROMODIFICATION OF THE MILLS PROCEDURE
FOR THE DETECTION OF PESTICIDES IN HUMAN TISSUES,
Enos, H. F., Biros, F. J., Gardner, D. T., Wood,
J. P.
PRINCIPLE:
A 0.5 gram sample of tissue is macerated in a micro tissue
grinder with acetonitrile. An aqueous solution of Na2SOit is added,
the pesticides are partitioned into hexane and the extract is
concentrated to 0.3 ml. Cleanup and partitioning are carried out
by successive elutions with 1% methanol in hexane through a micro
column of Florisil. Two fractions are collected, concentrated to
suitable volumes by evaporation in a modified micro Snyder assembly,
and subjected to GLC with electron capture detection.
III. MATERIALS AND REAGENTS:
1. MICROCOLUMN:
Place a small 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 gm of 60 to 100 mesh Florisil which has
been activated by the manufacturer at 1200°F. (Only PR grade
Florisil should be used for this method.) The column packing
is added in increments followed by a gentle tapping. Add
1.6 gm of sodium sulfate, granular, to the top of the column.
Wash the column with 50 ml of Nanograde hexane followed by
50 ml of Nanograde methanol. Dry and store columns in a 130°C
oven. The columns should be 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 time.
-------
Revised 12/2/74 Section 5, A, (2), (a) & (b)
Page 2
2. SODIUM SULFATE. ANHYDROUS, 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 yl and inject 5 yl into the gas chromatograph. The results
will indicate whether it is necessary to extract the batch of
sodium sulfate with hexane and methanol prior to storing in
the oven.
3. PESTICIDE QUALITY SOLVENTS:
Hexane, acetonitrile, methanol.
4. DISTILLED WATER:
Extract 8.0 ml with hexane. Concentrate the extract to 300 yl,
and inject 5 yl into the gas chromatograph. If extraneous
peaks occur, then the distilled water must be extracted with
hexane prior to use.
5. TISSUE GRINDER:
Dual tissue grinder Size 22 or 23 (Kontes Cat. No. K-885450).
6. MIXER:
Vortex Junior or equivalent.
7. CENTRIFUGE:
Capable of a speed of 2,000 rpm
8. EVAPORATIVE CONCENTRATOR:
Complete with modified micro Snyder column, 5 joint 19/22,
Kintes Cat. No. K-569250.
9. CONCENTRATOR TUBE:
Size 1025, Kontes Cat. No. K-57005Q.
10. CONCENTRATOR TUBE:
Size 2525, Special Order, Kontes Cat. No. K-570050.
-------
Revised 12/2/74 Section 5, A, (2), (a) & (b)
Page 3
11. TEST TUBE:
25 ml with I 19/22, joint with hooks, Special Order, Kontes Cat.
No. K-897900.
IV. 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 aldrin, 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: Run a complete reagent blank with each set of samples.
2. Centrifuge and pour supernatant into a 50-ml round bottom
test tube. Repeat extraction twice more, collecting supernates
in the test tube.
3. Add 25 ml 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-ml and
two 2-ml portions of hexane. Combine the extracts in a 10-ml
evaporative concentrator.
5. Concentrate the extract to 300 yl with the aid of a modified
micro Snyder column* and a 3-mm glass bead in the tube.
6. Proceed to Subsection V.
V. FLORISIL FRACTIONATION:
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) in
Subsection IV, to the top of the Florisil column with the aid
of a disposable pipet fitted with a rubber bulb. Begin imme-
diate collection of eluate in a 25-ml capacity concentrator tube.
*J. Burke et al., J.A.O.A.C. , 49 (5): 999-1033, 1966.
-------
Revised 12/2/74 Section 5, A, (2), (a) & (b)
Page 4
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,
aldrin, p_,p_'-DDE, p_,p_'-DDT, and p_,£'-DDT.
6. Collect a second fraction by eluting with a second 12 ml
portion of ~\% methanol in hexane. This fraction will contain
dieldrin, heptachlor epoxide, endrin, 8-BHC, Lindane, and
p_,p_'-DDD. (See Table 1.)
NOTE: A small amount of B-BHC, Lindane, and/or £,p_'-DDD may
appear in the first fraction.
7. Add 20 nanograms 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 yl,
respectively, and proceed with the GLC portion as outlined in
Subsection VII.
VI. ANALYSIS OF BRAIN:
Proceed with steps (1) through (4) as described under IV. SAMPLE
PREPARATION.
5. Concentrate the combined hexane extracts to 500 yl in a 25 ml
test tube fitted with a modified micro Snyder column and
using a 3 mm glass bead in the tube.
6. Add 0.3 ml Acetic Anhydride and 0.3 ml pyridine and incubate in
a water bath at 60 to 65°C for 1/2 hour.
7. Add 9 ml of 2% Na2$Qk anc' extract with 2 to 3 ml portions of
hexane.
8. Concentrate the combined extracts to 300 yl in a 10 ml evap-
orative concentrator fitted with a modified micro Snyder column
using a glass bead for a boiling chip.
9. Proceed as described under V. FLORISIL FRACTIONATION.
-------
Revised 12/2/74
VII. ANALYSIS OF HUMAN MILK:
Section 5, A, (2), (a) & (b)
Page 5
The basics of this procedure have been determined by
experience in a laboratory conducting intensive surveillance to
be wholly applicable to the analysis of human mother's milk.
A few modifications have proved critical, however, and these are
given in the following:
1. Follow Subsection IV, all steps as described but with one
precautionary comment. If the sample has been frozen, it
has been found advisable to use a supersonic disintegrator
to homogenize it after thawing.
2. Unlike cow's milk, no curd layer has been observed forming
on top; instead, there is sediment at the bottom of the
tissue grinder with generally a thin aqueous layer between
it and the solvent layer. The solvent layer is pipeted
after the first extraction, and the second extraction usually
gives a homogeneous liquid.
3. In countries where the use of DDT is permitted by law, the
chemist may find it advisable to dilute the final extract
to 1.0 ml or greater instead of a final volume of 300 pi as
specified in the final step.
VIII. GAS LIQUID CHROMATOGRAPHY:
Proceed with electron capture gas chromatography following
the general guidelines set forth in Section 4,A,(4) and making
sure that prevalent system sensitivity complies with the criteria
given in Misc. Note in Section 4,A,(4).
-------
Revised 12/2/74 Section 5, A, (2), (a) & (b)
Page 6
TABLE 1. ELUTION PATTERN OF SOME COMMON CHLORINATED AND ORGANO-
PHOSPHORUS PESTICIDES ON MICRO FLORISIL COLUMN.
12 ml hexane + Additional 12
12 ml 1% methanol ml 1% methanol
in hexane in hexane
Compound Fraction I Fraction II
Aldrin x
«-BHC x x
3-BHC x
Y-BHC x x
6-BHC x
DDA, methyl ester x
£,£'-DDD x x
£,£'-DDD x x
p_,£'-DDE x
£,£'-DDE x
p_,£'-DDT x
£,£'-DDT x
Diazinon x x
Dieldrin x
Endosulfan I & II x
Endrin x
Ethion x
Ethyl parathion x
Heptachlor x
Hept. epoxide x
1-Hydroxychlordene x
Malathion x
Methyl parathion x
Methoxychlor x
Nitrofen x x
Paradichlorobenzophenone x
Polychlorinated biphenyls x
Ronnel x
Toxaphene x x
-------
Revised 12/2/74 Section 5, A, (3), (a)
Page 1
>1 ANALYSIS OF HUMAN BLOOD OR SERUM
I. INTRODUCTION:
Because of its availability and probable diagnostic value with
regard to extent of both chronic and acute exposures 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. The Dale et al. method, as published,
was found to yield poor precision between laboratories, and in fact,
between chemists within a laboratory. However, a method including
these features is essential in the monitoring situation involving
analyses of large numbers of samples. The following procedure
utilizes only the direct solvent extraction principle of the Dale et
al. method. It is to be considered a general survey method for the
determination of chlorinated hydrocarbon pesticide levels in blood,
particularly DDT 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,(1) in this manual) be applied,
together with confirmatory 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-ml aliquot of serum is extracted with 6 ml 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 sep-
aration of the layers. A 5-ml aliquot of the hexane layer is
quantitatively transferred to an evaporative concentrator tube to
which is affixed a modified 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 pesti-
cide residue. A suitable aliquot is analyzed by electron capture
gas chromatography.
-------
Revised 4/8/75 Section 5, A, (3), (a)
Page 2
III. APPARATUS AND REAGENTS:
1. A rotary mixer so designed as to accommodate the 16 mm culture
tubes and which may be operated at a rotary speed of 50 rpm.
Fisher Scientific Company, Roto-Rack™, Cat. No. 14-456.
2. Gas chromatograph fitted with electron capture detector.
Recommended GLC columns and operating parameters are given
in Section 4,A.
3. Tubes, Culture, 16 x 125 mm, fitted with screw caps, size
15-415 with Teflon-faced rubber liners, Corning No. 9826.
4. Micro-Snyder column modified, with 19/22 f joint, Kontes No.
K-569251.
5. Concentrator tube, 10 ml, grad. 0 to 1 x 0.1 and 2 to 10 x 1,
19/22 I joint, size 1025, Kontes No. K-570050.
6. Syringe, 100 yl, Hamilton No. 710 or equivalent.
7. Vortex Genie mixer.
8. Pipet, Mohr type, 1 ml grad. in 0.01 ml increments. Corning No.
7063 or equivalent.
9. Pipets, transfer, 2, 5, and 6 ml Corning No. 7100 or the
equivalent.
10. Beads, solid, glass, 3 mm, Corning No. 7268 or the equivalent.
11. Six-place tube carrier, stnls. steel. May be fabricated at
local tin shop per attached sketch.
12. Water bath capable of holding temp, of 95 to 100°C.
13. Centrifuge with head to accommodate the Corning No. 9826 tube,
capable of speed of 2,000 rpm.
14. Hexane, distilled in glass, pesticide grade.
IV. SAMPLING:
After drawing sample from the donor (7 to 10 ml), it should be
transferred to a vial or tube fitted with a Teflon or foil lined
screw cap. DO NOT USE CAPS OF POLYETHYLENE OR RUBBER.
-------
Revised 6/77 Section 5, A, (3), (a)
Page 3
Place whole blood sample in the refrigerator for about 30 min-
utes for a settling period and then centrifuge for a sufficient time
for the separation of at least 3 ml of clear serum - generally
10 minutes at 2,500 rpm. Whether or not the analysis is to be con-
ducted immediately, it is desirable at this point to transfer the
2 ml sample aliquot to the 16 x 125 mm culture tube used for extrac-
tion. If analysis cannot be run immediately, place in refrigerator
at 2-5°C for periods of up to 24 hours before analysis. If time
interval to analysis exceeds 24 hours, the tube should be stored in
a deep freeze at -15 to -25°C. Stored in this manner, analysis may
be delayed for periods up to a month without undue effects on the
chlorinated pesticides present.
V. PROCEDURE:
1. Mix blood serum sample thoroughly and, with a volumetric pipet,
transfer 2 ml to a 15 ml round bottom culture tube.
NOTE: In case of the presence of a flocculent or sedimentary
material, it is strongly recommended that the sample
be centrifuged ca 5 minutes @ 2,000 rpm before pipetting
the 2 ml aliquot. Failure to observe this point may
result in poor reproducibility of replicated analyses
of the same sample.
2. Add 6 ml hexane 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 rpm and rotate for 2 hours.
NOTES: (1) This speed may vary from 50 to 44 rpm
but should be confined to this range.
(2) Unless the sample is extremely old, emulsion
formation should present no problem. In case
it occurs, centrifuge at 2,000 rpm 4 to 5 minutes,
or longer if necessary, to effect sufficient sep-
aration to permit withdrawal of the 5 ml aliquot
of clear extract.
4. With a volumetric pipet, transfer 5 ml of the hexane extract
to a 10 ml grad. concentrator tube, add one 3 mm glass bead,
and attach a modified micro-Snyder column. Evaporate the
extract in a steam or hot water bath at 100°C to a volume
slightly less than that which is estimated as appropriate to
accommodate (1) the current level of electron capture detector
sensitivity, and (2) the expected residue range in the
-------
Revised 6/77
5.
Section 5, A, (3), (a)
Page 4
particular sample. When working with general population blood
of low pesticide levels, it may be necessary to evaporate to
ca 0.5 ml.
NOTES: (1) With some experience the operator can complete
the evaporation step in less than 5 minutes.
The tube must be withdrawn from the water when
boiling agitation becomes too vigorous. Immersion
and withdrawal are alternated based on observa-
tion of boil agitation.
(2) Up to six tubes of extract may be evaporated
simultaneously by using the special rack shown
in Figure 2. Time and motion studies have shown
that the time required for the evaporation period
is equal to that required for a single tube.
(3) When working with blood from high exposure
donors, the 5-ml aliquot may require dilution
rather than concentration. This can be deter-
mined by a preliminary analysis of the 5-ml
aliquot.
(4) With lower concentrations, use higher degree of
concentration samples.
Allow the tube to cool (3 to 5 minutes), remove the micro-Snyder
column, and rinse down the sides of the tube and the column
joint with hexane. The volume used will depend on the desired
dilution.
NOTES: (1) When a minimal dilution is required after evap-
oration, a 100-yl syringe is useful in performing
the hexane rinse.
(2) To obtain a suitable extract concentration for
p_,p_'-DDE, it is generally necessary to adjust
the extract volume to a level in excess of 1 ml.
In this case, add hexane until the meniscus is
exactly at the 1-ml mark on the concentrator tube.
Then use a 1-ml Mohr pi pet for total volumes up
to 3 ml.
For larger volumes, use a 5-ml Mohr pi pet,
carefully measuring the volume of hexane delivered.
Above the 1 ml graduation mark, the concentrator
tube calibrations are not sufficiently accurate
for use in this analysis.
-------
Revised 6/77
6.
7.
Section 5, A, (3), (a)
Page 5
It is also good practice to check the graduation
marks up to 1 ml for all concentrator tubes used
in this analysis.
Stopper the concentrator tube and hold on the Vortex mixer,
set for high speed for ca 30 seconds for volumes of 6 ml or
less. It is safer practice to mix a full minute for larger
volumes.
Proceed with electron capture GLC observing the guidelines set
forth in Section 4,A,(4).
VI. CALCULATIONS:
The following equation is applicable when all volumes
specified in the method are followed precisely, with no exceptions:
Where
Example:
ppb =
a b x
c y
X 0.6
a = nanograms of pesticide in standard peak
b = height of sample peak
c = height of standard peak
x = total volume of final extract in microliters
y = microliters of extract injected
nanograms in standard peak = 0.3
height of sample peak = 80 mm
height of standard peak = 90 mm
total volume of final extract = 1,000 yl
volume of final extract injected = 5 pi
, 0.3 x 80 x 1000 n , „ nn.
ppb = 90 x 5 = 0.6 = 32 ppb
SPECIAL NOTE:
All analytical research and subsequent collaborative study
of the method was conducted with each laboratory following the
procedure exactly as written. In all probability, a serum sample
of less than 2 ml can be analyzed with confidence, provided the
same serum to hexane ratio is followed. The precision resulting
from the use of reduced volumes is not known, however. If such
deviation must be made, the final calculation may be accomplished
by using the following basic equation:
-------
Revised 12/2/74
Section 5, A, (3), (a)
Page 6
VII.
Where a, b, and c are the same as given for the simplified
equation
d = ml (or grams) in original sample
e = dilution factor obtained as follows:
ml of hexane added to serum X final extract volume (pi)
aliquot volume of extract (ml) X pi injected ~
Example: Assuming that the same serum used to illustrate
the simplified equation was available in a volume
less than 2 ml .
nanograms in standard peak
height of sample peak
height of standard peak
ml of serum in original sample
ml of hexane added to serum
final extract volume
volume of extract aliquot
injection volume
= 1
0.3
61.5 mm
90 mm
1.6
5
,000 yl
dilution factor (e) =
5 *
T" X 0
4
5
250
ml
yl
nnh - 0.3 X 61.5 X 250 _ „ .
Ppb -- 90 x 1.6 -- 32 ppb
REPORTING LIMITS - DETECTABILITY:
The Analytical Chemistry Committee has established the
following minimum reporting limits for chlorinated pesticides
in serum:
B-BHC, lindane, aldrin, heptachlor, heptachlor epoxide,
o_,jp_'-DDE, £,p_'-DDE, dieldrin --------- 1 part per billion,
Endrin, p_,jD_'-DDT, £,£'-DDD, £,£'-DDT-2 parts per billion,
If chromatographic peaks indicate the presence of any compound in a
quantity less than the minimum reporting level, the compound shall
be reported as trace (TR).
VIII. APPLICATION OF MILLS, ONLEY, GAITHER CLEANUP TO SERUM:
Some laboratories may wish to pool sera for Florisil cleanup and
an in-depth appraisal of the pesticides present. When this is indi-
cated, the following steps are taken:
-------
Revised 12/2/74 Section 5, A, (3), (a)
Page 7
1. Measure 50 ml of serum into a 1-L sep. funnel containing 190 ml
of CH3CN, 200 ml of aqueous 2% Na2SOit and 50 ml of hexane.
2. Stopper, shake funnel vigorously 2 minutes, and allow the layers
to separate.
3. Draw off the aqueous (lower) layer into a second 1-L sep. funnel
and percolate the hexane layer through a 2-in. column of
anhydrous Na2S04 into a 500-ml Kuderna-Danish flask fitted with
a 10-ml grad., evap. concentrator tube containing one 3-mm
glass bead.
4. Add another 50-ml portion of hexane to the aqueous solution in
the second 1-L separator; stopper and shake vigorously another
2 minutes. When layers have separated, draw aqueous layer
back into the first 1-L separator and percolate the hexane layer
through the Na2SO^ into the K-D flask. Repeat the extraction
twice more resulting in a total hexane extract of 200 ml.
5. Assemble K-D evaporator and concentrate extract to ca 3 ml.
Disassemble evaporator, rinsing tube joint with a small volume
of hexane, and dilute extract to exactly 5 ml. Stopper and
shake on Vortex mixer 2 minutes.
6. From this point on, follow the procedure outlined in
Section 5,A,(1) starting with Subsection VIII, Step 1 and
following through precisely as outlined.
-------
Revised 11/1/72
Section 5, A, (3), (a)
Page 8
FIGURE 1. ROTO-RACKR Mixer, variable speed
FIGURE 2. Evaporative concentrator tube holder, 6-place, stainless steel
r - 'I'-
}-~TT""
'L LJ
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-------
Revised 12/2/74 Section 5, A, (3), (b)
Page 1
DETERMINATION OF PENTACHLOROPHENOL (RAPID METHOD)
IN BLOOD
I. INTRODUCTION:
Pentachlorophenol (PCP) is an herbicide, defoliant, and antimi-
crobic chemical used throughout the United States as a preservative
agent for many products. Pentachlorophenol seems to be present
everywhere, appearing in municipal water supplies, wells, paints,
wood and paper products, and in blood and urine of every person now
being examined. The ubiquity of human exposure to this potentially
dangerous compound has prompted concern in the field of public health.
This interest has been stimulated by several recent industrial and
public intoxications which resulted in fatalities.
The method described herein incorporates portions of a method
currently in review by Rivers, and portions from a method by Cranmer
and Freal for PCP in urine.
REFERENCES:
1. Rivers, 0. B., Gas Chromatographic Determination of
PCP in Human Blood and Urine, Bull, of Envir. Contam.
& Toxicology, Vol. 8, No. 5, 294-296, 1972.
2. Cranmer, M., and Freal, J., Gas Chromatographic Analysis
of Pentachlorophenol in Human Urine by Formation of
Alkyl Ethers, Life Sciences, Vol. 9., Part II,
pp 121-128, 1970.
II. PRINCIPLES:
A rapid method is described for the determination of PCP based
on its conversion to a methyl ether after a 2-hour extraction of the
acidified sample in benzene. EC GLC is utilized for quantisation,
comparing sample peak against peaks from known standards, similarly
methylated.
III. APPARATUS:
1. Gas chromatograph with EC detection, fitted with either or both
columns of 4% SE-30/6% QF-1 and 5% OV-210. The 1.5% OV-17/
1.95% QF-1 should not be used.
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Revised 12/2/74 Section 5, A, (3), (b)
Page 2
2. Rotary mixing device, "Roto-Rack™", Fisher Scientific Company,
No. 14-057.
3. Tubes, culture, 16 x 125 mm, fitted with screw caps, Size 15-415
with Teflon-faced rubber liners, Corning No. 9826.
4. Pipets, transfer, 2, 3 and 6 ml, Corning No. 7100 or the
equivalent.
5. Pipets, Mohr type, 0.5 ml grad. in 0.01 ml, Corning No. 7063 or
the equivalent.
6. Flasks, vol., 10 ml.
7. Centrifuge, capable of spin velocity of 2000 rpm.
8. Vortex mixer.
IV. REAGENTS AND SOLVENTS:
1. Benzene, pesticide quality.
2. Hexane, pesticide quality.
3. Methanol, pesticide quality.
4. 2, 2, 4-Trimethylpentane, pesticide quality.
5. Acid, sulfuric, cone., reag. grade.
6. N_-Methyl-N_' -nitroso-N_-nitrosoguanidine, Aldrich Chemical Co.,
Inc., Milwaukee, WI.
7. Diazomethane methylating reagent:
Add 5 ml of 20% aqueous sodium hydroxide to a 15 ml test tube.
Place a volume of hexane, in excess of that to be used and not
less than 3 ml, on the 20% sodium hydroxide solution. Slowly
add N_-methyl-N_'-nitro-N_-nitrosoguanidine reagent to the hexane
in approximately 1 mg increments until a saturated hexane-
diazomethane solution, indicated by a constant yellow color, is
obtained. The reagent is ready for use only after diazoalkane
gas is no longer evolved.
NOTE: Use extreme caution when handling the skin irritant
diazoalkane reagent since both the reagent and the
diazoalkane gases are extremely toxic, carcinogenic
and potentially explosive. Diazoalkane generation
should be carried out in a high draft hood. Use of
safety goggles and disposable gloves is desirable and
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Revised 12/2/74 Section 5, A, (3), (b)
Page 3
close adherence to manufacturers' recommendations for
storage and handling is strongly recommended. Diazoal-
kane solutions should not be pipetted by mouth. It is
suggested that diazoalkane solution be prepared fresh,
as materials resulting in interfering peaks appear
during storage. The volume prepared should not be
greatly in excess of that required. The original
hexane-diazoalkane generating solution should not be
stored in ground glass stoppered containers nor in
bottles with visible interrior etching; however, no
hazard is involved in the culture tubes containing the
PCP benzene extract plus diazoalkane. Extended
exposure to air destroys the diazoalkane reagents.
8. Pentachlorophenol, analytical standard. Available from
Reference Standards Repository at Research Triangle Park, NC.
9. Preparation of Standard Solutions.
Dissolve 10 mg of PCP in 100 ml of benzene. Dilute 1 ml of
this solution to 100 ml with hexane. The resulting stock
solution has a concentration of 1 ng/yl.
React a 1-ml aliquot of the diluted stock solution with 0.25 ml
of the diazomethane reagent as described under Methylation.
The solution resulting from the derivatization reaction contains
800 pg of PCP per pi. Larger volumes may be used but strict
adherence to this ratio of the 1 ng/yl solution to alkylating
reagent should be maintained. The working standards, in a
range of 10 to 30 pg/yl, are prepared by diluting the deriva-
tized stock with isooctane.
V. SAMPLING:
Extreme care and precautionary measures should be taken to
insure freedom of the sample of contamination. The reader is
advised to carefully review the comments offered in the SAMPLING
Subsection IV of Method 5,A,(4),(a) pertaining to urine analysis
for PCP.
VI. PROCEDURE:
Extraction
1. In a 16 x 125 culture tube, combine 2 ml of blood serum and
6 ml of benzene.
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Revised 12/2/74 Section 5, A, (3), (b)
Page 4
NOTE: Because of the widespread prevalence of PCP, a
reagent blank consisting of 2 ml of pre-extracted
distilled water (Subsection VI, 4.) should be carried
through the entire procedure along with the sample(s).
2. Add 2 drops of cone. ^SO^, seal tightly with Teflon-lined
screw cap, and rotate for 2 hours at 50 rpm on the "Roto-Rack".
NOTE: If, after the extraction period, the layers do not
separate completely, centrifuge 5 minutes at 2,000 rpm.
3. Transfer 3 ml of the benzene (upper) layer to a 10-ml vol.
flask and proceed with methylation.
Methyl ati on
1. Add 0.3 ml of the methylating reagent (IV., 7.), stopper flask
and mix on Vortex for 2 minutes.
2. Allow to stand for 20 minutes and dilute to 10 ml volume with
isooctane or hexane.
3. Make an initial injection into the gas chromatograph of 5 yl to
determine the degree of dilution that may be required to obtain
peaks within 25% of the peak height response from one of the
working standards.
VII. MISCELLANEOUS NOTES:
1. Recovery studies by the author given in Table 1 indicated
recoveries over 90% for PCP concentrations of 190 ppb and
higher. The stated lower limit of detection is 10 ppb.
2. The method outlined here is relatively simple and rapid, and
utilizes equipment most of the laboratories have on hand. In
areas where the general population is continuously exposed to
PCP (for example, in Dade County (Miami), from framing of all
dwellings), blood serum levels in excess of 100 ppb are not
uncommon.
Little or no information is available concerning the levels
prevalent in the general population of the northern tier of
states in the U. S. where exposure to PCP should be far less
than that in the sub-tropical areas. Therefore, it cannot be
predicted at this time whether general population blood in
these northern areas might contain PCP residues approaching or
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Revised 12/2/74 Section 5, A, (3), (b)
Page 5
less than the stated minimum detectability of this method.
Should this prove to be the case in any laboratory, a modifi-
cation of the method contained in this manual for PCP in urine
C5,A,(4),(a)] might prove more suitable than the method
described here. That method, using a larger initial sample,
also incorporates a partitioning for removal of a portion of the
contaminants. Furthermore, using hexane as the extracting sol-
vent, it seems probable that less extraneous materials would
be extracted.
3. Use of the 1.5% OV-17/1.95% QF-1 column is not recommended in
this determination. On this column the relative retention
value for 2,4-D, methyl ester is identical to that of PCP
(methyl ester). Therefore, if the sample should contain 2,4-D
and/or PCP and 2,4-D, resolution by GLC would not be possible.
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 contami-
nated with PCP or other materials which may cause interferences.
Glassware should be washed with dilute NaOH solution followed
by deionized water and acetone rinse. Care should be taken not
to permit contact between wooden or paper materials and glass-
ware, as peg boards and some brands of absorbent paper products
have been found to contain PCP.
5. If the recommended volumes are used, calculations are simplified
and are as follows:
PCP(in ppb) in serum = pg/yl injected times 10.
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Revised 12/15/79 Section 5, A, (3), (b)
Page 6
TABLE 1. PERCENT RECOVERY OF PCP FROM SAMPLES
FORTIFIED BEFORE EXTRACTION
Sample PCP Found, ppma PCP Added, ppm PCP Recovered,
Blood Plasma 0.19 0.50 0.67
0.65
0.68
5.00 4.58
4.70
4.70
4.70
5.0 46.9
48.5
42.0
ppm Recovery %
96
92
98
88
90
90
90
93
97
84
Mean 92b
aLimit of detectability 0.01 ppm
Standard deviation ±4.5%
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Revised 12/15/79 Section 5, A, (4), (a)
Page 1
PENTACHLOROPHENOL (PCP) AND CHLORINATED
PHENOL METABOLITES OF PCP AND HCB
I. INTRODUCTION:
Pentachlorophenol (PCP) and its sodium and copper salts are well
known wood dressings, aquatic and terrestrial herbicides, and anti-
microbials used extensively throughout the United States. PCP seems
to be present everywhere, appearing in numicipal water supplies, wells,
paints, wood, and paper products. Human exposure may occur through
several routes, including inhalation of dusts, dermal absorption of
powders and solutions, and ingestion of residues present in food and
water. Due to the aqeous solubility of PCP salts, human elimination
occurs, at least in part, through the urinary system, providing a
convenient monitor for PCP exposure.
The following method for the determination of PCP in urine
includes a hydrolysis step that gives a much higher level for bio-
logically incorporated PCP than the previous method in this section
not specifying hydrolysis. The new procedure is highly selective and
more quantitative, and allows determination of PCP at low parts per
billion levels. Also described below is the multiresidue quantisation
and confirmation of several chlorinated phenol metabolites of PCP and
the chlorinated insecticide hexachlorobenzene (HCB).
The major metabolites from an HCB feeding study were identified
as PCP, tetrachlorohydroquinone, and pentachlorothiophenol. The
major metabolite of PCP was tetrachlorohydroquinone and a minor
metabolite was tetrachloropyrocatechol. Based on these results,
pentachlorothiophenol in urine can be used as an indicator of possible
exposure to HCB: PCP exposure would be indicated by a high level of
PCP and the presence of tetrachlorohydroquinone and tetrachloro-
pyrocatechol in urine.
The analytical methods have been tested on fortified urine
samples, rat urine, human general population urine, and urine from
a worker occupationally exposed to PCP.
REFERENCES:
1. Determination of Pentachlorophenol in Urine: The Importance of
Hydrolysis, Edgerton, T. R., and Moseman, R. F., J. Agr. Food
Chem. 27, 197 (1979).
-------
Revised 12/15/79 Section 5, A, (4), (a)
Page 2
2. MUItiresidue Method for the Determination of Chlorinated Phenol
Metabolites in Urine, Edgerton, T. R., Moseman, R. F., Under,
R. E., and Wright, L. H., J. Chromatogr. 170, 331 (1979).
II. PRINCIPLE:
PCP and chlorinated phenol metabolites of PCP and HCB are ex-
tracted with benzene after acidification of urine and hydrolysis.
The phenols are methylated with diazomethane before electron capture
gas chromatography. Cleanup and separation of methylated phenols
into groups is carried out on an acid alumina column. This step is
essential for determination at low ppb levels.
Compounds are confirmed by GLC-MS.
III. APPARATUS AND REAGENTS:
1. Tracer MT-220 gas chromatograph equipped with a 63Ni pulsed
linearized mode electron capture detector, or equivalent, operated
with the parameters given in Section VIII.
2. Anhydrous, granular sodium sulfate and sodium bisulfite, Soxhlet
extracted for 4 hours with hexane and oven dried at 130°C.
3. Acid alumina, Brockmann Activity I, Fisher Scientific Co.,
dried for 24 hours at 130°C and stored in a desiccator.
4. Potassium hydroxide and hydrochloric acid, reagent grade.
5. Benzene, diethyl ether, acetone, and hexane, pesticide grade
or equivalent.
6. N-Methyl-rf-nitro-N-nitrosoguandine (diazomethane), Aldrich
Chemical Co. CAUTION! This compound is a known carcinogen.
7. Preparation of methylating reagent:
a. Dissolve 2.3 grams of potassium hydroxide in 2.3 ml of
distilled water in a 125 ml Erlenmeyer flask and cool to
room temperature.
b. Add 25 ml of diethyl ether and cool the flask in the
refrigerator.
c. In a glovebox or high draft hood, add 1.5 grams of
IVmethyl-N'-nitro-lf-nitrosoguanidine in small portions to
the flask with vigorous shaking.
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Revised 12/15/79 Section 5, A, (4), (a)
Page 3
d. Decant the ether layer into a scintillation vial and store
in a freezer.
NOTE: Use EXTREME CAUTION when handling the skin irritant
diazoalkane reagent because both the reagent and
the diazolkane gases are extremely toxic, carcino-
genic, and potentially explosive. Diazoalkane
generation should be carried out in a high draft
hood. Use of safety goggles and disposable gloves
is desirable, and close adherence to manufacturer's
recommendations for storage and handling is strongly
recommended. Diazoalkane solutions should not be
pipetted by mouth. It is suggested that diazoalkane
solution be prepared fresh, as materials resulting
in interfering peaks appear after storage. Extended
exposure to air destroys the diazoalkane reagents.
8. Pentachlorophenol (99+%), 2,3,4,6-tetrachlorophenol; 2,3,5,6-
tetrachlorophenol; pentachlorothiophenol; 2,3,4,5-tetrachloro-
phenol, Aldrich Chemical Co. tetrachloropyrocatechol, Pfaltz
and Bauer. Tetrachlorohydroquinone, K and K Laboratories.
Recrystallize pentachlorothiophenol, tetrachlorohydroquinone,
and tetrachloropyrocatechol before use.
9. Preparation of PCP and other phenol standard solutions:
a. Prepare an analytical standard of 200 yg/ml for each phenol
in hexane and store at -15°C in a brown glass bottle.
b. Pipet a volume containing 10 yg of each phenol into separate
15 ml graduated centrifuge tubes.
c. Methylate the solutions of the phenols by adding, in a
high draft hood, 5 ml of diazomethane reagent (item 7 above)
to each tube.
d. Let the phenol standards stand for 1 hour.
e. Bubble nitrogen through the individual standard solutions
to remove any excess diazomethane.
f. Dilute the solution to the proper concentration for direct
EC GLC or subject to acid alumina column cleanup before
EC GLC.
-------
Revised 12/15/79 Section 5, A, (4), (a)
Page 4
NOTES:
1. A known amount of each phenol can be methylated as
a mixture rather than reacting the individual compounds,
The mixture is also allowed to stand one hour before
EC GLC determination.
2. Make urine fortifications from acetone dilutions of
the seven mixed phenol standards.
10. Glass wool.
11. Erlenmeyer flask, 125 ml.
12. Scintillation vial.
13. Culture tube, Teflon-lined screw-cap, 20 x 125 mm.
14. Centrifuge tubes, 15 ml.
15. Mechanical rotator.
16. Pipets, disposable Pasteur.
17. Pipets, volumetric.
18. Centrifuge.
19. Apparatus for concentration of solutions by nitrogen blow-down,
including water bath operated at 30°C.
20. Chromaflex column, size 22-9, Kontes 420530.
IV. SAMPLING:
It is mandatory that extreme care be taken in the preparation of
the glass containers and caps used to hold the sample and the manner
in which the sample is taken. Pentachlorophenol is very prevalent
in the environment, to such an extent that many commonplace materials
may contain levels sufficiently high to grossly contaminate a sample.
Paper products and wood frequently contain the compound, most par-
ticularly in subtropical and tropical areas where pressure treated
lumber is widely used in construction.
All sample containers must be scrupulously prepared by first
washing, then soaking in dilute NaOH followed by rinses with deionized
water and acetone. During drying, the interiors of bottles and caps
should be protected from air dust contamination, and must not be
allowed to contact wood or paper surfaces.
-------
Revised 12/15/79 Section 5, A, (4), (a)
Page 5
All bottle caps should be Teflon- or aluminum foil-lined. Under
no circumstances should the paper liner of the bottle cap be allowed
to come in contact with the sample. Paper-lined caps may be used
only if a layer of foil or Teflon is inserted to isolate the sample
from the paper liner.
V. EXTRACTION OF URINE:
NOTE: Before starting the analysis, the chemist should make
certain that all glassware used in the analysis has
been specially prepared as described in Section XIII,1.
1. Transfer 2 ml of urine to a Teflon-lined screw cap culture tube.
2. Add 100 mg of sodium bisulfite.
NOTE: Bisulfite is added to urine samples before hydrolysis
and to urine extracts after hydrolysis to act as a
reducing agent (see Subsection IX,2).
3. Acidify with 0.5 ml of concentrated hydrochloric acid.
4. Seal the tube and place in a boiling water bath for 1 hour with
periodic shaking to achieve hydrolysis.
NOTE: A hydrolysis time of 1 hour is necessary for the
maximum freeing of conjugated PCP in urine. Further
hydrolysis does not yield additional PCP.
5. Remove the tube and cool to room temperature.
6. Add an additional 100 mg of sodium bisulfite.
7. Extract the sample with 5 ml of benzene for 1 hour on a
mechanical rotator at 30-50 rpm.
8. Centrifuge the solution and transfer the benzene layer to an
aluminum foil-wrapped 15 ml centrifuge tube with a disposable
pi pet.
NOTE: Wrapping with aluminum foil minimizes the possible
effects of photodecomposition.
9. Repeat the benzene extraction and centrifugation (steps 7 and 8)
and add the second benzene extract to the wrapped centrifuge tube.
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Revised 12/15/79 Section 5, A, (4), (a)
Page 6
NOTE: The analysis of urine cannot be interrupted before the
methylation step or recoveries of pentachlorothiophenol,
tetrachloropyrocatechol, and tetrachlorohydroquinone will
be low and erratic. This is true even with the addition
of bisulfite.
VI. METHYLATION OF PHENOLS:
1. Concentrate the combined benzene extracts to a volume of
0.3-0.5 ml under a gentle stream of nitrogen in a 30°C water bath.
NOTE: The extract is analyzed at this point, before derivati-
zation, on a 5% DEGS column, which separates the two
tetrachlorophenols (2,3,5,6 and 2,3,4,6) as the free
phenols. These two phenols, when methylated, were not
separated on any of the GC columns tested (Table 1).
2. Methyl ate the phenols with 5 ml of diazomethane reagent, prepared
as described above in Subsection 111,7.
3. Let the methylated extract stand for 1 hour.
4. Concentrate the solution to ca 0.3 ml under a gentle nitrogen
stream.
5. Add 2 ml of hexane, and reconcentrate the solution to a volume
of 0.2-0.3 ml.
VII. ACID ALUMINA COLUMN CHROMATOGRAPHY:
1. Preparation of columns:
a. Loosely plug a size 22-9 Chromaflex column with a small
amount of glass wool.
b. Add 4.0 grams of acid alumina in small increments with
tapping.
c. Add 1.6 grams of anhydrous sodium sulfate on top of the
alumina.
d. Wash the adsorbents in the packed column free of interfer-
ences with 30 ml of hexane-benzene (60:40 v/v).
e. Thoroughly air dry the column and place in an oven at 130°C
overnight before use.
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Revised 12/15/79 Section 5, A, (4), (a)
Page 7
2. Cleanup and fractionation of methylated phenols:
a. Remove a prepared column from the oven and let cool to
room temperature.
b. Add 7 ml of hexane to the column.
c. When the solvent layer reaches the top of the sodium sul-
fate adsorbent, apply an aliquot of methylated sample or
methylated standard phenol mixture in 0.2-0.3 ml to the
column with a disposable pi pet. To accomplish quantitative
transfer of samples, rinse the centrifuge tube and pipet
with three 0.5 ml volumes of hexane.
d. Add an additional 3.5 ml of hexane, and collect and discard
the total 5.0 ml hexane fraction.
e. Elute the pentachlorophenol methyl ether with 20 ml of
hexanebenzene (90:10 v/v). 2,3,4,6-Tetrachlorophenol,
2,3,5,6-tetrachlorophenol, and pentachlorothiophenol also
elute in this Fraction I, if present in the extract.
f. Elute 2,3,4,5-tetrachlorophenol, tetrachloropyrocatechol,
and tetrachlorohydroquinone, if present, with 20 ml of
hexane-benzene (60:40 v/v) (Fraction II).
g. Adjust the fractions to an appropriate volume for EC GLC.
VIII. GAS CHROMATOGRAPHY:
Inject a portion, preferably 3-5 yl, of methylated PCP solution
into the gas chromatograph operated with the following parameters:
Column borosilicate glass, 1.8 m x 4 mm i.d.
Liquid phase 5% OV-210 coated on 80-100 mesh
Gas-Chrom Q
Column Temperature 160°C
Carrier gas argon-methane (95:5 v/v) flowing at
40 ml/minute
Detector temperature 300°C
Inlet 235°C
Transfer line 220°C
_ii
Detector pulsed mode EC, 5 x 10 amp full
scale
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Revised 12/15/79 Section 5, A, (4), (a)
Page 8
Under these conditions, 10 pg of PCP methyl ether gave a half-scale
deflection with a retention of 0.49 relative to aldrin. Retention
data for the seven phenol methyl ethers on 5% OV-210 and four other
GLC columns useful for confirmation purposes are given below in
Table 1. (See the Note in Subsection VI under item 1.)
TABLE 1. RELATIVE RETENTION DATA FOR METHYLATED
METABOLITES OF HCB AND PCP
Retention time relative to Aldrin
Metabolite 4%
6%
2,3,4,6-Tetrachlorophenol
2,3,5,6-Tetrachlorophenol
2,3,4,5-Tetrachlorophenol
Pentachlorophenol
Tetrachl oropyrocatechol
Tetrachl orohydroqui none
Pentachlorothiophenol
Se-30-
OV-210
0.23
0.23
0.38
0.46
0.45
0.48
0.95
1.5% OV-17-
1.95% QFI
0.33
0.33
0.51
0.55
0.55
0.56
1.06
5% OV-210
0.24
0.24
0.46
0.49
0.52
0.59
1.00
3% OV-1
0.22
0.22
0.34
0.44
0.42
0.42
0.91
5% DEGS*
1.21
1.13
1.66
2.58
—
--
— —
* Undervitali zed
Figures 1 and 2 illustrate the GLC separation on a 5% OV-210
column of the methyl ethers of the seven phenols after separation on
the acid alumina column.
IX. DETECTION AND RECOVERY DATA:
1. Recoveries of PCP from urine at fortification levels of 5 ppb
and greater averaged 90% when corrected for background PCP
(Table 2).
TABLE 2. RECOVERY OF PCP FROM URINE9
ppm added
1.0
0.3
0.1
0.003
0.01
0.005
% range
95.2-97.8
92.3-99.0
93.0-95.0
91.9-100.4
90.6-96.3
88.0-104.0
av.%
recov.
96.5
95.3
94.1
95.1
93.2
93.9
% rel.SDb
±1.1
±2.8
±0.8
±3.7
±2.5
±7.1
aFour determinations. SD, standard deviation.
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Revised 12/15/79 Section 5, A, (4), (a)
Page 9
2. Recoveries of phenol metabolites from urine fortified at
10 ppb-1 ppm are listed in Table 3. Recoveries below 80%
were obtained only for the two lowest concentrations of
tetrachloropyrocatechol, the lowest concentration of tetra-
chlorohydroquinone, and all levels of pentachlorothiophenol.
Addition of bisulfite and prompt execution of isolation
procedures provide the maximum recoveries of these compounds.
TABLE 3. RECOVERIES OF METABOLITES FROM FORTIFIED URINE
Four determinations for each measurement.
Metabolite
2,3,5,6-Tetrachlorophenol
2,3,4,6-Tetrachlorophenol
2,3,4,5-Tetrachlorophenol
Pentachlorophenol
Tetrachloropyrocatechol
Tetrachl orohydroqui none
Pentachlorothiophenol
ppm
Added
1.0
0.3
0.1
0.03
0.01
1.0
0.3
0.1
0.03
0.01
1.0
0.3
0.1
0.03
0.01
1.0
0.3
0.1
0.03
0.01
1.0
0.3
0.1
0.03
0.01
1.0
0.3
0.1
0.03
0.01
1.0
0.3
0.1
0.03
0.01
Range (%)
89.3-92.3
85.7-92.3
82.0-87.9
78.0-85.6
79.1-87.5
88.9-92.4
86.1-91.8
83.1-88.3
80.8-84.2
79.6-86.8
89.3-95.6
89.0-94.3
86.0-91.0
85.8-90.3
82.8-90.5
95.2-97.8
91.5-95.2
86.0-9.50
93.8-100.4
90.6-96.3
78.6-81.4
78.7-85.7
76.3-83.0
59.1-71.4
60.1-69.7
80.2-82.7
77.0-84.5
77.0-84.0
75.6-86.0
71.3-77.3
69.9-73.6
69.3-73.3
61.0-73.0
49.8-57.2
41.5-51.3
Average
Recovery (%)
91.1
88.8
85.3
82.3
82.8
90.9
88.9
86.0
82.5
82.6
93.1
91.8
88.2
87.4
85.6
96.5
93.4
92.0
97.2
93.2
80.1
81.6
79.8
65.6
63.7
81.5
81.4
80.9
80.4
74.6
71.9
71.1
66.4
53.3
47.3
Relative Stand.
Deviation (%)
±1.3
±2.7
+2.5
+3.3
+3.6
±1.5
+2.4
+2.5
+1.7
+3.2
±2.7
+2.3
±2.1
+2.0
±3.4
±1.1
+1.8
±4.1
+2.8
±2.5
+1.2
±2.9
±3.2
±5.4
±4.3
±1.0
±3.2
±2.9
±4.6
+2.5
±1.5
±1.7
+6.0
+3.3
±4.2
-------
Revised 12/15/79 Section 5, A, (4), (a)
Page 10
3. Method sensitivity was estimated to be 1 ppb for PCP and the
other phenols in urine. Column cleanup was essential for
determinations of PCP at levels below 30 ppb. When methods
without cleanup were tested, recoveries of less than 80%
were noted at fortification levels of 30 ppb or less.
4. The major metabolites from an HCB rat feeding study were tetra-
chlorohydroquinone, pentachlorothiophenol, and PCP. Minor rat
urinary metabolites were 2,3,5,6-tetrachlorophenol, 2,3,4,5-
tetrachlorophenol, and tetrachloropyrocatechol. Underivatized
2,3,5,6-tetrachlorophenol was separated from 2,3,4,6-tetra-
chlorophenol on a 5% DEGS column. The major metabolite isolated
from a PCP rat feeding study was tetrachlorohydroquinone. Minor
metabolites identified were 2,3,4,6-tetrachlorophenol and
tetrachloropyrocatechol.
5. PCP was identified in ten of the eleven urine samples from the
human general population, using the described analytical method.
Levels ranged from 1 to 80 ppb (Table 4). The presence of
2,3,4,6-tetrachlorophenol in the urine can be attributed to
its presence as an impurity in preparations of PCP. The only
measurable metabolites from the general population samples were
tetrachlorohydroquinone and tetrachloropyrocatechol. The
occupationally exposed worker contained a high level of PCP and
measurable levels of tetrachlorohydroquinone and tetrachloro-
pyrocatechol. As can be seen from these results, pentachloro-
thiophenol in urine can be used as an indicator of possible
exposure to HCB. PCP exposure would be indicated by a high
level of PCP and the presence of tetrachlorohydroquinone and
tetrachloropyrocatechol in the urine.
-------
Revised 12/15/79
TABLE 4. HUMAN URINE
Results in pptn.
Section 5, A, (4), (a)
Page 11
Sample
No.
1
2
3
4
5
6
7
8
9
10
11
12*
Penta-
chloro-
phenol
0.006
0.012
0.004
<0.001
0.080
0.004
0.015
0.012
0.009
0.038
0.018
3.60
Tetra-
chloro-
hydro
quinone
<0.001
<0.001
<0.001
<0.001
0.002
-------
Revised 12/15/79
X. CONFIRMATION BY CG-MS:
Section
Page 12
5, A, (4), (a)
1
2.
Confirm analytical results on a Finnigan Model 3200 quadrupole
mass spectrometer equipped with a Model 9500 gas chromatograph
and Model 6100 data system, or equivalent, with the following
parameters:
Reagent
Mode
methane
chemical ionization
Source temperature 120°C
Pressure
Electron energy
Emission current
GC column
Liquid phase
Column temperature
Carrier gas
Inlet temperature
Transfer line
Ion source
900 ym
110 eV
10 mA
borosilicate glass, 1.2 m x 2 mm i.d.
5% OV-210 on 80-100 mesh Gas-Chrom Q
90°C isothermal for 1 minute, then
programmed at 4°C per minute to 160°C
20 ml/minute (methane)
200°C
250°C
120°C
Chemical ionization using methane reagent gas produced fairly
strong M + 1 quasi-molecular ion isotope clusters, beginning
at m/e 245 for the three isomers of tetrachlorophenol, m/e 279
for PCP, m/c 295 for pentachlorothiophenol, and m/e 275 for
tetrachloropyrocathechol and tetrachlorohydroquinone. In
addition, a fiarly strong M + 1 quasi-molecular ion isotope
cluster beginning at m/e 240 was tentatively identified as
as an isomer of trichlorodihyroxybenzene from the PCP feeding
study samples. The phenolic metabolites in the urine from the
occupationally exposed worker were confirmed by GC-MS as
tetrachloropyrocatechol and tetrachlorohydroquinone.
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Revised 12/15/79 Section 5, A, (4), (a)
Page 13
XI. CONFIRMATION OF PCP BY R-VALUE:
Equilibrate each of the following solvents, acetonitrile,
methanol, and dimethylformamide, at a 1:1 v/v ratio with hexane
at room temperature for 24 hours. Pi pet 0.1 ml of a 0.5 ml hexane
solution containing the PCP-methyl ether into a 1 ml test tube.
Add, by means of a pipet, 0.1 ml of the hexane-equilibrated solvent
and thoroughly mix the two phases by means of a Vortex mixer for
approximately one minute. After the two phases separate, the
upper hexane layer is ready for GLC analysis. The £-value is
calculated as the concentration of PCP-methyl ether in the hexane
phase divided by the concentration that was determined to be in
the hexane before the partition. Determination of £-value from
standards in each laboratory must be carried out at the same time
as the unknown, because temperature and other variables affect
the partition coefficients.
The expected ^-values for the three solvent systems are:
1. Acetonitrile:hexane 0.62
2. Methanol:hexane 0.61
3. Dimethylformamide:hexane 0.44
XII. MISCELLANEOUS NOTES:
1. Great difficulty was encountered in finding a control urine
low enough in PCP content to use for fortification purposes.
A general population human urine with an average 4 ppb PCP
background was chosen for fortification purposes.
2. A comparison of PCP levels found in human urine samples by
the method described in this section with two other procedures
(including the one in this section in the last revision of
this Methods Manual) indicated as much as a 17-fold higher
result after hydrolysis.
3. Recoveries of 0.1-5 yg PCP and six phenolic metabolites of
either HCB or PCP through the acid alumina column ranged from
88 to 97%.
-------
Revised 12/15/79
XIII. ANALYTICAL QUALITY CONTROL:
Section 5, A, (4), (a)
Page 14
1. All reagents, including water, must be extracted with hexane
before use as they may be contaminated with PCP or other
materials that may interfere with analysis. Glassware should
be washed with dilute sodium hydroxide solution, followed by
deionized water and acetone rinses. Care should be taken not
to allow contact between wooden or paper materials and glass-
ware because peg boards and several brands of absorbent paper
products have been found to contain PCP.
2. Fortified urine samples should be analyzed along with each
series of actual samples to verify adequate recovery of PCP
and the other phenols of interest. Because of the ubiquity
of PCP, the "blank" used for fortification must be analyzed
and a correction must be made for the amount of PCP found.
3. A reagent blank consisting of 5 ml of pre-extracted distilled
water should also be carried through the entire procedure
along with the sample(s).
-------
Revised 12/15/79
Section 5,A,(4),(a)
Page 15
4 6 £
TIME , minutes
10
12
Fig. 1. Gas chromatogram of Fraction I from acid alumina
column of standard phenol methyl ether mixture:
(a) 2,3,5,6- and 2,3,4,6-tetrachlorophenol (See
Note, Section VI, 1); (b) pentachlorophenol; (c)
pentachlorothiophenol. Column, 5% OV-210 on 80-100
mesh Gas-Chrom Q. Oven temperature, 160°C. 5% Methane
in argon, flow rate 40 ml/minute.
-------
Revised 12/15/79
Section 5,A,(4),(a)
Page 16
DC
CC
LU
Q
CC
O
(J
4 6
TIME, minutes
10
Fig. 2. Gas chromatograms of Fraction II from acid alumina
column of standard phenol methyl ether mixture:
(a) 2,3,4,5-tetxachlorophenol; (b) tetrachlorocatechol;
(c) tetrachlorohydroquinone. Column, 5% OV-210 on
80-100 mesh Gas-Chrom Q. Oven temperature, 160°C.
5% Methane in argon, flow rate 40 ml/minute.
-------
Revised 1/4/71 Section 5, A, (4), (b)
Page 1
DETERMINATION OF BIS(jD-CHLOROPHENYL) ACETIC ACID (DDA)
IN HUMAN URINE
I. INTRODUCTION:
The analysis of blood, urine, and feces is of extreme importance
when studying transport and elimination of £,£'-DDT and p_,pJ-DDT -
derived metabolites. The examination of urine is of particular
interest because of the ease of collection and the anticipation of
fewer analytical problems than might be encountered with blood and
feces. Furthermore, a predominant metabolite of £,£'-DDT, £,£'-DDA,
is excreted in the urine. Excretion levels of this metabolite
have been established as sensitive indicators of exposure to £,£' -DDT
(Durham et al., 1965). However, a rapid, sensitive gas chromatographic
procedure for the analysis of this metabolite is desirable, particu-
larly one which gives accurate and precise data for low levels of
£,£'-DDA excretion. The following method was developed as a dual
analytical procedure to determine DDT and its polar and non-polar
metabolites in human urine (Cranmer et al., 1969). Utilizing
electrolytic conductivity or microcoulometric detection, the
procedure can be readily adapted for the exclusive determination of
£,£'-DDA excretion levels.
REFERENCES: Cranmer, M. F., J. J. Carrol, and M. F. Cope!and
(1969) Determination of DDT and Metabolites
Including DDA in Human Urine by Gas Chromatography.
Bull. Environ. Contamin. & Toxicol. 4_, 214.
Cueto, C., A. G. Barnes, and A. M. Mattson, (1956).
Determination of DDA in Urine Using an Ion
Exchange Resin. J. Agr. Food Chem. 4_, 943.
Durham, W. F., J. F. Armstrong, and G. E. Quinby
(1965). DDA Excretion Levels, Arch. Environ.
Health 1J_, 76.
II. PRINCIPLES:
Each sample of urine is thoroughly mixed with an equal volume of
2% acetic acid in hexane. Three such extractions are performed and
the combined extracts evaporated to near dryness taking care that no
residual traces of water or acetic acid remain. The dry extract is
treated with boron trifluoride-methanol reagent to convert free
£,£'-DDA to the methyl ester. After heating at 50°C for 30 minutes,
-------
Revised 1/4/71 Section 5, A, (4), (b)
Page 2
the reaction is quenched with water and the reaction mixture is then
extracted with three 5-ml portions of hexane. The combined hexane
extracts are volume adjusted and the p_,p_' -DDA methyl ester is
determined by microcoulometric and/or EC detection. An osmolality
correction factor is employed in reporting p_,pj -DDA excretion levels.
III. EQUIPMENT:
1. Gas chromatograph equipped with EC detector and microcoulometer
if available, and with the columns prescribed for the program.
2. Vortex-Genie mixer, Model 55Q-G or the equivalent.
3. Precision Systems Osmette, Model 20Q7 or the equivalent.
4. Culture tubes, screw caps with Teflon liners, 16 x 125 mm,
Corning No. 9826.
5. Culture tubes, screw caps with Teflon liners, 20 x 150 mm,
Corning No. 9826.
6. Separatory funnels, Teflon stopcocks, 60 ml, 125 ml and 250 ml.
7. Concentrator tubes, 10 ml and 25 ml capacity, graduated with f
19/22 ground glass joint (Kontes Glass Co., Cat. No. K-570050,
size 1025 (10 ml) and size 2525 (25 ml).
8. Modified microSnyder column, f joint size 19/22, Kontes No.
K-569251.
9. Kuderna-Danish flasks, 125 ml and 250 ml, Kontes No. K-570001.
10. Glass beads, solid, 3 mm.
11. Water bath(s), controllable at temperatures of 45°, 60°and 95°to
100°C.
12. Mohr pi pets, 5- and 10-ml.
13. Disposable pipets.
14. Filter tubes, 150 x 24 mm, Corning No. 9480 or the equivalent.
15. Micro Florisil column per specifications given in Section
5,A,(2),(a), page 1.
16. Test tubes, 25 x 200, with I glass stoppers, Corning No. 9810
or the equivalent.
-------
Revised 12/2/74 Section 5, A, (4), (b)
Page 3
IV. REAGENTS:
1. Acetic acid, glacial, reag. grade.
2. Hexane
3. Acetonitrile
All four solvents of pesticide quality.
4. Toluene
5. Methanol
6. Sodium sulfate granular, anhydrous, reag. grade.
7. A mixture of 2% acetic acid in hexane.
8. A solution of 1% methanol in hexane.
9. Boron trifluoride, reag. grade, lecture bottle size, The
Matheson Company, East Rutherford, N. J.
10. £,£'-DDA analyt. standard, available from EPA, Reference Standards
Repository, Research Triangle Park, N. C.
11. Preparation of esterification reagent:
Bubble boron trifluoride rapidly into cool methanol for
1 hour, stirring continuously by mechanical stirrer and
passing a slow stream of dry nitrogen over the surface of
the methanol to continuously purge the reaction flask.
A weight increase of a ca 10% should be observed during
the course of preparation of the reagent. Consistent results
should be obtained with reagent which is stored in tightly
capped bottles in the refrigerator for periods up to 2 weeks.
NOTE: An alternative to the preparation of the
methylating reagent is to purchase the com-
mercially prepared reagent. Applied Science
Laboratories markets a "BF3 METHANOL ESTER KIT"
consisting of 25 x 5 ml ampoules of 14% BF3/Methanol.
(Nanograde methanol will be substituted on request.)
12. Preparation of DDA-ME standard:
1. Weigh out approximately 25 mg of Bis- (p_-chlorophenyl )-
acetic acid (£,£'-DDA) into 25 x 200 mm glass-stoppered
round bottom test tube. (Item 16, Subsection III)
-------
Revised 12/2/74 Section 5, A, (4), (b)
Page 4
2. Dissolve the £,g_'-DDA, with the aid of a Vortex mixer, in
10.0 ml of BCl3-Methanol 10% w/v.
3. Place in steam bath for 30 minutes.
4. Remove from steam bath and quench reaction by addition
of 10 ml ice-cold distilled water.
5. Extract three times with 10 ml portions of hexane, filtering
each extract through sodium sulfate into previously tared
concentrator tubes.
6. Concentrate to small volume after each hexane extract.
7. After last hexane extract, rinse down sidewalls of the
concentrator tube with a small amount of hexane and then
evaporate just to dryness under gentle stream of nitrogen
at room temperature.
8. Place concentrator tube in desiccator and allow to equil-
ibrate.
9. Reweigh the concentrator tube to determine the amount of
DDA-methyl ester.
1 Recovery - mg of DDA(ME) x 28° x 100
h Recovery Mg Qf DDA x 2g4 x mu
10. The DDA-methyl ester is then quantitatively transferred to
50 ml volumetric flask with nanograde hexane to prepare
the DDA-methyl ester standard of approximately 1 mg/ml.
11. This DDA-methyl ester standard may then be further diluted
to give working standard of the desired concentration.
V. SAMPLE COLLECTION AND PREPARATION:
Urine collections are made in scrupulously cleaned, screw-cap
(Teflon or foil-lined) bottles to which 1 ml of toluene has been
added as a preservative. Donors may be requested to collect their
specimens immediately after arising in the morning. Pooled, 24 hour
urine specimens may be desirable in those cases where samples are
suspected or known to have p_,p_' -DDA concentrations approaching the
lower limit of detectability. The volume of urine extracted will
vary depending on exposure classifications. For analysis of the
urine of individuals classified as "normal", 20-50 ml should be
available.
-------
Revised 6/77 Section 5, A, (4), (b)
Page 5
In those cases where known or suspected exposure to £,p_'-DDT has
occurred, 5-10 ml of urine may be sufficient. The osmolality of
each specimen is determined shortly after receipt using a Precision
Systems Osmette. Samples to be stored prior to analysis should be
kept in a refrigerator.
VI. EXTRACTION:
A control sample of urine from an unexposed donor should be
carried through the entire procedure parallel with the sample(s) being
tested.
1. Place the urine sample in the extraction vessel of appropriate
type and size for the volume of sample and add an equal volume
of 2% acetic acid in hexane.
NOTE: A 5 ml sample can be extracted in a 16 x 125 mm
culture tube with Teflon lined screw cap. A 10 ml
sample will require the 20 x 150 mm tube. Volumes
of 15 to 20 ml and 25 to 50 ml may be extracted in
sep. funnels of 60 and 125 ml, respectively.
2. Shake vigorously for 2 minutes using hand agitation for
the sep. funnels or the Vortex mixer for culture tubes.
NOTE: Some emulsion may result from the vigorous shaking.
The test tubes may be centrifuged to break the
emulsion. If emulsions persist in sep. funnel or
tubes, add a few drops of acetonitrile.
3. The extraction is repeated twice more to insure complete
extraction of pesticides into the solvent phase. The method of
conveniently handling the repetitive extractions will depend
upon the initial volume of sample and subsequent total volume
of the three combined extracts. The following options are
based on this volume factor:
a. For 5 ml urine samples the 15 ml of combined hexane
extract is collected in a 25 ml grad. evap. concentrator
tube containing one 3 mm glass bead. The extract transfer
is made with a 5 ml Mohr pi pet. The cone, tube is fitted
with a modified microSnyder column and the extract is
concentrated in a boiling water bath to ca 2 ml.
b. A urine sample of 10 ml will result in a total combined
extract volume of 30 ml. In this case, transfer each
10 ml extract into a 50 ml grad. beaker by means of a
10 ml Mohr pipet. On a 45°C bath, evaporate the solvent
under a nitrogen stream to ca 5 ml. Cool beaker, add a
-------
Revised 12/2/74 Section 5, A, (4), (b)
Page 6
pinch of anhydrous Na2SOi+ and transfer concentrate to a
25 ml evap. concentrator tube, rinsing beaker with three
portions of 4 ml each of hexane. Proceed with concentration
as outlined in step a, above.
c. For initial urine samples of 15 to 25 ml which are
extracted in sep. funnels, evaporation in Kuderna-Danish
equipment is suggested. Draw off the aqueous (lower) layer
from the first extraction into a second sep. funnel and
filter the hexane extract through a filter tube containing
a 2-in. column of anhydrous ^SO^ into a 125 ml K-D flask
fitted with a 10 ml grad. evap. concentrator tube containing
one 3 mm glass bead. Add a like volume of the acetic
acid/hexane reagent to the urine phase in the second sep.
funnel, stopper, and shake vigorously 2 minutes. After
layer separation, draw off the aqueous layer into sep.
funnel No. 1 and the hexane extract through the N32S01+ filter
into the K-D flask. Similarly, repeat the extraction a
third time, conducting the extraction in sep. funnel No. 2.
Attach a Snyder column to the K-D flask, place lower cone.
tube in a boiling water bath and reduce extract to ca 2 ml.
d. For initial urine samples of 30 to 60 ml, the extraction is
conducted identically to that outlined in step c above except
that a 250 ml K-D flask is used to accommodate the larger
volume of combined extract.
4. The 25 ml evap. concentrator tubes will require two successive
rinses of ca 3 ml each with hexane to insure removal of any residue-
containing material adhering to the sides of the tube or around the
joint. After each 3 ml rinse, applied by disposable pi pet, the
extract is further concentrated down to ca 2 ml. The self flushing
action of the K-D assemblies should take care of this problem except
for a wash of the joint between K-D flask and evap. concentrator
tube. The final concentrated extract should be ca 2 ml.
5. The final concentrated extract of ca 2 ml will be in a 10 or 25 ml
evap. concentrator tube. This is placed in a 45° water bath and
reduced just to dryness under a dry nitrogen stream.
-------
Revised 6/77 Section 5, A, (4), (b)
Page 7
VII. ESTERIFICATION:
1. Add 2.5 ml of the methylation reagent to the dry extract in
the evap. concentrator tube. Place tube in a 50°C bath and
hold for 30 minutes.
NOTE: If the 14% commercial methylating reagent is used,
the volume of reagent may be reduced to 2.0 ml.
2. Quench reaction by adding 5 ml of dist. H20.
3. Add 5 ml hexane, stopper tube, and mix on Vortex 1 minute.
Allow layers to separate.
4. With a 5 ml Mohr pipet, transfer the hexane layer to a clean
25 ml evap. concentrator tube containing one 3 mm glass bead.
5. Repeat the extraction twice more with like volumes of hexane,
combining the three 5 ml extracts in the 25 ml evap. concen-
trator tube.
6. Attach a modified micro-Snyder column and reduce the volume of
the extract to ca 3 ml in a boiling water bath.
7. Remove tube from bath, cool, rinse joint with a small volume of
hexane applied with a disposable pipet, place tube under a dry
nitrogen stream and reduce extract volume to 0.3 ml.
VIII. FLORISIL FRACTIONATION:
1. Micro Florisil columns are prepared ahead of time and held in a
130°C oven until ready for use. Detailed instructions for
preparing the column are given in Section 5,A,(2),(a), page 1.
2. Remove micro column from oven and allow to cool to room temper-
ature, then prewet column with 10 ml of hexane, discarding
eluate.
3. Proceed with the elution as described in Section 5,A,(2),(a),
Subsection V, Steps 3 through 8.
NOTES:
1. After it has been established by trial that all of
the DDA is eluted in the second fraction and none in
the first fraction, the eluate from the first fraction can
be discarded if DDA is the sole compound of interest.
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Revised 6/77 Section 5, A, (4), (b)
Page 8
2. If a laboratory is running routine determinations as
for surveillance of a special group of donors, and is
conducting gas chromatography by microcoulometric detec-
tion only, the Florisil cleanup step may be eliminated.
However, the cleanup is necessary when the detection
technique is electron capture.
IX. GAS CHROMATOGRAPHY:
1. Urine from general population donors may be expected to yield
as little as 8 ppb of DDA. A 50 ml initial sample concentrated
to a final extract volume of 300 ul would yield an approximate
DDA concentration of 1.3 nanograms per micro!iter. A 10 yl
injection should produce a quantifiable peak via EC under
normal conditions. An exploratory injection of 25 yl for MC
detection will provide the operator with information suggesting
a lesser or greater injection volume to obtain a peak height
response of 10% or more FSD.
2. Compare the peak heights of the sample _p_,p_' -DDA methyl ester
with the peak heights produced by injection of a standard
solution of jD.,£'-DDA methyl ester of known concentration.
Correct the observed concentration levels of £_,£/ -DDA in the
urine samples to an osmolality of 1000 milliosmols by
multiplying the calculated value by a correction factor, K,
given by the following expression:
K = 100°
Observed Osmolality
3. The only potential pesticide interference to the DDA, methyl
ester, peak in the second fraction eluate would be from
dielodrin on the OV-17/QF-1 column operated at the prescribed
200°C. The SE-30/QF-1 and the OV-210 columns, when operated
at their prescribed parameters, should offer no overlap
problems. The OV-210 column is particularly recommended
because of its greater responsiveness.
The urine from high exposure donors would be expected to con-
tain a small amount of £,£' -DDE as compared to the DDA levels.
When the OV-17/QF-] and SE-30/QF-1 columns are operated at
the prescribed 200°C temperature, the peaks would overlap.
Complete separation can be obtained however, by operating at
170 C. The OV-210, operated at its prescribed temp, of 175-180'
should provide complete separation of the compounds.
-------
Revised 12/2/74 Section 5, A, (4), (c)
Page 1
DETERMINATION OF 2,4-D AND 2,4,5-T IN URINE
I. INTRODUCTION:
A number of derivatives of 2,4-dichlorophenoxyacetic acid (2,4-D)
and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) are applied exten-
sively as selective herbicides in the control of terrestrial and
aquatic broadleaf 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 agricul-
tural 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 monitoring 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 products 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% OV-210.
III. EQUIPMENT:
1. Gas chromatograph with EC detector fitted with a glass column
6 ft. x 1/4 in. o.d. packed with 4% SE-30/6% OV-210. Column
and instrumental parameters are those prescribed in Section 4A.
Injection port, transfer line and detector as maintained in
normal operation.
2. Chromatographic columns, Size 22, Kontes No. 420100.
3. Boiling water or steam bath.
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Revised 12/2/74 Section 5, A, (4), (c)
Page 2
4. Distilling column (condenser), 200 mm jacket, fitted with
tight glass stopper at top, Kontes No. 286810.
5. Circulating water pump.
6. Vortex mini-mixer.
7. Evaporative concentrator tubes, grad., 25 ml I 19/22, Kontes
No. 570050.
8. Conical centrifuge tubes, conical, grad., 15 ml with 5 stoppers,
Corning No. 8084 or the equivalent.
9. Disposable pipets, Pasteur, 9-in.
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.
15. Centrifuge capable of 2,000 rpm.
IV. REAGENTS:
1. Benzene, pesticide quality.
2. Hexane, pesticide quality.
3. Hydrochloric Acid, cone., 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 yl of water and 1 gram of silica gel to
a 125 ml Erlenmeyer flask. Stopper and rotate until
the water is evenly distributed throughout 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_-nitrosoquandine, Aldrich Chemical Co.
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Revised 12/2/74 Section 5, A, (4), (c)
Page 3
6. Distilled water. All distilled water used throughout
procedure must be benzene extracted.
7. Ethylating Reagent, Preparation:
a. In a 125 ml Erlenmeyer flask, dissolve 2.3 grams of KOH,
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 min.
c. In a VERY HIGH DRAFT hood, add 1.6 grams of N.-ethyl-N.1-
nitro-N-nitrosoguam'dine 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
screw cap. This may be stored for periods up to a week
at -18°C.
NOTES:
1. Because of demonstrated carcinogenicity and toxicity,
do not allow the nitrosoguam'dine of 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 EPA Reference standards Repository at Research
Triangle Park, NC.
9. Preparation of ethylated standard mixtures;
a. Weigh 20 mg of each of the two analytical standards into
separate 100 ml vol. flasks, dissolve, and make to volume
with benzene. These concentrated stock solutions will
contain 200 ng/yl each of the two compounds.
b. Transfer aliquots from each of the concentrated stock
solutions into a single 50 ml vol. flask in the following
volumes:
2,4-D 1.0 ml 2,4,5-T — 0.5 ml
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Revised 12/2/74 Section 5, A, (4), (c)
Page 4
c. Add diazoethane dropwise with a disposable pipet until a
definite yellow color persists.
d. Allow solution to stand 15 minutes, then bubble nitrogen
through the solution until yellow color disappears
(ca 5-10 minutes). THIS OPERATION MUST BE DONE IN A HIGH
DRAFT HOOD. Dilute to volume with benzene. This is the
alkylated stock standard mixture of the following concen-
trations:
2,4-D 4 ng/yl 2,4,5-T 2 ng/yl
e. Prepare an ethylated working standard mixture of highest
usable concentration by pipetting 5 ml of the alkylated
stock mixture (d. above) into a 50 ml vol. flask and make
to volume with benzene. This will yield a dilute mixture
of the following concentrations:
Alkylated 2,4-D 400 pg/yl
Alkylated 2,4,5-T -- —200 pg/yl
Injection of 5 yl of this mix into the gas chromatograph
will provide information on the final concentration range
needed for further diluted standards.
NOTE: These alkylated standards should be stored at
-18°C when not in use and discarded after one month.
V. EXTRACTION AND ALKYLATION:
A control sample of urine from an unexposed donor should be
carried through the entire procedure parallel with the sample(s)
being tested.
1. Pipet 1 to 5 ml of urine into a 25 ml evap. cone. tube.
NOTE: The precise volume is predicated on the expected
residue level.
2. Add dropwise a volume of cone. 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 1 hour, cooling the condenser with
circulating ice water.
4. Remove from bath, cool, and rinse inside walls and condenser
tip with 3 ml benzene.
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Revised 6/77 Section 5, A, (4), (c)
Page 5
5. Mix contents of tube for 2 minutes on a Vortex set at high
speed and then centrifuge at 2,000 rpm.
6. By means of a disposable pipet, carefully transfer the benzene
(upper) layer to a 15 ml centrifuge tube taking special care
not to transfer any water.
7. Repeat the extraction with another 3 ml 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 tube to stand 15 minutes, then bubble nitrogen through
the solution to remove excess reagent.
10. Concentrate the ethyl ated extract to ca 0.3 ml at room
temperature or on a 40°C water bath under a gentle stream of
nitrogen.
VI. SILICA GEL FRACTIQNATION:
Determination of Elution Pattern
The elution pattern of the ethylated compounds must be deter-
mined 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 wad of glass wool at the bottom of a Chromaflex
column and add 1 gram of the particularly deactivated silica
gel. Top this with 1/2 in. of anhydrous, granular
b. Prewash the column with 10 ml of hexane and discard the eluate.
c. When the surface level of the hexane reaches a point on the
column ca 2 cm from the top of the NaaSO^, add 0.3 ml of the
alkylated stock standard mixture (Subsection 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 yl from each fraction into
the gas chromatograph and calculate the percent of each com-
pound present in the fraction.
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Revised 6/77 Section 5, A, (4), (c)
Page 6
A typical elution pattern is shown in the table:
Eluting Solvents 2,4-D 2,4,5-T
5 yg 2 yg
20% Benzene-Hexane 0 0
40% Benzene-Hexane 0 0
60% Benzene-Hexane 0-2% 20-25%
80% Benzene-Hexane 98-100% 75-80%
100% Benzene 0 0
Sample Fractionation
a. Prepare a chromatographic column of silica gel as described on
the previous page and prewash column with 10 ml of hexane,
exactly as described, discarding the elute.
b. Transfer the concentrated extract to the column, rinsing
centrifuge tube with two successive portions of 5 ml each
of 20% benzene/hexane, collecting the elute.
NOTE: If chlorinated phenols are present they should
elute in this fraction.
c. 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 he is able to obtain a consistent
altered pattern, some appropriate revision in the
eluate collection instructions may be indicated.
VII. GAS GHROMATOGRAPHY:
Inject into the gas chromatograph 5 to 10 pi of the 20% fraction
for the determination of the phenols and 5 to 10 yl of the combined
60-80% fraction for the determination of the chlorophenoxyacetic
acids. Injections of 5 to 10 yl can also be made from fractions
which have been concentrated to 5 ml, if necessary, in case of lower
levels of exposure. The elution pattern of the 2 compounds extracted
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Revised 6/77 Section 5, A, (4), (c)
Page 7
from a fortified urine sample must be established as described below.
The limits of detectability for 2,4-D and 2,4,5-T are 0.05 and
0.01 ppm, respectively. Quantification is conducted by mathematical
comparison of sample peaks against peaks resulting from the injection
of working standard (Subsection IV,9,e).
The retention values, relative to aldrin, of the two ethylated
compounds on the SE-30/QF-1 column at 200°C are:
2,4-D 0.51 2,4,5-T 0.78
VIII. MISCELLANEOUS NOTES:
1. Recovery runs are essential for the operator to determine 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 ml vol. flask. Dilute to volume with
benzene without ethylating. Transfer a 2 ml aliquot to a 15 ml
grad. centrifuge tube and add an equal volume of 1 N NaOH.
Mix well and allow to stand for 10 minutes, agitating from time
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.
2. 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 must be decreased (use
spiked control urine, not standard compounds to determine
pattern).
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Revised 12/15/79 Section 5, A, (5), (a)
Page 1
DETERMINATION OF KEPONE IN HUMAN BLOOD
AND ENVIRONMENTAL SAMPLES
I. INTRODUCTION:
Kepone (chlodecone) is a pesticide added to bait or other inert
material to control banana and potato pests and to serve as a
potent ant and roach killer. It is an ingredient in about 55 commer-
cial pesticide formulations used in the United States and other
countries. The following methods describe the analysis of Kepone in
human blood, air, river water, bottom sediments, and fish as carried
out by the EPA Health Effects Research Laboratory, Research Triangle
Park, NC.
REFERENCES:
1. Electron Capture Gas Chromatographic Determination of
Kepone in Environmental Samples, Moseman, R. F., Crist,
H. L., Edgerton, T. R., and Ward, M. K., Arch. Environ.
Contam. Toxicol. 6_, 221 (1977).
2. A Micro Technique for Confirmation of Trace Quantities
of Kepone, Moseman, R. F., Ward, M. K., Crist, H. L.,
and Zehr, R. D., J. Agr. Food Chem. 26_, 965 (1978).
3. Analytical Methodology for the Determination of Kepone
Residues in Fish, Shellfish, and Hi-Vol Air Filters,
Hodgson, D. W., Kantor, E. J., and Mann, J. B., Arch.
Environ. Contam. Toxicol. 1_, 99 (1978).
4. Mass Spectrometric Analyses and Characterization of Kepone
in Environmental and Human Samples, Harless, R. L., Harris,
D. E., Sovocool , 6. W., Zehr, R. D., Wilson, N. K, and
Oswald, E. 0., Biomed. Mass Spectrom. 5_(3), 232 (1978).
5. Preliminary Report on Kepone Levels in Human Blool from
the General Population of Hopewell, VA, U.S. EPA, HERL,
Research Triangle Park, NC, March 3, 1976.
II. PRINCIPLE:
Samples are extracted, and extracts are cleaned up by chroma-
tography on a micro Florisil column, base partitioning, or gel
permeation chromatography. The Kepone is determined by electron
capture gas chromatography with multiple columns. Confirmation of
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Revised 12/15/79 Section 5, A, (5), (a)
Page 2
Kepone in samples is possible by several procedures: (1) chemical
derivatization that converts Kepone to mi rex followed by further
cleanup and analysis by EC GLC; (2) use of the halogen selective
Hall conductivity GLC detector; or (3) chemical-ionization mass
spectrometry (methane reagent gas) coupled with gas chromatography.
III. SAMPLE COLLECTION:
Samples of water, sediment, soil, ice, and sludge are collected
in 1-quart (0.946 L) Mason jars previously washed and solvent treated
according to procedure given in Section 3,A of this Manual. Jar lids
should be lined with Teflon or aluminum foil. Fish samples are
wrapped in foil and frozen along with other solid samples.
All samples should be refrigerated as soon as possible after
collection and the refrigeration maintained until the start of the
analysis.
The following methods are intended for the analysis of Kepone
residues in human blood, air (sampled with Hi-Vol filters), water
(and ice), sediment, soil, sludge, fish, and shellfish.
IV. APPARATUS AND REAGENTS:
See Section 5,A,(2),(a), III for materials and reagents for the
micro Florisil cleanup method. Additional requirements follow:
1. Gas chromatograph fitted with a DC or pulsed linearized mode
electron capture detector. GLC columns, boroscilicate glass,
1.8 m x 4 mm i.d., packed with 3% OV-1 or 1.5% OV-17/1.95%.
OV-210 on 80-100 mesh silanized support, operated with specific
parameters given under Gas Chromatography, Section XII.
Criteria for high sensitivity in the GLC system, as set forth
in Section 4,A,(4), page 4 for EC detection should be carefully
noted. Alternative columns for confirmation are 4% SE-30/6%
OV-210, 5% OV-210, and 5% OV-1.
NOTE: It should be noted that on the 4% SE-30/6% OV-210
column, Kepone and p_,p_'-DDD co-elute. This may prove
troublesome if the column promotes conversion of
p_,p_'-DDT to £,p_'-DDD.
2. Culture tubes, 125 x 15 mm and 77 x 15 mm, with Teflon-lined
screw caps.
3. Chromatographic columns, Chromaflex 22-7, Kontes Glass Co.
4. Pipets, Pasteur disposable.
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Revised 12/15/79 Section 5, A, (5), (a)
Page 3
5. Mechanical rotator producing a tumbling action at ca 50 rpm.
6. Centrifuge tubes, conical, 15 ml, graduated.
7. Apparatus for evaporation of solutions held in a 60°C water
bath under a gentle stream of purified nitrogen gas.
8. Pipet, Mohr, 10 ml, graduated in 0.1 ml increments.
9. Centrifuge tube, 50 ml, graduated, with screw cap.
10. Tube, round bottom, 50 ml, with screw cap.
11. Separatory funnels, 125 ml and 1000 ml, with Teflon stopcock.
12. Soxhlet extraction apparatus, size 50 x 250 mm.
13. Erlenmeyer flask, 125 ml, glass-stoppered.
14. Food chopper, Hobard, Model 84142.
15. Dual! tissue grinder, number K-885450, Kontes Glass Co.
16. Polytron homogenizer, Brinkmann Instruments.
17. Volumetric flask, 100 ml capacity.
18. Waring Blender.
19. Son/all Omni-Mixer, Type OM.
20. Vacutainer tube, holder, and needle blood collection system.
21. 3-Ball Snyder column.
22. Analytical pesticide standards, prepared from analytical grade
Kepone, available for qualified laboratories from the Reference
Standards Repository, ETD, HERL, EPA, Research Triangle Park,
NC. Prepare stock solutions in pesticide quality benzene,
and prepare the final working solution from an intermediate
dilution with 1% methanol in benzene. The use of 1-2% tnethanol
in benzene is mandatory for all standards and samples in order
to obtain maximum electron capture GLC response.
23. Solvents, all of pesticide quality.
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Revised 12/15/79 Section 5, A, (5), (a)
Page 4
24. Sodium sulfate, reagent grade, Soxhlet extracted for 6 hours
with pesticide quality benzene or methylene chloride and oven
dried at 130°C before use.
V. ANALYSIS OF HUMAN BLOOD:
1. Collect blood samples in Vacutainer. Samples should contain
no anti-clotting agent. Prepare a homogeneous sample by
breaking up the clots with a flat-end glass rod of a diameter
nearly as great as the inside diameter of the collection tube.
2. Using an open tipped Pasteur pipet, weigh 2.0 g of blood into
a culture tube with a Teflon-lined screw cap.
NOTES:
1. Small blood clots tend to clog the tip of a normal dispos-
able pipet. For this reason, break off the tip to provide
a greater inside diameter.
2. From this point on, run spiked blood and reagent blanks
through the entire procedure in exactly the same manner
as the samples.
Add 6 ml of hexane-diethyl ether (1:1 v/v) to the tube and
cap securely.
Shake the tube on a mechanical rotator at 30-50 rpm for 30
minutes.
Centrifuge the extraction mixture at 3000 rpm for ca. 5 minutes
to eliminate emulsions.
Remove the solvent layer with a disposable pipet and transfer
to a clean 15 ml graduated centrifuge tube.
Repeat the extraction with an additional 6 ml of the hexane-
diethyl ether (1:1 v/v), combining the extracts in the 15 ml
centrifuge tube.
Evaporate the solution in the centrifuge tube to 0.20 ml under
a gentle stream of nitrogen.
Carry out Florisil column cleanup as follows:
a. Prepare and activate a micro column containing 1.6 grams
of 60-100 mesh Florisil (activated by the manufacturer at
1200 C) topped by 1.6 grams of anhydrous sodium sulfate as
described in Section 5,A,(2),(a) ,111, 1 and 2.
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Revised 12/15/79 Section 5, A, (5), (a)
Page 5
NOTE: Prewash the column with 30 ml benzene-methanol
(1:1 v/v) rather than with hexane followed by
methanol as specified in Section 5,A,(2),(a).
Let the column air dryothoroughly before overnight
oven activation at 130°C.
b. Remove the prepared column from the oven and cool. Prewet
the column with 10 ml of methanol-benzene-hexane (2:4:94
v/v) and discard the eluate.
c. When the solvent level reaches the top of the Na2S04, trans-
fer the total sample extract to the column with a disposable
pipet, rinsing the tube with three 0.5 ml portions of the
same methanol-benzene-hexane solvent and adding these to
the column with the same pipet. Begin to collect the column
effluent in a clean 15 ml centrifuge tube as soon as the
addition of the sample is begun.
NOT E: The sample aliquot should not exceed 500 mg
contained in a 0.2-0.3 ml volume.
d. Elute the column with an additional 5.5 ml of this solvent,
added to the column using a 10 ml pipet. The first 7.0 ml
collected to this point (Fraction I) is discarded. This
fraction should contain the PCBs, mirex and several
additional chlorinated pesticides, if present.
e. Add 30 ml of methanol-acetonitri1e-benzene-hexane
(1:2:4:93 v/v) and collect the effluent in a clean 50 ml
centrifuge tube. This is Fraction II, which contains the
Kepone. Dieldrin and endrin, if present in the sample, will
be partially recovered in this fraction.
NOTE: Recovery of Kepone at very low levels (5-30 ng)
through a Florisil column is only semiquantitative.
At higher amounts, recovery is at least 90%.
Concentrate the solution under a stream of nitrogen to an
appropriate volume for injection of a 5 yl sample into
the EC GLC.
NOTES:
At a screening level of 1 ppb, a 2.0 g sample would
contain 2.0 ng (assuming 100% recovery), so the in-
jected aliquot must contain a fraction of this final
solution consistent with the sensitivity of the EC GLC
system for Kepone.
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Revised 12/15/79 Section 5, A, (5), (a)
Page 6
2. Care must be taken to ensure that the final solution
contains ca. 1-2% methanol.
VI. ANALYSIS OF WATER:
1. Using a 50 ml graduated cylinder, transfer 50 ml of vigorously
shaken water sample into a 125 ml separatory funnel and add 5 ml
of pesticide grade benzene.
2. Stopper and shake the flask for 2 minutes, let the layers separate,
and drain the water (lower) layer back into the 50 ml cylinder.
3. Percolate the benzene portion through a small amount of granular
sodium sulfate into a 15 ml centrifuge tube.
4. Transfer the water in the cylinder back into the separatory funnel,
rinsing the cylinder with two portions of 2.5 ml each of benzene
and collecting these rinses in the separatory funnel.
5. Repeat Steps 2 and 3 once more and then discard the water layer.
6. Concentrate the combined benzene extract in the centrifuge tube
under a gentle stream of nitrogen at ambient temperature to a
volume appropriate for EC GLC, adjusting the final solution to
contain a concentration of 1-2% methanol.
7. If injection of the sample indicates need for cleanup of the
extract, proceed with micro Florisil column chromatography
as described above for the analysis of blood. Base partitioning
was found unnecessary in our laboratory for the water samples
analyzed. If it should be considered necessary as an adjunct
to the micro Florisil chromatography, it would be carried out
at this point as follows:
a. Evaporate the sample extract just to dryness under a
gentle stream of nitrogen.
b. Add 10 ml of hexane and 10 ml of 5% aqueous sodium hydroxide
solution to the tube containing the sample.
c. Vortex mix the sample for about 30 seconds and let the phases
separate.
d. Discard the hexane layer, and extract the aqueous alkali
solution with at least two 10 ml portions of diethyl ether or
until the aqueous phase remains clear.
e. Transfer each ether extract with a disposable pipet and com-
bine in a 50 ml centrifuge tube.
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Revised 12/15/79 Section 5, A, (5), (a)
Page 7
f. Evaporate the ether just to dryness under a stream of
nitrogen, and dissolve the residue in an appropriate
amount of benzene containing 1-2% methanol.
g. Determine Kepone by injection of 5 yl of solution into the
EC GLC.
NOTES:
1. Tests with spiked samples indicate that recoveries of
Kepone by the sodium hydroxide partitioning method
approximate 90%.
2. Potassium hydroxide cannot be substituted for sodium
hydroxide.
3. Standards and samples stored in solutions containing
methanol require hydrolysis of the extract with 2 ml
of 2 N hydrochloric acid at 80°C for 1 hour before base
partitioning in order to minimize losses of Kepone par-
titioned into hexane.
VII. ANALYSIS OF SEDIMENT. SOIL, AND SLUDGE:
1. After thawing, mix the sample well and air dry on a large
watch glass.
2. Soxhlet extract 20 grams of air dried (24-48 hours) sample for
16-18 hours with 300 ml of methanol-benzene (1:1 v/v) solvent.
3. Attach a 3-ball Snyder column to the boiling flask and reduce
the volume of extract to ca 75 ml.
4. Quantitatively transfer the extract to a 100 ml volumetric flask
and dilute to volume with benzene.
NOTE: Samples with Kepone levels below 0.5 ppm usually
require cleanup by Florisil column chromatography
(Subsection V,9) and/or by base partitioning (Sub-
section VI,7).
VIII. ANALYSIS OF AIR:
1. Remove a portion (ca. 60 mm x 60 mm) of the Hi-Vol sample paper,
representing ca. 1/12 of the total collected sample.
2. Extract by shaking in a 125 ml Erlenmeyer flask for 5 minutes
with 100 ml of methanol-benzene (1:1 v/v). A 50 ml screw cap
centrifuge tube with 50 ml of solvent may be used as an alterna-
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Revised 12/15/79 Section 5, A, (5), (a)
Page 8
tive for the extraction.
3. Filter the sample extract through Whatman No. 1 filter paper in
a Buchner funnel to remove the glass fiber.
4. Concentrate the extract to an appropriate volume for EC GLC.
If the GLC scan indicates a high level of background interference,
it will be necessary to conduct a micro Florisil cleanup
(Subsection V,9), particularly if the Kepone level is below
1 ng per cubic meter of air.
IX. ANALYSIS OF FINFISH:
1. Remove the entrails of fish and handle as a separate sample by
the technique given below. Prepare a homogeneous fish tissue
sample with a Hobart food chopper. If necessary, store the
fish tissue in a freezer at -10°C until the time of analysis.
2. Grind a 25 gram subsample in a 500 ml mortar with sufficient
anhydrous sodium sulfate (ca. 100 grams) to dry the sample.
3. Transfer the sample to a pre-extracted thimble and Soxhlet
extract with 300 ml of diethyl ether-petroleum ether (1:1 v/v)
for 12-16 hours.
4. Replace the extractor tube with a Snyder column, and concentrate
the extract to approximately 50 ml using the same heating mantle
as during the extraction.
5. Transfer the concentrated extract to a 100 ml volumetric flask
using benzene-methanol (99:1 v/v).
6. Make a screening injection into the gas chromatograph to determine
if micro Florisil column cleanup is necessary. If so, proceed
as follows:
a. Prepare Florisil columns as described in Subsection V,9,
a and b.
b. Substitute 10 ml of petroleum ether for methanol-benzene-
hexane (2:4:94 v/v) as the wash solvent.
c. Apply 0.5 ml of sample solution to the column, equivalent
to 500 mg of original sample.
d. Rinse the sample tube with two 0.5 ml portions of Solvent I
(25 ml of petroleum ether).
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Revised 12/15/79 Section 5, A, (5), (a)
Page 9
e. Add these two rinsings and the rest of Solvent I to the
column and collect as Fraction I in a 50 ml centrifuge tube.
f. Elute the column with 40 ml of Solvent II (methanolacetoni-
trile-benzene-hexane, 1:2:4:93 v/v) and collect in a 100 ml
centrifuge tube. This fraction should contain ca. 80% or
more of the Kepone.
g. Adjust Fraction II to an appropriate volume by nitrogen
blowdown or steam temperature solvent evaporation, being
sure that the final injection solvent consists of at least
1% methanol in benzene.
NOTES:
1. An alternate extraction procedure consists of extrac-
tion of a homogenate with 25 ml of toluene-ethyl
acetate (1:3 v/v) using a Polytron tissue homogenizer.
2. Additional or alternative cleanup procedures for fish
extracts can include base partitioning and/or gel
permeation chromatography.
X. ANALYSIS OF FINFISH LIVERS AND ENTRAILS:
1. Homogenize large samples in a Sorvall Omni-Mixer at high speed
with acetonitrile for ca 2 minutes. When only a small amount
of sample is available, macerate the sample (ca 500 mg) in a
motor driven Duall tissue grinder with 2.5 ml of acetonitrile.
2. Separate the macerated tissue from the solvent by centrifugation.
3. Remove the solvent by pipet and place in a 50 ml centrifuge tube.
4. Repeat the maceration/extraction twice, combining the extracts in
the same centrifuge tube.
5. Mix the combined extract with 25 ml of 2% aqueous Na2SO[+
and partition against 5 ml of benzene.
6. Repeat the extraction twice more with 2 ml portions of benzene.
7. Combine the three benzene extracts in a 10 ml concentrator tube
and evaporate to 0.1 ml with a gentle stream of nitrogen.
8. Wash the walls of the tube with an additional 0.5 ml of benzene
and concentrate by the same procedure to 0.1 ml.
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Revised 12/15/79 Section 5, A, (5), (a)
Page 10
9. If a screening injection into the gas chromatograph indicates
the need for micro Florisil column cleanup, follow Subsection IX,
6, a-g, exactly.
XI. ANALYSIS OF SHELLFISH:
1. Approximately six clams or oysters are thawed, shucked, and
drained. Homogenize the composited meat sample in a blender at
high speed for 2 or 3 minutes. If the sample is to be stored
before analysis, transfer to a glass container and place in a
freezer at -10°C.
2. Blend a 10-30 gram subsample of the homogenate with 50 ml of
acetonitrile at high speed in a blender.
3. Vacuum filter the extract through sharkskin paper, without
making any attempts to remove the solid particulate matter on
the sides of the blender.
4. Scrape the sides of the blender jar with a spatula, allowing
the solid material to fall to the bottom of the jar.
5. Perform a second extraction with an additional 25 ml of
acetonitrile and pass the extract through the same filter.
6. Transfer the combined extracts to a 1 L separatory funnel
containing 300 ml of distilled water, 5 ml of aqueous
saturated Na2S04, and 50 ml of benzene, and shake for 2 minutes.
7. Discard the aqueous layer and wash the benzene layer twice with
50 ml of distilled water.
8. Pass the extract through a funnel containing ca 5 grams of
anhydrous Na2SO^, concentrate by nitrogen blowdown, and make
an exploratory injection into the gas chromatograph.
9. If micro Florisil column cleanup is indicated, follow Subsection
IX,6 with 25 ml of petroleum ether as Solvent I, 12 ml of ben-
zene followed by 12 ml of methanol-benzene (1:9 v/v) as
Solvent II, and 24 ml of methanol-benzene (9:9 v/v) as Solvent
III. Fractions II and III contain the eluted Kepone from the
column.
10. Fractions II and III are analyzed separately. If Fraction III
contains more Kepone than Fraction II, it is possible that more
Kepone can be recovered from the column by another elution with
Solvent III. If time is crucial, Fractions II and III can be
combined and analyzed as one fraction. Figure 1 illustrates
chromatograms of the various micro Florisil column fractions
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Revised 12/15/79 Section 5, A, (5), (a)
Page 11
from a shellfish sample. A sample co-extractive having the same
retention time as Kepone can be seen in the Fraction I
chromatogram (A).
XII. GAS CHROMATOGRAPHY:
1. The parameters of carrier gas flow rate and column temperature
depend on the column selected. If one assumes that a column
of 1.5% OV-17/1.95% OV-210 is used, typical parameters would
be as follows:
Column temperature 200°C - 210°C
Nitrogen flow rate 60-80 ml/minute
Tritium EC detector 210-215°C, DC mode at 80-85%
standing current, linear range up to
100 pg of Kepone injected
Ni detector 250°C, DC mode at 90% standing
current, linear range up to 100 pg
of Kepone injected
63Ni linearized 1 na, argon containing 5% methane
detector carrier gas flow rate 80 ml/minute,
purge gas 10-20 ml/minute, linear
response up to 500 pg of Kepone
injected
Inlet 235°C
Transfer line 220°C
2. Determine the Kepone concentration at an appropriate quantitative
screening level (e.g., 5 ppb for blood) with a suitable column
and electron capture detection. All samples and standards should
contain 1-2% methanol to obtain maximum EC response.
NOTE: Methanol has been found to enhance and stabilize the
electron capture gas chromatographic response of Kepone
dissolved in nonpolar solvents. The response reached a
maximum at approximately 1-2% methanol content; an
increased percentage of methanol did not elevate the
response. This was apparently due to the formation of
the corresponding hemiketal, which is less likely to
adhere to surfaces in a microliter syringe. It was
found that Kepone could not be removed from a syringe
when injected in hexane or benzene containing no methanol.
Solvents which allowed maximum response without the
-------
Revised 12/15/79 Section 5, A, (5), (a)
Page 12
presence of methanol were: acetone, ethyl acetate,
and toluene-ethyl acetate (1:3 v/v).
3. Confirm the presence of Kepone in all samples above this level
on both OV-17/OV-210 and the OV-1 GLC columns or other alterna-
tive, equivalent columns. The relative retention times of
Kepone on several liquid phases are listed in the following
table:
Oven temp. Carrier flow
Liquid phase RRT* (C) rate (minimum)
3% OV-1
5% QF-1
5% OV-210
4% SE-30/6% OV-210
1.5% OV-1 7/1. 95% OV-210
2.91
2.81
2.65
2.61
2.58
2.85
180
200
180
180
200
200
70
70
45
60
70
60
*relative to aldrin
4. Samples containing 5-50 ppb levels of Kepone may be further
confirmed by chemical derivatization and are treated as
described in Subsection XII.
5. Additional confirmation can be obtained by use of the more
specific Hall conductivity GLC detector system and GLC-MS in the
chemical iom'zation mode with continuous monitoring of four
ions in the quasi molecular region (Miscellaneous Notes 1
and 2).
XIII. RECOVERY AND RESPONSE:
1. The choice of solvents is critical in the extraction, cleanup,
and analysis for Kepone. Also, unless great care is taken to
exclude water from solvents, the compound exists in solution
as the hydrate. Aliphatic hydrocarbon solvents such as hexane
are poor solvents for this pesticide; methanol, acetone, and
benzene are better solvents. At ambient temperature methanol
-------
Revised 12/15/79 Section 5, A, (5), (a)
Page 13
apparently reacts with Kepone to form the hemiketal. Combined
gas chromatography-mass spectrometry (GLC-MS) indicates that
the hydrate and hemiketal forms revert to Kepone in the
injection port of a gas chromatograph at temperatures above 200°C.
Due to the polar nature of Kepone, it was difficult to remove
it from substrate using nonpolar extraction solvents such as
petroleum ether or hexane. In general, the most effective
extraction solvent systems were me'thanol-benzene (1:1 v/v)
and toluene-ethyl acetate (1:3 v/v).
2. Kepone does not quantitatively elute from some of the Florisil
columns used in the standard multiresidue methods. For example,
the Mills, Onley, Gaither macro Florisil system (Subsection
5,A,1) only partially elutes Kepone (< 8% recovery). A further
problem with this system is that the Kepone elutes in both the
15 and 50% fractions. The recovery of Kepone through the micro
Florisil column system listed for chlorinated pesticides in
human or animal tissue and human milk (Subsection 5,A,2) is
not quantitative. Minor modification of the solvent system
(replacement of hexane by benzene) allows for good separation
with good quantitative recovery. The solvent systems presented
in these methods allow the best possible removal of co-extractives
from extracts, while permitting acceptable recoveries of Kepone.
3. The limit of quantification of the method for Kepone was ca 5 ppb
in blood, 40 ppt in water, 0.1 ng/m3 in air, and 10 ppb in
sediment, soil, sludge, fish, and shellfish.
4. The completeness of extraction of Kepone from all sample types
was validated by exhaustive extraction of the sample with
additional solvent systems and by comparison with spiked standard
reference samples for the specific substrate. Recovery of Kepone
from blood at 5-10 ppb was generally greater than 85%. At
Kepone levels below 5 ppb, it is very difficult to assign a
quantitative value with any degree of confidence. This uncertain-
ty is caused by interferences from co-extractives and a greater
variability in recovery at lower levels. For these reasons, the
lower practical limit of reporting for blood is 5 ppb, and
samples below this level are reported as nondetectable.
5. Recovery of Kepone from spiked water samples was generally 90%
or greater.
6. The recovery of Kepone from sediments and soil exceeded 90%.
Additional extraction of soil and sediment samples after acidifi-
cation yielded no significant (< 1%) increase in Kepone.
-------
Revised 12/15/79 Section 5, A, (5), (a)
Page 14
7. Typical extraction efficiency of Hi-Vol air filters spiked with
a total of 1 yg of Kepone was greater than 90%.
8. Extraction and cleanup efficiencies for Kepone in fish were in
the range of 85-95%, depending on the specific method variation.
Recoveries of Kepone standards and spiked fish samples, using
the gel permeation cleanup method, were greater than 90%.
9. Typical overall recovery after extraction and cleanup of shell-
fish spiked at 0.6-0.8 ppm was approximately 80%.
XIV. CONFIRMATION DERIVATIZATION:
The procedure for qualitative confirmation of Kepone by chemical
derivatization is as follows:
1. Transfer the cleaned up sample extract (Florisil column
Fraction II) to a 16 mm x 77 mm screw-cap culture tube.
2. Evaporate the solvent just to dryness under a gentle nitrogen
stream.
3. Add ca 200 mg of anhydrous, reagent grade phosphorus pentachloride,
50 mg of anhydrous, reagent grade aluminum chloride, and 3.0 ml
of carbon tetrachloride to the tube, close with a Teflon-lined
screw cap, and place in a heating block at 145°C for 3 hours.
NOTES:
1. At this point, reagent blanks and Kepone standards in
approximately the same amount as is expected in the
sample extracts should be derivatized in parallel with
samples.
2. The presence of aluminum chloride in the reaction mixture
improves the completeness and reproducibility of the
derivative formation. The mode of action of aluminum
chloride is not yet known.
3. Remove the tube, cool to room temperature, and open.
4. Add 3 ml of distilled water, reclose the tube, and
shake for 2 minutes.
5. After phase separation, transfer 2.0 ml of the lower
carbon tetrachloride layer to a clean 5 ml graduated
centrifuge tube, using a disposable pipet.
-------
Revised 12/15/79 Section 5, A, (5), (a)
Page 15
6. Evaporate the solvent in the 5 ml tube just to dryness under
a stream of nitrogen.
7. Using a disposable pipet, transfer the residue to a micro
Florisil column (Subsection V,9) with the aid of three 0.5 ml
portions of hexane.
NOTE: Florisil cleanup is necessary when the total amount of
Kepone is less than 25 ng. When more than 200 ng is
present, the derivatized extract can be analyzed without
Florisil cleanup. In this case, take special care to
evaporate all the carbon tetrachloride before dissolving
the residue in hexane for injection into the electron
capture gas chromatograph.
8. Elute the mi rex from the column with an additional 8.5 ml
of hexane, collecting the total effluent in a 15 ml centrifuge
tube.
NOTE: Any mirex present in the original sample would have been
eluted in the first (discarded) Florisil fraction of
the earlier cleanup (Subsection V,9). This ensures
that mirex recovered in this step was formed from
conversion of Kepone.
9. Concentrate or dilute the eluate so that a 5 yl injection
into the EC GLC, operated under the conditions in Section
XII, gives a mirex peak within the predetermined linear
range of the electron capture detector. Note that only 2/3
of the derivatized sample extract was taken in Step 5 to be
carried through the micro Florisil column cleanup.
NOTES:
1. The overall average percent conversion of 10-1000 ng
amounts of Kepone derivatized in quadruplicate was 104%,
with a relative standard deviation of 8.2%.
2. Derivatization of other common chlorinated hydrocarbon
pesticides produced no interfering GLC peaks at the
retention time of mirex.
XV. MISCELLANEOUS NOTES:
1. The Hall micro electrolytic conductivity detector (Section 4,C)
is operated in the oxidative chlorine mode with a furnace
temperature of 830°C (nickel reaction tube), reaction gas (air)
flow rate of 100 ml/minute, helium flow rate of 65 ml/minute,
-------
Revised 12/15/79 Section 5, A, (5), (a)
Page 16
and methanol conductivity solvent flowing at 0.4-0.6 ml/minute.
The GLC column is 1.8 mm x 2 mm glass, containing 3% OV-101
and operated at 200°C.
2. For coupled GLC-MS, a Finnigan model 3200 GLC/MS EI-CI system
with model 6000 data control system was employed in the multiple
ion detection mode (MID), monitoring ions with m/e - 488.7,
490.7, 492.7, and 494.7. The operating conditions were:
methane reagent carrier gas, 1000 p ion source pressure, 80°C
ionizer temperature, and 70-100 eV. A 1.8 m x 2 mm glass
GLC column containing 3% OV-1 was operated at 210°C with an
injection temperature of 240°C. Simultaneous monitoring of
the four ions ar,d the characteristic GLC retention time of
Kepone adds substantial confidence to the identification of
residues.
3. The derivatization procedure converts Kepone to mirex. The
analyst should, therefore, be certain that no mirex was carried
through the cleanup steps before derivatization. The micro
Florisil column should elute any mirex present in the first
(discarded) fraction. The absence of mirex in the Fraction II
eluate can be easily established by permitting sufficient
development of chromatograms to elute mirex.
4. The Autoprep 1001 GPC system (Section 5,B) was used for the
additional removal of lipids from fish tissue extracts with
the following parameters before Florisil column chromatography.
Column 230 mm x 25 mm i.d., packed with
200-400 mesh Bio-Beads SX-3
Solvent toluene-ethyl acetate (1:3 v/v)
Pumping rate 3.5 ml/minute
Discard volume 0-72 ml
Collect volume 73-113 ml
Wash volume 114-154 ml
Recovery of Kepone standards through GPC averaged about 95%.
Recovery of Kepone from actual fish samples at 0.1 ppm levels
ranged from 80-86% after GPC plus Florisil column cleanup.
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Revised 12/15/79 Section 5, A, (5), (a)
Page 17
XVI. ANALYTICAL QUALITY CONTROL:
1. Sufficient control and spiked standard reference materials should
be used to assure the validity of analytical results.
2. Elution patterns for Florisil columns should be carefully
established by each analyst. These can vary appreciably in
different areas and even to some extent between analysts in
the same laboratory.
3. Analytical standards should be validated by cross-reference
analysis of additional preparations of analytical grade Kepone
with agreement within ±5% of the established purity.
-------
Revised 12/15/79
Section 5,A, (5),(a)
Page 18
LU
cn
2
O
CL
CO
LU
cr
cr
LU
Q
cr
o
o
LJ
or
10 15 20
TIME, minutes
I
25
30
Figure 1. Chronatogram of shellfish micro Florisil fractions: A.
Fraction I (no Kepone elution in this fraction); B. Fraction
II; and C. Fraction III. Injection of 100 yg equivalent of
shellfish. Column: 3% OV-1, nitrogen flow rate: 60 ml/minute.
-------
Revised 12/15/79 Section 5, B
Page 1
CLEANUP BY GEL PERMEATION CHROMATOGRAPHY (GPC)
I. INTRODUCTION:
Gel permeation chromatography is useful for the cleanup of
biological extracts for residue analyses of low molecular weight
organic compounds and pesticides. Large molecules are excluded
from the pores of the gel and elute first. Later fractions con-
taining low molecular weight organic compounds of interest can be
collected and analyzed by analytical chromatography, such as GLC or
HPLC. The self-regeneration characteristics of GPC allows sequential
sample analyses on the same column and consequently automation.
GPC adds another dimension to cleanup by using molecular size instead
of partition and adsorption coefficients, which in some cases may
not be favorable.
The original gel permeation chromatography (GPC) system using
BioBeads SX-2 crosslinked polystyrene gel (EPA Pesticide AQC Manual,
Section 7,M) has undergone extensive revision by the authors and other
investigators. The automated system has been commercially available
from Analytical Biochemistry Laboratories, Inc., Columbia, MO,
since 1974 (Figure 1).
The first GPC applications involved the separation of fish lipid
and nonionic chlorinated hydrocarbons from fish tissue desiccated
with anhydrous sodium sulfate and extracted with 6% diethyl ether
and petroleum ether. Mobile phases composed of toluene-ethyl acetate
(1:3 v/v), ethyl acetate, methylene chloride, and mixtures of methyl-
ene chloride-cyclohexane containing from 5 to 50% methylene chloride
have been found useful for cleanup of pesticides and other organic
pollutants from plant and animal tissue extracts. Recently,
Biobeads SX-3 has been used by some workers in place of SX-2.
Biobeads SX-3 eluted with toluene-ethyl acetate (1:3 v/v) has
been successful in removing lipid interferences from fish extracts
for subsequent electron capture GLC analysis of Kepone. If PCB,
dieldrin, or endrin interferences were observed by EC GLC, the
extract was sufficiently lipid free to allow reproducible liquid
adsorption column chromatography to isolate Kepone (Section 5,A,(5),
(a)). A similar system was described by Fehringer for the analysis
of polybrominated biphenyl residues in dairy products.
Hopper and Hughes found that methylene chloride-cyclohexane
(10:90 v/v) resulted in increased resolution of lipids and pesticides
observed in a total diet survey composed of fatty products such as
-------
Revised 12/15/79 Section 5, B
Page 2
butter, mayonnaise, shortening, and margarine. Pesticides
monitored included nonionic chlorinated hydrocarbons, organo-
phosphates, and carbamates. Kuehl and Leonard, from the U. S.
EPA, Duluth, Minnesota Environmental Research Laboratory, investi-
gated various solvent mixtures of methylene chloride and cyclo-
hexane ranging from 100% methylene chloride to 10% methylene
chloride-cyclohexane. The 50:50 v/v mixture of methylene chloride-
cyclohexane gave the best resolution of lipids and polar-nonpolar
low molecular weight organics for gas-liquid chromatographic-mass
spectroscopic analyses. Recoveries were demonstrated on 27 compounds,
including Aroclors, HCB, DDT, and PCP. Fifty-seven compounds
were identified in fish tissue by fractionation and GLC MS analysis.
Crist and Moseman (Sections 4,C,(3) and 12,A) used gel
permeation chromatography for additional cleanup of certain human
biological extracts having an adverse effect on the performance of
the Hall detector due to excessive lipid material.
Investigation by Shofield et^ al_. revealed the cleanup capability
of GPC used in conjunction with crop, vegetable, and fruit extracts
for organophosphates. Satisfactory recoveries were observed for
parent compounds as well as their oxidized analogs. The retention
volumes of alkyl and aryl organophosphates could be decreased by
increasing the percentage of methylene chloride in cyclohexane,
Leight et^ aj_. reported the applicability of GPC to cleanup or
organophosphate, triazine, and carbamate pesticide residues.
Pflugmacher and Ebing have done extensive elution profile character-
ization on various gels and elution solvents and have typified
106 pesticides including organophosphates, carbamates, thioureas,
triazines, phenoxy acids, and phenoxy acid esters. Fujie and
Fullmer determined 0.05 ppm residues of a new synthetic pyrethroid
insecticide in samples containing oil and lipid by EC GLC after
GPC and Florisil column cleanup.
REFERENCES:
1. Procedure for the Analysis of Nonionic Chrlorinated Pesticides
in the Lipid of Poultry, Swine, Beef, Soybeans and Corn Prepared
for Gas Chromatographic Analysis by Gel Permeation Chromatog-
raphy, Brookhart, G., Johnson, L. D., and Waltz, R. H., Abstracts
of FACSS, 3rd Annual Meeting, p. 207 (November 15-19, 1976).
2. Effects of Graded Levels of Toxaphene on Poultry Residue
Accumulation, Egg Production, Shell Quality and Hatchability
in White Leghorns, Bush, P. B., Kiher, J. T., Page, R. K.,
Booth, N. H., and Fletcher, 0. J., J. Agr. Food Chem., 25_,
928-932 (1977).
-------
Revised 12/15/79 Section 5, B
Page 3
3. Determination of Polybrominated Biphenyl Residues in Dairy
Products, Fehringer, N. V., J. Assoc. Off. Anal. Chem., 58,
978-982 (1975).
4. Determination of FMC 33297 Residues in Plant, Animal, and
Soil Matrices by Gas Chromatography, Fujie, G. H., and
Fullmer, 0. H., J. Agr. Food Chem., 26>, 295-398 (1978).
5. Gel Permeation Chromatographic System; An Evaluation,
Griffitt, K., and Craun, J., J. Assoc. Off. Anal. Chem., 57_,
168 (1974).
6 An Improved GPC System for Pesticides in Fats, Hopper, M. L.,
and Hughes, D. D., FDA Kansas City Field Office, Total Diet
Laboratory, Information Bulletin No. 1958, pp. 1-6 (May, 1976).
7 Kepone Analysis and Other Applications Using Automated Gel
Permeation Chromatography Cleanup, Johnson, L. D., Shofield,
C. M., and Waltz, R. H., presented at Third Annual Symposium
of the Analytical Instrumentation Discussion Group and the
American Chemical Society, Central Arkansas Section (April 29-30,
1976).
8. Automated Gel Permeation Chromatographic Cleanup of Animal and
Plant Extracts for Pesticide Residue Determination, Johnson,
L. D., Waltz, R. H., Ussary, J. P., and Kaiser, F. E., J. Assoc.
Off. Anal. Chem. 59_, 174-187 (1976).
9. Evaluation of Gel Permeation Chromatography for the Separation
of Carbamate Pesticide Residues from Vegetable Extractives,
Krause, R., presented at the 89th Annual Meeting of the
Association of Official Analytical Chemists, Washington, DC
(October 13-16, 1975).
10. Isolation of Xenobiotic Chemicals from Tissue Samples by Gel
Permeation Chromatography, Kuehl, D. W., and Leonard, E. N. ,
Anal. Chem., 5£, 182 (1978).
11. Isolation and Identification of Polychlorinated Styrenes in
Great Lakes Fish, Kuehl, D., Hopperman, H., Vieth, G., and
Glass, G., Bull. Environ. Contam. Toxicol., 1_6_, 127-132 (1976).
12. Methylene Chloride/Chclohexane Solvent System Applied to
Automated Gel Permeation Cleanup of Residue Samples for
Organophosphate, Triazine, and Carbamate Pesticides, Leicht, R.,
Schofield, C. M., Johnson, L. D., and Waltz, R. H., Analytical
Biochemistry Laboratories, Inc., Applications Report.
-------
Revised 12/15/79 Section 5, B
Page 4
13. Evaluation of the Elution Behavior of Some Classes of Pesti-
cides in Gel Chromatography, Pflugmacher, J., and Ebing, W. J.,
Chromatogr., 151, 171-197 (1978).
14. Cleanup of Vegetable, Straw, and Forage Plant Samples for
Organophosphate Residue Analysis Utilizing Methylene Chloride/
Cyclohexane Solvent System with Automated Gel Permeation
Chromatography, Shofield, C. M., Johnson, L. D., Ault, J. A.,
and Waltz, R. H., presented at the 29th Pittsburgh Conference,
Cleveland, OH (February 27-March 3, 1979).
15. Automated Gel Permeation - Carbon Chromatographic Cleanup of
Dioxins, PCPs, Pesticides and Industrial Chemicals, Stalling,
D., Johnson, J., and Huckens, J., Environmental Quality and
Safety, Supplement Volume III, lectures held at the IUPAC
Third International Congress of Pesticide Chemistry, Helsinki
(July 1-9, 1974).
16. Approaches to Comprehensive Analysis of Persistent Halogenated
Environmental Contaminants, Stalling, D., Smith, L., and
Petty, J., presented at ASTM Committee D-19 Symposium on
Measurement of Organic Pollutants in Water and Wastewater,
Denver, CO (June 19-20, 1978).
17. Cleanup of Pesticide and Polychlorinated Biphenyl Residues in
Fish Extracts by Gel Permeation Chromatography, Stalling, D.,
Tindle, R., and Johnson, J., J. assoc. Off. Anal. Chem., 55,
32-38 (1972).
18. Apparatus for Automated Gel Permeation Cleanup for Pesticide
Residue Analysis, Tindle, R., and Stalling, D., Anal. Chem.,
44, 1768-1773 (1972).
II. ELUTION PATTERNS AND RECOVERY DATA FOR PESTICIDES:
The data in the following table consists of elution profiles
for some common pesticides from the AutoPrep 1001 gel permeation
chromatograph. All data are from a 50 gram, 30 x 2.5 cm column of
BioBeads SX-3 gel, with a flow rate of approximately 5.0 ml/minute
unless otherwise noted.
The elution profiles were compiled by personnel at Analytical
Biochemistry Laboratories, Inc. To be certain of maximum recovery
and accurate quantitation of samples, analysts should calibrate
their particular columns under local conditions with standards of
the pesticides of interest.
-------
Revised 12/15/79 Section 5, B
Page 5
The following symbols are used throughout the Table:
( ) - Method of detection:
1. GLC/Electron Capture
2. GLC/Sulfur-Phosphorus Emission Detector
3. High Pressure Liquid Chromatography/UV Detector
4. GLC/Alkali Flame lonization Detector
* - Not fractionated but recovered quantitatively within
these parameters
** - GPC column - 30 grams, 2.5 x 17.5 cm column of
BioBeads SX-3
N.T.- Not Tested
-------
Revised 12/15/79
Nom'onic Chlorinated Compounds
1. Aldrin [1]
2. a-BHC [1]
3. a-Chlordane [l]
4. Y-Chlordane [1]
5. p,p'-DDD [1]
6. p,p'-DDE [1]
7. o,p'-DDT [1]
8. p,p'-DDT [1]
9. Dieldrin [l]
10. Endrin [l]
11. Heptachlor [l]
12. Heptachlor Epoxide [l]
13. Hexachloro Benzene (HCB) [1]
14. Lindane [1]
15. Methoxychlor [l]
16. Mi rex [1]
17. Toxaphene [l]
PCBs and PBBs
1.
2.
3.
4.
5.
Arochlor
Arochlor
Arochlor
Arochlor
PBB (hexa
1016
1
1
1
)
242
254
260
[1]
[1]
[1]
[1]
[1]
Organophosphates
1. Acephate [2]
2. Azodrin [4]
3. Dasanit (PSSO) [2]
4. Dasanit (POSO) [2]
5. Dasanit (POS02) [2]
6. Dasanit (PSS02) [2]
7. DDVP (Vapona) [2]
8. Diazinon [2]
9. Dimethoate [2]
10. Dioxathion [2]
11. DiSyston (Parent-PSS)
12. DiSyston (POS) [2]
13. DiSyston (PSSO) [2]
14. DiSyston (PSS02) [2]
15. Dursban [2]
16. Dyfonate [2]
17. Ethion [2]
18. EPN [2]
[2]
Elution Volume (ml )
with Methylene Chloride-
Cyclohexane (15:85)
110+150
150+190
120+170
120+170
160+210
110+160
110+150
120+170
140+180
130+170
110+150
120+170
120+160
160+210
1 40+200
90+1 40
1 20+240*
1 20+240*
1 20+240*
120+240*
1 20+240*
N.T.
200+320
150+240
140+230
140+220
160+250
160+250
1 00+200
80+160
140+240
140+190
110+160
110+160
100+160
160+210
120+210
130+210
110+160
150+220
Section
Page 6
Elution Volume
with Toluene-
Ethyl Acetate (
110+130
90+130
100+120
100+120
90+120
110+160
110+130
110+130
110+130
110+130
100+130
110+130
1 20+1 50
100+130
1 00+1 20
110+130
100+140
N.T.
1 1 0+1 40
100+150
1 1 0+1 40
140+180
N.T.
N.T.
80+1 1 0
N.T.
N.T.
N.T.
90+1 20
90+120
N.T.
N.T.
100+130
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
5, B
(ml)
1:3)
-------
Revised 12/15/79
Section 5, B
Page 7
19. Fenthion [4]
20. Gardona [2]
21. Malathion [2]
22. Methamidophos [4]
23. Methidathion [2]
24. Naled [2]
25. Paraoxon [2]
26. Parathion (Ethyl) [2]
27. Parathion (Methyl) [2]
28. Phosdrin [2]
29. Pirmiphos (Methyl) [2]
30. Ronnel [2]
31. Ruelene [2]
32. Thimet [2]
33. TrUnion [2]
Carbamates
1. Aminocarb [3]
2. Carbaryl [3]
3. Carbofuran [3]
4. Carbofuran, 3-keto [3]
5. Carbofuran 3-OH [1]
6. Captafol [3]
7. Mesurol [3]
8. a-Napthol [3]
9. Zectran [3]
Tnioureas
1. Cotoran [3]
2. Diuron [3]
3. Fenuron [3]
4. Monuron [3]
Synthetic Pyrethriods
liPermetrin (Both Isomers)
2. Pydrin [1]
Triazines
1. Atrazine [1]
2. Bladex [1]
3. Simazine [1]
Elution Volume (ml )
with Methylene Cnloride-
Cyclohexane (15:85)
130+200
120+180
1 20+200
140+260**
1 20+240
1 20+1 90
155+235
120+230
190+250
140+210
100+160
130+210
120+210
110+170
140+180
1 40+21 0
210+280
160+220
1 20+230
240+310
160+260
170+220
290+350
130+200
160+230
210+280
1 60+240
200+280
.] 130+190
110+170
140+200
150+300
160+210
Elution Volume (ml )
with Toluene-
Ethyl Acetate (1:3)
N.T.
N.T.
90+1 1 0
N.T.
N.T.
N.T.
N.T.
90+1 20
90+1 20
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
-------
Revised 12/15/79 Section 5, B
Page 8
Elution Volume (ml) Elution Volume (ml)
with Methylene Chloride- with Toluene-
Cyclohexane (15:85) Ethyl Acetate (1:3)
Other Chlorinated Compounds
1. Kepone [1] N.T. 100+190
2. 2,4-D Esters:
Isopropyl [1] N.T. 90+120
Butyl [1] N.T. 90+120
PGB [I] N.T. 80+110
Butoxyethanol [1] N.T. 80+120
Iso Octyl [1] N.T. 80+120
Ethyl Hexyl [1] N.T. 80+120
Methyl [1] N.T. 90+130
3. Silvex Esters:
Propylene Glycol [1] N.T. N.T.
Methyl [1] N.T. N.T.
4. 2,4,5-T Esters:
Isopropyl [1] N.T. 90+130
Butyl [1] N.T. 90+130
Ethyl Hexyl [1] N.T. 90+130
Iso Octyl [1] N.T. 90+120
Methyl [1] N.T. 90+130
Miscellaneous Compounds
T;Metribuzin (parent) 110+160** N.T.
2. Metribuzin (DA) 100+170** N.T.
3. Metribuzin (DADK) 140+200** N.T.
4. Metribuzin (DK) 140+180** N.T.
5. Treflan 100+140 90+110
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Revised 12/15/79 Section 5, B
Page 9
III. CLEANUP OF ADIPOSE TISSUE:
See Section 8,M,j of the EPA Pesticide AQC Manual for a
description of the theory, equipment, column preparation and
operation, and procedure for the GPC cleanup of adipose tissue
using toluene-ethyl acetate (1:3 v/v) as the mobile phase.
This procedure has, for example, been used in the EPA HERL
laboratory for cleanup in the identification of polychlorinated
terphenyls in human adipose tissue at trace levels by GLC MS
(Wright, L. H., Lewis, R. G., Crist, H. L. Sovocool, G. W.,
and Simpson, J. M., 0. Anal. Toxicol., 2_, 76 (1978)).
J. A. Ault of Analytical Biochemistry Laboratories has
described the use of cyclohexane-methylene chloride (85:15 v/v)/
BioBeads SX-3 GPC cleanup coupled with a deactivated mini-alumina
column for blended feed-grade fat samples containing low molecular
weight fatty acids and glycerides. In this improved procedure, the
GPC eluate as it emerges from the column is directed through a
1.5 gram column of alumina (deactivated with 5% water) in the neck
of a powder funnel.
The preparation of the powder funnel/alumina column (Figure 2)
is as follows:
1. Place a plug of glass wool in the end of the powder
funnel neck and rinse with 5 ml cyclohexane-methylene
chloride (85:15 v/v).
2. Pour approximately 1.5 grams of 5% water-deactivated
alumina (v/w) into the funnel.
The reservoir of the powder funnel must be large enough to
hold approximately 100 ml of solvent because the alumina restricts
solvent flow. After all solvent has passed through the funnel,
the alumina should be rinsed with 15 ml of cyclohexane-methylene
chloride (85:15 v/v) to wash any remaining residues into the
collection vessel. The samples are rotary vacuum evaporated,
transferred to culture tubes with 5 ml of petroleum ether, air-
stream evaporated to 1 ml, and analyzed by EC GLC.
Figures 3 and 4 compare gas chromatograms of a pesticide-
containing fat sample prepared by GPC alone with that treated by
GPC plus alumina. Figure 3 shows interferences from lipids and
phthalates, while the modified procedure chromatogram (Figure 4)
indicates adequate cleanup to quantitate chlorinated pesticides.
The allows mark peaks that correspond to 0.08 ppm dieldrin and
0.12 ppm endrin, respectively, which match the values obtained
-------
Revised 12/15/79 Section 5, B
Page 10
from a previous independent analysis of this same sample. Average
recovery of 16 compounds was 89% using this new technique, with
percent coefficient of variation of lindane, p_,p_'-DDE, and
dieldrin of 8%, 10%, and 7%, respectively.
-------
Revised 12/15/79
Section 5,3
Page 11
PULSE DAMPED
PUMP
DIGITAL
CONTROLLER
(mi
_J_ ROTARY VALVE
1 rnfjioni
SOLVENT
RESERVOIR
(81)
SAMPLE STORAGE
LOOPS(5ml)
24 POSITIONS
EFFLUENT DISTRIBUTOR
SOLVENT
ELECTRICAL
SAMPLE/^MPLE
LOADI/INTRODUCTION
U VALVE (SIV)
CONTROLLER
BYPASS
DRAIN
I
GPC
COLUMN
COLLECT/DUMP
SOLENOID VALVE
WASTE
RESERVOIR
'COLLECTION
BOTTLES(23)
Fig. 1. Schematic diagram of the automated GPC AutoPrep 1001.
-------
Revised 12/15/79
Section 5,B
Page 12
SAMPLE
COLLECTION
TUBE
ALUMINA FUNNEL
COLLECTION —-?
VESSEL ^
ALUMINA
GLASS WOOL
Fig. 2. Alumina adsorption cleanup column.
-------
Revised 12/15/79
Section 5,3
Page 13
T
-------
Revised 12/15/79
Section 5 ,3
Page 14
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Revised 12/15/79 Section 6, A, (1)
Page 1
ORGANOPHOSPHORUS PESTICIDES AND METABOLITES IN
HUMAN TISSUES AND EXCRETA - GENERAL COMMENTS
AND ANALYSIS OF INTACT PESTICIDES
The pesticide residue chemist is sometimes called on to consult
and perform analyses pertaining to poisoning by organophosphorus
pesticides. Such cases may come about through accidental exposure,
attempted homicide or suicide, or by small children ingesting the
material, thinking it to be a confection.
In a suspected OP poisoning case, time is of the essence if the
chemist is to obtain any of the original or parent compound that was
ingested or cutaneously absorbed. The OP compounds are relatively
unstable as contrasted to the organochlorine pesticides. Therefore,
the chemist will find it most advantageous to work closely with the
pathologist to expedite the delivery to the laboratory of tissues,
gastrointestinal contents, blood, and urine so that analytical
work can be started at once. In a matter of hours the parent com-
pound, per se, may no longer be detectable in the sample substrates.
The analysis of intact OP pesticides in tissue or blood from sus-
pected poisoning victims is described at the end of this section.
The information so obtained may be sufficient to advise the
medical team of the identity of the intoxicant, but the chemist
may be provided with the rather rare opportunity to conduct in-
depth studies of the metabolic pathways of the pesticide degradation
in the patient, and to document all findings for supplementation to
existing knowledge.
In cases resulting from low exposure or in high exposure cases
many hours after exposure, the probability is greatly reduced of
detecting the parent compound in the body fluids or tissue. The
chemist is then faced with the problem of selecting appropriate body
fluids for analysis, and very importantly, selecting an analytical
method appropriate for the potential breakdown metabolite. A
knowledge of the metabolism of these pesticides will assist the
analyst in making these decisions.
The several analytical methods included in this section provide
some selections for assessing exposure to the OP compounds. For
example, a phenolic metabolite of methyl and ethyl parathion is
para-nitrophenol. The analytical method for detection and quan-
titation in the urine is described in Section 6,A,(2),(b) of this
section. The formation of salts of dimethyl or diethyl phosphate,
thiophosphate, and dithiophosphate results from hydrolysis of
-------
Revised 12/15/79 Section 6, A, (1)
Page 2
various OP compounds. These metabolites appear in the urine and
may be assayed by the procedure described in Section 6,A,(2),(a).
The OP compounds exert an inhibiting effect on blood cholinesterase.
A sensitive procedure for measuring cholinesterase activity in the
blood is provided in Section 6,A,(3),(a).
The block diagram given in Appendix VI provides some guidelines
for selection of methodology, not only for the OP compounds, but
for other suspected exposure to pesticidal compounds.
Determination of Intact OP Pesticides in Tissue or Blood
1. Homogenize 1 gram of liver, brain, or fat with 1 ml of water.
Mix the homogenate with 10 volumes of acetone and centrifuge.
2. Mix serum or whole blood with 10 volumes of acetone and
centrifuge.
3. Inject either centrifugate directly into a gas chromatograph
equipped with an FPD and operated as described in Section
6,A,(2),(a). If there has been significant exposure, the
sensitivity will be more than adequate for detection,
identification, and quantisation.
4. For the analysis of samples with low levels (< 0.1 ppm), the
acetone supernatant is evaporated to 1/10 of the original
volume of acetone, and column cleanup is carried out as follows:
a. Saturate the concentrated sample with sodium chloride
and extract with 15 ml of hexane.
b. Evaporate the organic layer to 0.3-0.5 ml under nitrogen
gas.
c. Prepare a size 22 Kontes Chromaflex column packed with
2.5 cm of anhydrous ^SO^ on top of 1 gram of silica
gel that has been deactivated by adding 1 ml of water
to each 10 grams of activated silica gel.
d. Rinse the column of hexane and transfer the evaporated
sample quantitatively.
e. Elute the column with 10 ml of hexane (Fraction I).
f. Elute next with 10 ml of an appropriate solvent (e.g.
hexane-benzene (80:20 v/v) that will completely recover
the pesticide(s) of interest (Fraction II)). The elution
pattern of the column must be established with pesticide
standards.
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Revised 12/15/79 Section 6, A, (1)
Page 3
g. Evaporate Fraction II to 0.2-0.4 ml and inject a 5 yl
aliquot into the FPD equipped gas chromatograph.
-------
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Revised 6/77 Section 6, A, (2), (a)
Page 1
METHOD FOR DETERMINATION OF METABOLITES OR HYDROLYSIS PRODUCTS
OF ORGANOPHOSPHORUS PESTICIDES IN HUMAN URINE, BLOOD,
AND OTHER TISSUES
I. INTRODUCTION:
The metabolism and urinary hydrolysis of Organophosphorus (OP)
pesticides in mammals results in the excretion of a variety of alkyl
phospates. These include the salts of dimethyl or diethyl phosphate
and phosphorothioate, and phenylphosphonates. The gas chromatographic
separation and quantification of such products in urine may be of
value in estimating the extent of exposure to the parent organo-
phosphorus pesticide. The procedure (1) permits the determination
of four alkyl phosphates derived from most of the common OP pesti-
cides and three phosphonate metabolites of the insecticide leptophos
(Phosvel). Recovery data and limits of detectability for all 10
derivatives formed by these metabolites and analysis of urine samples
from individuals exposed to OP pesticides are reported.
The new analytical procedure (1) is based on a previous method
described in the 12/2/74 revision of this Manual (2-6). Though a
small fraction of phosphonate metabolites are recovered by the
acetonitrile-diethyl ether extraction step in the previous method,
these compounds are recovered with ion-exchange resins from urine.
The new method will, therefore, monitor phosphonate metabolites of
leptophos in addition to alkyl phosphates, and additionally provide
higher recoveries, decreased gas chromatographic interferences,
and faster analyses than the former procedure. It has been applied
with success to analysis of whole blood, serum, and other tissue
samples as well as to urine.
REFERENCES:
1. Extraction and Recovery or Organophosphorus Metabolites
from Urine Using an Anion Exchange Resin, Lores, E. M.,
and Bradway, D. E., J. Agr. Food Chem., 25, 75 (1977).
2. Determination of Metabolic and Hydrolytic Products of
Organophosphorus Pesticide Chemicals in Human Blood and
Urine, Shafik, T. M., and Enos, H. F., J. Agr. Food
Chem. 17., 1186 (1969).
3. Characterization of Alkylation Products of Diethyl Phos-
phorothioate, Shafic, T. M., Bradway, D. E., Biros, F. J.,
and Enos, H. F., J. Agr. Food Chem. 18_, 1174 (1970).
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Revised 6/77 Section 6, A, (2), (a)
Page 2
4. A Cleanup Procedure for Determination of Low Levels of
Alkyl Phosphates, Thiophosphates, and Dithiophosphates in
Rat and Human Urine, Shafik, T. M., Bradway, D. E., and
Enos, H. F., J. Agr. Food Chem. 19, 885 (1971).
5. A Method for Confirmation of Organophosphorus Compounds
at the Residue Level, Shafik, T. M., Bradway, D. E., and
Enos, H. F., Bull. Environ. Contam. Toxicol. 6, 55 (1971).
6. Human Exposure to Organophosphorus Pesticides. A Modiefied
Procedure for the Gas-Liquid Chromatographic Analysis of
Alkyl Phosphate Metabolites in Urine, Shafik, T. M.,
Bradway, D. E., Enos, H. F., and Yobs, A. R., J. Agr.
Food Chem. 21_, 625 (1973).
7. Gas Chromatographic Detector for Simultaneous Sensing of
Phosphorus and Sulfur containing Compounds for Flame
Photometry, Bowman, M. C., and Beroza, M., Anal. Chem. 18,
1174 (1968).
8. Comparison of Cholinesterase Activity, Residue Levels, and
Urinary Metabolite Excretion of Rats Exposed to Organo-
phosphorus Pesticides, Bradway, D. E., Shafik, T. M., and
Lores, E. M., J. Agr. Food Chem., 25., 1353-1358 (1977).
9. Malathion Exposure Studies. Determination of Mono- and
Dicarboxylic Acids and Alkyl Phosphates in Urine, Bradway,
E. E., and Shafik, T. M., J. Agr. Food Chem., 25,
1345_1344 (1977).
II. PRINCIPLE:
Organophosphate metabolites or hydrolysis products in urine are
extracted quantitatively with an ion exchange resin. The metabolites
are subsequently derivatized with diazopentane reagent on the resin.
This reagent is used rather than the more common diazoethane because
the derivatives of metabolites and interfering inorganic phosphate
are more easily resolved. The derivatives are determined by gas
chromatography with flame photometric detection. If very low levels
of alkyl phosphate metabolites are present, further cleanup by silica
gel fractionation is required to remove interfering substances such
as the triamyl phosphate derivative of inorganic phosphate.
-------
Revised 12/15/79 Section 6, A, (2), (a)
Page 3
III. APPARATUS:
1. Tracor model 222 gas chromatograph with flame photometric
detector operated in the phosphorus mode. The detector was
equipped with a Spectrum 1020 noise filter and a variable
power supply (Power Designs, Inc.). A Valco switching valve
#CV 4 HT was interfaced between the GLC column and FPD
detector to allow interchange of the column effluent and
nitrogen purge gas (the flow rates of which should be equal
to maintain a steady recorder baseline).
NOTE: The principal purpose of the switching valve with
the original FPD is to vent solvent and prevent flame
blowout when injections are made. The new configuration
of the FPD permits injections of >10 yl without
extinguishing the flame. However, the switching valve
is still used to permit operation of two different col-
umns or to vent large peaks or column bleed. Also,
the column can be silylated or Carbowax treated without
running the effluent through the detector.
2. Gas chromatographic column-borosilicate glass, 1.8 m x 4 mm i.d.,
packed with 5% OV-210 on 80-100 mesh Gas-Chrom Q support.
Prepare and condition the column by Carbowax deposition treat-
ment as described in Section 4,B.
NOTE: An alternative column, which may be used for confirma-
tion of identity of peaks, is a 1.8 m x 4 mm i.d.
borosilicate glass column packed with 4% SE-30/6%
OV-210 on 80-100 mesh Gas-Chrom Q. Condition in a
similar manner.
3. Centrifuge tubes, 13 ml capacity, conical, graduated, with f
ground glass stoppers.
4. Pipets, disposable glass, Pasteur type, 9 in. length, fitted
with rubber bulbs.
5. Pipets, disposable glass, 5 ml capacity, for use as ion
exchange columns.
6. Vortex-Genie mixer.
7. IEC centrifuge, Model EXD, explosion proof, operated at
2000 rpm.
8. Culture tubes, glass, 16 x 150 mm.
9. Pi pet, 0.1 ml capacity, graduated in 0.01 ml units.
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Revised 12/15/79 Section 6, A, (2), (a)
Page 4
10. Pipets, assorted capacities, to be used in combination with
appropriate volumetric flasks for preparation of standard
solutions.
11. Bottles, reagent, narrow mouth, 1 oz. capacity, with polyseal
screw caps (A. H. Thomas 2203-C bottles and 2849-E caps).
12. Column, chromatographic, size 23 (Kontes 420100).
13. Nitrogen evaporator with water bath maintained at 40°C
(organomation Associates).
14. Exhaust hood with minimum draft of 150 linear feet per
minute.
IV. REAGENTS:
1. Diethyl ether, AR grade, containing 2% ethanol (Mallinckrodt
0850 or equivalent).
2. All other solvents are pesticide quality, distilled from
all-glass apparatus.
3. Silica gel, Woelm, activity grade I (ICN Pharmaceuticals, Inc.),
activated at 130°C for 48 hours and stored in a desiccator.
4. Potassium hydroxide, pellets, AR grade.
5. N-amyl-N_'-nitro-N_-nitrosoguanidine (Aldrich Chemical Co.).
6. Anion exchange resin, Amberlite CG-400 AR, 100-200 mesh
(Mallinckrodt 3345), in the chloride form or BioRad AGlxS,
100-200 mesh (BioRad Laboratories, Richmond, CA), in the
chloride form.
7. Hydrochloric acid, reagent grade, approximately 37%.
8. Glass wool, pre-extracted with pesticide grade methylene
chloride.
9. Diazopentane reagent - Preparation:
a. Dissolve 2.3 g of KOH in 2.3 ml of distilled water in
a 125 ml Erlenmeyer flask. When solution is complete,
cool in a freezer for 30 minutes.
b. Add 25 ml of cold diethyl ether, cover flask mouth with
foil, and cool in a -18°C freezer for 15 minutes.
-------
Revised 6/77 Section 6, A, (2), (a)
Page 5
c. In a very high draft hood, add 2.1 g of j^-amyl-N_' -nitro-
j^-nitrosoguanidine to the flask in small portions over a
period of a few minutes, swirling the flask vigorously
after each addition.
d. Decant the ether layer into a 1 oz. reagent bottle fitted
with a Teflon lined screw cap. This may be stored at
-20°C for periods up to a week.
NOTES:
1. Because of the demonstrated carcinogenicity and
skin irritating characteristics, do not allow the
nitrosoguanidine or_ the diazoalkane tp_ come i_n_ contact
with the skin. Wear disposable vinyl gloves and
safety goggles while handling. Avoid breathing
vapors. Working inside a radiological glove box,
if possible, is strongly recommended.
2. Do not use ground glass stoppered bottles or bottles
with visible interior etching. Avoid strong light.
10. Standards
The names of various phosphate compounds will be abbrev-
iated from this point on to conserve space (see Table 1).
The organophosphorus potassium salts are supplied by American
Cyanamid Corporation and phosphonate standards by Velsicol
Chemical Corporation. Certain diethyl and dimethyl phosphate
and phosphorothioate standards are available in 100 mg incre-
ments from the EPA Repository in Research Triangle Park, NC
(See EPA-600/9- 76-012 for a listing of available standards).
a. Prepare stock solutions of all standards except DMP at
1 mg/ml levels in acetone. Prepare DMP at the same
level in water.
b. Quantitatively make a 1:10 dilution of the stock solutions
(to 0.01 mg/ml (10 yg/ml)) with acetone to prepare the
working standards. These may be used for spiking control
urine samples to be carried through the procedure for
comparison with actual samples. To spike at 0.1 ppm,
add 1 yg of compound (0.1 ml of the working standard) to
10 ml of urine; to spike at 1 ppm, add 1 ml to 10 ml of
urine (see also Section XI).
c. To alkylate phosphate and phosphonate standards in the
absence of urine proceed as follows:
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Revised 6/77 Section 6, A, (2), (a)
Page 6
(1) Weigh accurately 10 mg of each standard into 13 ml
centrifuge tubes.
NOTE: As an alternative, pipet 0.1 ml of each
working standard (equivalent to 1 yg of
compound) into a 13 ml centrifuge tube,
add 1 ml of acetone, and proceed with
steps (2)-(5). If necessary, make further
final dilutions to prepare the required
standards.
(2) Add to each tube one drop of 2 M HC1 to convert
the salts to the corresponding free acids.
(3) Allow the standards to stand for one hour before
being derivatized with diazopentane.
(4) Add sufficient diazopentane reagent to produce a
persistent yellow color (2-5 ml usually suffices),
mix, and allow to stand for one hour with occasional
mixing of the solution.
NOTE: If, at any time during this period, the
yellow color disappears, add more diazo-
pentane.
(5) Remove excess reagent by adding a solution of formic
acid-benzene (1:99 v/v) dropwise until the yellow
color just disappears. Avoid an excess of this
reagent.
(6) Dilute the solution(s) to exactly 10 ml with acetone,
stopper, and mix thoroughly. Store in the freezer
in glass stoppered containers when not in use.
d. These alkylated concentrated stock standards may be diluted
individually or prepared as mixtures at an intermediary
concentration range. For example, a 1:100 dilution of a
concentrated standard will yield a solution of 10 ng/yl.
e. Working Standard Mixtures:
(1) Dilute each of the intermediary stock mixtures in a
1:50 ratio with acetone.
(2) To establish that the working standard mixtures are in
a proper concentration range, observe the recorder
response resulting from the injection of 5 yl of each
-------
Revised 6/77 Section 6, A, (2), (a)
Page 7
into the gas chromatograph.
Photometric tubes vary somewhat in sensitivity and
it may prove necessary to either further dilute or
to prepare higher concentrations of the working
standards. Injection volumes may be varied from 5
to 25 yl.
V. SAMPLE COLLECTION:
The sampling schedule is a most important portion of this total
project if meaningful data are to be obtained from a study of the
urinary metabolites of the OP pesticides. If any type of surveil-
lance or monitoring program is to be implemented, there must be a
highly coordinated relationship between the chemist performing the
analysis and the individual who plans the sampling schedules.
It is strongly recommended that both individuals obtain copies of
references 3, 4, and 5 as background material, and, if possible,
discuss the proposed project with the senior authors of the publi-
cations cited.
This method, as a tool for determining the exposure index of
the subject individual sampled, is considerably more sensitive to
low levels of OP exposure than the ChE method given in Section
6,A,(3),(a) which measures the depression in blood cholinesterase.
In deciding on a sampling schedule, the time of day of taking
the urine sample should be coordinated to the donor's working
schedule since the urinary levels of alkylphosphate metabolites
will vary with the time of sampling and the type of OP pesticide
under study. Generally, the highest concentration of urinary metab-
olites is found from four to eight hours after the time of exposure.
As a general rule, the best time to collect a urine sample is at
the end of the work day.
VI. SAMPLE PREPARATION AND EXTRACTION:
1. Store urine in a freezer until ready for analysis. When the
urine sample is thawed, mix well, centrifuge, and discard
solids.
2. Pi pet 1 ml of urine into a 13 ml centrifuge tube.
NOTE: At this point, a sample of control urine from an
unexposed donor should be started and carried through
the entire procedure. The donor should be an individ-
ual known to have no contact with OP pesticides for at
least a week.
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Revised 6/77 Section 6, A, (2), (a)
Page 8
3. Add 10 ml of acetone to precipitate some interfering compounds,
including a large portion of the inorganic phosphate.
NOTES:
a. One of the major factors influencing the recovery of
metabolites from urine was found to be the urineracetone
ratio used to precipitate the interfering compounds. In
order to determine the optimum ratio, several urine:
acetone ratios were investigated for each metabolite.
One ml of urine was used throughout the experiment, and
the volume of acetone was varied from 3 to 10 ml. As
shown in Table 2, no single ratio was optimum for all
alkyl phosphates and phosphonates. The lower amounts of
acetone seem to favor some compounds, while the higher
amounts seem to be best for others. When a specific alkyl
phosphate is being sought, the urine:acetone ratio which
gives the best recovery for that compound should be used.
However, for a general screening method, a compromise ratio
must be selected. The 1:10 ratio was selected for general
use and gave cleaner chromatograms than the other ratios
that were tried.
b. The pH of urine-acetone mixture was investigated and found
to have little effect on the recovery of the metabolites.
The time required for complete derivatization was also
checked, and it was found that, while most of the reaction
was complete within one hour, increased yields could be
obtained with overnight waiting periods.
4. Mix well with the Vortex mixer and centrifuge.
5. Prepare an ion exchange column as follows:
a. Weigh one gram of ion exchange resin and slurry in
0.1 M HC1.
b. Add the slurry to a 5 ml disposable pi pet which has a
plug of glass wool in the tip.
c. Rinse the column with 5 ml of 0.1 M HC1 followed by
ca 50 ml of distilled water.
6. Transfer the supernatant from the centrifuge tube to the column
using a disposable pipet, being careful to avoid any particles
of residue.
7. Rinse the residue in the tube with 2 ml of acetone, centrifuge,
and again transfer the supernatant to the column.
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Revised 6/77 Section 6, A, (2), (a)
Page 9
8. Allow the column to drain as much as possible.
9. Using a rubber suction bulb, blow the resin out of the column
into a culture tube. Rinse the empty column with ca 1 ml of
acetone and add to the culture tube.
10. To remove the metabolites from the resin, pi pet 0.05 ml of
6 N HC1 into the culture tube. Allow to stand one hour with
occasional mixing of the resin.
VII. ALKYLATION:
1. After one hour, slowly add diazopentane reagent to the resin in
the culture tube until a yellow color persists in the super-
natant.
NOTES:
a. A large excess of diazopentane must be avoided since it
is a major source of background interference.
b. Be sure to do this work in a high draft hood.
2. Allow the reaction to proceed one hour, with occasional mixing.
NOTE: Add more diazopentane reagent if at any time during
this period the yellow color disappears.
3. With a disposable pi pet, transfer the supernatant to a 13 ml
graduated centrifuge tube, and wash the resin with small
portions of acetone until a total volume of 10 ml is obtained.
4. Inject this solution into the gas chromatograph and compare
to standards. If the level of alkyl phosphate metabolites
appears too low for quantification, concentrate the sample
to ca 0.2 ml under a nitrogen stream and chromatograph through
silica gel. This procedure is used to remove interferences
and quantify levels as low as 0.01 ppm. Alkyl phosphonate
metabolites cannot be separated from interfering compounds by
silica gel chromatography.
VIII. SILICA GEL COLUMN CHROMATOGRAPHY:
1. Prepare silica gel as follows: Partially deactivate 10 g of
silica gel by shaking 2 hours with 2.0 ml distilled water.
Transfer 2.4 g to a size 23 chromatographic column with a small
wad of glass wool at the bottom. Top the column with ca 2 g
of anhydrous sodium sulfate and prewash with 10 ml of hexane.
(See Note on next page)
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Revised 6/77 Section 6, A, (2), (a)
Page 10
NOTE: Because elution patterns may vary from one laboratory
to another depending on the temperature and relative
humidity, each laboratory should establish an elution
pattern of standards and spiked control urine samples
under local conditions before analysis of samples.
2. Add 3 ml of hexane to the 13 ml centrifuge tube (Step 3 in
Subsection VII), and transfer the sample to the column with
a disposable pipet. Rinse the tube with two 2 ml portions of
hexane and add to the column with the same pipet. Discard all
eluate to this point.
3. Place a 25 ml concentrator tube under the column and add 15 ml
of methylene chloride to the column. This is Fraction I,
which contains DMTP and DETP.
4. Add 15 ml of acetone-methylene chloride (1:99 v/v) and discard
this eluate. This eluate contains most of the triamyl phos-
phate, the thiolate derivatives, and other interfering
substances.
5. Place another 25 ml concentrator tube under the column and add
20 ml of acetone-methylene chloride (3:97 v/v). This is
Fraction II, which contains DMP and DEP.
IX. GAS CHROMATOGRAPHY:
1. For alky! phosphates, the operating conditions of the gas
chromatograph are:
Column temperature 140°C
Injection block temperature 200°C
Detector temperature 175°C
Nitrogen (carrier) flow rate 40 ml/minute
Hydrogen flow rate 50-60 ml/minute
Air flow rate 80-90 ml/minute
For the phosphonates, raise the column oven temperature to
160°C.
NOTES:
The temperature of the transfer block and the switching
valve therein on the MT-222 chromatograph, which cannot
be adjusted independently, ran ca 15° higher than the
inlet on the instrument used. No oxygen is used with the
model FPD employed with this procedure.
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Revised 6/77 Section 6, A, (2), (a)
Page 11
b. If an MT-220 chromatograph is used, transfer line and
switching valve temperatures of 200°C are recommended.
Since at least two different models of the FPD detector
are in use, each laboratory should set the conditions
of their FPD in accordance with the manufacturer's
instructions.
2. Inject 5-25 yl of the 10 ml eluate from the ion exchange
resin or Fractions I and II from the silica gel column if
cleanup was carried out. Also, inject appropriate volumes of
working standards (Subsection IV, 10) for comparison with
samples.
3. Dilute or concentrate the sample depending on the chromatographic
response from these initial injections.
4. Correct the amounts of metabolites found by subtracting the
heights of interfering peaks found in the chromatogram of the
control urine sample.
5. Correct results further based on recoveries from spiked control
urine samples carried through the procedure.
6. Peak heights obtained in the phosphorus mode are used for
comparison of working standards, samples, controls, and
spikes (see Subsection XI).
NOTES:
a. DMTP and DETP isomerize upon alkylation, producing
thionates and thiolates (Table 1). Quantification is
based on the respective amyl thiolate derivatives
because of greater interferences with the thionate peaks.
b. Any small amount of inorganic phosphate not precipitated
by acetone or removed by silica gel column cleanup elutes
from the GC column as triamyl phosphate with a late
elution time (Ry > 20 minutes). An injection schedule
can be adopted such that several sample injections are
made in close succession, timed so that all peaks to be
measured elute between the groups of interfering triamyl
phosphate peaks.
See Figure 1 for chromatograms of amyl derivatives of alky!
phosphates and Figure 2 for chromatograms of phosphonate
samples. In both cases, no cleanup step was carried out.
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Revised 6/77 Section 6, A, (2), (a)
Page 12
7. Do not inject Silyl 8 on a column directly connected to the
FPD. If the column needs reconditioning, use the switching
valve to vent the column effluent during the conditioning
period.
NOTES:
a. After extended use of the gas chromatographic system,
extraneous peaks may appear, chiefly from the accumula-
tion 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 yl
of diazopentane solution periodically (2 weeks). If
underivatized compounds show up as peaks following the
diazopentane injection, recondition the column.
b. Quantification is based on the amyl derivatives DMAP,
DEAP, DMAPTh, and DEAPTh. Some inorganic phosphate is
extracted and converted to triamyl phosphate.
c. Confirmation and Specificity.
(1) The ability to interchange the sulfur and phosphorus
filters in the single detector, or the use of the
base assembly for dual phototube operation with both
filters (7), greatly enhances the specificity of
this method. Suspected thiophosphate can be con-
firmed using the sulfur filter by increasing the
concentration of the compound injected into the gas
chromatograph by a factor of 5 to 10, if interference
in the sulfur mode from urinary components is not
excessive.
(2) Confirmation of any particular compound can be
accomplished by preparing the hexyl derivative.
The method described for diazopentane is followed
except that N-hexyl-N.1 -nitro-N^-nitrosoguandine is
used as the diazoalkane precursor.
(3) Further confirmation is achieved using the silica
gel column when the sulfur-containing derivatives
are eluted in the methylene chloride fraction and
the non-sulfur derivatives are eluted in the
acetone-methylene chloride (3:97 v/v) fraction.
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Revised 6/77 Section 6, A, (2), (a)
Page 13
X. MISCELLANEOUS NOTES:
1. This method has been applied to media other than urine.
Alkyl phosphates have been determined in whole blood, serum,
stomach contents, and in liver from poison cases. Whole blood
or serum is mixed with 10 parts of acetone, and analysis is
continued as in Subsection VI,4. Tissue and other solid
samples are first ground with equal parts of water (i.e., 0.5 ml
water to 0.5 g sample). Ten ml of acetone is added for every
one gram of slurry, and analysis is continued as in Subsection
VI,4.
2. Since a minimum of 8 to 10 samples per day can be analyzed, the
procedure is useful in a monitoring program or in clinical
laboratories where the rapid classification of poisoning agents
is essential.
3. DMP and DEP are the two most prevalent compounds detected.
Whenever DETP shows up, DEP will invariably be present, and
when DMTP shows up, DMP will invariably be present.
4. In a high exposure situation or a poisoning case resulting from
malathion, DMP, DMTP, and DMDTP (0_,0_-dimethyl phosphorodi-
thioate) may all be found. The latter compound is also alkyl -
ated by diazopentane, and the derivative will elute in
Fraction I from the silica gel column. Its retention time
on the GLC column will be between the derivatives of DMP and
DEP.
5. Two recently published papers are related to this procedure:
a. Blair, D., and Roderick, H. R., J. Agr. Food Chem. 24,
1221 (1976).
b. Daughton, C. G., Crosby, D. G., Garnas, R. L., and
Hsieh, D. P. H., J. Agr. Food Chem. 24_, 236 (1976).
The first paper describes the use of a cation exchange resin
for isolation of urinary DMP, but the method is applicable only
to DMP in the absence of other alkyl phosphates. Methyl
derivatives are utilized which are highly volatile and difficult
to resolve by GLC.
The procedure in the second paper employs XAD-4 adsorbent to
remove alkyl phosphates from aqueous media. A partitioning
between water and ethyl acetate is included, which results in
low recovery (ca 50%) of DMP. Diazomethane is again used for
derivatization, leading to poor gas chromatographic resolution.
-------
Revised 6/77 Sections, A, (2), (a)
Page 14
6. Table 3 shows recovery data obtained from spiked urine samples
without silica gel cleanup. The recovery data are based on a
1:10 urine:acetone ratio for the alky! phosphates, and on a
1:5 ratio for the phosphonates. The low recovery of 0_,0_-dimethyl
0_-amyl phosphorothionate, DMTP, is due to an interfering peak
that seems to come from the diazopentane reagent. However,
since the other isomer, 0_,0_-dimethyl S_-amyl phosphorothiolate,
DMPTh, can be quantified, the original level of 0_,0_-dimethyl
phosphorothioate can be determined (3).
7. Of the compounds investigated, only 0_-methyl phenylphosphono-
thioate, MPTPn, could not be quantitatively determined owing
to an interfering peak that could not be resolved by GLC or
removed by silica gel cleanup. The interference, which comes
from the urine, can be reduced enough to allow qualitative
determinations by using a 1:10 urine:acetone ratio.
8. The recovery of the metabolites varied from one urine sample
to the next. Morning and afternoon urine samples were obtained
from five donors to check the variability of the method. These
samples were spiked with alkyl phosphates at 0.5 and 1.0 ppm.
As shown in Table 3, the variation, which was possibly due to
the variation in the salt content of different urines, was a
little more than desirable.
9. The variability of urine is also a major factor influencing the
limit of detectability, which is below 0.1 ppm for the alkyl
phosphates. When the level of interference is low, urine
samples may be evaporated to 1 ml for detection of very low
levels of metabolites. If lower limits of detection are re-
quired, or if the interference level is too high, the sample
must be carried through silica gel fractionation. The limit of
detectability for the phosphonates ranged from 0.04 ppm for
the 0-methyl phenylphosphonic acid, MPPn, to 0.15 ppm for
phenylphosphonic acid, PPn. Cleanup was found to be of little
help with the phosphonates because the interference is not
removed by silica gel.
10. The peak that interferes with the determination of MPTPn can be
seen in the control urine chromatogram in Figure 1. The control
urine chromatogram in Figure 2 shows the peak that interferes
with the determination of DMP. However, the level of inter-
ference was generally low enough to allow quantification of
levels above 0.1 ppm. With a highly efficient 5% OV-210
column (5000 theoretical plates), it is possible to obtain
baseline separation of the alkyl phosphates. With less effi-
cient GLC columns, the silica gel fractionation is required to
separate the phosphate derivatives.
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Revised 6/77 Section 6, A, (2), (a)
Page 15
XI. ANALYTICAL QUALITY CONTROL:
Because the method is complex, routine analyses must be
validated by conducting simultaneous analyses of spiked SPRM's
(spiked reference materials). For occasional routine analyses, one
SPRM should be analyzed with the unknown and in exactly the same
manner. When large numbers of samples are analyzed, run at least
one SPRM for every nine samples.
Because instability of a spiked urine sample precludes advance
preparation of a large sample of SPRM for periodic analysis, prepare
each SPRM as needed. The following procedure is suggested:
1. Prepare aqueous standard solution mixtures at 1.0 and 0.1 ppm
containing each of the following compounds: DMP, DEP, DMTP,
and DEPT. Use the spiking solution described in Subsections
IV,10,a. and b. to prepare these solutions or proceed as
follows: Weigh 10 mg of each into a one L volumetric flask,
making to volume with water and shaking thoroughly. Dilute
10 ml of this solution to 100 ml. This will be STANDARD
MIXTURE A. Transfer 10 ml of Mixture A to a 100 ml volumetric
flask and make to volume with distilled water. This will be
STANDARD MIXTURE B. Mixtures A and B will have the respective
concentrations of 1.0 and 0.10 ppm.
2. Divide both mixtures into several screw cap test tubes or vials
and freeze immediately. Do not fill the tubes over half full,
and lay on side during freezing to reduce probability of
cracking the glass.
3. When ready to conduct an SPRM analysis, thaw one tube (of each
concentration) and draw a 0.1 ml aliquot to spike 1.0 ml of
control urine (from an unexposed donor) contained in a 15 ml
centrifuge tube.
NOTE: If previous experience indicates that one of the
two concentrations (0.1 or 1.0 ppm) will closely
match the expected concentration in the unknown, only
one concentration may be needed. Without such knowl-
edge, prepare both concentrations.
4. In another centrifuge tube carry along a 1.0 ml unspiked urine
sample from the same control donor.
5. Into another tube pipet 0.1 ml of the standard alone.
6. Proceed with the analysis of the two urine samples (spiked and
control) as described in the procedure above.
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Revised 6/77 Section 6, A, (2), (a)
Page 16
7. To the tube containing the standard alone, add one drop of
6 N HC1, 1.0 ml of methanol and 2.0 ml of diazopentane, or
a sufficient volume to give a persistent orange color. Mix
well and allow to stand one hour (see Section IV,10,c).
8. Calculate recoveries by comparing the chromatographic data
of the spiked samples with those of the corresponding standard.
-------
Revised 6/77
Section 6, A, (2), (a)
Page 17
TABLE 1. A LIST OF STANDARD METABOLITES AND THEIR DERIVATIVES
Standard
0,0-dimethyl phosphate Na
0,0-diethyl phosphoric acid
0,0-dimethyl phosphorothioate K
0,0-diethyl phosphorothioato K
0-methyl phenylphosphonic acid
0-methyl phenyl phosphonothioatp
phenylphosphonic acid
* Compound forms two derivatives
** Phosphonates are from leptophos
Abbre-
viation
Derivative(s)
DMP
DEP
DM1P
DMPTh
DETP
DEPTh
0,0-dimethyl Q-amyl phosphate
0,0-diethyl 0-amyl phosphate
p_,p_-dimethyl 0-amyl phosphorothionate
0,0-dimethyl S-amyl phosphorothiolate
0,0-diethyl 0-amyl phosphorothionate
0,0-diethyl S-amyl phosphorothiolate
MPPn
MPTPn
MPPnTh
PPn
0_-methyl 0-amyl phenylphosphonate
0-niethyl 0-amyl phenylphosphonothionate
0-methyl S-amyl phenylphosphonothiolate
0,0-diamyl phenylphosphonate
-------
Revised 6/77
Section 6, A, (2), (a)
Page 18
TABLE 2. COMPARISON OF PERCENTAGE RECOVERY WITH
VARIOUS URINE:ACETONE RATIOS
DMTP
DETP
DMP
DEP
DMPTh
DEPTh
MPTPn
MPPn
PPn
Avg Recovery3
1:3
b
b
b
b
b
b
Interference
92 (6)
71 (6)
With Ratios of
1:5
90 (2)
81 (2)
85 (3)
65 (3)
Not analyzed
Not analyzed
Interference
92 (6)
82 (6)
1:10
39 (20)
77 (20)
85 (20)
105 (20)
95 (20)
87 (20)
74 (2)
44 (2)
79 (2)
figure in parentheses represents the number of determinations
^Background too high to be useful
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Revised 6/77 Section 6, A, (2), (a)
Page 19
TABLE 3. RECOVERY, VARIABILITY, AND DETECTION LIMITS
OF STANDARD DERIVATIVES
Compound
DMTP
DETP
DMP
DEP
DMPTh
DEPTH
MPTPn
MPPn
PPn
0.1
51
87
74
97
--
--
--
97 ±
87 ±
% Recovered3 When
ppm 0.5 ppm
36 + 14
77 ± 10
85 ± 20
106 ± 15
97 ± 23
87 + 9
--
7 95 ± 14
11 86 ± 18
Spiked
1.
42
76
85
105
93
87
74
90
79
With
0 ppm
± 13
± 11
± 15
± 13
± 8
± 7
± 8
± 22
Limit of Detection
Without Cleanup (ppm
0.1
0.1
0.05
0.05
0.1
0.1
0.15
0.04
0.07
± Standard deviation
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Revised 6/77
Section 6, A, (2), (a)
Page 20
I
£
A-.
Figure 1.
Figure 2.
Figure 1. Chromatograms of alkyl
phosphates injected into a gas
chromatograph without cleanup.
A. Alkyl phosphate standards,
0.5 ng each. B. Urine extract
from dichrotophos poison case.
C. Extract of human urine spiked
with 0.5 ppm of IMP and DEP.
D. Control human urine.
Figure 2. Chromatograms of phosphonate
samples injected into a gas chromato-
graph without cleanup. A. Phenyl-
phosphonate standards, 0.5 ng each.
B. Extract of urine from rats fed
leptophos at 0.01 ID™ Control rat
urine. :>u*
-------
Revised 1/4/71 Snecti°n 6> A>
Page 1
DETERMINATION OF PARA-NITROPHENOL (PNP) IN URINE
I. INTRODUCTION:
Urinary PNP, the phenolic metabolite of parathion, methyl para-
thion, and EPN has been measured for years as an indicator of
exposure to these organophosphorus pesticides. The Elliott spectro-
photometric method is only semi-specific, requires a minimum of
10 yg of PNP, possesses marginal accuracy at low levels and is some-
what lengthy. The following gas chromatographic method yields
acceptable analytical results at 50 ppb level and requires less than
two hours for analysis. This method, in fact, deviates little from
the Elliott method except for an added cleanup step and the deter-
minative procedure.
REFERENCES:
1. Elliott, J. W., K. C. Walter, A. E. Penick, and
W. F. Durham (1960). "A Sensitive Procedure for
Urinary para_-Nitrophenol Determination as a Measure
of Exposure to Parathion." J. Agr. Food Chem. 8_, 111.
2. Cranmer, M. F. (1970), "Determination of p-Nitrophenol
in Human Urine." Bull. Environ. Contamin. and Toxicol.,
Vol. 5, No. 4, 329-332.
II. PRINCIPLES:
A small volume of urine is hydrolyzed with hydrochloric acid
to free the PNP from the bound or adsorbed state. The hydrolyzed
urine is made alkaline and extracted with benzene-ether to minimize
co-extraction of interferences in the subsequent determinative
extraction. The urine is then re-acidified and extracted with
benzene-ether. The extract is dried, a suitable aliquot removed
and the PNP converted on the column to the less polar and more
volatile trimethylsilyl ether during the gas chromatographic
determinative step.
III. APPARATUS:
1. Gas chromatograph with electron capture detector fitted with a
column of 1.5% OV-17/1.95% QF-1 prescribed in the program.
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Revised 1/4/71 Section 6, A, (2), (b)
Page 2
2. Centrifuge tubes (distill, recvr.), grad., 12 ml, with f 14/20
outer joint, Kontes, #288250.
3. Stoppers, glass, flathead, f, 14/20, Kontes #850550.
4. Reflux condensers, equipped for water cooling with lower J inner
joint the same size as test tubes. Kontes #282200.
5. Pipets, Mohr, 0.5 ml, grad. in 0.01 ml, Kimble #37023 or the
equivalent.
6. Pipets, Mohr, 3 ml, grad. in 0.05 ml, Kimble #37023 or the
equivalent.
7. Pipets, Mohr, 10 ml, grad. in 0.1 ml, Kimble #37033 or the
equivalent.
8. Pipets, Transfer, 1 ml, Kimble #37000 or the equivalent.
9. Block tube heater, "Dri-Thermolyne," constant temp. 100°C, or
comparable block heater with holes of appropriate size to
accommodate centr. tubes.
10. Vials, glass, with screw caps, 15 x 45 mm, 1 dram, Kimble
#60910.
11. Cap liners, Teflon, size 13, Arthur H. Thomas Co. #2849-04.
IV. REAGENTS:
1. Hydrochloric acid, cone., A. R. grade.
2. Sodium hydroxide, A. R. grade, aqueous solutions of 0.1 N and
20%.
3. Benzene, pesticide quality.
4. Diethyl ether, pesticide quality.
5. Benzene-ether mixture - 80:20 (v/v).
6. Sodium sulfate, A. R. grade, anhydrous, granular.
7. Hexane, pesticide quality.
8. Hexamethyldisilizane reagent, 20% in hexane.
9. Hexamethyldisilizane reagent, 10% in hexane.
-------
Revised 1/4/71 Section 6, A, (2), (b)
Page 3
10. p-Nitrophenol standard solution of appropriate concentration
range in hexamethyldisil izane - hexane solution (10:90 v/v).
PNP standard-Eastman stock number EK192. The suggested
concentration range for the working standard is 5 to 25 pg/yl .
V. HYDROLYSIS. EXTRACTION AND CLEANUP
1. With a 3 ml Mohr pipet, transfer 2.7 ml of urine into a 12 ml
glass stoppered cent, tube and attach tube to a stoppered water
cooled condenser.
NOTE: At this point, a reagent blank consisting of 2.7 ml
of distilled water and a control sample of 2.7 ml of
urine from an unexposed donor should be carried through
the entire procedure along with the suspect sample(s).
2. Add with a grad. 0.5 ml Mohr pipet, exactly 0.30 ml of cone.
HC1 and reflux the mixture for 1 hour with tube inserted in
"Dri-Thermolyne" block heater.
NOTE : During the refluxing period, the condenser must be
stoppered and cooled by water circulation.
3. Remove assembly from heat and rinse down condenser with 2 ml
of 0.1 N NaOH. Cool tube and adjust to a pH of 11 or higher
with 0.4 ml of 20% NaOH solution.
4. Add 5 ml of the 80:20 benzene-ether reagent, stopper tube, and
shake vigorously 1 minute.
5. Remove as much as possible of the benzene-ether (upper) layer
with a disposable pipet and repeat extraction one more time with
another 5 ml of the benzene-ether, discarding the benzene-ether
extract from both extractions.
6. Reacidify the urine to pH 2 or lower with ca 0.2 ml cone. HC1 ,
add 5.4 ml of the benzene-ether solvent, stopper tube, and
shake vigorously 1 min.
7. Using a disposable pipet, carefully transfer as much of the
solvent (upper) layer as possible into a second centrifuge tube,
taking care that no aqueous phase is included.
8. Add 0.5 grams anhydrous ^SO^, stopper tube, and shake
vigorously 1 min. to remove traces of moisture from the solvent
extract.
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Revised 6/77 Section 6, A, (2), (b)
Page 4
9. Transfer 1 ml of the dried benzene-ether extract to a 1 dram
glass vial with a Teflon lined screw cap, then add 1 ml of 20%
hexamethyldisilizane in hexane. Cap vial and shake vigorously
1 min.
VI. GAS CHROMATOGRAPHY:
Before injecting the sample extract, pre-condition the column
with several repetitive injections of the PNP/HMDS-hexane standard
(Subsection IV,10). This serves the dual purpose of (1) providing
a quantitative standard peak, and (2) conditioning the column prior
to sample injection.
NOTES:
1. During the course of sample injections the column must be
monitored to determine whether all the PNP injected is being
converted on-column to PNP-TMS. This is done by injecting
HMDS-hexane (10:90 v/v) without PNP. If a PNP peak is
produced, it is indicated that the column has adsorbed PNP
and requires further conditioning with HMDS.
2. The author reported recoveries greater than 90% for PNP
levels down to 25 ppb in urine.
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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 correlated
with the decrease of acetylcholinesterase activity in the nervous
system which in turn is accompanied by an increase in the concen-
tration of acetylcholine. 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 acetylcholine by the enzyme cholinesterase overcomes
many of the undesirable features of other methods. This method
does not utilize buffers, is temperature and atmospherically con-
trolled, and has easily calculated units. In addition, the substrate
and enzyme concentrations can be adjusted and maintained at levels
which allow optimal enzyme activity.
REFERENCES:
1. Michel, H. 0. An electrometric method for the determin-
ation of red blood cell and plasma cholinesterase activity.
J. Lab Clin. Med. 3^; 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 acetyl-
cholinesterase 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
cholinesterase present in the blood fraction reacts with the AChI
releasing acetic acid as illustrated in the following:
-------
Revised 11/1/72 Section 6, A, (3), (a)
Page 2
C C
C+-N-C-C-0-C-C C+-N-C-C-OH + C-C-OH
C C
Acetylcholine Choline Acetic Acid
(ACh) HA
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, thermistor 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 rpm.
4. Aspirator for connection to suction pipet.
5. Pipet, volumetric, 2 ml.
6. Pipets, measuring, 0.2, 0.5 and 5.0 ml, Corning 7064 or the
equivalent.
7. Centrifuge tubes, 5 ml, glass stoppered, Corning 8061 or the
equivalent.
*Manufacturers 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.
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Revised 11/1/72 Section 6, A, (3), (a)
Page 3
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 primary standard,
available from the National Bureau of Standards, Washington, D.C.
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 1-liter vol. flask,
dissolving and making to volume in freshly boiled dist. water.
3. Titrant standard solutions prepared from sodium hydroxide, reag.
grade, Fisher S-318 or the equivalent, 98.7% NaOH.
a. Stock solution (1.0 N NaOH): Weigh 4.053 grams NaOH,
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 NaOH): Pipet 1.0 ml of the 1.0 N
solution into a 100-ml vol. flask and make to volume with
freshly boiled dist. water. Standardize using potassium
acid phthalate as the primary reference.
4. Standardization of titrant working solution.
This solution should be restandardized each time 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 exact
normality is known, 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
neutralization of each ml of the P.A.T. solution will require
0.001 meq of NaOH. The NaOh working solution contains 0.01 meq/ml
Therefore, 0.1 ml of the working titrant = 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 instru-
ment operating temperature to 37°C.
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Revised 11/1/72 Section 6, A, (3), (a)
Page 4
b. Set titrant delivery to appropriate position for 2.5 ml
delivered 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:
M = 2.0 ml x 0.001 meg/ml _ 0.4 , ,
N RU x 0.005 ml/RU RU meq/ml
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.
5. Acetylcholine iodide is available from Calbiochem Company,
P. 0. Box 54282, Los Angeles, California 90054.
Substrate solution - Weigh 0.7510 grams of AChI into a 25-ml
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
acetylcholine 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 sample. If a few hours delay
is unavoidable, 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 rpm.
b. Pipet plasma into clean tubes for storage.
NOTE: If a part of this sample will eventually be analyzed by
EC GLC, the tube cap should be ground glass or screw
cap with Teflon or foil liner.
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Revised 11/1/72 Section 6, A, (3), (a)
Page 5
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
colution by vacuum pipet.
f. Repeat steps d. and e. The RBC mass is now ready for assay.
VI. PREPARATION OF RBC HEMOLYSATE:
1. Pipet 1.8 ml of dist. water into a 5 ml conical centrifuge
tube and then pipet in 0.2 ml of packed red blood 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
solution up into the bore of the pipet repeatedly until all
adhering cells are washed into the water.
3. Stopper tube and 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 manu-
facturer's instructions. This is best done by using two buffers,
one below and 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% NaCl solution
and 0.15 ml of plasma, (or 4.2 ml of 0.9% NaCl 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.
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Revised 11/1/72 Section 6, A, (3), (a)
Page 6
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 .
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.
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
From formula (1), N = f^ff-,= gf^y = 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 yM/min/ml (first replicate)
48.5 x 0.1222 = 5.927 yM/min/ml (second replicate)
Plasma 2: 30.5 x 0.1222 = 3.727 yM/min/ml (only one replicate
shown)
B. Alternative method, without using factors:
Substituted:
Plasma ! : RU °5 m1/RU = ml/minute = 46'5 * °-005 = 0.0775
ml/min.
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Revised 11/1/72 Section 6, A, (3), (a)
Page 7
ml/minute x Normality = meq/minute = 0.0775 x 0.011
= 0.000853 meq/minute
meq/minute x 1000 yM/meq = yM/minute = 0.000853 x 1000
yM/meq
= 0.853 yM/minute
= 5.687 yM/min/ml
IX. MISCELLANEOUS NOTES:
1. Because of the variety of instruments available, operating
instructions 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 standardization, plus the s.c.heme 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 anticoagu-
lants 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 yM/min/ml - Plasma
8.0 yM/min/ml - Red Cells
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Revised 11/1/72 Section 6, A, (3), (a)
Page 8
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: yM/min/ml.
8. Standard sources of enzyme are available from Sigma and are
useful in intralaboratory quality control.
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Revised 11/1/72
Section 6, A, (3), (a)'
Page 9
'FIGURE 1. Sample of strip chart of pH-stat assay of cholinesterase
activity.
30.5 Recorder units(RU)
One minute
Pen direction
f 5h.O RU
tttistttttttta
Standardization of
0.01 N NaOH against
potassium acid
phthalate, three
replications
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Revised 11/1/72 Section 6, A, (3), (a)
Page 10
TABLE 1 . FACTORS FOR THREE MINUTE TITRATIONS
Normality NaOH
Solution
0.
0095
0096
0097
0098
0099
0100
0101
0102
0103
0104
0105
These factors are
Red cell factor
Plasma factor
Factor
0.
3167
3200
3233
3267
3300
3333
3367
3400
3433
3467
3500
derived as follows:
= ml/min x meq/ml x
0.05
= ml/min x meq/ml x
Plasma ChE Factor
0.
1055
1067
1078
1089
1100
1111
1122
1133
1144
1156
1167
1000 yM/meq
1000 yM/meq
0.15
These factors are valid only if a 0.5-ml syringe is used for titrant
delivery, so that the ml/min factor in the equation becomes
0.005, or 0.00167 ml/min.
3
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Revised 11/1/72 Section 7, A
Page 1
DETERMINATION OF 1-NAPHTHOL IN URINE
I. INTRODUCTION:
Humans exposed industrially to the insecticide carbaryl
(1-naphthyl-N-methyl carbamate) excrete relatively large quantities
of 1-naphthol, conjugated either as the sulfate or glucuronide.
Quantitative determination of 1-naphthol in human urine has been
generally accomplished using a colorimetric procedure. This method
lacks both the sensitivity and specificity necessary for deter-
mining the relatively small amounts of 1-naphthol excreted in the
urine of agricultural workers exposed to low levels of this insecti-
cide.
The 1-naphthol resulting from the hydrolysis of carbaryl has
been used as an indirect measure of the residue level of the parent
insecticide 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 publication, this procedure was utilized to determine
a number of carbamate insecticides. However, it was indicated
that further modification 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 monochloroacetate derivative.
This, coupled with a silica gel cleanup results in a method sensi-
tivity down to 20 ppb of 1-naphthol.
REFERENCES:
1. Shafik, M. T., Sullivan, H. C., Enos, H. F., Bull of
Environ. Contamin. & Toxic., Vol. 6, No. 1, 1971
pp 34-39.
II. PRINCIPLES:
A small sample of urine is subjected to acid hydrolysis.
The 1-naphthol present is extracted in benzene and derivatized
with chloroacetate anhydride solution. After silica gel cleanup,
the resulting 1-naphthyl chloroacetate is quantitatively determined
by EC,GLC, comparing sample peaks against peaks obtained from pure
1-naphthol standard, similarly derivatized.
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Revised 11/1/72 Section 7, A
Page 2
III. APPARATUS:
1. Gas chromatograph equipped with EC detector and fitted with a
6' x 1/4 " o.d. column of 1.5% OV-17/1.95% QF-1. Operating
parameters for the column are those prescribed in Section 4,A
of this manual.
2. Chromatographic column (Chromaflex), size 22, Kontes #420100.
3. Evap. concentrator tubes grad., glass stoppered, 25 ml, I 19/22,
Kontes #570050.
4. Distilling column (condenser), 200 mm jacket, Kontes #286810,
fitted with tight glass stopper at top.
5. Centrifuge capable of 2000 rpm that will accept metal shield,
I. E. Company No. 367.
6. Volumetric flasks, 10 and 100 ml.
7. Pipets, Mohr, 0.2, 0.5 and 10 ml, Corning 7064 or the equiv-
alent.
8. Pipets, transfer, 2, 3 and 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 f glass
stoppers, Corning 8084 or the equivalent.
12. Circulating water pump.
13. Boiling water or steam bath.
IV. SOLVENTS AND REAGENTS:
1. Benzene, pesticide quality.
2. Hexane, pesticide quality.
3. Pyridine, Spectro Grade, Eastman #13098.
4. Chloroacetic anhydride, Eastman #335 - prepare a 2% solution in
benzene and hold no longer than one week.
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Revised 11/1/72 Section 7, A
Page 3
NOTE: The chloroacetic anhydride must be dry and
therefore should be stored in a desiccator as
it is extremely hygroscopic.
5. Hydrochloric Acid, reag., cone.
6. Sodium sulfate, anhydrous, granular - preextract on a Soxhlet
with benzene for about 50 discharge cycles, remove excess
solvent and store in 130°C oven.
7. Sodium sulfate, 3% solution in dist. water. Use preextracted
Na2S(V
NOTE: Deionized 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 and 1.0 N NaOH solutions.
10. Silica gel, Woelm, activity grade I, Waters Associates, Inc.,
DO NOT SUBSTITUTE.
11. Preparation of silica 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 1.5% water in the following manner: Add the
necessary volume of water to a 125-ml 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 distributed throughout the
adsorbent. Allow to equilibrate for 2 to 3 hours with periodic
shaking. Chromatographic columns are prepared just prior to
use.
12. 1-Naphthol, Eastman #170 or Reference Standards Repository,
EPA, Research Triangle Park, NC.
13. Preparation of standard solutions;
a. Stock Standard I. Weigh 20 mg of 1-naphthol into a 100-ml
vol. flask, dissolve and dilute to volume with benzene.
This is the cone, stock of 200 ng/pl and may be held
several months at -18°C.
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Revised 12/2/74 Section 7, A
Page 4
b. Stock Standard II. With a 0.5-ml Mohr pi pet, transfer
0.25 ml of stock standard I to a 50-ml vol. flask and
make to volume with benzene. This intermediary stock
standard of 1 ng/yl 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,
carefully 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
rpm 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 con-
centrations of 1-naphthyl chloroacetate of 0, 10
and 50 pg/yl.
d. Prepare another intermediary stock standard (III) of
derivatized 1-naphthol (1-naphthyl chloroacetate) by
transferring 0.5 ml of stock standard I to a 25-ml evap.
concentrator tube and dilute to 5 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-ml vol. flask, rinsing tube with several
portions of benzene and finally making to volume with
benzene. From this derivatized stock of 1 ng/yl concentra-
tion of 1-naphthyl chloroacetate, dilutions may be made
and used to check the derivatized working standards
finalized in Step c, (5) above.
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Revised 6/77 Section 7, A
Page 5
NOTE: The derivatized standards are relatively
stable. Assuming no solvent losses from repeat-
ed 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. HYDROLYSIS, EXTRACTION AND DERIVATIZATION:
When the following procedure is started there should be no
interruption until the final derivatized extract is obtained in
Step 10. It is highly desirable that a sample of control urine from
an unexposed donor be carried along parallel with the sample(s)
under test.
1. Pi pet 5 ml of urine into a 25 ml evap. concentrator tube,
add 1 ml of cone. 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 be tightly
stoppered.
3. After removal 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 cone, tube in centrifuge and spin for 10 minutes at
2,000 rpm.
6. Carefully transfer as much of the benzene (upper) layer as
possible to a clean 25 ml concentrator tube, using a dis-
posable 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.
8. Wash benzene extract with two 3 ml portions of 3% Na2S04
solution, centrifuging and discarding each successive
aqueous layer.
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Revised 6/77 Section 7, A
Page 6
9. To the combined benzene extract add 2 ml of 2% chloroacetic
anhydride solution and 0.1 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 Subsection 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 circumstances should final volume be
permitted to go below 0.3 ml.
VI. SILICA GEL FRACTIONATION:
1. Place a small wad of glass wool at the bottom of a Chromaflex
column and add 1 gram of the partially deactivated silica gel.
Top this with ca 1/2-inch of anhydrous, granular Na2SO[+.
2. Prewash the column with 10 ml of hexane, discarding the eluate.
3. When the surface level of the nexane reaches a point on the
column ca 2 cm from the top of the Na2SOit transfer the con-
centrated benzene extract to the column with a disposable pi pet
and rinse tube with two portions of 0.5 ml of 20/80 benzene/
hexane solvent applied with another disposable pipet, dir-
ecting 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 1-naphthyl chloroacetate derivative.
Finally, adjust volume of extract to exactly 10 ml with benzene.
NOTES:
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-ml conical grad. centrifuge tube mix 2 ml
of 1.0 H_ NaOH and 2 ml of the diluted, underivatized
standard described in the NOTE in Step b, of
Subsection IV, 13.
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Revised 6/77 Section 7, A
Page 7
b. Stopper tube and mix 2 minutes on a Vortex mixer.
Allow to stand 10 minutes and centrifuge 5 minutes
at 2,000 rpm.
c. Pipet aliquots of 0.1, 0.25 and 0.5 from the aqueous
layer (sodium naphthoxide solution) into separate
25 ml grad. evap. concentrator tubes.
d. From this point on, conduct the hydrolysis and
derivatization as previously described in Subsection
V, starting with Step 1, under Hydrolysis, Extraction
and Derivatization, ending at Step 4 under Silica Gel
Cleanup. The final extracts contained in the three
15-ml centrif. tubes should have concentrations of
10, 25 and 50 pg/yl of derivatized 1-naphthyl
chloroacetate. Recovery data are obtained by
chromatographing these extracts against the deriva-
tized working standards.
2. At no time during the elution should the liquid level in
the column be allowed to drop below the top surface of
the Na2S04 bed.
VII. GAS CHROMATOGRAPHY:
After adjusting operating parameters of the gas chromatograph
to the values prescribed in Section 4,A of this manual, commence
injections of derivatized sample and standard extracts. Assuming
an average background current, it should be possible to quantify as
little as 50 picograms of the derivatized 1-naphthol. Using the
1.5% OV-17/1.95% QF-1 column, the relative retention value for
1-naphthyl chloroacetate should be ca 0.92 with respect to aldrin.
VIII. 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 pro-
cedure for spiked control urine samples is as follows:
a. In a 15-ml conical grad. centrifuge tube mix 2 ml of
1.0 N NaOH and 2 ml of the diluted, underivatized
standard described in the NOTE in Step b, of Subsection
IV, 13.
b. Stopper tube and mix 2 minutes on a Vortex mixer. Allow
to stand 10 minutes and centrifuge 5 minutes at 2000 rpm.
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Revised 6/77 Section 7, A
Page 8
c. Pipet aliquots of 0.1, 0.25 and 0.5 from the aqueous
layer (sodium naphthoxide solution) into separate 25 ml
grad. evap. concentrator tubes.
d. From this point on, conduct the hydrolysis and derivati-
zation as previously described in Subsection V, starting
with Step 1, under Hydrolysis, Extraction and Derivatiza-
tion, ending at Step 4 under Silica Gel Cleanup. The final
extracts contained in the three 15 ml centrif. tubes should
have concentrations of 10, 25 and 50 pg/yl of derivatized
1-naphthyl chloroacetate. Recovery data are obtained
by chromatographing these extracts against the derivatized
working standards.
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Revised 12/15/79 Section 8, A
Page 1
SAMPLING OF PESTICIDES IN AIR
I. INTRODUCTION:
Pesticides in air represent an important class of toxic
pollutants, which may have chronic deleterious effects on human
health and the ecological balance. In 1963, the President's
Science Advisory Committee recommended that the air be continuously
monitored for pesticides. The authority for such monitoring in the
U. S. has been granted to the Environmental Protection Agency under
the Clean Air Act as amended in December 1970, and the Federal
Insecticide, Fungicide, and Rodenticide Act as amended in 1972.
Presently, there are only limited data concerning the contam-
ination of the atmosphere by pesticides, especially in urban areas.
Information relating to transport or ambient trends is even less
available. Such information must be obtained before total air
quality can be defined and before the threat of atmospheric pesti-
cidal pollutants to the general populace and the ecosystem can be
determined.
The determination of pesticides in the ambient air is a
formidable task. There are hundreds of pesticides registered for
use in the U. S., many of which are potential air pollutants.
These pesticides may exist in air as vapors, aerosols, or adsorbed
on suspended particulate matter; thus, their collection is compli-
cated. Pesticides are usually present in air at levels far lower
than those found in crop residues for which most methods of analysis
are designed; hence, their detection is difficult. Metabolites and
degradation products of pesticides, which are sometimes considerably
more toxic than the parent pesticide, are, of course, at even lower
atmospheric concentrations.
Most of the existing data concerning the nature and degree of
contamination of the ambient atmosphere by pesticides was
collected over the period from 1970 to 1972 by the EPA. The sam-
pling method utilized was based on impingement in ethylene glycol,
which was expensive and cumbersome to use and did not provide an
adequate sample size to permit subnanogram per cubic meter
detectabilities for most pesticides. Sections 8,B and 8,C in
the 6/77 revision of this Manual were based on collection of samples
in ethylene glycol. During the past several years, EPA has
developed a high volume air sampler that is believed to better serve
the needs for pesticide ambient air monitoring. This sampling
device and others for indoor air sampling, crop re-entry monitoring,
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Revised 12/15/79 Section 8, A
Page 2
and workplace and personnel monitoring are described in this
subsection, and analytical methodology for the pesticides collected
in these samplers are detailed in Section 8,B.
REFERENCES:
1. Evaluation of Polyurethane Foam for Sampling of Pesti-
cides, Polychlorinated Biphenyls, and Polychlorinated
Naphthalenes in Ambient Air, Lewis, R. G., Brown, A. R.,
and Jackson, M. D., Anal. Chem. 49., 1668-1672 (1977).
2. Sampling and Analysis of Airborne Pesticides, Lewis, R. G.,
in Air Pollution from Pesticides and Agricultural Pro-
cesses, R. E. Lee, Jr., (Ed.), CRC Press, 1976, pp. 52-94.
3. Protocol for Assessment of Human Exposure to Airborne
Pesticides, Lewis, R. G., Analytical Chemistry Branch,
U. S. EPA, ETD, HERL, Research Triangle Park, NC 27711
(1978).
4. Sampling Methodologies for Airborne Pesticides and
Polychlorinated Biphenyls, Lewis, R. G., MacLeod, K. E.,
and Jackson, M. D., Paper No. 65, Chemical Congress,
ACS-Chemical Society of Japan, Honolulu, Hawaii, April 2-6,
1979.
5. Sources of Emissions of Polychlorinated Biphenyls into
the Ambient Atmosphere and Indoor Air, MacLeod, K. E.,
EPA-600/4-78-022, March, 1979. Analytical Chemistry
Branch, ETD, HERL, Research Triangle Park, NC 27711.
II. AMBIENT AIR SAMPLING:
1. Descriptions of Ambient Air Samplers
For ambient (uncontaminated) air, sufficiently large samples
must be taken to permit detection and measurement at ultratrace
levels (pg/m3 to ng/m3). Such sampling should be performed
over an entire diurnal cycle if results are to be representative
of the average quantities of the substances normally present
in the atmosphere.
a. The sampler developed by EPA has been referred to as the
modified SURC sampler, since it is similar to a high
volume pesticide air sampler designed for EPA by Syracuse
University Research Corporation. The device uses a Hi-Vol
pump and shelter, and draws air through a glass fiber
filter (to collect particulate matter) and a solid sorbent
cartridge (to trap vapors) at sampling rates up to 280
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Revised 12/15/79 Section 8, A
Page 3
liters/minute. The sampler can be used with a wide
variety of sorbents in a manner that permits their con-
tinual re-use. It is designed for low cost and simple
operation. The sampler has been demonstrated to efficient-
ly collect a number of organochlorine and organophosphate
pesticides, and it is presently being evaluated for
carbamates.
A standard aluminum Hi-Vol sampler housing is modified by
replacing the sheet aluminum support plate with one that
is 9 mm thick. A second support plate is added approxi-
mately 25 cm from the bottom of the sampler to lend
strength. The top plate forms the support for the blower
unit and pesticide collection module as well as the
necessary plumbing.
All parts and how they are connected are shown in Figure 1.
A variable power transformer is provided to adjust the
vacuum pulled by changing the motor speed. This prolongs
the life of the motor. The flow is measured by two
devices: a Dickson recorder, which keeps a continuous
record of the flow versus time, and a venturi (Barco
Model BR-12402-08-31) - Magnehelic gauge (Dwyer Instruments
Model 2100), used to set the flow rate of the sampler when
in operation. The exhaust duct is required to stop re-
cycling of the air.
b. PCBs have been collected on polyurethane foam by
sampling 3.4-200 m3 of air with Bendix Hurricane dual
speed pumps (National Environmental Instruments, Inc.,
Warwick, RI 02888, Cat. No. 16003) at flow rates of
0.1-0.5 m3/minute.
2. Descriptions of the Sampling Modules
a. The SURC sampling module is shown assembled (a) and
exploded (b) in Figure 2. The basic module consists of
a 4-inch (i.d.) by 2-inch (i.d.) (10 cm x 5 cm) glass
process pipe reducer (Kimax 6650, size 4, or equivalent -
Part 1 in Figure 2). This part is approximately 18 cm
long. Standard glass pipe fittings (parts 2 and 3) are
used on each end (2-inch and 4-inch connectors). At the
smaller end, a stainless steel screen (3.9 openings/cm2)
is cut to fit and installed to hold the polyurethane foam
plug (4) or other sorbent in place. A piece of stainless
steel screen (1.5 openings/cm2) is cut and installed at
the larger end. This holds either the glass fiber filter
(5) in normal operation or wool felt filter for controlled
introduction of vapors of the test compounds. When foam
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Revised 12/15/79 Section 8, A
Page 4
is used as the sorbent, the larger opening screen may
be used on both ends. The SURC module is shown in
place on the sampler in Figure 1. The lower pipe fitting
(part 3) is tightened down on a 2 inch stainless steel
flange.
b. EPA - SWRI Sampling System - The sampling system was
developed by Southwest Research Institute and was sub-
stantially modified and improved by EPA to allow use of
a variety of sorbent types. It is shown in Figure 3. The
same sampler described under Item 1 above is used. There
are two parts to the sampling system: the sampling module
or cartridge and the air-tight cartridge holder.
Sampling cartridge - A 65 mm borosilicate glass tube is
cut to 125 mm in length. An indentation is formed 20 mm
from one end (bottom) to provide a rim to retain a 25 mesh
or larger stainless steel screen to hold the sorbent. The
cartridge can then hold a polyurethane foam plug, porous
(macroreticular) beads or other solid sorbents, or liquid
coated beads.
This entire cartridge can be placed in a Soxhlet extractor
for removal of substances collected in air. Vacuum
drying at 30°C to 40°C restores the sorbent for reuse
within several hours. The cartridge is shown in Figure 3
(Part a).
Cartridge holder - The basic cartridge holder is shown
both assembled (b) and disassembled in Figure 3. Part 2
screws down on to Part 1 and silicone rubber (GLC septum
sheet stock, Supelco Catalog No. 2-04626) gaskets (c) on
both ends form an air-tight seal. Part 3, a 10-mesh
stainless steel screen, holds either the glass fiber filter
(d) or the felt pad. Part 4 holds the filter or pad in
place. Part 1 is tapped and threaded on the bottom to
attach to the 1/2 inch NPT inlet of the high volume air
pumping system shown in Figure 1.
c. Bendix Hurrican Pump filter holder - The standard 10 cm
filter holder is modified by attaching a cylindrical
chamber, 25 cm long x 5 cm i.d., behind the filter holder
with epoxy cement. Place two foam flugs, 5.5 cm diameter
x 8 cm thick, in the chamber and a 10 cm diameter glass
fiber filter in front of them in the filter holder.
Connect the sampler to the Bendix pump by a 7.6 meter
length of Flexaust CWC hose.
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Revised 12/15/79 Section 8, A
Page 5
3. Calibration of Air Sampler
Refer to Figure 1 for a schematic diagram of the sampler.
The needle valve is for test purposes only and is not used
in normal operation. The red tap of the venturi goes to the
high pressure side, and the green tap goes to the low pressure
side of the Magnehelic gauge. A gasket goes between each
floor flange and the base, and between the Hi-Vol housing and
the base. Check these for leaks before starting.
Calibration procedure:
a. Attach the calibration venturi (figure 4] in place of the
sampling module and tighten securely.
b. Connect a 4"-0-4" slack tube Hg manometer to the taps of
the calibrated venturi. Make sure the manometer is
zeroed and level. Mark this manometer to indicate that it
is to be used only with the audit venturi.
c. Zero the Dickson recorder (tap the face) and the Magnehelic
gauge.
d. Turn the power transformer to 100 volts and turn the switch
to ON. Allow the Hi-Vol motor to warm up for several
minutes before readings are taken.
e. Record the ambient temperature in °C on the data form.
f. Record the barometric pressure in mm Hg.
g. Open the ball valve fully (the pointer on either zero
mark). Record the audit venturi DP, the Dickson reading,
and the Magnehelic reading.
h. Close the indicator valve slightly until the Magnehelic
drops approximately 5 inches (13 cm) and record a new
set of readings.
i. Repeat step (h) until five spaced sets of readings are
obtained.
j. Remove the calibrated venturi.
k. Prepare a calibration chart of flow rate versus meter
readings as shown in Figure 5.
To calibrate Bendix high volume pumps, force the exhaust
air through a restricting orifice (supplied with the pump)
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Revised 12/15/79 Section 8, A
Page 6
and measure the resulting back pressure by a gauge placed
directly ahead of the orifice. A gauge calibrated by
the manufacturer to read directly in ft3/minute air flow
is available.
4. Descriptions of Sampling Media (Sorbents)
Two types of sampling media are recommended for use with the
modified SURC sampler: polyurethane foams and granular solid
sorbents. Foams may be used separately or in combination with
granular solids in either sampling module described previously.
With the EPA-SURC module, the sorbent may be extracted and
reused (after drying) without unloading the cartridge.
Polyurethane foam (PUT) - Use polyether-type polyurethane foam
[density No. 3014 (0.0225 grams/cm3), or equivalent]. This is
the type of foam generally used for furniture upholstery,
pillows, and mattresses. It is white and yellows on exposure
to light. Use 7.6 cm (3 in.) sheet stock and cut from it
cylindrical plugs that fit under slight compression in the
glass cartridge or module, supported by the wire screen. For
the SURC module, the plugs should be 5.5 cm in diameter and
fitted into the lower 5 cm (i.d.) chamber of the module. For
the EPA-SWRI cartridge, the plug diameter should be 6.0 cm.
Granular solids - Porous (macroreticular) chromatography
sorbents are recommended. Examples are Chromosorb 102, 20 to
40 mesh (Johns-Manville, Denver, CO); Porppak R, 50 to 80 mesh
(Waters Associates, Mil ford, MA); Amber!ite XAD-2, 16 to 50
mesh (Rohm and Haas Co., Philadelphia, PA); Tenax - GC, 60 to
80 mesh (Enka N. V., The Netherlands); and Florisil PR-grade,
16 to 30 mesh (Floridin, Pittsburgh, PA). Pore sizes and mesh
sizes must be selected to permit air flow rates of at least
200 liters/minute. Approximately 25 cm3 of the sorbent is
recommended. The granular solids may be "sandwiched" between
two layers of polyurethane foam (a 60 mm diameter x 50 mm foam
plug on top and a 60 mm diameter x 25 mm PUF plug on the bottom)
to prevent loss during sampling and extraction (Figure 6).
5. Preparation of Sampling Media
a. Prepare sorbent for initial cleanup before use. For foam,
cut an appropriate size cylindrical plug with a cutting
tool and place in a Soxhlet extractor. For granular or
porous polymeric solids, add to pre-extracted Soxhlet
thimble and place in the extractor.
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Revised 12/15/79 Section 8, A
Page 7
b. Extract with 5% diethyl ether in r[-hexane (glass distilled,
pesticide quality or equivalent) or other appropriate
solvent(s) for 14-24 hours at ca 4 cycles/hour.
NOTE: To determine the blank value of each plug,
extract twice for periods of 7-12 hours; con-
centrate the second solvent, pass through an
alumina column, and analyze by GLC (see Section
8,B).
c. Dry the sorbent under vacuum at 75°C.
d. Place the sorbent into glass sampling modules. For loose
solids, the appropriate volume (e.g., 25 ml) should be
measured and the corresponding weight recorded.
e. Place the sampling module in a sealed container or
wrap in hexane-rinsed aluminum foil until ready for use.
6. Determination of Sampling Efficiencies for Specific
Pesticides
a. Pesticide Retention Efficiency
No air sampler may be used for assessing of atmospheric
concentrations of any compound without first determining
the efficiency of the sampler to trap and retain the com-
pound. Determine retention efficiencies by multiple
injections of micro!iter volumes of the pesticide of
interest in jv-hexane directly into the sorbent trap. After
a one hour drying period, place the fortified trap in
front of a second trap in the sampling system. Pump
ambient air through the train for the length of time and
volume to be used in the sampling (i.e., for the high
volume system, 24 hours at 200-250 liters/minute) to
determine breakthrough to the second trap. Exclude
airborne particulate matter by means of a glass fiber
prefiIter.
b. Pesticide Collection Efficiency
Determine collection efficiencies by vaporizing individual
compounds or mixtures into the intake of the air sampler
under study. Replace the glass fiber prefilter with a
pre-extracted wool felt filter (weight 14.9 mg/cm2,
thickness 0.6 mm), which is then fortified with the
pesticide of interest before pulling ambient air through
it and, subsequently, the vapor trap(s). Add dropwise
hexane solutions containing microgram amounts of the test
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Revised 12/15/79 Section 8, A
Page 8
compounds to the filter in amounts of 1 ml or less, and
evaporate the solvent before the filter is attached to
the sampling module. After 24 hours of air flow, analyze
the filter and sorbent trap(s) individually (Section 8,B).
Make at least one blank determination with unfortified
filters simultaneously to correct for airborne inter-
ferences and possible contamination or losses from the
analytical methodology.
Perform these tests outdoors with unaltered ambient air
(in a rural, nonindustrialized area) whenever possible.
When required, filter the intake air through a PUF trap
to remove interfering contaminants.
All pesticidal compounds used for establishing sampling
efficiency should be of the highest purities obtainable.
Purities should be checked before use. All solvents
should be of pesticide quality or equivalent.
Conduct at least six independent trials for each test
compound in order to provide statistical data. Acceptable
standard deviation values will depend on the nature of the
pesticide. For example, for the less volatile, more
chemically stable, and more easily analyzed pesticides,
higher precision and accuracy of results will be expected.
A sampling efficiency of 75% should, in general, be
considered satisfactory for a collection medium. For the
more easily trapped pesticides such as DDT and mirex,
sampling efficiencies should be essentially quantitative.
Reuseability of sorbents is considered important; as a
guideline, at least six months of repeated use should be
expected before loss in sampling efficiency is noted. The
sorbents selected are also expected to vary little in
trapping and retaining test compounds with changes in
temperature and humidity.
7. Collection of Air Samples
A modified SURC air sampler may be operated at ground level
or on roof tops. In urban or congested areas, it is recom-
mended that the sampler be placed on the roof of a single-story
building. The sampler should be located in an unobstructed
area, at least two meters from an obstacle to air flow. The
exhaust hose should be stretched out tn the downwind direction,
if possible. The sampler should be operated for 24 hours in
order to obtain average daily levels of airborne pesticides.
(Air concentration may fluctuate with time of day, temperature,
humidity, wind direction and velocity, and other climatological
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Revised 12/15/79 Section 8, A
Page 9
conditions.) On and off times and weather conditions during
the sampling period should be recorded. Air flow readings
should be taken (from the Magnehelic gauge) at the beginning
and end of each sampling period. The chart from the Dickson
recorder should be examined to note the occurrence and duration
of any power failure and any change in sampling rate during
the period. Blower motor brushes should be inspected fre-
quently and replaced, as necessary. An electrical power source
of 110 VAC, 15A is required.
The glass sampling cartridge and glass fiber filter (on the
modified SURC module) should be removed from the sampler
with forceps and clean, gloved hands and immediately placed in
a sealed container(s) for transport to the laboratory.
Similar care should be taken to prevent contamination of the
filter and vapor trap when loading the sampler.
8. Results and Discussion
The greatest value of the high volume collection system is that
it provides a large sampling of air (at least 300 tn3/24 hours).
Thus, even with poorly trapped compounds, sufficient quantities
can be collected to detect very low air concentration. For
efficiently collected compounds, detection limits can be
extended to the subpicogram-per-cubic-meter range, and
sufficient quantities can often be trapped in 24 hours to pro-
vide for mass spectrometric confirmation.
See Section 8,B for collection data on pesticides and PCBs.
III. SOURCE SAMPLING:
Contaminated, or source-related, atmospheres generally
present less problems with respect to either the sampling process or
analytical measurement because of the higher levels of pesticide
present. However, source sampling often requires special sampling
equipment that is portable, battery-powered, or is otherwise
commensurate with specific sampling needs. Often it is also not
practical (or desirable) to collect 24-hour samples. Thus a
relatively high-flow device, which may also need to be portable and/
or battery operated, may be required.
Monitoring atmospheres inside domiciles or workplaces requires
a sampler that is unobtrusive and operates quietly, does not get in
residents' or workers' way, and places little or no time or financial
demands on the site owner to maintain. Similar requirements are
made on devices used to monitor inspired air. They need to be worn
on the person; hence, must be battery-operated, light weight,
comfortable and quiet. Ideally, they should sample air at flow rates
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Revised 12/15/79 Section 8, A
Page 10
similar to normal human respiration. Since indoor levels are
generally much higher than outdoor levels, due mainly to pest control
measures exercised inside domiciles and places of employment, small,
low volume air samplers may be used. Sampling rates in the 1 to 10
liters/minute range are adequate and can be provided with any of
several personal air sampling pumps on the market. These pumps can
be operated on batteries for up to 8 hours or for longer periods if
attached via a charging unit to 110 VAC house current. Personal
sampling devices are discussed further in Section IV.
1. High Volume Source Sampler
A high volume sampler developed for the U. S. Army and manu-
factured by Environmental Research Corporation (ERCO), a
subsidiary of Dart Industries, St. Paul, MM, is shown in
Figure 7. Air is drawn at flow rates up to 185 liters/minute
through either or both of two parallel 15 cm composite filter
pads comprised of Poropak R sandwiched between two layers of
glass fiber mat.
The major advantage of the ERCO sampler for source air moni-
toring is that is provides a relatively large sample size with
short sampling times. It is compact and light weight, which
makes it highly portable. The model studied was equipped for
either AC or DC power and could be operated on a heavy duty
automobile battery at flow rates up to 160 liters/minute. The
greatest disadvantage of the system is the high cost of the
composite filter pads, which cannot be reused.
2. Low Volume Indoor Source Samplers
a. Pumps:
MSA Monitaire™ Sampler, Model S, Catalog No. 458475 and
charger No. 456059. Mine Safety Appliances Company,
600 Penn Center Boulevard, Pittsburgh, PA 15235
or
DuPont Constant Flow Sampling Pump, Model P4000A
(includes charger), Catalog No. 66-241 (Figure 8).
DuPont, Applied Technology Division, Wilmington, DE 19898.
Both of these small, battery operated pumps are capable
of pumping air through an 18 mm diameter x 50 mm cylindri-
cal PUF plug at 2.5 to 4 liters/minute for at least 8 hours
with a fully charged battery pack. The DuPont pump has
the advantage that it will automatically adjust its
pumping rate to compensate for changes in flow resistance
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Revised 12/15/79 Section 8, A
Page 11
(e.g., due to accumulation of particulate matter at the
intake of the collection module). It also operates more
quietly than the MSA and can be programmed to stop sam-
pling after a prescribed period.
b. Collection Devices:
Any cartridge capable of holding a cylindrical plug of
polyurethane foam (approximate volume 15 to 20 cm3) or 5
to 10 cm3 of granular sorbent can be used. Several col-
lection modules are shown in Figure 9, along with a
portable pump.
Module a is a Teflon bottle containing a 2.5 cm diameter
x 5 cm foam plug preceded by a 2 cm diameter glass fiber
filter (Gelman Type A or MSA CT-75428) mounted in the cap.
The hose attachment is constructed from a plastic hose
connector pressed tightly through a hole in the bottom of
the bottle and sealed with Teflon tape.
Module b_ is an open glass tube, 18 mm i.d. x 50 mm, drawn
down to a 7 mm o.d. open tip on one end for attachment
to the plastic tubing. The foam plug is cut slightly
oversized for a compression fit. This module has no pro-
vision for separate collection of particulate matter.
Module £ is a standard filter holder (e.g., MSA No. 92944)
for dust collection only. Either glass fiber (Gelman Type
A or MSA CT-75428) or PVC membrane filters (e.g., MSA
Type FWS-B), 37 mm in diameter, may be used.
Modules a_ and b_ are most suitable for use with granular
sorbents. It is suggested that small cylinders of poly-
urethane foam be inserted before and after the granular
sorbent to retain the latter in place.
A glass tube, 2 cm in diameter and 7.5 cm long (tapered
the last 3 cm) has also been used for the sampling cart-
ridge with MSA and DuPont pumps. A small foam plug, 4 cm
long x 2 cm diameter is placed in this tube, and the en-
tire tube is wrapped in hexane-rinsed foil for transport.
For sampling, the tube is connected to the MSA pump by
a length of Tygon tubing.
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Revised 12/15/79 Section 8, A
Page 12
c. Preparation and Analysis of Sorbents and Glass Fiber
Filters
Follow the same basic procedures described in Subsection
11,5. Scale down volumes for the smaller plugs or
quantities of granular sorbents used. Smaller Soxhlet
extractors will cycle more frequently (e.g., 8 cycles/
hour). Because efficient extraction of pesticide from
glass module ID (Figure 9) will probably not be achieved
with the sorbent in place, extract the foam and sorbent
separately. Cut glass fiber filters to size, wrap loosely
in aluminum foil, heat to 315°C in a muffle furnace over-
night to remove any organic material, and place in a
desiccator until use.
d. Calibration of Air Sampler
For low volume samplers, a simple soap bubble meter is
adequate for calibration. The commercial calibration unit
shown in Figure 10 is available from Mine Safety
Appliances (Catalog No. 457629). It consists of a one
liter bubble tube assembly, manometer, needle valve,
stop watch, and voltmeter (for battery test).
When polyurethane foam alone is used, as in module b_,
(Figure 9), the sampling pump may be calibrated without
the module attached. However, the additional use of a
prefilter or granular sorbents causes sufficient pressure
drops across the sampling module to require calibration
with the loaded module in place. In all cases, it is
suggested that the loaded sampling module be installed
during calibration, or there may be very large differ-
ences between the pump flow meter reading and actual
flow achieved through the module.
For calibration, the pump and sampling module should be
attached to the top of the bubble meter upstream of the
needle valve and manometer (location f_ in Figure 10).
Some means of adapting the intake face of the module into
the calibration system must be devised. For module JD,
laboratory "bubble" tubing (3/4 to 3/8 inch) may be used.
(A suggested source of the latter is Sherwood Medical
Industries, Argyle, NY.) Allow the pump to operate a
few minutes, then set the manometer to read 2 inches
(5 cm) of vacuum by adjusting the needle valve. Squeeze
the rubber bulb at the bottom of the bubble meter to
introduce soap bubbles. Use the stop watch to time the
passage of soap bubbles between the calibration marks
(one liter). Adjust the pump to achieve the desired flow
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Revised 12/15/79 Section 8, A
Page 13
rate or generate a plot of flow rate versus the pump
rotameter reading.
The DuPont Company markets a calibration unit (Catalog
No. 66-242-f-l) especially designed for the Model P4000A
pump. It includes a bubble tube, flow rate meter, and
pressure drop meter (Figure 11).
Pumps may also be calibrated by displacing water from an
inverted 2 liter graduated cylinder during a measured
length of time.
e. Determination of Sampling Efficiencies for Specific
Pesticides
Measured quantities of pesticides in a volatile solvent
such as ji-hexane are placed in a U-tube, which is
attached to the sampling module. The U-tube is immersed
in a heating bath, which is carefully controlled to slowly
volatilize the pesticide. After the sampling period
(which whould be as long as that anticipated in actual
monitoring studies), the amount of pesticide remaining
in the U-tube and that collected by the sorbent is
determined to establish collection efficiency. Sampling
periods should be 4 to 8 hours.
f. Collection of Air Samples
For determination of pesticide residues indoors, air
samples should be taken in as many locations as necessary
to achieve a profile of the distribution throughout the
building. In houses with forced-air heating and/or
air conditioning, air concentrations will tend to be
equilibrated, although there will probably be areas in
rooms where circulation is impaired. Unlike the situation
in outdoor air, there should be little diurnal variation
in pesticide levels. Concentrations may vary more widely
in houses without air circulating devices, and may also be
weather dependent (i.e., depend on whether windows and
doors to the outdide are open or closed).
Nearly all domiciles and many other buildings are given
preconstruction termite treatment. This results in a slow
release of the insecticide over very long periods of time
(at least up to 25 years). In buildings where circulation
is poor, airborne termitacide levels may be higher in
basements or ground floors than on other floors. In
"plenum houses", the crawl space under the house serves
as the plenum in the air destribution system, which con-
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Revised 12/15/79 Section 8, A
Page 14
tributes substantially to the transport of termitacide to
other portions of the dwelling.
Kitchens and bathrooms are favorite areas for insects such
as roaches, ants, and silverfish; consequently, the
application of insecticides in these areas is common
practice. The chemicals are usually applied in baits or
in slow-release formulations, so that pesticides may be
emitted into the air for many months after treatment.
Similarly, crack and crevice treatment for pest control is
popular in commercial buildings.
The design of the structure and history of its past control
treatment should be taken into account when planning an
air monitoring project. Several samplers should be used
at once to obtain a distribution profile of pesticide
levels in the building. Normally, an 8 hour sampling
period at about 2 liters/minute is sufficient to obtain
an adequate sample for analysis. In this period, about
one m3 of air is sampled, which should provide a detection
limit of 0.1 yg/m3 or lower for most pesticides. This
level is one-tenth that of the National Institute of
Occupational Safety and Health proposed standard of
1 yg/m3 for a 10 hour work day, 40 hour work week expos-
ure to carcinogenic compounds. Although the portable
pumps described earlier in this section are designed to
operate for 8 hours on fully charged battery packs, house
current (through the battery charger) should be used when
available to assure more uniform pumping rates during the
sampling period.
The air intakes of the sampling modules should be placed
one or two meters above floor level and oriented down-
ward or horizontally. If oriented upward, non-respirable
pesticide loaded dust may be collected. If pesticide
residues on household dust particles appear to be very
significant, a prefilter should always be used.
IV. WORKPLACE AIR - PERSONNEL MONITORING:
Inhalation of airborne dust and vapors containing high concen-
trations of pesticides constitutes a serious hazard to pest control
operators, pesticide formulators, and other persons occupationally
involved in agricultural industry. Respiratory exposure can be
best assessed through the use of a personal monitor worn on the body
while working in areas of high pesticidal contamination.
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Revised 12/15/79 Section 8, A
Page 15
1. Air Sampling Devices
The small sampling units described in the previous section
are designed for personal monitoring. They are battery oper-
ated and can be worn on the body.
The MSA pump weighs 870 grams and may be worn comfortably on
the waist belt. The DuPont pump is also designed to be
attached to a waist belt, but is rather heavy (1.2 kg with
battery packs that are required for 8 hours of operation).
DuPont markets a smaller constant flow unit that weighs only
400 grams, but it draws only 200 ml/minute at full flow (no
resistance). In order to achieve the sensitivity in the 0.1
to 1 yg/m3 range for many pesticides, flow rates of 1 to 3
liters/minute are needed, particularly for sampling periods
that are necessarily shorter than 8 hours. The sampling modules
are attached to the shirt collar or lapel to monitor air in
the breathing zone. The intake should be oriented downward to
exclude large dust particles, which may not enter the nostrils.
It has been pointed out that estimation of respiratory expos-
ure in areas of high pesticide concentration is accurate only
for true gases, due to the probable lack of uniform dispersion
of particulate matter in the breathing zone. The aerodynamics
of respiration through the nostrils is difficult to duplicate
with an air sampler. Most sampling devices also will not
differentiate between particles that would be trapped in the
nasopharynx and the smaller respirable particles that reach
the lungs. However, a small cyclone sampler that separates
and discards nonrespirable particulates (above 10 ym in dia-
meter) is marketed by Mine Safety Appliances. The unit,
called the Gravimetric Dust Sampling Kit (MSA 456241), can be
attached to the collar or lapel and is designed to sample at
three calibrated flow rates (2.0, 1.8, and 1.6 liters/minute).
Respirable particulate matter collected in a filter cassette
may be analyzed for pesticide content. A separate vapor
trap (and pump) could be worn for comparative data.
2. Preparation and Handling of Samples
Pre- and post-treatment of sampling devices and analytical
procedures should be identical to those described in the
preceding sections. Special care should be exercised to avoid
contamination of samples in the field. Improper handling
of the collection module before or after the sampling period
could easily deposit a microgram of the material being moni-
tored (or interfering substance) on the sampling medium, which
would result in a false positive analysis of 1 yg/m3. There-
fore, the collection modules should be loaded in the laboratory
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Revised 12/15/79 Section 8, A
Page 16
and sealed in hexane-rinsed aluminum foil or a clean, sealed
glass jar before transport to the field. An analyst should
carefully install the sampling device and instruct the wearer
not to touch or disturb it. The analyst should be present at
the end of the sampling period to remove the module, place it
in a sealed container, and transport it back to the laboratory.
3. Sampling times should be commensurate with known or anticipated
exposure times. If potential exposure to airborne pesticides
is intermittent or brief, sampling should be performed only
during those periods. If exposure is continuously uniform
throughout the work day, sampling may be conducted for only a
portion of the day and the result extrapolated to estimate the
total exposure for the entire work period. If exposure is not
uniform but occurs for regular periodic cycles during the work
day, sampling should be conducted over the entire work day to
obtain an accumulated total exposure assessment. The moni-
toring program selected should, of course, be the result of
careful planning in order to provide a realistic assessment of
worker exposure.
4. Estimation of Inspired Quantities
Since the sampling rates achievable with small, battery oper-
ated pumps are substantially lower than the respiratory rates
of most workers, monitoring data must be extrapolated on the
basis of estimated lung ventilation values to obtain an assess-
ment of total exposure. Table 1 gives the average respiratory
rates and their normal ranges for men and women at rest and at
work. Values would be lower for children and elderly people.
Ideally, pulmonary function test (PFT) measurements should be
made on the worker while performing the job in order to deter-
mine the exact respiratory rate. Since PFT equipment and
personnel trained in its operation are not likely to be
available, a subjective estimation must be made of the breath-
ing rate if an approximation of the total quantity of pesticide
inspired is desired. To the untrained eye, it may sometimes be
difficult to differentiate between light and heavy work.
Estimates of average respiration rates likely to be encountered
among persons occupationally exposed to pesticides have been
made by H. R. Wolfe based on many visual observations over
many years. These estimates, which are given in Table 2, are
subjective but may be better than inexperienced judgments.
They should not be used a priori unless the data are appro-
priately qualified. Also, unless the sampler used can differ-
entiate between respirable and nonrespirable particulate
matter, it cannot be assumed that the quantity of pesticides
collected is proportional to the total inspired into the lungs.
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Revised 12/15/79 Section 8, A
Page 17
TABLE 1. NORMAL RESPIRATORY RATES FOR HUMANS
Respiratory Rates in Liters per Minute
Level of
Activity
Rest
Light Work
Heavy Work
Adult
Avg.
7
29
60
Male
Range
6-10
27-31
50-90
Adult
Avg.
4
16
24
Female
Range
4-7
16-17
17-32
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Revised 12/15/79 Section 8, A
Page 18
TABLE 2. SUBJECTIVE ESTIMATIONS OF RESPIRATORY
RATES FOR PESTICIDE WORKERS
Estimated
Respiratory Rate
Work Situation (L/min)
Agricultural Workers: Adu1t Adult
Male* Female**
Sprayer Using Hand Gun and Dragging Hose 50-67 22-25
Driver of Tractor Pulling Spray Equipment .... 18 10
Fruit Thinner or Picker 29-30 16
Flagger for Aircraft Spray Application 18 10
Pesticide Formulation Plant Workers:
Bagger (Filling small bags - 2 to 5 Ib.) 29-30 16
Bagger (Filling large bags - 50 Ib.) 32-33 17
Stacker (Stacks 50 Ib. bags or pallets) 33-42 17-20
Bagger & Stacker (Filling and stacking 50 Ib.
bags or pallets) 33-42 17-20
Boxer (Packing small bags into snapping boxes) . . 30-32 16-17
Fork Lift Operator 20 12
Mixer (Emptying bags of dry pesticide into
hopper for blending) 33 17
Worker Cleaning Inside of Hoppers and Bins .... 33 17
*Based on numerous visual observations and the respiratory rates
given in Table 1.
**Calculated as the percent of the male rate using data in Table 1.
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Revised 12/15/79
Section 8, A
Page 19
RAIN SHELTER
PARTICULATE
FILTER
SILICONS
GASKETS
FILTER SUPPORT
SCREEN
GLASS VAPOR TRAP
SORBENT
SUPPORT
SCREEN
METAL SAMPING
MODULE
SILICONS
GASKETS
1/2 INCH NIPPLE
MAGNEHELIC
GAUGE,0 100 IN
VARIABLE
TRANSFORMER
DICKSON
PRESSURE
RECORDER
Figure 1. EPA high volume ambient air sampler for pesticides, PCBs and
other organic ccsnpounds.
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Bevised 12A5/79
Section 8, A
Page 20
SORBENT
SUPPORT SCREEN
FILTER SUPPORT
SCREEN
30 40
SCALE, cm
Figure 2. The SURC Sampling Module, assembled (a) and disassembled (b). Part 1 is a 4-in x 2-in glass process
pipe reducer. Parts 2 and 3 are stainless steel pipe fittings with Teflon inserts. Part 4 is a
5.5-cm x 7.6-cm polyurethane foam cylinder and Part 5 is a Gelman Type A glass fiber filter (it is
installed under Part 2).
-------
Revised 12/15/79
Section 8fA
Page 21
Figure 3. EPA High-Volume Sampling Madule. (a) Sampling cartridge; (b) Assembled module containing cartridge
and prefilter; (c) Silicone rubber gaskets; (d) Glass fiber pre-filter; (e) Support Screen;
(f) Silicone rubber "0"-ring. Part 1 - Cartridge receptacle. Part 2 - Prefilter adapter.
-------
Revised 12/15/79
Section 8,A
Page 22
Cut
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Calibration plot for modified
-------
Revised 12/15/79
Section 8, A
Page 24
65 mm x 125 mm
GLASS
CYLINDER
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Figure 6. Dual sorbent vapor trap.
-------
to
-a co
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ua o
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ro o
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Figure 7. A high-volume air sampler developed for the U. S. Army by Environmental Research
Corporation (ERGO). Shown to the right are the composite filter pads used to trap
airborne pesticides.
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-------
Revised 12/15/79
Section 8, A
Page 27
a
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Figure 9. Personal sampling pump and three collection modules, (a) Polyurethane foam with particulate filter in
modified Teflon bottle, (b) polyurethane foam in glass holder, and (c) filter holder for dust collection only.
-------
Eevised 12/15/79
Section 8, A
Page 28
H
Figure 10.
D
Calibration unit for Personal Sampling Pumps. (Courtesy of Mine Safety Appliances)
(a) Manoneter, (b) soap bubble meter, one liter, (c) rubber bulb, (d) stop watch,
(e) needle valve, (f) pump being calibrated, (g) voltmeter, 0-10 V, and (h) soap
solution reservoir.
-------
Revised 12/15/79
Section 8, A
Page 29
D
CONSTANT FLOW SAMPLE CALIBRATOR
B
Figure 11. Calibration unit for DuPont Personal Sampling Pumps: (a) Soap bubble meter, 500 ml; (b) Soap
solution reservoir; (c) Flow rate meter; (d) Pressure drop meter; (e) Pump being calibrated.
-------
Revised 12/15/79 Section 8, B
Page 1
ANALYSIS OF PESTICIDES IN AIR
I. INTRODUCTION:
The analytical scheme described in this section presupposes
collection of the samples by one of the procedures described in
Section 8,A. The methodology replaces that reported in the last
revision of this Manual, which was based on collection of pesticides
in ethylene glycol.
REFERENCES:
1. Direct Chromatographic Determination of Carbamate Pesti-
cides Using Carbowax 20M-Modified Supports and the Electro-
lytic Conductivity Detector, Hall, R. C., and Harris,
E. E., J. Chromatogr. 169, 245-259 (1979).
2. Analysis of Pesticide Residues by Chemical Derivatization.
II. J^-Methylcarbamates in Natural Water and Soils, Coburn,
J. A., Ripley, B. D., and Chau, A. S. Y., J. Assoc. Off.
Anal. Chem. 5_9, 188-196 (1976).
3. Sources of Emissions of PCPs into the Ambient Atmosphere and
Indoor Air (EPA-600/4-78-022, March 1979), MacLeod, K. E.,
Analytical Chemistry Branch, U. S. EPA, ETD, HERL, Research
Triangle Park. NC.
4. Separation of PCBs, Chlordane, and £p_'-DDT from Toxaphene by
Silicic Acid Column Chromatograp-y, Bidleman, T. F.,
Matthews, J. R., Olney, C. E., and Rice, C. P., J. Assoc.
Off. Anal. Chem., 61_, 820-828.
5. A One-Step Method for the Determination of Carbamate Pesti-
cides by Derivatization with a-Bromo-2,3,4,5,6-Pentafluoro-
toluene (EPA-600/4-79-036, September, 1979), Jackson, M. D.,
Soileau, S. D., Sovocool, G. W., and Sachleben, S.,
Analytical Chemistry Branch, U. S. EPA, ETD, HERL, Research
Triangle Park, NC.
6. Evaluation of a Commercial Instrument for Chlordane and
Heptachlor Sampling (USAF-75M-12, August, 1975), Thomas,
T. C., and Jackson, J. W., Environmental Health Laboratory,
McClellan AFB, CA.
(See also references listed in Section 8,A)
-------
Revised 12/15/79 Section 8, B
Page 2
II. PRINCIPLE:
Sampling media are Soxhlet extracted with hexane-diethyl ether
(95:5 v/v). Chlorinated pesticides and PCPs are measured by EC GLC
after column chromatographic cleanup on alumina. PCBs are separated
from technical chlordane and other pesticides by column chromatography
on silicic acid deactivated with 3% distilled water.
III. EQUIPMENT:
1. Gas chromatograph, Tracer 222 or 560, equipped with linearized
63Ni FPD, and electrolytic conductivity detectors, or equivalent.
2. Rotary vacuum evaporator, e.g., Buchii, with 250, 500, and 1000
ml round bottom flasks.
3. Centrifuge tubes, 15 ml, graduated.
4. N-evap apparatus for evaporation of solvent under a gentle
nitrogen stream, Organomation Corp., Northborough, MA, with a
40°C water bath. '
5. Extractor, Soxhlet, 1000, 500, and 250 ml.
6. Separatory funnel, 500 ml.
7. Buchner filtration apparatus.
8. Cleanup microcolumn, 10 cm x 5 mm i.d. disposable pipet or
Chromaflex column, size 22, 20 cm x 7 mm, Kontes, Vineland,
NJ, K 420100-0022.
9. Chromatoflo chromatography column, 25 cm x 9 mm i.d.,
Pierce # 29020, equipped with a Teflon mesh support membrane,
Pierce # 29268, lower end plate, adapter, and 500 ml solvent
reservoir (Ace # 5824-10).
IV. REAGENTS:
1. Solvents, glass distilled, pesticide quality, or equivalent.
2. Diethyl ether, analytical reagent grade, Mallinckrodt # 0850,
containing 2% ethanol.
3. Pesticide standards and commercial PCB mixtures, 98-100% pure,
obtainable from the Pesticide Repository, U. S. EPA, ETD, HERL,
Research Triangle Park, NC (MD-69).
-------
Revised 12/15/79 Section 8, B
Page 3
4. Individual PCBs, obtainable from RFR Corp., Hope, RI.
5. Alumina, basic, 60 mesh, Alfa Products. Adjust to Brockmann
activity IV by adding 6% (w/w) distilled water to the adsorbent
in a flask, stoppering, and shaking well; allow to equilibrate
for at least 15 hours before use. Discard after two weeks.
6. Sylon CT (dimethyldichlorosilane in toluene), Supelco,
Bellefonte, PA.
7. Potassium hydroxide, analytical reagent grade; prepare hydrolysis
solution by dissolving 10 grams in 100 ml of methanol in a low
actinic flask; discard when discoloration first appears.
8. Sulfuric acid, analytical reagent grade, 50% aqueous solution.
9. Sodium sulfate, analytical reagent grade, Soxhlet extracted
with pesticide grade benzene and oven dried before use.
10. Potassium carbonate, analytical reagent grade.
11. Pentafluorobenzyl (PFB) bromide reagent, 1% (v/v); prepare by
dissolving 1 ml of reagent (Pierce Chemical Co., Rockford, IL,
No. 58220) or a-bromo-2,3,4,5,6-pentafluorotoluene (Aldrich
Chemical Co., Milwaukee, WI) in 100 ml of acetone in a low
actinic volumetric flask. Prepare fresh every 2-3 weeks.
Caution: the reagent is a strong lachrymator!
12. Nitrogen gas, dry, purified.
13. Silica gel, grade 950, Davison Chemical Co., Baltimore, MD,
deactivated by adding 1.5% (w/w) distilled water and mixing for
2 hours. Store in a tightly stoppered container in a desiccator.
14. Silicic acid, Mallinckrodt AR, 100 mesh; heat at 130°C for at
least 7 hours and cool to room temperature in a desiccator; to
deactivate, weigh into a bottle, add 3% (w/w) distilled water,
seal tightly, shake well, and place in a desiccator for at least
15 hours. Discard any adsorbent not used within one week.
V. EXTRACTION OF SAMPLING MODULE:
1. Place the sampling medium (Section 8,A) in a Soxhlet extractor,
handling with forceps rather than hands.
NOTE: After sampling, the glass fiber filters and foam plugs
should have been wrapped in aluminum foil until analysis.
Use plugs and filters carried to the field along with
those employed for sampling as controls.
-------
Revised 12/15/79 Section 8, B
Page 4
2. Extract with an appropriate volume of rv-hexane-acetone-diethyl
ether (47:47:6 v/v) for 16-24 hours at 4 cycles per hour for
the large Soxhlets and 8-12 hours at 8 cycles per hour for the
smaller Soxhlets.
NOTE: As examples, extract large foam plugs in 1000 ml Soxhlet
extractors with a total of 300-750 ml of solvent, and
smaller plugs and filters in 500 ml Soxhlets with
200-350 ml.
3. Remove the boiling flask to a rotary evaporator and reduce the
solvent volume to approximately 5 ml.
4. Transfer the concentrate to a 15 ml graduated centrifuge tube
with rinsing.
VI. DETERMINATION OF OC1 PESTICIDES AND PCBs:
1. Reduce the volume in the 15 ml tube to below 1 ml by careful
evaporation under a gentle stream of nitrogen at room tempera-
ture.
2. Carry out alumina cleanup as follows:
a. Place a small plug of preextracted glass wool in the
Chromaflex column and wash with 10 ml of hexane.
b. Pack the column with 10 cm of activity grade IV alumina.
c. Transfer the sample from the centrifuge tube to the top
of the column; rinse the tube three times with 1 ml
portions of n-hexane, adding each rinse to the column.
d. Elute the column at a rate of ca 0.5 ml per minute with
10 ml of rv-hexane, collecting the eluate in a 15 ml centri-
fuge tube.
e. Adjust the final volume of the eluate to 10 ml for gas
chromatographic analysis.
3. When necessary, separate PCBs from technical chlordane by
silicic acid chromatography as follows:
a. Place 3 grams of deactivated silicic acid in a Chromatoflo
column assembly.
b. Wash the column with hexane.
-------
Revised 12/15/79 Section 8, B
Page 5
c. Place the sample, concentrated to less than 1 ml, on
the column and add 130 ml of hexane to the reservoir.
d. Apply nitrogen pressure to the column to increase the
flow rate to ca 1 ml/minute.
e. Collect the eluate in three fractions: Fraction I (0-30 ml)
contains all the HCB and Aroclor 1254 and most of the
Aroclor 1242; Fraction II (31-50 ml) contains the remain-
der of Aroclor 1242, p_,£'-DDE, some of the p_,p_'-DDT and
toxaphene, and the early eluting peaks of technical
chlordane; Fraction III (51-130 ml) contains the remainder
of the technical chlordane, including all of the cis- and
trans-chlordane £,p_'-DDT, and 30% of the toxaphene.
f. Elute dieldrin, p_,p_'-DDD, 6% of the toxaphene, and the
remaining pesticides with 15 ml of dichloromethane.
g. Adjust the fraction volumes and analyze by GLC.
4. Blank values of unused plugs determined by extraction and
alumina cleanup of the extract should be equivalent to < 1 pg/m3.
VII. DETERMINATION OF OP PESTICIDES:
1. Adjust the final volume in the centrifuge tube as required.
2. Inject directly without cleanup into the gas chromatograph
equipped with an FPD detector.
NOTE: OP compounds are retained by the alumina column,
necessitating their analysis without this cleanup.
VIII. DETERMINATION OF CARBAMATE PESTICIDES:
1. Adjust the final volume in the centrifuge tube as required.
2. Inject directly into the gas chromatograph containing a 3%
OV-101/Ultra Bond 20M column and electrolytic conductivity
detector.
3. As an alternative to direct analysis, determine carbamates by
EC GLC after chemical derivatization with a-bromo-2,3,4,5,6-
pentafluorotoluene as follows:
a. Exchange the 5 ml of solvent in the concentrated extract in
the rotary evaporator flask (Subsection V,3) for methylene
chloride by careful evaporation just to dryness and re-
dissolving of the residue.
-------
Revised 12/15/79 Section 8, B
Page 6
b. Add 2 ml of 10% (w/v) methanolic potassium hydroxide to the
methylene chloride solution and hydrolyze at room tempera-
ture overnight.
NOTE: Hydrolyze and derivatize a mixture of standard
carbamates of interest in exactly the same way
in parallel with samples.
c. Transfer the hydrolysis solution, by washing with 50-60 ml
of distilled water, into a 500 ml separatory funnel and add
50 ml of methylene chloride.
d. Shake briefly and discard the methylene chloride.
e. Acidify to pH < 2 with ca 0.3-0.5 ml of 50% sulfuric acid.
f. Extract the hydrolysis solution with two 50 ml portions of
benzene, and dry the benzene by suction filtration through
a 10 gram sodium sulfate column into a 250 ml round bottom
flask.
g. Evaporate the benzene to 1-2 ml on a rotary evaporator with
a water bath at 40°C.
h. Transfer to a 15 ml centrifuge tube by rinsing with 5-6 ml
of acetone.
i. Add 25 yl of 5% aqueous potassium carbonate and 100 yl of
1% PFB bromide solution to the centrifuge tube.
j. Stopper, shake, and react at room temperature for at least
3 hours or at 60°C for 30 minutes (loosely stoppered if
heated).
NOTE: See Miscellaneous Note 1 for a similar derivatiza-
tion procedure combining hydrolysis and deriva-
tization into one step.
4. Cleanup and fractionation of carbamate derivatives:
a. Add 2 ml of isooctane to the derivatized solution and
evaporate to 1 ml in a 35-40°C water bath with dry nitrogen
gas.
b. Repeat the isooctane addition and evaporation to 1 ml.
c. Prepare a cleanup micro column by adding 1 gram of
deactivated silica gel to a disposable pipet or Chromaflex
column.
-------
Revised 12/15/79
Section 8, B
Page 7
d. Prewet the column with 5 ml of hexane, and place the
isooctane solution containing the derivatives into the
column when the level of the wash liquid just reaches top
of the bed.
e. Wash the centrifuge tube with 1 ml of hexane and add this
solution to the column.
f. Wash the column with 5 ml of hexane-benzene (95:5 v/v).
This fraction containing excess reagent is discarded.
g. Next elute the column in turn with 6 ml of hexane-benzene
(75:25 v/v) (Fraction I), 8 ml of hexane-benzene (25:75
v/v) (Fraction II), and 10 ml of pure benzene (Fraction
III), collecting each eluate in a clean centrifuge tube.
Each eluent is added after the previous one has just
reached the top level of the column.
NOTE: Determine the elution pattern of the PFB ether
derivatives of the carbamates of interest on the
silica gel column under local laboratory conditions.
The compounds studied by Coburn et al. eluted as
follows:
PFB ether
derivative
Fraction I
Recovery,
Fraction II
Fraction III
Propoxur
Carbofuran
3-Ketocarbofuran
Metmercapturon
Carbaryl
Mo bam
84-89
97-100
96-99
93-97
94-97
12-15
0-2
0-3
2-5
2-4
96-98
Obtained by comparing the peak areas of 5 samples
passed through the silica gel columns with 3
samples not fractionated.
h. Concentrate the eluate fractions as needed and analyze by
EC GLC.
-------
Revised 12/15/79 Section 8, B
Page 8
IX. GAS CHROMATOGRAPHY:
1. Determine OC1 and OP pesticides on a 183 cm x 4 mm i.d. glass
column packed with 1.5% OV-17/1.95% OV-210 and/or 4% SE-30/6%
OV-210 ono80-100 mesh Gas Chrom Q; column, 200°C; injection
port, 215°C; nitrogen carrier gas, 60-85 ml/minute; electron
capture detector for OC1 pesticides and P-mode FPD (200°C) for
OP pesticides.
2. Determine PCBs by EC GLC under the above conditions on a similar
column packed with 3% OV-1 on Gas Chrom Q at 180°C. Alterna-
tively, use columns containing 3% OV-225 on Supelcoport, 80-100
mesh or 4% SE-30/6% OV-210 on Gas Chrom Q, 100-200 mesh at
200°C.
3. Determine carbamates on a 103 cm x 2 mm i.e. silanized (Sylon
CT) glass column packed with 3% OV-101 on Ultra Bond 20M (RGC
005, RFR Corp., Hope, RI) [Section 4,A(7)H; column and injec-
tion port, 170-185°C; helium carrier gas, 25 ml/minute; N-mode
Hall electrolytic conductivity detector (Section 4,C):
reductive mode with nickel wire catalyst and strontium hydroxide
scrubber; conductivity solvent, water-isopropanol (85:15 v/v);
hydrogen reaction gas, flow rate 80 ml/minute; furnace tempera-
ture, 720°C; inlet temperature, 10°C above the column temper-
ature; transfer line, 200°C.
4. Determine carbamate PFB ether derivatives by EC GLC on a 183 cm
x 4 mm i.d. glass column containing 3% (w/w) OV-225 on 80-100
mesh Chromosorb W (HP). An alternate column for Fractions II
and III of the silica gel cleanup column is 3.6% (w/w) OV-101/
5.5% (w/w) OV-210 on acid washed, dimethyldichlorosilane
treated Chromosorb W. Use the following operating conditions:
injector temperature 205°C, column 190°C, detector 280°C;
5% methane-argon carrier gas flow rate 50 ml/minute + 20 ml/
minute purge for the OV-101/OV-210 column, 30 ml/minute + 20 ml/
minute purge for OV-225; EC detector in pulsed mode with
electrometer settings of 55 V2 90 psec pulse rate, 8 ysec
pulse width, and 6.4-1.6 x 10 9 amp full scale attentuation.
Relative retention times of PFB ether derivatives on the two
columns are as follows:
-------
Revised 12/15/79 Section 8, B
Page 9
RRT RRT ,
Derivative OV-101/OV-210 OV-225
Propozur 0.43 0.41
Carbofuran 0.64 0.63
3-Ketocarbofuran 1.15 1.13
Metmercapturon 1.26 1.28
Carbaryl 1.38 1.31
Mobam 1.48 1.31
a Relative to aldrin 2.7 minutes
Relative to aldrin 7.9 minutes
Quantitate by comparison of peak areas against chroma tograms
of derivatized standard carbamate phenols. The standard
derivatives are synthesized as follows:
a. React each carbamate phenol with a 10-fold molar excess of
PFB bromide in acetone and a 10-fold molar excess of
methanolic KOH.
b. Reflux for 2 to 3 hours, cool, and remove the solvent on
rotary evaporator.
c. Dissolve the product in benzene and wash the benzene twice
with equal volumes of 0.1 M
d. Dry the benzene, using suction, by passing it through a
10-20 gram column of anhydrous Na2S04.
e. Remove the benzene on a rotary evaporator and recrystallize
from hexane or methanol .
Inject 5 yl or another appropriate volume of the sample extract
or cleanup column eluate into the gas chroma tograph.
Record chromatograms under the above parameters and measure
retention times relative to aldrin or another suitable refer-
ence standard.
-------
Revised 12/15/79 Section 8, B
Page 10
7. Compare the relative retention time of each component of
interest against those of the corresponding primary standard.
8. Quantitate peaks in the usual way, i.e., by measuring peak
heights to the nearest mm when the base width is <1 cm or
via peak areas by integration or triangulation for broader peaks.
9. Confirm results as required by combined GLC/MS or some other
appropriate procedure (EPA Pesticide Analytical Quality Control
Manual, Chapter 8).
10. Commercial PCB mixtures are quantitated by comparisons of the
total heights or areas of GLC peaks with the corresponding
peaks in the standard used. The absolute retention times on
the 3% OV-1 column for the peaks used were as follows:
Aroclor 1242 - 2.39, 2.65, 3.11, 3.33, 3.94, 4.37, 4.67, 5.59,
and 6.25 minutes.
Aroclor 1254 - 3.81, 4.28, 4.61, 5.55, 6.68, 7.76, 8.23, 9.83,
11.47, and 13.67 minutes.
With the SE-30/OV-210 column, the total peak heights of the
peaks shown in Figure 1 can be used for quantitation.
Make Aroclor standards by dissolving the Aroclor in isooctane,
and prepare dilutions in hexane. Store stock solutions in brown
bottles at -10°C. Remake working standards periodically from
these and store in a refrigerator when not in use.
X. RECOVERY DATA:
Measurements made with six polychlorinated biphenyls using the
dual sorbent trap (Section 8,A) are shown in Table 1. The dichlor-
obiphenyl and one of the two trichlorobiphenyls tested appeared to
be more efficiently collected by all of the dual traps than by the
trap containing only PUF. Preferential vaporization of the more
volatile PCBs from the fortified felt pad (trace B, Figure 2) and
more efficient trapping of the less volatile PCBs by the PUF alone
were found to occur. Studies completed to date with a variety of
pesticides have been less conclusive. Essentially no differences were
observed between the sorbent systems for the eleven pesticides shown
in Table 2. Work is yet to be completed with a- and fc~-BHC and
several carbamates.
The ERCO high volume sampler has been shown to be efficient for
malathion collection and has been evaluated for heptachlor and
chlordane, methyl parathion from treated foliar survaces, diazinon
vapors, chlorpyrifos, and the carbamate propozur (Table 3). In all
-------
Revised 12/15/79 Section 8, B
Page 11
cases, collection efficiencies were determined over only short
sampling periods (2-4 hours). Due to the thinness of the filter pads
(3 mm), significantly longer sampling periods would be expected to
lead to potential breakthrough of sorbed vapors. Earlier collection
efficiency data for separate mixtures of OC1 and OP pesticides by
volatilization from a wool felt filter into a tandem pair of foam
plugs is shown in Tables 4 and 5.
The retentions of Aroclor 1242 and Aroclor 1254 on PUF after
4 hours at 4 liters/minute air flow are given in Table 6. This
determination was made by injecting the PCB mixtures in hexane solu-
tion into the foam, allowing it to air dry at ambient temperatures
for one hour, then placing it into module b_ (Section 8,A, Figure 3)
and pulling prefiltered air through it into a second PUF trap of the
same dimensions. Both traps were extracted after 4 hours to deter-
mine the amount of PCBs remaining in the first and that displaced to
the second trap. As expected, retentions were better than those
found for the same type PUF when exposed to 24 hours of air flow at
225 liters/minute in the high volume EPA sampler.
Collection efficiencies were measured by vaporizing known
quantities of the test compounds or mixtures into the PUF. These
data are presented in Table 7, along with comparative values for the
high volume EPA sampler. Again, the lower flow rates and shorter
sampling times appear to favor the low volume sampler. Gas chromato-
grams in Figure 3 show the selective volatilization of the lower
boiling components of technical chlordane from the vapor generator
and the nonuniform trapping of those vapors by the foam.
The efficiency of the recovery process was checked by spiking
foam plugs with Aroclor standards and then carrying them through the
sample extraction and cleanup procedure. A syringe or pipet was
used to fortify the small plugs with a known amount of Arochlor 1242
and Aroclor 1254 in hexane. After allowing the hexane to evaporate,
the plugs were extracted in the usual manner and the extracts were
concentrated, run through alumina, and analyzed. Four small
fortified plugs gave 77% ±11% recovery for the Aroclor 1242 and
100% ± 18% for Aroclor 1254 when spiked with 100 ng of each of these
standards.
In summary, polyurethane foam in both high and low volume air
samplers, used in conjunction with the analytical procedures described
in this section, serves well for the determination of low levels of
many PCBs and relatively nonvolatile pesticides in indoor and outdoor
air. The use of tandem traps does not always improve collection
efficiencies, despite this expectation.
-------
Revised 12/15/79 Section 8, B
Page 12
IX. MISCELLANEOUS NOTE:
An alternate one-step method for the determination of carbamate
pesticides by derivatization with a-bromo-2,3,4,5,6-pentafluorotoluene
has been devised by M. D. Jackson et^ aj_. (Reference 5). The pro-
cedure, which combines the alkaline hydrolysis and derivatization
steps, was tested on 23 carbamate pesticide standards, 18 of which
formed gas chromatographable derivatives using the standard EPA GLC
parameters. Selected products were studied by GLC MS, which indica-
ted that those carbamates hydrolyzing to give phenolic intermediates
formed derivatives with one fluorine on the PFB ring displaced by
an ethozide ion via aromatic nucleophilic substitution. The deriva-
tization procedure and GLC relative retention and sensitivity values
follow: Details of methods and further results (Linearity of EC
response, quantity of derivative to give 50% FSD, storage of deriva-
tives, background interferences, mass spectra) are given in the com-
plete report available from the above address.
Procedure
a. Pipet one ml of alcoholic potassium hydroxide, 0.1 ml of
derivatizing reagent, and one ml of carbamate standard into
a 15 ml culture tube with a Teflon lined screw cap.
b. Place the culture tube in a preheated (95 ±1°C) tube block
heater for two hours.
NOTE: The length of time and temperature are critical,
for overheating can cause an increase in the for-
mation of extraneous gas chromatographic peaks.
c. Remove, allow to cool at room temperature, and add 5 ml of
distilled water and 4 ml of jvhexane to the culture tube.
d. Place the culture tube on a tube rotator (60 rpm) for two
minutes and, at the end of this time, transfer the rv-hexane
layer to a 15 ml centrifuge tube.
e. Add an additional 4 ml of n_-hexane to the culture tube, and
place in the tube rotator for an additional two minutes.
f. Combine the r^-hexane layer with the previous ivhexane extract.
g. Bring the final volume of the centrifuge tube to 10 ml with
ri-hexane. The sample is now ready for further cleanup or gas
chromatographic analysis.
GLC Results - See Table 8
-------
vised 12/15/79
TABLt 1. HIGH-VOLUME COLLECTION EFFICIENCIES OF PCB CONGENERS
ON FOAM/GRANULAR SORBENT COMBINATIONS
Section 8, B
Page 13
% Collection on Foam/Sorhent Combinations
PCB
4,4'-di
?,4,5-tri
2,4',5-tri
2,2',5,51-tetra
2,2' ,4,5,5'-penla
2, 2', 4, 4' ,5,5'-hexa
Calc
Air Cone.
(ng/m3)
2-20
0.2-2
0.2-2
0.2-2
0.2-2
0.2-2
Foam
Alone
62
36
86
94
92
86
Chromo 102
(20/40)
82
80
81
81
79
84
After 24 hr. at 225
Porapak R
(50/80)
R2
87
89
88
92
92
L/im'n.
XAD-2
(16/50)
96
91
93
88
96
95
Tenax GC
(60/80)
85
-
80
81
84
85
Florisil
(16/30)
111
92
88
92
97
93
-------
Revised 12/15/79
Pesticide
Aldrin
p,p'-DDE
p,p'-DDT
Mirex
Tech. Chlordane
a-Chlordane
y-Chlordane
Diazinon
Methyl Parathion
Ethyl Parathion
Malathion
TABLE 2. HIGH-VOLUME COLLECTION EFFICIENCIES OF PESTICIDES ON
FOAM/GRANULAR SORRENT COMBINAF10NS
Calc Air
Cone.
(ng/m3)
0.3-3.0
0.6-6.0
1.8-18.0
1.8-18.0
15-150
1.5-15.0
1.5-15.0
3.0-30.0
1.8-18.0
3.6-36
0.9-9.0
Foam
Alone
?8
89
83
93
73
114
1?6
63
91
96
97
Section 8, B
Page 14
% Collection on Foam/Sorhent Combinations
After 24 hr. at 225 L/min.
Chromo 102 Porapak R XAO-2 Tenax GC Florisil
(20/40) (50/80) (16/20) (60/80) (16/30)
34
83
77
94
85
108
104
72
82
85
88
35
93
89
95
74
96
91
59
72
72
78
33
135
138
132
87
102
96
71
80
81
89
-
71
69
78
73
100
93
76
87
86
91
40
138
119
123
97
98
100
72
83
83
81
-------
Revised 12/15/79
Section 8,
Page 15
TABLE 3. ERCO SAMPLER COLLECTION EFFICIENCIES
AT 183 L/MIN. FOR 2 HRS.
Compound
Methyl Parathion
Diazinon
Chlorpyrifos
Propoxur
Chlordane
Heptachlor
Calc
Air Cone.
(yg/m3)
0.02-150
0.11-2.2
0.02-0.22
4.5
2.9-5.5
0.8-4.0
Collection
Efficiency
(%}
105
93
77
54
69-72*
66-98*
At 145-170 L/min. for 2 to 4 hrs. (Thomas & Jackson)
-------
Revised 12/15/79 Section 8, B
Page 16
TABLE 4. COLLECTION EFFICIENCIES OF POLYURETHANE FOAM
AT 225L/MIN FOR CHLORINATED PESTICIDES VS
AIR CONCENTRATIONS
Y-BHC
Aldrin
£,p>DDE
2., P.1 -DDT
Mi rex
Calc.
Air Cone.
(ng/m3)
0.15
0.08
0.03
1.50
0.30
0.06
3.10
0.60
0.30
0.10
9.20
1.84
0.92
0.37
9.20
0.60
0.30
0.12
Efficiency
(%)
53.2
38.4
55.0
58.5
35.3
50.2
95.7
96.2
104.8
101.0
114.7
94.6
93.6
83.0
103.7
100.4
98.7
105.4
Statistical
Data
n a
6
5
6
2
9
5
3
7
7
3
3
8
7
2
3
7
6
5
6.4
14.0
20.7
2.1
24.1
9.4
9.0
16.4
24.5
27.6
12.6
13.4
9.0
21.2
11.6
7.1
5.6
5.7
-------
Revised 12/15/79
Section 8, B
Page 17
TABLE 5. AVERAGE COLLECTION EFFICIENCIES OF POLYURETHANE
FOAM FOR ORGANOPHOSPHORUS PESTICIDES AT 225L/MIN
AND 184 L/MIN
Pesticide
Diazinon
Methyl parathion
Malathion
Parathion
Air
Volume
(m3)
326
265
265
326
265
265
326
265
265
326
265
265
Calc.
Air Cone.
(ng/m3)
30.7
18.9
3.8
18.4
11.3
2.3
36.8
22.6
4.5
9.2
5.7
1.1
% Collected
70.4
91.0
75.5
73.6
73.3
71.9
87.2
76.6
81.2
84.8
70.3
65.8
Statistical
Data
n a
5
6
6
5
5
4
5
5
4
5
5
4
3.97
16.19
14.40
4.56
6.52
4.12
33.40
12.47
14.68
4.15
4.70
3.75
-------
Revised 12/15/79
Section 8,
Page 18
TABLE 6. RETENTION OF PCB MIXTURES ON POLYURETHANE FOAM
Initial
Quantity
PCB Mixture (yg)
% of Original Recovered
After 4 hr. at 4 L/min
1st Plug* 2nd Plug
*Into which initial quantity was injected.
Total
Aroclor 1242
Aroclor 1254
2
2
87
99
9
0.4
96
99
TABLE 7. COMPARISON OF COLLECTION EFFICIENCIES OF
POLYURETHANE FOAM IN LOW- AND HIGH-VOLUME
SAMPLES
Compound
or
Mixture*
Aroclor 1242
Aroclor 1254
Aroclor 1260
tech. Chlordane
a-Chlordane
y-Chlordane
% Collected
4 hr. at
2.5 L/min
99
90
117
84
108
101
After:
24 hr. at
225 L/min
76
85
100
73
114
126
* Introduced as vapors. Calculated air concentrations for duration of
sampling periods were 1 to 2 yg/m3 for the low-volume sampler and
0.01 to 0.02 yg/m3 for the high-volume sampler.
-------
Revised 12/15/79
Pesticide
Section 8, B
Page 19
TABI E 8. r,AS CHROMATOGRAPHY OF CARBAMATE PFS1
Relative Retention Time
1 2
Derivatization
Linearity, yq"
Aldicarb
Aminocarb
Barban
Benthiocarb
Carbaryl
Carbofuran
CDEC
Chloropropham
Dismedipham
Formetanate-HCl
Karbutilate
Meobal
Methiocarb
Methomyl
Penmedipham
Promecarb
Propoxur
Thiophanate methyl
0.56
2.0
2.0
0.91
4.7
2.1
0.64
0.39, 2.9
2.8
1.7
2.5
1.2
3.7
0.37, 0.71
1.3e
1.8, 2.3e
2.8
1.4
1.2
3.0
0.60
1.9
3.0
1 .00
3.6
d -
0.62
0.406, 3.0
1.8
2.2
2.1
1.0, 1.2e
3.1
0.40
5e
1.5, 1.6e
1.8
1.4
1.2
3.3
0.25
1
1-100
1-100
100-1000
10-1000
1-1000
2-1000
0.1-1000
10-1000
100-1000
100-1000
100-1000
10-1000f
1-1000
1-100
100-1000
10-1000f
10-1000
io-ioof
PFBB DERIVATIVES
0.080
1.6
20
0.65
0.20
0.70
0.024
0.72
6.3
4.7
9.7
0.091
0.35
0.031
18
0.14
0.080
0.0087
0.040
0.49
33
0.48
0.18
d
0.024
0.72
8.4
4.1
9.9
0.096
0.30
0.026
14
0.14
0.081
0.012
Minimum Detection level, ng
1 2
a Relative to aldrin (1.0) on (1) 1.5X OV-17/1.95°/, OV-210 or (2) 4% SE-30/6% OV-210 on 80-100 mesh
Gas Chrom 0 at 220°C
b Checked over a concentration range from 0.1-1000 ng/ul, if possible, or to the limits of detection;
allowances of _* 157. were tolerated in determining the linearity range.
c 10%full scale deflection with 63Ni FC detector at 350°C, nitrogen carrier gas, 100 ml/minute,
columns (1) and (2) as in a
d Characterization was not attempted on the SF-30/OV-210 column due to high background interference
e Major peak
f Linearity could not he confirmed below 10 yq Hue to blank peak interference
-------
RELATIVE PEAK HEIGHT
a
H-
c.
en S
u> 3
o ;~i
"
O
fc'
C7i
^ f1
? I
5r
tr,
CJ1
N)
CO
o
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33 33 33
O O O
O O O.
O O O
33 33 33
N3 NJ NJ
O> 01 £»
p ^ JSJ
pop
in cji bi
o o o
CQ CQ CO
"O I/)
PJ 0)
tQ O
fD r+
«j.
ro o
o 3
oo
-------
Revised 12/15/79
Section 8,B
Page 21
x
10
12
14
IB
TIME/WIN.
Figure 2. Electron capture gas chromatograms showing collection of poly-
chlorinated biphenyls or polyurethane foam at 225 L/min. Trace
A is of the mixture of PCS congeners in n-hexane, B is of the resi-
due remaining on the wool felt pad ^fter 24 hr (at one-half the at-
tenuation) and C is the residue colL-ted by the foam.
-------
TECHNICAL CHLORDANE
STANDARD
RESIDUE IN GENERATOR
1
TRAPPED ON FOAM
Figure 3- Electron capture gas chromatograms showing selective vaporization of technical chlordane
and non-uniform trapping efficiencies of the components on polyurethane foam (after 4 hrs
at 4 L/min).
ji
CO
a
01
•^J
VO
-O
cu (D
-------
Revised 12/4/74 Section 9, A
Page 1
POLYCHLORINATED BIPHENYLS
INTRODUCTION
All chromatographers with experience in the analysis of biological
materials are only too familiar with problems involving "artifact"
peaks which, based on their retention characteristics, could be identi-
fied as aldrin, dieldrin, heptachlor, DDT or one of its metabolites
and/or some other common pesticides. Several years ago one series of
compounds came to light as a contributory source of a great deal of
this confusion. These were the polychlorinated biphenyls. The first
report of detection in the environment came from Sweden in 1966, and a
year later from the United States, despite the fact the materials have
been used for 40 years.
The prime manufacturer in the United States of these products is
the Monsanto Chemical Company. A series of PCB's have been marketed
under the trade name of Aroclor. A company bulletin listed many
products in which the materials could be used as plasticizers, flame
retardants, insulating fluids, or to impart some other useful quality.
Among these products were natural and synthetic rubber, electrical
products, floor tile, printer's ink, coatings for paper and fabric,
brake linings, auto body sealants, paints, varnishes, waxes, asphalt,
and many adhesives and resins. The PCB's were at one time recommended
by Monsanto for mixing with chlorinated insecticides to suppress their
vaporization and extend their persistence. At the time of this current
printing we understand that only two of the Aroclor compounds are being
produced.
Since the first U. S. environmental detection of the PCB's in
peregrine falcon eggs in 1967, their presence has been reported in
many segments of the environment. To mention a few, gull eggs in
San Francisco Bay, human milk in Colorado, human adipose tissues from
many parts of the U. S., fish from several areas, fresh and saltwater.
We are presenting some information on the PCB's in this manual
primarily to alert chromatographers to the ever potential presence
of these contaminants in routine samples, particularly of adipose
tissue. A method has been reported by Amour and Burke for separating
and PCB's from the common chlorinated pesticides. A reprint of this
method is included in Section 9,C. Typical chromatograms have been
obtained for five Aroclors, along with numerical data for relative
retention and response on the two working columns of the program.
These data are presented in Subsections 9,E and 9,F.
-------
Revised 12/4/74 Section 9, A
Page 2
Manipulation of the analytical procedures for PCB's is somewhat
more difficult than that of a number of the other methods in this
manual. However, competent residue chemists should experience no
sustained difficulty in coping with the procedures.
-------
Revised 6/77 Section 9, B, (1)
Page 1
DETERMINATION OF PCBs IN HUMAN MILK
MACRO METHOD
I. INTRODUCTION:
The analytical procedure described in this section was modified
and used in a survey, conducted by the Colorado Fnidemiologic
Pesticides Studies Laboratory, Colorado State University for measuring
the levels of PCBs in mother's milk. The survey was made under a
contract that the University had with the EPA Office of Pesticide
Programs. The method was evaluated in the EPA Analytical Chemistry
Branch, ETD, HERL, Research Triangle Park, and found to be satisfactory
for obtaining an approximation of PCB levels. It should be stressed
that the method, in common with other existing PCB analytical proced-
ures, provides only a semi-quantitative approximation for finger-
printing the multicomponent family of isomeric PCB compounds and is
not absolutely quantitative. The approximation levels of PCBs ob-
tained from the method must be interpreted with great care. Section
9,B,(2) presents alternative micro methods for analysis of mother's
milk as well as confirmation procedures for the PCBs.
REFERENCES:
1. Residues of Organochlorine Pesticides and Polychlorinated
Biphenyls and Autopsy Data for Bald Eagles, 1971-72,
Chromartie, E., et al., Pestic. Monit. J. 9_, 11 (1975).
2. Method for Separating PCB's from DDT and its Analogs,
Armour, J. A., and Burke, J. A., J. Assoc. Offic. Anal.
Chem. 53^, 761 (1970); see also Section 9,C.
II. PRINCIPLE:
The procedure consists of isolating the fat from the milk,
extracting PCBs from the fat, cleanup of the extract, and electron
capture GLC for determination. A weighed milk sample is extracted
with acetone and hexane, PCBs are transferred to the hexane layer by
adding sodium sulfate solution, and the hexane is dried by passage
through a sodium sulfate column. Part of the sample is used for a
lipid determination, and the rest is partitioned with acetonitrile and
then taken through a Florisil column fractionation. Identification and
quantification are done by GLC using an electron capture detector and
two columns with different resolution characteristics. Further confir-
mation of PCBs and pesticides can be obtained by GLC with electrolytic
-------
Revised 6/77 Section 9, B, (1)
Page 2
conductivity detector (Cl mode) and GLC MC of pooled samples.
III. SAMPLE COLLECTION:
1. Samples are manually expressed by participants into glass tubes
equipped with plastic screw caps and Teflon liners. The filled
tubes are kept frozen (-10°C) until the time of extraction.
2. Pertinent data must be obtained from donors by the hospital nurse
doing the sampling, and careful selection of donors must be made
by the field epidemiologist in charge of the program to achieve
the goals of any survey. Important data include age and geograph-
ical location of each donor, urban or rural location of the
hospital, and pesticide usage levels in the donor's area.
IV. EQUIPMENT:
1. Gas chromatograph, such as a Tracer 220 or equivalent, equipped
with 63Ni or3H electron capture detector. If desirable for
confirmation of residue identity, an electrolytic conductivity
detector can be used.
GLC columns, borosilicate glass, 183 cm x 4 mm i.d., packed with
1.5% OV-17/1.95% OV-210 and 4% SE-30/6% OV-210, both coated on
Gas Chrom Q, 80-100 mesh, operated with the specific parameters
given under Gas Chromatography, Section X. These packings are
available from most gas chromatography supply houses, e.g.,
Applied Science Laboratories, Inc., Supelco, Inc., etc.
2. Centrifuge bottles, 200 ml, with Teflon-lined screw caps, 3.8 cm
diameter.
3. Glass wool, Pyrex, precleaned by rinsing three times with
petroleum ether and acetone.
4. Centrifuge, capable of 2000 rpm.
5. Separatory funnels, 125 ml, 500 ml, and 1000 ml.
6. Chromatographic columns, 300 mm x 25 mm o.d., with Teflon stop-
cocks, with or without fitted glass plates, size 241, Kontes
420530.
7. Flasks, round bottom, short neck, Pyrex, 250 ml and 500 ml.
8. Kuderna-Danish (K-D) evaporative concentrator, 250 ml or 500 ml
flask, Kontes 57001; 3-ball Snyder column, Kontes 503000; 1/2 inch
steel springs, Kontes 662750; 10 ml graduated concentrator tubes,
size 1025, Kontes 570080.
-------
Revised 6/77 Section 9, B, (1)
Page 3
9. Modified micro-Snyder column, 19/22, Kontes K-569251.
10. Glass beads, 3 mm plan.
11. Disposable pipets.
12. Volumetric flasks, 100 ml.
13. Beakers, 50 ml, Griffin.
14. Pipets, 20 ml, class A.
15. Ovens, capable of regulation to 130°C and 37°C.
16. Evaporation apparatus utilizing a nitrogen gas stream.
17. Mixer producing a tumbling action at ca 50 rpm (Fisher Roto-Rack
or equivalent).
All glassware is cleaned according to Section 3,A.
V. REAGENTS:
1. Diethyl ether, AR grade, containing 2% ethanol, peroxide free,
Mallinckrodt 0850 or equivalent.
2. jvHexane, acetone, acetonitrile, and petroleum ether of
pesticide quality.
3. Eluting mixture, diethylether-hexane (6:94 v/v). Dilute 60 ml
of diethyl ether to 1000 ml with hexane. The solution should be
kept no longer than 24 hours after preparation.
4. Florisil, 60-100 mesh, PR grade, stored at 130°C until used
(see Sections 3,D and 5,A,(1), p. 3 for special comments).
5. Sodium sulfate, anhydrous, reagent grade, granular Mallinckrodt
8024, prewashed or Soxhlet-extracted with hexane prior to use.
Prepare sodium sulfate solutions with hexane-extracte deionized
water.
6. Silicic acid, SilicAR CC-4 Special, Mallinckrodt 7086, prewashed
and activated prior to use as described below in the method.
7. Filter paper, Whatman No. 1, rinsed in hexane. Check the final
background and Soxhlet extract the paper if required.
8. PCB and pesticide standards, obtained from the EPA repository,
HERL, ETD, Research Triangle Park, NC.
-------
Revised 6/77 Section 9, B, (1)
Page 4
VI. SAMPLE EXTRACTION:
1. Thaw and thoroughly mix the whole milk sample.
2. Weigh 4.5 to 24.3 g into a clean, dry centrifuge bottle.
3. Add enough pre-cleaned glass wool to adhere to the coarse precip-
itate of the milk solids.
4. Add 100 ml of redistilled acetone to the bottle, shake manually
for one minute, and then centrifuge at 1500 rpm for ca 2 minutes.
5. Transfer the acetone to a 500 ml separatory funnel, filtering
through Whatman No. 1 filter paper or prerinsed glass wool.
6. Extract the milk precipitate with two 25 ml portions of acetone,
shaking but not centrifuging.
7. Combine all three extracts in the 500 ml separatory funnel,
following the procedure in step 5.
8. Add 50 ml of hexane to the coarse precipitate of milk solids,
shake, centrifuge, decant, and combine with the acetone in the
500 ml separatory funnel. Repeat with 50 ml more of hexane.
9. Add 125 ml of 2% aqueous sodium sulfate solution to the 500 ml
separatory funnel.
10. Shake the funnel manually for 1 minute, allow the phases to
separate, and discard the lower (aqueous) layer.
11. Repeat steps 9 and 10, again discarding the lower layer.
12. Place 3 inches of sodium sulfate into a size 241 column. Wash
the column with 100 ml of hexane and discard the hexane. As
the last of the hexane wash just reaches the top of the sodium
sulfate, drain the hexane extract from the 500 ml separatory
funnel into the column. Allow this extract to sink into the
sodium sulfate; then add 100 ml of hexane to the column. Collect
all of the eluate in a clean, dry 250 ml concentrator flask.
13. Reduce the volume in the concentrator flask to ca 10 ml and
transfer quantitatively, using a clean disposable pipet, to a
100 ml volumetric flask. Dilute to volume with hexane.
14. Pipet a 20 ml aliquot, representing 1/5 of the original milk
sample, from the flask into a clean, dry 50 ml beaker. Evaporate
the solvent under a nitrogen stream and place the beaker in a
37°C oven overnight for a lipid determination. (Caution: Remove
-------
Revised 6/77 Section 9, B, (1)
Page 5
all hexane before placing beaker in oven.)
NOTE: See Miscellaneous Note 1 for the determination and
calculation of lipids.
15. Concentrate the remaining 80 ml of sample and transfer quan-
titatively to a 125 ml seperator funnel. Adjust the volume of
hexane to 15 ml.
VII. LIQUID-LIQUID PARTITIONING:
1. Add 30 ml of acetonitrile, previously saturated with hexane,
to the 125 ml separatory funnel and shake vigorously for
2 minutes.
2. After phase separation, draw off the acetonitrile layer into a
1 L separatory funnel containing 550 ml of 2% sodium sulfate
solution and 100 ml of hexane.
3. Repeat extraction of the hexane layer in the 125 ml funnel three
more times in a similar way, combining all acetonitrile extracts
in the 1 L funnel.
4. Stopper the 1 L funnel, invert, vent off pressure, and shake
for 2 minutes, releasing pressure periodically as required.
Allow the phases to separate and discard the lower (aqueous)
phase.
5. Wash the hexane phase with two additional 100 ml portions of
2% sodium sulfate solution, discarding the aqueous washings.
6. Transfer the hexane layer to a K-D evaporator.
7. Attach a 3-ball Snyder column over the evaporator and place in
a water bath at 90-100°C. Approximately 4 cm of the concentrator
tube should be below the water surface.
8. Concentrate the extract to ca 5 ml, and rinse down the sides
of the evaporator and ground glass joint with a total of 3 ml
of hexane.
9. Reconcentrate to ca 5 ml under a gentle stream of nitrogen
at room temperature.
-------
Revised 6/77 Section 9, B, (1)
Page 6
VIII. FLORISIL COLUMN FRACTIONATION:
1. Prepare a chromatographic column containing 10 cm (after
settling) of activated Florisil topped by 4 cm of sodium
sulfate. Place a small wad of class wool at the bottom of
the column to plug the glass tube and retain the adsorbent.
NOTE: The small amount of Florisil needed for proper
elution should be determined for each different
lot by elution of analytical standards.
2. Prewash the column with 100-200 ml of hexane, and discard.
NOTE: From this point on through the elution process, the
solvent level should never be allowed to fall
below the top surface of the sodium sulfate layer.
If air is introduced into the column, channeling may
occur, causing an inefficient column. Each solution
is added to the column just as the previous one reaches
the top of the bed.
3. Immediately transfer the ca 5 ml of extract from the evaporator
tube onto the column, using a 5 ml Mohr pipet or long dispos-
able pipet. Allow the sample to sink into the column.
4. Rinse the evaporator tube with two successive 5 ml portions of
petroleum ether, carefully transferring each portion to the
column with the pipet.
NOTE: Delivery of the extract by pipet directly onto the
column precludes the need to rinse down the inside
column walls.
5. Prepare a complete 500 ml K-D evaporative assembly with 10 ml
concentrator tube. Place one glass bead in the tube.
6. Commence elution with 200 ml of the 6% eluting mixture at a
rate of 5 ml per minute, collecting the eluate in the K-D
assembly. After collection of the fraction, concentrate as in
Subsection VII, step 7.
NOTE: If determination of more polar chlorinated pesti-
cides in addition to PCBs is desired, place a second
500 ml K-D assembly under the column and continue
elution with 200 ml of diethyl ether-petroleum ether
(15:85 v/v). Pesticides eluting in these fractions
are listed in Table 1, Section 5,A,(1).
-------
Revised 6/77 Section 9, B, (1)
Page 7
7. Remove the K-D assembly from the bath and cool to ambient
temperature.
8. Disconnect the collection tube from the D-D flask and care-
fully rinse the joint with a small amount of hexane.
9. Attach a modified micro-Snyder column to the collection tube,
place back in the water bath, and concentrate the solution to
1 ml.
NOTE: If preferred, this concentration can be done at
room temperature under a gentle nitrogen stream.
10. Remove the tube from the bath and cool to ambient temperature.
Disconnect the tube and rinse the joint with a little hexane.
IX. SILICIC ACID COLUMN FRACTIONATIQN:
Silicic acid is used for separating PCBs from DDT and some of
its analogs also present in the Florisil eluate. The method is a
modification by Cromartie et al. (1) of the Armour and Burke
(2) procedure, which eliminates use of Celite and air pressure to
speed the column elution.
1. Prepare the adsorbent as follows:
a. Prewash SilicAR CC-4 three times with acetonitrile-hexane-
methylene chloride (1:19:80 v/v). Approximately 320 ml
of wash solution is used for each 125 g of silicic acid.
b. Activate the washed adsorbent in a 130°C oven for 24 hours.
c. Remove the adsorbent from the oven, transfer to a
stoppered Erlenmeyer flask, and cool to room temperature.
d. After cooling, add enough distilled water to a weighed
portion of adsorbent to give a 3% deactivated material
(3 ml water per 100 g silicic acid). Stopper at once.
e. Shake the flask for 1 hour on a mechanical shaker, and
then allow an additonal 30 minutes of equilibration before
use.
2. Pack the column as follows:
a. Place 20 g of deactivated silicic acid in a beaker and
add enough hexane to form a slurry.
-------
Revised 6/77 Section 9, B, (1)
Page 8
b. Pour the slurry into a pre-rinsed size 241 glass column
to which 13 mm of anhydrous sodium sulfate has been added.
c. Allow the silicic acid to settle and add 13 mm of sodium
sulfate on top to prevent the surface of the acid column
from being disturbed when applying the sample.
d. Rinse the column with 50 ml of petroleum ether, discarding
the eluate.
3. When the last of the wash just reaches the top of the bed, trans-
fer the sample from the collection tube onto the column using
a disposable pipet.
NOTE: Observe the same precaution as in the note under
step 2 of the preceding subsection.
4. After the sample has entered the column, elute with 400 ml
of petroleum ether at a flow rate of 5 ml per minute.
5. Collect the effluent in two separate fractions. The first
100 ml is Fraction I, in which hexachlorobenzene, mirex, and
some other chlorinated pesticides are collected. The remaining
300 ml is Fraction II, which contains PCBs and most of the DDE.
6. Concentrate Fraction II to a suitable definite volume for
analysis of PCBs. Fraction I is not analyzed unless it is
suspected that some of the PCBs may be eluting in it or if
determination of the pesticides in Fraction I is of interest.
NOTE: It is wise to spotcheck Fraction I for the presence
of PCBs from time to time. The performance of each
batch of silicic acid can be evaluated by elution of
standard PCBs and pesticides through a column, as will
be done for analyses.
X. GAS CHROMATOGRAPHY:
1. The extent of concentration (or dilution) of the eluate is
dependent on the PCB concentration in the sample being analyzed
and the sensitivity and linear range of the EC detector being
used. Further concentration will be required for detection
with an electrolytic conductivity detector.
2. All samples should be chromatographed on at least two different
GLC columns with EC detection to enhance the qualitative
aspects of the determination.
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Revised 6/77 Section 9, B, (1)
Page 9
NOTE: Further confirmation can be obtained by pooling
the 6% Florisil eluates and performing GLC with mass
spectroscopy. Pooled samples can also be confirmed
by electrolytic conductivity (Cl mode) detection for
the presence of PCBs as well as chlorinated pesticides.
3. Primary identification and quantification of PCBs is based on
the calculation of all peaks and comparison to an Aroclor 1254
standard. If there is evidence that erroneous data may result
from the interference of extraneous compounds, use for quanti-
fication only those peaks in the sample chromatogram that are
free of interferences. Compare these to the corresponding peaks
in the standard chromatogram of Aroclor 1254 (see Section XII
and Miscellaneous Note 2).
NOTE: It is likely that standard Aroclor 1254 will yield a
chromatogram most closely resembling the array of peaks
observed in actual samples. If the sample matches the
chromatogram of another Aroclor standard more closely,
this compound should be used for quantitative comparisons.
See Section 9,E for typical chromatograms of different
PCBs.
4. Operating parameters for electron capture GLC are:
Temperatures - injector 220-225°C
columns 200°C
detector 250-300°C (53Ni) or
205-210°C (3H)
Carrier gas - highly purified nitrogen
Flow rates - 60-80 ml per minute for OV-17/OV-210
100-120 ml per minute for SE-30/OV-210
(at 40 psig)
For the electrolytic conductivity detector the temperature
parameters are:
injector 245°C
columns 200°C
furnace 820°C
block 230°C
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Revised 6/77 Section 9, B, (1)
Page 10
XI. SENSITIVITY AND RECOVERY RESULTS:
The 6LC sensitivity limit for PCBs is 20 ppb theoretical and
50 ppb practical based on the whole milk sample weight.
The 50 ppb sensitivity limit was determined in the following
manner: Based on the sensitivity of the instrument involved in the
analyses and the assumption that background interference was absent,
the theoretical detection limit of the method was established as
22 ppb. The average background interference exhibited by the reagent
blanks was then added to the theoretical detection limit to give a
value of 33 ppb. As a final safety margin, the practical detection
limit was established as 50 ppb. Any lower values encountered in
the actual samples are reported as "trace."
Three quality assurance samples and a blank (control) were pre-
pared using goat's milk and were analyzed by the procedure at the
Colorado State University Laboratory and also at a second laboratory.
The fortification levels and average recoveries from at least
duplicate analyses on a whole milk basis were as follows:
Sample Content ppb PCBs Reported (and % Recovery)
Colorado Laboratory Laboratory 2
1. Aroclor 1254, 200 ppb 184 (92%)137 (68%)
2. Aroclor 1254, 150 ppb 121 (81%) 92 (61%)
3. HCB, 50 ppb; heptachlor
epoxide, 60 ppb; p_,p_'-DDT,
100 ppb; £,p_'-DDE, 80 ppb;
PCBs, none 0 not analyzed
4. Control 2 14
XII. DISCUSSION OF THE METHOD AND RESULTS:
The average recovery of the Colorado lab shown in the above
tabulation for the two PCB samples was 87%, compared to 65% for
the second laboratory. The higher recovery results were not
unexpected because of the greater familiarity of the Colorado group
with their own method. The results of the two laboratories on
these split samples are considered to be quite comparable and indi-
cate that the procedure is useful for approximating the level of
PCBs in human milk. If known, fortified samples are analyzed with
each batch of actual samples; the percent recovery of the samples
can be corrected for the recovery of the spikes (see Section XIV,4).
The approximation levels of PCBs reported above were referenced
to a commercial standard of Aroclor 1254. Due to the many early
eluting gas chromatographic peaks in the reagent blanks and the partial
carry-over of p_,p_'-DDE into the PCB fraction, a maximum of only five
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Revised 6/77 Section 9, B, (1)
Page 11
out of a total of thirteen GLC peaks for the Aroclor 1254 standard
obtained for these chromatograms using the specified packed columns,
could be used for the quantifications. In many cases, only the last
three eluting GLC peaks could be used in these fingerprinting methods.
The GLC traces generated with the electron capture detector and
the two specified GLC columns for the milk samples prepared by the
macro Colorado method (and the micro procedure in Section 9,B,(2)}
resembled in appearance only the commercial Aroclor 1254 standard.
On the contrary, as confirmed by the mass spectrometric analyses, the
human milk samples contained a higher percentage of the hexachloro-
biphenyl isomers than did the standard Aroclor 1254. The fingerprint
chroma togram of each extract resembled the reference standard of
Aroclor 1254, but the sample extracts did not contain the equivalent
isomeric PCBs. The present inadequacies of the "State of the art"
methods using packed GLC columns are the inability to separate and
to reference the individual isomers present in the sample extract.
As noted earlier, this method can provide only a semi -quantitative
approximation of PCB amounts. Mathematical conversions of such
approximation levels of PCBs on a whole milk basis to those on a
total fat basis yield numbers with little analytical significance.
Extreme care must be applied in considering such approximation
levels as indicators of the absolute identity and quantity of PCBs
present in human milk.
XIII. MISCELLANEOUS NOTES:
1. The percentage of lipids is calculated as follows: The beaker
should be weighed before adding the 20 ml aliquot of sample
and again after the hexane has evaporated, leaving the lipid
in the beaker. The difference in weight is the weight of the
lipid. The weight of the lipid is then divided by the weight
of sample in the 20 ml portion to give percent lipid. For a
7.0 g sample, the 20 ml portion would contain 1.4 g sample,
and the remaining 5.6 g would be used for PCB analysis.
Example: Lipid + Beaker: 27.3937 g
Dry Beaker: 27.3652 g
Lipid: 0.0285 g
Total milk sample = 7.00 g ?n n
Sample in 20 ml = 7.00 x - 1.40 g
= 2.04% lipid.
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Revised 6/77 Section 9, B, (1)
Page 12
2. An example of the calculation of ppb PCBs on a lipid basis
is as follows:
Original milk sample = 7.00 g
Milk sample carried through PCB analysis = 7.00 x ^fp =5.60g
Final sample volume = 5.00 ml
Volume injected for GLC - 5.00 pi _.,.__
Whole milk sample injected = 5.60 x ^^ = 5.60 mg
Sample peak height(s) (mm) x pg/mm from standard =
pg in sample peak(s)
pg in sample peak(s) , n~n . , .,, ,
5.60 mg sample = ppb PCB on whole milk basis
ppb PCB on whole milk basis x ^^ = ppb PCB on lipid basis
It should be understood that this conversion increases the
numerical value but not the analytical significance of the
results. An analytically insignificant approximation level
of 30-80 ppb of PCBs on a whole milk basis becomes 50 times
as great or 1.5 to 4.0 ppm (assuming 2% lipid as above) on
a total fat basis. Values at this or lower levels have
no significant analytical meaning.
XIV. ANALYTICAL QUALITY CONTROL:
1. If the procedure is being used in a monitoring program, the
thoroughness of the personnel collecting the samples in obtaining
pertinent data from the donors should be checked periodically.
If necessary, further training should be provided.
2. Likely sources of contamination leading to a high reagent blank
are the filter paper, glass wool, and sodium sulfate required
by the method. These should be thoroughly precleaned with
pesticide grade solvents as directed under Subsections IV and V.
3. Each sample analyzed required a total volume of ca 2000 ml of
solvent. Care must be utilized in concentration of such a large
volume to the final 1-5 ml volume for analysis.
4. A suggested in-house QC program involves running one blank and
one fortified sample for every set of 10 human milk samples.
a. The reagent blank is carried through the entire procedure
without addition of milk. Any background interferences
displayed by the blank are subtracted from the levels
observed in the set of human milk samples.
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Revised 6/77 Section 9, B, (1)
Page 13
b. The spiked sample was prepared from 7.0 g aliquots of
goat's milk that had been stored in a frozen state at
-10°C. Prior to analysis, the aliquot was thawed in a
40°C water bath and spiked with 1.0 ml of an acetone
solution containing 200 ng per ml of Aroclor 1254. On
a whole milk basis, this represented a PCB concentration
= 28'6 ppb"
c. The spiked sample is taken through the whole procedure.
d. Typical average recovery from the spiked goat's milk has
been 77%, and the average amount of background PCBs has
been 78 ng, which is equivalent to 11.0 ppb for a 7.00 g
milk sample.
e. Recoveries of the set of actual samples can be corrected
for the 77% average recovery from the spiked milk by
multiplying each individual recovery by JJ—Q •
f. At irregular intervals, a goat's milk control sample is
analyzed in addition to the reagent blank and spike.
Any detectable levels of PCBs would be subtracted from
the spiked sample before calculating its recovery level.
5. If an outside, independent source of spiked PCB reference
material (SPRM) is available to the laboratory using the
procedure, these SPRMs should be used as blind QC checks on
analytical performance.
6. Before handling actual samples, laboratory personnel should be
guided through the method at least four times by an experienced
worker. This should involve analyzing a duplicate of a sample
that had already been analyzed by the experienced worker. If
the results are acceptable at this point, the individual is
allowed to do an additional set of four spiked samples in
duplicate without the aid of the experienced worker. After
demonstrating adequate results, the individual should be
proficient enough to handle actual samples.
-------
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Revised 6/77 Section 9, B, (2)
Page 1
. DETERMINATION OF PCBs IN HUMAN MILK
MICRO METHODS
I. INTRODUCTION:
This section describes alternative methods for the analysis
of PCBs in human milk developed by the Analytical Chemistry Branch
of the EPA Environmental Toxicology Division, Health Effects
Research Laboratory, Office of Research and Development, at Research
Triangle Park, NC, to complement and confirm the macro procedure in
Section 9,B,(1). The procedure utilizes chromatography on a micro
silicic acid column to further resolve PCBs. As with the method in
Section 9,C,(1) these methods are not capable of accurately
identifying and quantifying absolute levels of PCBs, but provide semi-
quantitative results.
II. PRINCIPLE:
A 0.5 g milk sample is extracted with acetonitrile, residues
are partitioned into hexane, and the hexane is concentrated and
eluted through a micro Florisil fractionation/cleanup column. The
eluate fraction containing PCBs is concentrated and eluted through
a micro silicic acid column for further separation of PCBs and
chlorinated pesticides. The column eluate containing PCBs is con-
centrated and analyzed by GLC with electron capture detection.
A perchlorination method can be used to confirm the presence and
amounts of PCBs.
III. EQUIPMENT AND REAGENTS:
Since this procedure is a combination of methods described in
Sections 5,A,(2),(a) and 9,B,(1), readers should refer to the
respective subsections on EQUIPMENT and REAGENTS in these Sections.
Additional items only will be listed here.
1. a. Centrifuge tube, 40 ml, Corning 8122.
b. Caps, molded screw, with Teflon liners, size 24-410
(Corning 9999).
NOTE: Do not use caps which come with Corning 8122
tubes. The rubber liners may contain contaminants.
2. Centrifuge tube, 15 ml.
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Revised 6/77 Section 9, B, (2)
Page 2
IV. SAMPLE EXTRACTION:
1. Extract a 500 mg sample of milk with three 2.5 ml portions of
acetonitrile in a tissue grinder, and centrifuge at 2000 rpm
after each extraction in the grinder.
NOTES:
(1) A reagent blank and a fortified sample are started at
this point and run through the complete procedure.
(2) Consult Notes 1 and 2 in Section 5,A,(2),(a),VII.
2. Combine the supernatants in a 40 ml screw cap centrifuge tube.
3. Add 25 ml of 2% aqueous sodium sulfate solution to the combined
supernatants and mix on a Vortex mixer.
4. Extract the aqueous acetonitrile mixture in turn with one
5 ml and two 2 ml portions of hexane.
5. Transfer each extract with a disposable pipet and combine in
a 15 ml centrifuge tube.
6. Concentrate the hexane solution to a volume of 300-500 yl under
a gentle stream of nitrogen.
V. FLORISIL FRACTIONATION:
1. Prepare, prewash, and activate a micro Mills Florisil column
according to Section 5,A,(2),(a),111.
2. Remove the column from the oven and allow to cool to room
temperature.
3. Prewet the column with 10 ml of hexane and discard the eluate.
NOTE: From this point on, the solvent level should never
be allowed to drop below the top of the bed or
channeling may occur and degrade the resolution.
4. Transfer the sample onto the column with a disposable pipet and
begin collection of the eluate in a 25 ml concentrator tube.
5. Rinse the sample tube with two 0.5 ml portions of hexane, added
with a disposable pipet, and transfer each rinse to the column
with the same disposable pipet.
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Revised 6/77 Section 9, B, (2)
Page 3
6. Elute the column with an additional 10.5 ml of hexane followed
by 12 ml of methanol-hexane (1:99 v/v). All 24 ml are collected
in the same concentrator tube and represent Fraction I.
NOTE: Fraction I contains PCBs and those pesticides listed
in this fraction in Table 1, Section 5,A,(2),(a).
7. Elute the column with an additional 12 ml of methanol-hexane
(1:99 v/v), collecting the eluate in a second concentrator tube.
NOTE: This fraction contains heptachlor epoxide and may be
checked for any overlap of PCBs. See Table 1, Section
5,A,(2),(a) for the elution pattern of other pesticides
from the micro Florisil column.
8. Concentrate Fraction I to 300-500 yl under a gentle stream of
nitrogen.
VI. SILICIC ACID FRACTIONATION:
1. Prepare 3% deactivated silicic acid as described in Section
9,B,(1),IX, and prepare a column as follows:
a. Place one gram in a small beaker and add enough hexane
to form a slurry.
b. Add a small glass wool plug and 10 mm of anhydrous sodium
sulfate into a prewashed Chromaflex column, and then pour
in the slurry.
NOTE: Do not allow the solvent level to go below the
top of the column bed.
c. Add another 15 mm of anhydrous sodium sulfate after the
silicid acid has settled.
2. Rinse the column with 10 ml of petroleum ether, discarding
the eluate.
3. As soon as the last of the rinse reaches the top of the column,
transfer the sample onto the column with a disposable pipet and
start collecting the effluent in a 15 ml centrifuge tube.
4. Rinse the sample tube with two 0.5 ml portions of petroleum
ether, and add the rinses to the column using the same disposable
pipet.
5. Collect a total of 4 ml of petroleum ether for Fraction I.
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Revised 6/77 Section 9, B, (2)
Page 4
6. Change the collection tube and continue elution until a total
of 10 ml of petroleum ether is collected (Fraction II).
NOTE: Fraction I will contain hexachlorobenzene while
Fraction II will contain PCBs and some DDE.
7. Concentrate Fraction II under a gentle stream of nitrogen to
a suitable volume for EC GLC.
VII. GAS CHROMATOGRAPHY:
Proceed with electron capture gas chromatography following
the general guidelines set forth in Section 4,A,(4) and the specific
parameters and procedures in Section 9,B,(1),X. The volumes of
sample and standards injected and degree of eluate concentration must
reflect the smaller sample size originally taken for the micro method
compared to the macro method. Calculations of results are made in
a manner similar to that explained in Section 9,B,(1), Miscellaneous
Notes 1 and 2. If results are to be reported on a total lipid basis,
a separate milk sample is taken for the determination of percentage
lipids.
VIII. RESULTS AND DISCUSSION:
Results obtained by the Research Triangle Park Analytical
Chemistry Branch with the micro silicic acid procedure on the
same fortified goat's milk samples cited in Section 9,B,(1),XI were
162 ppb for the 200 ppb sample (81% recovery) and 139 ppb for the
150 ppb sample (93% recovery). The control yielded 31 ppb PCBs
(all results are averages of at least two analyses). In general,
the micro method yielded somewhat higher recovery values than did
the Colorado macro method for a series of samples analyzed for both.
For 10 samples, the macro method gave an average of 66 ppm of PCBs
(18-184 ppb range) and the micro method 103 ppb (46-243 ppb range).
All results were higher by the latter method except for one sample,
which gave the same results by both methods. These results were
substantiated by high resolution mass spectroscopic analysis of
samples cleaned up by both the macro and micro methods.
Because of the lesser quantities of reagents and smaller glass-
ware required, the reagent blank problem was in general not as
great with the micro method. The time required for preparing a set
of samples with the micro method was considerably less than for
the macro method. However, confirmational analyses of the micro
method extracts were limited owing to the small sample size (see
Miscellaneous Note 1). The micro method seemed to respond, although
with wide degrees of variations, to lower levels than did the
macro method.
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Revised 12/15/79 Section 9, B, (2)
Page 5
It must be emphasized again that the micro and macro methods
are both semi-quantitative, yielding approximate PCB values.
Extreme care must be applied in considering such approximation
levels as indicators of the absolute identity and quantity of
PCBs in human milk.
IX. MISCELLANEOUS NOTES:
1. Chemical derivatization by perchlorination to yield decachloro-
biphenyl (DCB) followed by EC GLC has been used to confirm PCBs
in selected samples of human milk which had been cleaned up by
either the macro or micro methods. The procedure, as reported
by Crist, H. L. and Moseman, R. F., in J. Assoc. Off. Analy.
Chem., 6£, 1277 (1977), follows:
a. Transfer a cleaned-up sample equivalent to as much as 500 mg
of milk to a 1.5 ml glass tube fitted with a Teflon screw
cap (Mini-Actor, Applied Science Laboratories). Standards
and blanks should be run with each set of samples.
b. Evaporate the solvent to 0.02-0.03 ml under a gentle
nitrogen stream. Add ca 0.25 ml of chloroform and
evaporate again to 0.02-0.03 ml. Repeat chloroform
addition and evaporation twice more to ensure complete
removal of hydrocarbon solvent.
NOTES:
1. Hydrocarbon solvents react with SbCl5 to form a
carbonaceous mass.
2. Avoid complete dryness of the sample to prevent
loss of PCBs with a lower degree of chlorination.
c. Add by pipet exactly 0.2 ml of antimony pentachloride
(J. T. Baker).
d. Tightly cap the tube and place in a sand bath at 160-170°C
for 16 hours.
e. Remove the tube, allow to cool, and carefully remove the
cap. Add ca 0.5 ml of 6 M hydrochloric acid to the tube
to deactivate excess reagent.
f. Transfer the mixture to a 15 ml centrifuge tube with a
disposable Pasteur pipet.
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Revised 12/15/79 Section 9, B, (2)
Page 6
g. Rinse the reaction tube with an additional 0.5 ml of the
HC1 and three 0.5 ml portions of hexane, transferring all
rinses to the centrifuge tube with the same disposable
pipet. Rinse the reaction tube cap with several drops of
acid and hexane in the same manner to complete transfer
of the sample.
h. Extract the DCB from the acid phase into the hexane by
mixing for 30 seconds on a Vortex mixer.
i. After phase separation, transfer the hexane to a clean
15 ml centrifuge tube using a new disposable pipet. Repeat
the extraction twice with 1.0 ml portions of hexane.
j. Wash the combined hexane extracts with 1.0 ml of distilled
water for 30 seconds, followed by 1.0 ml of 10% aqueous
sodium bicarbonate solution. Discard both aqueous wash
phases.
k. Reduce the hexane volume to ca 0.25 ml under a gentle
nitrogen stream.
1. Remove a prewashed and activated micro Florisil column
from the oven (Section 5,A,(2),(a),111) and let cool.
m. Prewet the column with 10 ml of hexane. After draining
the wash solvent to the top of the bed, transfer the sample
quantitatively to the column with a disposable pipet,
rinsing with two 1 ml portions of hexane and collecting the
eluate in a 15 ml centrifuge tube.
n. Elute DCB from the column with a total of 7 ml of hexane.
NOTE: Elution patterns for DCB should be verified by
the analyst to take into account variation in
Florisil activity.
o. Reduce the eluate to an appropriate volume.
p. Inject a sample into an electron capture gas chromatograph
fitted with a 1 m x 4 mm i.d. column of 5% OV-210 at
200°C and a carrier gas flow rate of 60 ml per minute. DCB
has a retention time of ca 8 minutes under these conditions.
q. Determine the amount of DCB in the sample. Convert to the
desired Aroclor by multiplying DCB found by the quotient
obtained from dividing the average molecular weight of the
Aroclor by 499, the molecular weight of DCB.
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Revised 12/15/79 Section 9, B, (2)
Page 7
NOTE: Since all Aroclors have an average molecular
weight less than 499, levels of PCBs expressed
as a particular Aroclor will always be less than
than calculated for DCB.
The perchlorination method gave values of 145 ppb for a 200 ppb
fortified milk standard and 102 ppb for a 150 ppb standard cleaned
up by the macro method (after correction for a 37 ppb reagent blank),
based on the amount of Aroclor 1254 that would produce an equivalent
amount of DCB. This indicates that the method is capable of providing
a further semi-quantitative approximation of PCBs in human milk
samples. Results are not expected to be totally consistent between the
EC GLC fingerprinting method using Aroclor 1254 as reference standard
and DCB method because only later eluting peaks are useful for
quantitative approximation in the former (because early peaks of less
chlorinated isomers would be overlapped by impurities and would not
resemble the standard chromatogram), whereas all isomer peaks will
contribute to the production of DCB. The greatest inadequacy of
the perchlorination technique is the inability to determine the
isomeric identity and distribution in an unknown and the total
conversion efficiency of each of the different isomers to DCB.
The following table shows comparisons between results obtained
for human milk samples by the perchlorination method and a minia-
turized silicic acid cleanup procedure preceding EC GLC. The
results indicate reasonably good agreement at the low concentrations
present.
X. ANALYTICAL QUALITY CONTROL:
General aspects of quality control for the micro method are
similar to those presented for the macro method in Section 9,B,(1),XIV,
with details modified to reflect the smaller sample size taken through
the procedure. A typical SPRM to be analyzed along with actual samples
would contain 15 ng/500 mg - 30 ppb.
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Revised 12/15/79 Section 9, B, (2)
Page 8
POLYCHLORINATED BIPHENYLS IN HUMAN MILK EXTRACTS (500 mg)
Sample
lb
2C
3
4
5
6
7
8
9
10
PCBs found, ppm (as Aroclor 1254)a
Perchlor
0.12
0.14
0.05
0.06
0.08
0.06
0.06
0.04
0.14
0.07
Micro
0.15
0.17
0.04
0.07
0.09
0.06
0.06
0.05
0.19
0.10
a Reported on whole milk basis.
Goat milk fortified with 0.15 ppm Aroclor 1254.
c Goat milk fortified with 0.20 ppm Aroclor 1254.
Samples 3-10 were human milk samples.
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Revised 12/15/79
Section 9, B, (2)
Page 9
Figures 1 and 2 illustrate a human milk sample before and after
perchlorination. Perchlorination combined with the micro extraction
and cleanup method is particularly well suited for confirmatory
purposes when the amount of sample is limited. The cleanup achieved
allows perchlorination of the sample without excessive lipid inter-
ference with the reaction.
1
1 1 1 1 1 r
10 12 14 16 18 20 22 24
T Ifv* L mm
Fig. 1. Chromatogram of human milk extract; 5 y1/1.0 ml injected:
4% SE-30/6% OV-210 column; oven temperature 205°C;
carrier gas flow 60 ml/minute.
-------
Revised 12/15/79
Section 9,B,(2)
Page 10
TIME, mm
Fig. 2. Chromatogram of perchlorinated human milk extract
(0.14 ppm as Aroclor 1254); 5 yl/8.0 ml injected;
OV-210 column; oven temperature 205 C; carrier gas
flow 60 ml/minute.
X. ANALYTICAL QUALITY CONTROL:
General aspects of quality control for the micro method are
similar to those presented for the macro method in Section
9,B,(1), XIV, with details modified to reflect the smaller
sample size taken through the procedure. A typical SPRM to
be analyzed along with actual samples would contain
15 ng/500 mg = 30 ppb.
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Revised 12/2/74 Section 9, C
Page 1
SEPARATION OF SOME POLYCHLORINATED BIPHENYLS FROM CERTAIN
ORGANOCHLORINE PESTICIDES
I. INTRODUCTION:
Polychlorinated biphenyls (PCB) are a group of chemicals with
industrial applications. They are stable (resistant to alkali and
acid) and persistent; their residues have been found in wild life.
Most Aroclors actually consist of many different chlorobiphenyls,
although some, partially or totally, consist of members of another
group of compounds, chloroterphenyls.
The various components of PCB residues are partially or com-
pletely recovered through multiresidue methodology for organochlorine
pesticides; they are eluted from the Florisil column by the 6% ethyl
ether/petr ether eluant. PCB residues exhibit complex gas chromato-
graphic 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 determina-
tion of some organochlorine pesticides. Likewise, the presence
of certain organochlorine pesticides can interfere with the deter-
mination of PCB.
NOTE: The polychlorinated terphenyls (PCT) are also recovered
through the multiresidue methodology used for the analysis
of organochlorine pesticides and PCB. However, the PCT
elute from the GLC column much more slowly than either the
pesticides or PCB and so do not interfere with the determin-
ation. 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 chloro-
terphenyl components.
A number of procedures have been proposed for dealing with the
various pesticide PCB combinations encountered. These include the
si lie 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
quantisation of both pesticides and PCB without their separation from
one another on the silicic acid column. Other combinations of PCB
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Revised 11/1/72 Section 9, C
Page 2
and pesticides must be separated before quantisation; still other
combinations cannot be separated by silicic acid, yet cannot be
determined in the presence of one another. The relative amounts of
residues of pesticides and PCB may also influence the decision on
whether or not to perform silicic acid separation prior to quanti-
tation. Even when the residues will not be completely separated by
this technique, its use may be the best means of achieving quantita-
tive estimation of the residues when one chemical is present in much
larger amounts than the other.
REFERENCES:
1. Armour, J., and Burke, J., JAOAC 53^ 761-767 (1970).
2. Masumoto, H. T., JAOAC, in press.
3. Pesticide Analytical Manual, Vol. 1, Section 251, U. S.
Food & Drug Admin.
II. PRINCIPLES:
The silicic acid column chromatographic procedure given here
permits separation of DDT and its analogs from some of the PCB,
including those with which they interfere. The silicic acid is
standardized before use by addition of enough water to effect the
best possible separation between p_,p_'-DDE and Aroclor 1254. The
interfering PCB are eluted with petroleum ether from a column of the
standardized silicic acid. The DDT compounds, most other organo-
chlorine 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 CSection
5,A,(I)} 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. APPARATUS:
1. Chromatographic column 400 x 22 mm 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.
3. Kuderna-Danish Assembly as follows:
Evaporative concentrator flask - Kontes Catalog No. K-570000,
500-ml capacity, lower joint 19/22 f, upper joint 24/40 J;
Snyder column - 3 ball, lower joint 19/22 f, upper joint 24/40 f;
-------
Revised 11/1/72 Section 9, C
Page 3
Tube - 10 or 15 ml capacity, 19/22 f upper joint.
4. Air pressure regulator - for pressure reduction to deliver ca
1 Ib. psig; air must be clean and dry.
5. Separatory funnel - Used for column eluant reservoir, 250 ml, with
Teflon stopcock, 24/40 I 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 pre-
pared 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 sub-
stances. If electron capturing substances are extracted by
petroleum ether, treat as follows: Slurry Celite with 1 +1
hydrochloric acid - H20 while heating on steambath; wash with
H20 until neutral; wash successively with several portions each
of methanol and acetone (to remove H20); then ethyl acetate
and petr ether. Remove solvents by suction and 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.
3. Silicic acid - Place silicic acid to depth of about 1 inch in
open beaker and heat for a minimum of 7 hours, but preferably up
to 24 hours, in 130°C oven to remove water. After heating, place
beaker in desiccator and allow to cool to room temperature.
Quickly weigh silicic acid into glass-stoppered bottle and add
3% H20 by pipette (97 g silicic acid + 3 ml H20 = 3% H20).
Stopper bottle tightly and seal with tape to insure that container
-------
Revised 6/77 Section 9, C
Page 4
is air tight. Shake well until all H20 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 yg Aroclor 1254 and 3 yg
£,£'-DDE in hexane. Elute as described and determine recoveries
in each eluate. Inadequate separation of PCB from jD_,£'-DDE will
require that further testing be done with other heated batches
of silicic acid, treated with different amounts of H20 as needed
to achieve the desired separation. Increments of 0.25% or 0.5%
more or less H20 are recommended for the testing. More H20 is
required when the initial test results show PCB eluting in the
polar solvent with the p_,p_'-DDE; less H20 when £,£'-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 stored in a desiccator between uses. Desired activity
remains for about 5 days.
VI. SEPARATION OF PCBs FROM ORGANOCHLORINE PESTICIDES:
1. Weigh 5 g Celite, then 20 g activated silicic acid and
combine in 250 ml beaker. Immediately 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.
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 pressure 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.
NOTE: Do not allow column to go dry or to crack at any
time during the procedure. Close stopcock when air
pressure is not being applied. At this point, column
of adsorbent should be firm and should not lose its
shape if tipped.
-------
Revised 11/1/72 Section 9, C
Page 5
4. Place 250 ml 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 re-
quired 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 equiv-
alent be placed on the silicic acid column.
5. Add aliquot carefully to column being careful not to disturb
top of adsorbent.
6. Apply slight air pressure until solvent level is ca 3 mm
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 mm
point above adsorbent.
7. Position a 250 ml 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 ml/min.
is established. Continue elution until eluate volume in the
graduate is exactly 250 ml.
8. Quantitatively transfer eluate to a 500 ml Kuderna-Danish
evaporator fitted with a 5 ml evap. concentrator tube. Rinse
graduate with small portions of pet. ether.
9. Place a second 500 ml 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 mm above
adsorbent and add 200 ml of CH3CN-hexane-CH2Cl2 (1:19:80) eluant
to upper reservoir. Open stopcock and slowly reapply air
pressure, continuing elution until all of eluant passes through
column into the C-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.
-------
Revised 6/77 Section 9, C
Page 6
NOTE: The first (petr ether) eluate should contain the PCB's
and the combined solvent eluate should contain the
chlorinated pesticides.
VII. GAS CHROMATOGRAPHY AND INTERPRETATION:
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 GLC 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 more 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 judgment on the part of the analyst.
GLC with halogen specific microcoulometric or electrolytic
conductivity detection is often preferable as means of quantitation.
This type of detection system also provides confirmatory evidence for
the identification of the residue as PCB.
-------
Revised 11/1/72
Section 9, C
Page 7
TABLE 1. PESTICIDES AND OTHER CHEMICALS RECOVERED THROUGH SILICIC ACID
COLUMN CHROMATOGRAPHIC SEPARATION OF SOME POLYCHLORINATED
BIPHENYLS (PCB) FROM CERTAIN ORGANOCHLORINE PESTICIDES. a
Petroleum Ether Eluate
Acetonitrile, Methylene Chloride,
Hexane Eluate
Aldrin
Aroclor
Aroclor
Aroclor
Aroclor
Aroclor
Aroclor
Aroclor
Aroclor
1221
1242
c,d
12481
1254
1260
1262
4465
5460C
hexachlorbenzene
mirex6
octachloro-dibenzo-p-dioxin
polychlorinated naphthalenes^
2,3,7,8-tetrachloro-dibenzo-p-dioxins
Aroclor
Aroclor
Aroclor
Aroclor
Aroclor
1221
1242
1248[
5442'
5460
c,d
BHC (all isomers)
chlordane (technical)
£_,£_'-DDE
p_,£'-DDT
£,p_'-DDT
dieldrin
endrin
heptachlor
heptachlor epoxide
1indane
Perthane9
£,R'-TDE
toxaphene
Method tested only with chemicals listed.
Divides between the two eluates. The earliest (GLC) eluting peaks in any
of these Aroclors are the most likely to elute in the polar eluate.
°Divides between the two eluates.
Aroclors 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 Chrom Q at 240°C, 120 ml N2/min. (Wieneke, W.,
private communication, Jan. 1972).
eMirex may be separated from Aroclors 1260 and 1254 by collecting the first
100 ml petr ether separately. This fraction will contain the mires.
(Gaul, J., private communication, July 13, 1971).
Method tested with commercial polychlorinated naphthalenes: Halowaxes
1014, 1099 (Armour, J., and Burke, J., JAOAC Jrt, 175-177 (1971).
9Krause, R. T., in press. JAOAC.
-------
-------
Revised 1/4/71 Section 9, D
Page 1
SEMI-QUANTITATIVE ESTIMATION OF POLYCHLORINATED BIPHENYLS
IN ADIPOSE TISSUE
I. INTRODUCTION:
The incidence of certain of the polychlorinated biphenyls
(PCB's) in human adipose tissue has become quite common in very recent
years although the compounds have been in use nearly 40 years. It
seems probable that improved methods of detection may well account for
the prevalence of the current observations.
The prime manufacturer in the United States of these products is
the Monsanto Chemical Company. A series of the PCB's are marketed
under the trade name of Aroclor. A company bulletin lists many
products in which the materials may be used as plasticizers, flame
retardants, insulating fluids, or to import some other useful quality.
Among these products are natural and synthetic rubber, electrical
products, floor tile, printer's ink, coatings for varnishes, waxes,
asphalt and many adhesives and resins. The PCB's have also been
recommended by Monsanto for mixing with chlorinated insecticides to
suppress their vaporization and extend their kill-life.
The Aroclor series of compounds are identified by numbers such
as 1242, 1248, 1254, 1260, and so on. The last two digits of the
formulation indicate the percentage of chlorine. To date, the two
compounds which have predominantly appeared in adipose tissue samples
are Aroclor 1254 and 1260. The method presented here essentially is
a modification of the method developed by Mulhorn et. al., (1), pro-
vides a convenient means of separating these compounds from the
common chlorinated pesticides, confirming identification, and approx-
imating the concentration. In addition to Aroclor 1254 and 1260, the
method is also applicable to aroclor 1262 and 1268.
Thin layer chromatography provides a sound approach for the
semi-quantisation of the stated PCB's as the various compounds of
the series have similar Rf values and therefore, produce a single
spot.
REFERENCES:
1. Mulhorn, Cromartie, Reichel, and Bel isle
Semi-Quantitation of Polychlorinated Biphenyls in Tissue
Samples by Thin Layer Chromatography presented at the
84th meeting of the AOAC, October 12-15, 1970 in
Washington, D. C. #8
-------
Revised 1/4/71 Section 9, D
Page 2
2. Private communication from Monsanto
Tentative Procedure for the Determination of Airborn
Polychlorinated Biphenyls.
3. Pionke, Chesters, and Armstrong Dual Column and
Derivative Techniques for Improved Specificity of Gas-
Liquid Chromatographic Identification of Organochlorine
Insecticide Residues in Soil Analyst October 1969, 94,
pp 900-903.
4. W. W. Sans - Multiple Insecticide Residue Determination
Using Column Chromatography, Chemical Conversion, and Gas-
Liquid Chromatography 0. Ag. Food Chem 1_5 Jan-Feb 1967,
pp 192-198.
II. PRINCIPLES:
Adipose tissue is subjected to extraction by pet. ether,
acetonitrile partitioning, and Florisil cleanup. A portion of the
resulting 6% ethyl ether/pet, ether eluate, in concentrate form, is
treated with KOH to effectuate dehydrochlorination of DDT and ODD to
their olefins, thus eliminating the problem of separating these
pesticides from the PCB's. Oxidative treatment is then applied to
convert any interfering DDE to p_,£'-dichlorobenzophenone which has an
Rf value different from the PCB's. The PCB's are then determined by
thin layer Chromatography.
III. APPARATUS:
1. Gas chromatograph fitted with E. C. detector (this equipment is
not mandatory for this specific method unless assessment of
the pesticides is required).
2. Evap. concentrator tubes, 10 ml, size 1025, Kontes #570050.
3. Evap. concentrator tubes, 25 ml, size 2525, Kontes #570050.
4. Modified micro-Snyder columns, 5 19/22, Kontes #569251.
5. Glass beads, 3 mm plain, Fisher #11-312, or the equivalent.
6. Pipets, disposable (Pasteur), 9-inch length.
7. Pipets, spotting, 10 pi, Kontes #763800.
8. All equipment specified in section 5,A,(1) of this manual for
the extraction and cleanup of adipose tissue.
-------
Revised 1/4/71 Section 9, D
Page 3
9. Equipment specified in Section 12,B of this manual to conduct
thin layer chromatography.
10. A bath of white mineral oil and heating device with sufficient
control to hold bath at 100°C., ±2°. A beaker resting on a
rheostatically controlled electric hot plate may be used.
11. Steam or hot water bath adjustable to 95 to 100°C.
12. Vortex mixer, variable speed.
IV. REAGENTS AND SOLVENTS:
1. Hexane, pesticide quality.
2. Benzene, pesticide quality.
3. Ethanol, absolute.
4. Methanol, absolute.
5. Acetic acid, glacial, reag. grade.
6. Chromium trioxide, cryst., reag. grade.
7. Potassium hydroxide, pellets, reag. grade.
8. Silver nitrate, cryst., reag. grade.
9. Developing solvent 5% benzene in hexane.
10. Aluminum oxide G (Merck).
11. Alcoholic KOH, 2.5% w/v of KOH in ethanol - As this reagent should
be prepared fresh each day of use, it is convenient to prepare
only a small quantity. One pellet (ca 80 ml) is dissolved in
3 ml of ethanol. Five minutes of vigorous mixing should suffice
to complete solution.
12. Oxidizing solution - 1.5 grams of Cr03 is added to 1 ml of
distilled water. Finally add 59 ml of glacial HAC. This
solution should be suitable for a month's use.
13. TLC plate coating - Dissolve 1 gram of AgNOs in 4 ml of distilled
water and add 56 ml of methanol. Mix this with 30 grams of
A1203 -G and prepare 8" TLC plates as described in Section 12,B
of this manual.
-------
Revised 6/77 Section 9, D
Page 4
14. Analytical reference standards of the series of Aroclor
compounds available from Perrine Repository.
15. a. Stock Standard solution, Aroclor 1260. Weigh 50 mg,
dissolve in benzene and dilute to 50 ml. Concentration is
1 mg/ml.
b. From the stock standard, prepare four working standards
of 25,50,100 and 400 ng/yl, using hexane as the diluent.
V. EXTRACTION AND CLEANUP:
An adipose tissue sample of sufficient size to yield 3 grams of
pure fat is prepared, extracted, and carried through acetonitrile
partitioning and Florisil cleanup as described in Section 5,A,(1) of
this manual, altering the latter procedure only by using a 25 ml
evap. concentrator tube for the final evaporation. For the purpose
of the following procedure, only the concentrate from the 6% ethyl
ether/pet, ether eluate is needed as the PCB's are eluted in this
fraction. Pipet off an aliquot representing 5% of the extract for
such direct GLC analysis as may be required. Use the 95% remaining
in the 25 ml evap. concentrator tube for dehydrochlorination.
VI. DEHYDROCHLORINATION:
1. Attach a modified micro Snyder column to the concentrator tube
and concentrate the extract to 1 ml or less in a 100°C water
or steam bath.
2. Cool and remove micro Snyder column.
3. Remove the volatile solvent under a stream of nitrogen at room
temperature and add 2 ml of alcoholic KOH.
4. Reattach the modified micro Snyder column and immerse tube in
a 100°C oil bath for 30 minutes.
NOTE: Do_ not attempt to use a hot water or steam bath for
this purpose.
5. Remove tube from oil bath, allow to cool to room temp, and add
2 ml of dist. water and 5 ml of hexane. Stopper and mix
vigorously on a Vortex mixer for 30 seconds.
6. Allow layers to separate and, with a disposable pi pet, carefully
transfer the hexane layer to a 25 ml evap. concentrator tube.
7. Add 5 ml portions of hexane for two additional extractions as
described above in Steps 5 and 6.
-------
Revised 6/77 Section 9, D
Page 5
8. Adjust the final volume exactly to 19 ml, stopper and mix
vigorously on Vortex mixer for one minute.
9. Transfer 1 ml (representing 5% of the original extract) to
a second 25 ml evap. concentrator tube, add exactly 10 ml of
dist. water, stopper, mix thoroughly on the Vortex, and set
aside for direct injection of the hexane layer for GLC
assessment.
10. Add a 3 mm glass bead to the first 25 ml evap. concentrator tube
containing the remaining 90% of the hexane extract, attach a
modified micro Snyder column and boil down to 1 ml or less in
a steam or hot water bath.
11. Take tube from bath, allow to cool and remove column. Place the
tube under a nitrogen stream and evaporate to dryness at room
temperature.
VII. OXIDATION:
1. Add 2 ml of the oxidizing solution to the tube, attach a modified
micro Snyder column and immerse tube in the 100°C oil bath for
30 minutes.
2. Remove tube from oil bath, allow to cool, and add 10 ml of
dist. water and 3 ml of hexane. Stopper and mix vigorously on
Vortex for 30 seconds.
3. Allow layers to separate and carefully transfer the hexane layer
to a 10 ml evap. concentrator tube with a disposable pipet fitted
with rubber bulb.
4. Add 3 ml portions of hexane for two additional extractions as
described above in Steps 2 and 3.
NOTE: If GLC analysis for the ctichlorobenzophenone is
required, adjust the volume of extract to exactly
9 ml, stopper, mix vigorously on Vortex 30 seconds
and transfer 0.5 ml to a 25 ml evap. concentrator
tube. Add 9 ml of dist. water, stopper, mix vigor-
ously and hold for direct injection of the hexane
extract into the GLC.
5. Add one 3 mm glass bead to the tube, attach a modified micro
Snyder column and concentrate the extract to 0.3 ml in a boiling
water bath.
6. Remove, allow tube to cool, rinse column joint with ca 2 ml of
hexane, stopper and hold on Vortex at medium speed for 30 seconds.
-------
Revised 6/77 Section 9, D
Page 6
7. Place tube under a nitrogen stream and evaporate just to
dryness at room temp. Add exactly 0.1 ml of hexane, stopper
and mix on Vortex for 1 minute.
NOTE: From a 3.0 gm. sample of pure fat, assuming that
aliquots were removed for dichlorobenzophenone and
GLC before and after dehydrochlorination, the sample
weight equiv. in this final 100 yl of extract is
25.5 mg per microliter.
VIII. THIN LAYER CHROMATOGRAPHY:
1. On one 8 in. T.L. plate, spot 10 yl each of the four working
standards of Aroclor 1260 and also 10 yl of the concentrated
extract from Step 7 under the OXIDATION subsection above.
2. Develop the plate in 200 ml of a solution of 5% benzene in
hexane to a previously scored line 150 mm from the spotting
line.
3. Remove plate from tank and allow solvent to evaporate.
4. Expose plate in the U. V. box until the sample spot is clearly
visible.
5. Remove plate from U. V. box and, by visual comparison of sample
spot intensity to the intensities of the various standard spots,
estimate the number of nanograms represented by the sample spot.
NOTE: The operator should be comparing varying degrees of
intensity of a gray shading. If the sample spot is
black, the indication is an excessive concentration
of sample, and quantitative comparisons are not
possible. In this case, some quantitative dilution of
the sample extract is required to reduce the spot
intensity to a level comparable with the standards.
IX. MISCELLANEOUS NOTES:
1. Any p_,£_'-DDT present in the sample may be measured by GLC
quantitation of the £,£' -DDE peak before and after dehydro-
chlori nation. Also, any o_,p_'-DDT present in the sample may be
quantitated by measurement of the p_,p_'-DDT peak before and
after dehydrochlorination.
2. While it may be possible to detect and estimate lower levels,
an arbitrary limit of ca 1.0 ppm has been tentatively established
for this procedure.
-------
Revised 6/77 Section 9, D
Page 7
3. Recovery studies have indicated a precision of ±50% for this
procedure when using Aroclor 1260 as the reference standard.
-------
Revised 11/1/72
Section 9, E
Page 1
4%SE-30/6%OV-210
Chromato^rc-s of three ARCCLOR3 en colunn of
\& SE-30 / 6% OV-?10. Col inn ter.p. 200°C.,
carrier flow 60 rl/ir,in., •%! detector, electrom.
attenuation on sn 5-2 10 x l6j dotted line a
mixture of chlorinated pesticides, identity and
injection concentration given below:
1. Dja?,inon
2. Heptachlor -
3. Aleirin
h. Kept.Koox. -•
5. PjP'-UDE
6, Dieldrin
- 1.5 ng 7. o,p'-DDT — 0.2k
- 0.03 8. pjp'-DDD — ,2U
- ,0li5 9. p,p'-L;Dr — .30
- .0? 10. Dilan — .75
- .09 11. Methoxychlor .60
- .12
AROCLOR 1221
6 ng injection
-------
CD
4%SE-30/6%OV-210
Chromato^ra-ns of three AROCLORS on column of
h% SE-30 / 6% OV-210. Colurnn temp. 200°C.,
carrier flow 60 ml/min., •'H detector, electrom.
attenuation on an E-2 10 x 16; dotted line a
mixture of chlorinated pesticides, identity and
injection concentration given below:
AROCLOR 1248
6 ng injection
1. Diazinon
2. Heptecolor -
3. Aldrin
li. hept.Epox. -•
5. p,p'-LDE
6. Dieldrin
— 1.5 ng 7. o,p'-DDT ~ 0.2k ng
- 0.03 8. p,p'-DDD — 02i|
- eOii5 9. p,p'-DDT — .30
— .0? 10. Dilan — .75
- .09 11. Kethoxychlor .60
- .12
AROCLOR 1260
4 ng injection
AROCLOR 1254
CD
a.
ro
"O CO
CU fD
UD O
fD r+
-------
Revised 11/1/72
Section 9, E
Page 3
Chromatogra-s of three ARCCLOKS on cclunn of
1.5-0 OV-1? / 1.95,c QF-1. Coioin tcr.ip. 200°C.,
carrier flow 60 nl/nin., ^H detector, elcctrcneter
attcn» 10 x 16 on an E-2; dotted line a mixture
of chlorinated pesticide compounds, identity and
injection concentration given below:
1.5%OV-17/1.95%QF-1
1.
2.
3.
U.
5.
6.
Diazinon
HepUchlor «
Aldrin
Hept.Epcx. —
Dieldrin
1.5 ng 7. o,p'-CM — 0.2U ng
0.03 8. p,p'-DDD — ,2U
.OU5 9. P,P'-BDT — .30
.09 10. Dilan — .75
,09 11. Kethoxychlor .60
.12
AROCLOR 1232
7ng injection
AROCLOR 1221
6 ng injection
AROCLOR 1242
5 ng injection
-------
1.57oOV-17/1.95%QF-l
Chromatoerans of three AROCLORS on columnof
1.5> OV-17 / 1.95',- QF-l. Column temp. 200 C.,
ca-rier flow 60 nl/min., 3n detector, electrometer
atten. 10 x 16 on an E-2; dotted line a mixture
of chlorinated pesticide compounds, identity and
injection concentration given below:
1. Diazinon
?. HeptEchlor -
3. Aldrin
U. Hept.Kpox. -
5. pjp'-EfiB
6. Dieldrin
5 ng 7. o,p'-DDT - 0.2U ng
.- 0.03 8. P,p'-SDD — .2U
- .OU5 9- pfp'-UDT — .30
.. .09 10. Lilian — «75
- .09 11. Methoxj^chlor .60
.12
1
AROCLOR 1248
4 ng injection
(D
n>
o.
AROCLOR 1260
3 ng injection
AROCLOI! 1254
S ng >n|M(i«n
©
®
®
-o oo
&i fD
ta o
OS rh
-------
F
1/4/71
Section 9, F
Page 1
Retention Values, Relative to Aldrin and Response Values, Relative to the
Major Peak of Six of the Aroclor Compounds (poly-chlorinated biphenyls).
Column: Pyrex glass , b-ft., 4 mm. i.d., l.S'i OV-17/1.9S°6 QF-1,
200 C Column Temp. Carrier flow 60 ml/min.
Detector: Electron capture, JH, parallel plate, 210°C.
Misc.'
Pesticides
Phosdrin
Thimet
Diazinon
rleptachloi
6-EHC
Aldrin
"3 77"
7^n~*r +-1- „ ,-.
L C'.Z Gl.iiJ.vll
ttept.
•Epoxide —
Malathion
Parathion
p,p'-DDE
Dieldrin
Endrin
o,p'-DDr
jp.p'-DDD
p,p'-DDT
Ethion
Dila-i I
Dilan II
Mpthcx/-
CiUor
.RRR2
0.32
.50
.63
*
.83
.92
1.00
J. . — ~»
1.54
1.63
1.81
2.23
2.40
2.93
3-. 17
3.49
4.18
4.44
5.7
6.4
8.3
i
i n
#1221
?RR2 RPH3-
0.27 0.19
.34 .03
.40 1 .05
.43 .27
.43 1 Z. 00
.62 .05
1
.82 .04
.91 1 .02
1
!
1
1
1
1
1
1
1
1
1
I
1
1
i
I
1
I
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1
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i
1
1
!
i
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i
t
/•'1232
RSR RPH
0.27 0.17
.33 .03
.40 .07
.43 .27
.47 Z. 00
.61 .38
.73 .20
.80 .85
'.90 '.33
.95 .26
1.03 .13
1.22 .20
1.30 .19
1 4 A t -
A . *r-r . — -J
1.54 .27
1.83 .19
2.26 .03
2.43 .05
2.78 .07
3.13 .02
3. 57 .04
4.04 .03
i
! !l
(71242
RRR RPH
0.48 0.34
.63 .41
.74 .22
.82 1.00'
.90 .38
.97 .32
1.05 \23
1.24 .23
1.331" .21
-, 4,1 i r
J. . -f^ . j. ^
1.57 .29
1.88 .21
2.52 .03
2.85 .04
3.67 .02
#1248
RRR RPH
0.48 0.08
.63 .34
.74 .15
.83 1. 00
.92 .31
.98 .24
1.06 .68
1.26 .62
1.34 .56
1 1 ~,
J..^' .T*. I
1.58 .96
1.88 .65
2.21 .13
2.32 .17
2.50 .24
2.85 .32
3.18 .24
3.66 .36
4.12 .05 L
!
i!l?.5'4
RRR RPH
1.04 0.23
1.24 .14
1.34 .08
1.43 .07
1.S6 .29
1.62 .33
1.80 .58
1.97 .13
2.20 .19
2.29 .36
2.47 .68
2.82 1. 00
3.16 .53
3.60 .50
4.08 .66
4.42 .08
5.28 .11
5.95 .09
6.4 .09
8.4 '.05
i
I
1
i
#1260
RRR 1 RPH
1
i
1
1
i
1
1 " '
I
1
1
l
1
1
|
1
1
1
i
l
I
1.61 '0.09
1.78 j .16
1
l
- 1
2.53 j .39
2.81 1 .42
3.20 1. 00
3.50 I;".S2
4.10 | .74
4.38 1 .53
5.0 j .07
5.4 ! .38
5.7 j .18
1
6.4 I .90
8.4 ' .^3
I
!
1
^Relative retention ratios given for some camion pesticides for comparison purposes.
RRR - Means the. retention relative to aldrin.
RPH - Means the peak height response-relative to that of the tallest peak shown ;n
^talic3. ' .
Indicates chlordane peaks eluting in the appropriate area.
-------
1/4/71
Section 9, F
Page 2
Retention Values, Relative to Aldrin and Response Values, Relative to the'
Major Peak of Six of the Aroclor Compounds (poly-chlorinated biphenyls)
Column: Pyrex glass, 6-ft., 4 mm. i.d., 4°, SE-30/6% QF-1, 200°C Column temp.
Carrier flow 70 ml/min.
Detector: Electron capture, JH, parallel plate, 210°C.
Misc.'
Pesticides
•Phosdrin
2,4-D(ME)
Thimet
Simazine
Lindane
o-BHC
2,4-DCBE)
Heptachlor
Ronnell
Aldrin
M
Parathion
Hept.
Epmri dp
p,p'-DDE
Captan
o.p'-DDD
Dieldrin
o.p'-DDT
Endrin
p,p'-DDD
Thiodan II
p.p'-DDT
Trithion
Dilan I
Methoxy-
_chlor
Dilan IT
RRR2
0.32
*
.44
.47
.54
.60
.69
X
.78
.83
.91
1.00
1.34
*1.43
1.82
1.94
1.98
2.12
2.39
2.42
2.55
2.72
3.12
' 3.2C
4.4C
4.6
5.1
i
#1221
, 1 3
IRR I RPH
0.24 1 0.37
.32 ! .07
.34 1 .13
.39 i .40
.44 1 Z.OC
1
|
.55 I .04
.60 J .05
.73 | .06
1
i i
j 1
#12 3 2
RRR RPH
0.25 0.26
.32 1 .07
.35 , .13
.39 I .33
.42 Z.CC
1
.55 .37
.62 .28
.72 [ -.70
.79 1 .31
.82 | .27
.90. 1 .14
1.02 .16
1.09 I ,20
J_ T^C. (\1
1.31 1 .24
1.48 , .19
1.77 1 .03
1.96 j .04
1
1
I
1
2.27 .05
1
1
1 '
1
1
!
!
i
1
1
i
. . I
#1242
RRR RPH
0.34 0.04
.38 .08
.43 .52
.47 .03
.55 .54
.61 .38
.72 Z.£0
.77 .43
.81 .36
.89 .18
1.00T .22
1.07 | .27
ii^1 n?
i ' "
1.30 1 .30
1.47 ! .23
1.77 1 .03
1.90 j .03
2.02 1 .03
I
I
2.27 .02
2.69 .03
;
#1248
RKR 1 RPH
1
1
I
1
0.441 0.14
!
.55! .42
.62 | .25
.73' .99
1
.80t .34
.83{ .36
.90j .61
1.03 .66
1.10| .68
1 18 1 .10 i
Z.35! Z.tftf
1.50 .78
1.801 .19
1.92 .25.
!
1
l
2.24| .07
2.30J .27
1
1
1
2.73| .18
.1
!
3.121 .06
I
!
1
1
1
1
1
1
!
1
1
#1254
RRR RPH
0.90 | 0.38
1.02' .25
1.08| .14
1" - "I "'-
I
1.291 .79
1.48 .95
1.751 .53
1.87] .83
2.00| .09
2.14J .28
z.n\ z.00
i
i
2.51| .78
2.62 .38
i
1
1
2.991 .91
1
1
3.551 .12
i
4.201 .10
4.66, .12
5.5-11 .05
1
#1260
RRR 1 RFIL
' 1
|
1
1
|
1
|
1
1
|
1
1
|
1
1
|
1
1
1
1 '
i
1.30 10.10
1.52 | .17
1.80 | .07 .
1.93 j .08
2.02..J .36
2.18 ' .40
1
1
I'
2.59 'Z..00
1
[ 2.81 ! .35
3.10 1 .67
3.39 j .59
3.71 I .05
3.95 | .4,:
t
4.83 ,' .92
5.77 1 .30
6.2 j .20
Relative retention ratios given tor some common pesticides for comparison purposes.
RRR - Means the retention relative to aldrin.
"RPH - Means the peak height response relative to that of the tallest peat shown in
*Indicates chlgrdane peaks eluting in the 'anpi oxnuate area.
-------
Revised 6/77
Section 9,F
Page 3
Retention values, relative to aldrin and response values, relative to the major peak, of six
of the Aroclor compounds (polychlorinated biphenyls).
Column: Pyrex glass, 5 ft., S/32" i.d., 5% OV-210, 200°C column temp., carrier flow 45 ml/min.
Detector: Electron capture, *H, parallel plate, 205°C.
Miscellaneous
Pesticides
Diazinon
S-BHC
1-Hydroxychlordene
Kept. EyoxiJc
Dimethoate
p,p' -DDE
o.p'-DDT
Malathion
M. Parathion
p,p'-DDT
Methoxychlor
Dilan II
'•MR
0.73
.94
'1.3C
1 ">-
1.7J
1.81
2.2
2.32
2.44
3.25
4.7C
.
8.4
4RRA
#1221
2RRR ( 3RPH
0.33i 0.27
.41 .10
.46 .31
.SI1 1.00
.61 .02
.72 .06
.79 .08
.88 .03
.92 .03
•
•
'
.014
i
1
#1232
RRR , RPH
0.33 '0.19
.42 , .07
.46 ; .24
.52 '1.00
.62 i .30
.70 1 .20
.80 \ .84
.88 ' .13
.93 , .59
1.12 | .29
1.29 i .27
1.41 ' .54
|-
1
1.62 ' .15
i
1.83 I .03
1.96 [ .07
1
2.32 ' .13
I
1
2.82 i .05
2.99 , .07
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I.0097
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#1242
RRR ! RPH
i
i
0.47 iQ.Ol
.53 ,' .43
.62 ' .50
.71 ' .22
.81 [-1.00
.88 i .16
.94 ; .66
1.12 | .29
1.29 ; .17
1.41 ' .52
1
i
1.62 i .17
i
i
i
i
- 1
1.97 ' .04
i
1
2.32 ' .08
i
i
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2.86 , .04
I
1
1
I
1
1
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1
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i
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#1248
RRR ,' RPH
i
i
1
O.S2I 0.08
.60, .23
.69! .11
.80 i .58
.86', .05
.92' .68
l.OSi .54
1.28J .49
1.371 1.00
1.471 .06
1.591 .24
i
1.771 .10
1
i
1.92] .22
1
2.281 .32
1
1
1
2.78' .19
2.96i .08
3.601 .02
4.32! .02
i
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I
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.1
#1254
RRR J RPH
i
I
1
I
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1
1
i
I
i
0.911 0.25
l.OSi .13
1.27 .38
1.391 .26
1.46* 1.00
1.
1
1.77, .41
1
1
- 1
1.92| .75
i
2.28J .77
2.40J .57
2.63i .11
2.79J .20
2.93" .80
3.63, .21
4.291 .13
i
i
5.32J .06
i
.025
(
#1260
RRR i RPH
1
i
I
i
i
•
i
I
1
t
i
,
1.28 ' 0.05
i
1.49( .20
t
1.72' .04
i
1.86( .44
1
2.06! .48
i
2. 44' .89
2.66, .22
i
2.33 ' 1.00
3.58} .43
1
4.41, .76
5.38J .34
6.5 - .05
8.0 ' .tnl
i 1
i
1.027 j
1
i
I
Relative retention ratios given for some coiimon pesticides for comparison purposes.
Indicates the retention ratio, relative to aldrin.
Indicates the peak height response relative to that of the tallest peak shown in italics.
Indicates the peak height response of the tallest peak relative to that obtained from an
equivalent amount of aldrin.
-------
Revised 12/15/79 Section 9, F
Page 4
Retention indices for all 210 possible chlorinated biphenyls
on 13 GC liquid phases have been tabulated in the following
reference:
Identification of the Individual Polychlorinated Biphenyls
in a Mixture by Gas-Liquid Chromatography, Albro, P. W.,
Haseman, J. K., Clemmer, T. A., and Corbett, B. J.,
J. Chromatogra., 136, 147 (1977).
-------
Revised 12/15/79 Section 9, G
Page 1
DETERMINATION OF TCDD RESIDUES IN HUMAN MILK,
BEEF LIVER, FISH, WATER, AND SEDIMENT
I. INTRODUCTION
The highly toxic compound 2,3,7,8-tetrachlorodibenzo-p_-dioxin
(TCDD) may be formed as a by-product in manufacturing processes
utilizing tetrachlorobenzene to produce trichlorophenol. Under very
basic, high temperature, high pressure conditions, 1,2,4,5-tetra-
chlorobenzene is hydrolyzed to the 2,4,5-trichlorophenate. Acidifi-
cation yields the phenol. Unfortunately, a condensation can take
place in this reaction resulting in formation of TCDD. Herbicides
containing esters of 2,4,5-trichlophenoxyacetic acid (2,4,5-T)
manufactured from trichlorophenol have been found to contain trace
amounts of TCDD. TCDD has been recognized as an extremely toxic
(oral LD50 0.6 mg/kg, guinea pig), teratogenic compound that is
stable in biological systems. The toxicological properties of TCDD
have been well documented.
Because of its toxicity and occurrence as a trace contaminate in
chemical products, it is necessary to analyze for TCDD at picogram/
gram (low and sub-parts per trillion, ppt) levels, which are below
the usual limits of detection for pesticide residue analysis. The
analysis of human, biological, and environmental samples for possible
TCDD contamination in the ppt concentration range is complicated by
the presence of many interfering components ranging from naturally
occurring compounds to industrial pollutants, such as PCBs, and the
agricultural chemicals DDT, DDE, etc.
An extremely efficient and specific analytical cleanup procedure
is a prerequisite for ppt TCDD analysis specifying GLC MS detection
techniques. The GLC MS detection technique must be ultra sensitive
and also highly specific because of the required low ppt detection
limits. High resolution glass capillary column GLC interfaced with
high resolution MS multiple ion selection analysis provides the
required GLC resolution, MS sensitivity, and specificity for TCDD
analysis in the 0.02-100 ppt concentration range.
Described below are the sample preparation procedures and
capillary column GLC HRMS techniques developed and currently applied
by EPA laboratories for isolation, detection, quantification, and
confirmation of TCDD residues in human milk, beef liver, fish, water,
and sediment extracts. The results of quality assurance samples and
samples with unusual contamination are also discussed.
-------
Revised 12/15/79 Section 9, G
Page 2
REFERENCE:
Sample Preparation Procedures and Gas Chromatography/Mass
Spectroscopic Methods of Analysis for TCDD, Harless, R. L.,
Oswald, E. 0., and Wilkinson, M. K., Analytical Chemistry
Branch, U.S. EPA, HERL, ETD, Research Triangle Park, NC
27711. Submitted for publication to Anal. Chem.
II. PRINCIPLE:
Tissue, milk, water, soil, and sediment samples are subjected to
an "acid-base" sample preparation procedure involving saponification
with hot caustic followed by extraction with hexane, washing with
concentrated sulfuric acid, cleanup by alumina column chromatography,
and capillary column GLC/high resolution mass spectrometric multiple
ion selection (GLC HRMS) analysis for TCDD residues. Fish tissue is
subjected to a "neutral" cleanup procedure that is similar except
that extraction is carried out with acetonitrile, and cleanup by
solvent partitioning and Florisil column chromatography precedes the
alumina column cleanup. 37C1-TCDD is added to all samples as an
internal standard or marker to monitor and determine the analytical
cleanup procedure efficiency.
III. SAFETY PRECAUTIONS:
TCDD is toxic and can pose grave health hazards if used improper-
ly. Techniques for handling radioactive and infectious materials are
applicable to TCDD. Only qualified individuals who are trained in
laboratory procedures and familiar with the dangers of TCDD should
handle this substance. Females of childbearing age should not work
with this material.
A good laboratory practice involves routine physical examinations
and blood checks of employees working with TCDD. Also, facial
photographs using oblique photoflood lighting should be periodically
taken to detect chloroacne, which is an early sign of overexposure.
IV. EQUIPMENT:
1. Gas chromatograph, Varian Model 2700, equipped with an SE-30
WCOT glass capillary column, 30 m x 0.25 mm, i.d. The capillary
column yielded an efficiency of 113,000 effective plates measured
at the 35C1-TCDD peak. Splitless injection incorporating
n-tetradecane was employed.
2. Mass spectrometer, Varian 311 A, interfaced to the chromatograph
so as to ensure maximum transfer efficiency. The spectrometer
was equipped with a turbo-molecular vacuum pumping system,
combination chemical ionization (CI) and electron impact (EL)
-------
Revised 12/15/79 Section 9, G
Page 3
ion source, and a Varian eight channel hardware (manual control)
multiple ion selection (MIS) device. This vacuum system easily
accommodated the 5 ml/minute helium flow from the GLC MS inter-
face and did not contribute detectable background contamination.
The MIS device was operated in the normal coupled electric mode
(jumping the acceleration voltage). Each MIS channel was
equipped with individual controls for selecting the acceleration
voltage, measuring range, output signal bandwidth, compensation
for background contamination, and intergration rate. The
intensities of the selected masses were monitored in a time
division multiplex system, setting alternately to each of the
selected masses and recording their intensities simultaneously
on an eight channel Soltec recorder. The adjustable integration
rate, 0.01 second to 1 second was sufficient to accurately
reproduce capillary column peaks two seconds wide at half height.
Alternative Instrumentation: Current MS instrumentation used by
other laboratories (EPA contract laboratories) for TCDD analysis
include (1) AEI MS-30, (2) AEI MS-50, and (3) Varian CH-5DF.
These instruments are interfaced with a packed column gas chro-
matograph and use high resolution MS double ion monitoring
techniques.
The general requirements for the GLS MS instrumentation are:
(1) Packed or preferably capillary column GLC introduction
of the sample.
(2) High resolution (10,000 minimum) MS mass analysis.
(3) Ultra high sensitivity (1 to 50 pg TCDD quantification
standards).
3. Pasteur pipets, 5.75 inches (14.6 cm) x 0.5 cm i.d.
4. Glass column, 50 cm x 11 mm, equipped with a Teflon stopcock
and removal glass tip.
5. Desiccator, equipped with Drierite, which can accommodate
adsorbent-packed Pasteur pipets.
6. Reflux condenser, water cooled, equipped with 100 ml boiling
flasks.
7. Separatory funnels, 250 ml.
8. Evaporation apparatus including a 12 ml distillation receiver,
micro-Snyder column Kontes K-569251, and steam bath.
-------
Revised 12/15/79 Section 9, G
Page 4
9. Filter funnel.
10. Filter tube, glass, 16 cm x 42 mm.
11. Glass column, 39 cm x 11 mm i.d., with a 125 ml reservoir and
Teflon stopcock.
12. Kuderna-Danish (K-D) evaporative concentrator, 250 ml.
13. Chromaflex sample tube, 2 ml, graduated, Kontes K-422560.
14. Glass tubing, 7 cm x 3 mm i.d.
15. Blender, Waring, or equivalent.
16. Magnetic stirrer.
17. Hotplate, explosion proof.
18. Mills type concentrator tube, Kontes K-570050.
V. REAGENTS:
1. 2,3,7,8-Tetrachlorodibenzo-p_-dioxin (TCDD), 37C labeled, isotopic
purity >98% 37C1, Eco-Control, Inc., 71 Rogers St., Cambridge,
MA 02142.
2. TCDD analytical standard, Dow Chemical Co., Midland, MI; ITT
Research Institute, Chicago, IL; and Eco-Control, Inc.
3. Hexane, acetone, benzene, methylene chloride, ethyl alcohol,
acetonitrile, Mallinckrodt Nanograde.
4. Carbon tetrachloride, Fisher ACS grade, 0.01% water maximum;
a greater water content can cause TCDD to elute in the incorrect
fraction.
5. Alumina, neutral, activity grade 1, Woelm.
6. Florisil, 60-120 mesh, suitable for pesticide residue analysis
by the criteria in Section 3,D, activated at 225°C for 24 hours
before use.
7. Sodium carbonate, sodium sulfate, and ammonium chloride,
Mallinckrodt AR grade, Soxhlet extracted overnight with methylene
chloride and dried at 200°C.
8. Potassium hydroxide and sulfuric acid, Mallinckrodt AR grade.
-------
Revised 12/15/79 Section 9, G
Page 5
9. Glass wool, pre-extracted with methylene chloride.
10. Water, passed through a column of activated carbon and distilled.
11. Nitrogen for solvent evaporation, Zero Grade, Liquid Air, Inc.,
New Orleans, LA.
12. Dry ice.
VI. PREPARATION OF CHROMATOGRAPHIC CLEANUP COLUMNS:
1. Alumina
a. Prewash and dry a disposable Pasteur pi pet and plug the tip
with glass wool.
b. Pack the pipet with 4.5 cm of neutral alumina and top the
column with 0.5 cm of anhydrous, granular sodium sulfate.
c. Wash the column with 4 ml of methylene chloride and force the
residual solvent from the column with a stream of dry
nitrogen.
d. Store the prepared columns in an oven at 225°C at least
24 hours.
e. Before use, equilibrate the oven-activated columns to room
temperature in a desiccator over Drierite.
2. Florisil
a. Pack a 500 x 11 mm glass column with 15 grams of activated
Florisil.
b. Pack a 2.5 cm layer of anhydrous sodium sulfate on top of the
Florisil.
c. Hold at 225°C until ready for use (a minimum of 24 hours).
d. Cool the column to near room temperature and prewash with
100 ml of hexane.
VII. SAMPLE PREPARATION AND CLEANUP - ACID-BASE PROCEDURE:
1. Lean Tissue
a. Grind tissue samples to obtain a homogeneous sample.
-------
Revised 12/15/79 Section 9, 6
Page 6
b. Weigh a 10-20 gram sample into a 100 ml boiling flask and
add 20 ml of ethyl alcohol and 40 ml of 45% KOH solution.
c. Add 5-10 ng of 37C1-TCDD standard solution.
d. Attach the flask to a water cooled reflux condenser and
heat under reflux with stirring for 2.5 hours.
e. Cool and transfer the solution to a 250 ml separatory funnel.
f. Rinse the boiling flask with 10 ml of ethyl alcohol followed
by 20 ml of hexane, and add to the separatory funnel.
g. Extract the solution with four 25 ml portions of hexane and
combine the hexane extracts.
2. Adipose Tissue
a. Grind or render adipose samples, if necessary, to obtain a
representative sample free of connective or other tissue.
b. Add 5-10 ng of 37C1-TCDD to 10 grams of sample.
c. Add 15 ml of distilled water to the sample. Extract and
continue as described in Subsection 1, b-g, for lean tissue.
3. Milk
a. Add 2.5 ng of 37C1-TCDD standard solution to 10-20 grams of
milk.
b. Extract the sample as described in Subsection 1, b-g, for
lean tissue.
4. Water
a. Fortify one kilogram of a well mixed water sample (including
particulate matter, if present) with 2.5 ng of 37C1-TCDD
standard solution.
b. Extract the sample with three 100 ml portions of methylene
chloride.
c. Evaporate the combined extracts to near dryness in a K-D
concentrator with an attached Snyder column utilizing a steam
bath. Complete the evaporation to dryness by placing the
tube in a warm water bath under a gentle stream of dry
nitrogen.
-------
Revised 12/15/79 Section 9, G
Page 7
d. Transfer the residue to a separatory funnel with several
rinsings of hexane totaling 100 ml.
e. Wash the hexane solution with 50 ml of 1 N KOH solution
followed by concentrated ^SO^ as described in Subsection 6
on cleanup.
5. Soil and Sediment
a. Fortify 10-20 grams of well mixed sample with 2.5 ng of
37C1-TCDD.
b. Extract as described in Subsection 1, b-g, for lean tissue.
c. After refluxing and cooling, decant the solution into a
separatory funnel through a filter funnel packed with glass
wool .
d. Rinse the boiling flask and filter funnel with two 10 ml
portions of ethyl alcohol followed by 20 ml of hexane.
e. Extract the combined solution with four 25 ml portions of
hexane that had previously been used to rinse the boiling
flask and filter funnel.
6. Cleanup
a. Wash the combined hexane extracts, obtained as describe
above in Subsections 1-5, with 25 ml of 1 N KOH solution
followed by four 50 ml portions of concentrated
b. Add 25 ml of water and shake. Neutralize the water and
hexane layers by addition of powered Na2C03 in small portions
with mixing until C02 evolution ceases.
c. Discard the aqueous layer, and dry the hexane layer by
passage through the 39 cm x 11 mm i.d. glass column contain-
ing 10 cm of anhydrous powered Na2C03.
d. Transfer the hexane concentrate to an alumina column,
prepared as described in Subsection VI, 1, that was prewetted
with one ml of hexane.
e. Wash the column with 6 ml of CCl^ and discard the wash.
f. Elute the column with 4 ml of methylene chloride and collect
in a 21 ml distillation receiver.
-------
Revised 12/15/79 Section 9, G
Page 8
g. Cap the distillation receiver with a micro-Snyder column
and evaporate the methylene chloride just to dryness on a
hot water or steam bath.
h. Add two separate 2 ml portions of hexane to the distillation
receiver and evaporate each just to dryness.
i. Dissolve the residue in 3 ml of hexane and chromatograph on
a second alumina column as just described.
j. Evaporate the methylene chloride eluate from the second
column just to dryness.
k. Add 2 ml of benzene to the receiver and concentrate to
ca 100 yl.
1. Transfer the benzene solution quantitatively to a 2 ml
graduated Chromaflex sample tube.
m. Carefully concentrate the benzene ca 100 yl under a gentle
stream of dry nitrogen. Quantitatively transfer with two
25 yl benzene rinses to a glass tube (7 cm x 3 mm i.d.)
that is sealed at one end.
n. Carefully concentrate the extract to 60 yl and flame seal
the tube. Store below 0°C until analysis by GLC MS.
VIII. SAMPLE PREPARATION AND CLEANUP - NEUTRAL PROCEDURE:
1. Extraction of Fish Tissue
a. Grind fish tissue to obtain a homogeneous sample.
b. Place a 15 gram sample and 150 grams of anhydrous granular
sodium sulfate in a blender jar and blend for one minute.
c. Blend next with 50 gram portions of dry ice until the sample
is thoroughly powered.
d. Transfer the powder to an Erlenmeyer flask and add 10 ng of
37C1-TCDD directly onto the powder.
e. Rinse the blender jar with acetonitrile.
f. Add the rinsings plus enough additional acetonitrile to the
flask to make a total of exactly 150 ml.
g. Mix vigorously on a magnetic stirrer for 2 hours.
-------
Revised 12/15/79 Section 9, G
Page 9
h. Filter the mixture through a glass filter tube containing
30 grams of anhydrous granular sodium sulfate.
2. Cleanup
a. Partition exactly a 100 ml aliquot of the acetonitrile
extract, representing 10 grams of the original sample, with
50 ml hexane that is saturated with acetonitrile. Draw
the acetonitrile (bottom) layer into a 500 ml separatory
funnel.
b. Partition the hexane layer with two 100 ml portions of
acetonitrile saturated with hexane followed by one 50 ml
portion of the same solvent and combine with the above
acetonitrile (350 ml total volume).
c. Partition the combined acetonitrile layers with 10 ml of
hexane saturated with acetonitrile.
d. Draw the acetonitrile layer into a 500 ml flat bottom 20/40
I Florence flask and concentrate to ca 10 ml under a Snyder
column on an explosion-proof hotplate.
e. Transfer the concentrate by repeated rinsings with a total
of 200 ml of hexane to a K-D apparatus with a 10 ml Mills
tube attached. Concentrate each of 5-10 ml on a hot water
or steam bath.
f. Transfer the hexane concentrate to a Florisil column,
prepared as described in Section VI,2, using three 5 ml
portions of hexane.
g. Elute the column with 100 ml of hexane-methylene chloride
(90:10 v/v) and discard this eluate.
h. Elute with 100 ml of hexane-methylene chloride (75:25 v/v)
and collect in a Kuderna-Danish evaporator equipped with
a 100 ml Mills tube.
i. Concentrate to ca 3 ml on a hot water or steam bath.
j. Dissolve the concentrate in 100 ml of hexane and again
evaporate to ca 3 ml.
k. Transfer the concentrate to an alumina column and proceed
with chromatography as described in the acid/base procedure
(Subsection VII,6), but use only one alumina column.
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Revised 12/15/79 Section 9, G
Page 10
IX. CAPILLARY COLUMN GC/HRMS MULTIPLE ION SELECTION ANALYSIS:
1. Tune the magnet current to perfluorokerosene (PFK) m/e 318.9793
and adjust the spectrometer from 5000 to 9000 mass resolution.
2. Monitor the ESA voltage and use in calculating the exact
acceleration voltage required for the masses m/e 327.8847
, m/e 321.8936 C12Hlt0235C1337C1 , and m/e 319.8965
3. Introduce the calculated values of MIS channels 2, 3, and 4.
4. Inject 2 yl of TCDD quantification standard, 500 pg/yl 37C1-TCDD
(labeled 2,3,7,8-tetrachlorodibenzo-p_-dioxin, 37C1 , isotopic
purity greater than 98%) and one pg/ pi TDCC, and 0.5 yl of
n-tetradecane (keeper) into the capillary column (on-column
splitless injection) maintained at 80°C.
5. Rapidly turn the GLC oven manual temperature control to 265°C
exactly 6 minutes after injection of the sample; the manual
control provides an accurate and rapid heating rate of 34°C/
minute.
6. Close the solvent vent valve exactly 14 minutes after injection
of the sample.
7. Optimize the MS sensitivity for the source operating pressure,
6 x 10-6 Toor, using PFK m/e 318.9793.
8. Initiate the MIS analysis 16 minutes after injection of the
sample.
9. Adhering to a strict (stopwatch) time schedule of events, the
GLC HRMS experimental retention time for TCDD was 23 minutes +_
15 seconds with the following GLC and MS parameters:
30 m SE-30 WCOT glass capillary column
injection port temperature, 260°C
GLC transfer line into the MS ion source, 255°C
ion source temperature, 240°C
variable acceleration voltage 3 kV maximum
electron energy, 70 eV
filament emission, 1 mA
mass resolution 5,000-10,000
multiplier gain, greater than 106
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Revised 12/15/79 Section 9, G
Page 11
X. COC1 LOSS ANALYSIS:
1. Tune the magnet current to PFK m/e 254.9856.
2. Introduce the exact acceleration voltages required for TCDD
masses m/e 256.9327, m/e 258.9298, m/e 319.8965, m/e 321.8936,
and m/e 327.8847 into respective MIS channels (M+—COC1 peak,
m/e 258.9298, is used to confirm TCDD structure).
3. Perform the analysis, adhering to the previously described time
schedule of events.
4. Observe the GLC HRMS five channel simultaneous response for
37C1-TCDD and TCDD and record at the correct GLC retention time
for TCDD.
XI. ELEMENTAL COMPOSITION ANALYSIS:
1. Adjust the mass spectrometer for 10,000 mass resolution using
PFK m/e 318.9793 as reference.
2. Initiate the peak matching analysis, adhering to the exact time
schedule of events utilized in the GLC HRMS MIS analyses.
3. Display alternately the reference mass and the exact mass range
of interest and view simultaneously on the MS oscilloscope.
XII. DETECTION AND RECOVERY RESULTS AND DISCUSSION OF METHODS:
1. Glass capillary columns enhanced the GLC HRMS method of analysis
because (a) they provided the required resolution of complex
mixtures into individual components before they entered the mass
spectrometer; (b) the narrow band width of the TCDD component
enhanced MS sensitivity; (c) direct coupling of the capillary
column to the mass spectrometer ensured maximum transfer effi-
ciency; (d) capillary column bleed rate was low, therefore
background contamination was minimized and MS sensitivity was
enhanced.
2. The requirements imposed on the mass spectrometer used as a GLC
detector in these analyses were (a) extremely stable electronic
circuits; (b) ultra high sensitivity; (c) specific mass detection.
The requirements were satisfied by optimizing all components
that influenced sensitivity, noise, and mass resolution. The
MIS response for a quantification standard, 37C1-TCDD and TCDD,
is shown in Figure 1.
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Revised 12/15/79 Section 9, G
Page 12
3. Precision and Accuracy of GLC HRMS Technique
When adjusted for 7500 mass resolution and used as a GLC detec-
tor in MIS analyses, the mass spectrometer will give positive
responses for those components eluting from the gas chromato-
graph that yield a molecular or fragment ion in the range +_
30 millimass units of TCDD masses m/e 319.8965, m/e 321.8936,
and m/e 327.8847. This minimizes the interference from
contaminating components, thus yielding responses that are
reproducible and linear relative to the amount of TCDD injected.
Two concentration ranges, 0.2-2 pg and 2-10 pg, were used to
provide the most efficient and accurate quantification of TCDD
because of the extremely high sensitivity and manual control
attenuation used in these analyses. Sample extracts should be
diluted or concentrated as required for quantification purposes.
The reproducibility of peak height response for TCDD standards,
1 pg/yl or 5 pg/yl, during daily operation was +_ 20%. The TCDD
m/e 320 and m/e 322 chlorine isotope ratio ranged from 0.75-0.95
to 1. The variation from the theoretical chlorine ratio, 0.8 to
1.0, was attributed to the MIS integration rate, very narrow
capillary column GLC peaks, and the small amount of TCDD being
analyzed.
The GLC HRMS peak matching accuracy for known elemental composi-
tions was determined to be +_ 2 millimass units at 9500 mass
resolution with PFK as reference. The reference mass and TCDD
mass were observed to be exactly superimposed on the mass
spectrometer oscilloscope at the exact GLC HRMS retention time
of TCDD.
4. Recovery of 37C1-TCDD and Measurement of TCDD
The MIS simultaneous peak height responses (m/e 328, m/e 322,
and m/e 320)"of sample and sample fortified with a known amount
of 37C1-TCDD and TCDD were used to determine the sample prepara-
tion procedure efficiency (percentage recovery), TCDD residue
level, and limit of detection. The criteria utilized for
confirmation of TCDD are shown in Table 1. The percentage
recovery experimental value was used to correct the TCDD residue
level and limit of detection for 37C1-TCDD recovery losses. A
minimum acceptable percentage recovery (50%) was established for
reporting TCDD analyses. The infrequent analyses exhibiting
less than 50% recovery were discarded. TCDD results were not
corrected for recovery values greater than 100%. Recovery
values between 100 and 135% were attributed to interference from
PCBs and unidentified contamination. Due to the widespread
distribution of PCBs, the accuracy of 37C1-TCDD determination
primarily depends on the sample preparation procedure efficiency
and specificity, and the capillary column GLC resolution of
-------
Revised 12/15/79 Section 9, G
Page 13
components. The MS mass resolution, ca 45000, required to
separate 37C1-TCDD m/e 327.8847 and PCB m/e 327.8758 is not
feasible owing to MS limitations. For occasional and highly
contaminated sample extracts, the 37C1-TCDD m/e 328 peak height
was determined utilizing the PCB m/e 326 peak height to
calculate the PCB contribution to the m/e 328, a mixture of
37C1-TCDD and PCB.
5. Limit of Detection
The limit of detection was defined as the quantity of TCDD that
would provide a signal to noise ratio greater than 2.5:1 with
clearly defined peak shapes (m/e 320 and m/e 322) in the proper
isotopic ratio. The limit of detection varied from sample to
sample because of percentage recovery, sample size, matrix
effects, and electronic noise present in the time frame of
measurements.
6. Isotopic Purity of 37C1-TCDD Fortification Standard
The 37C1-TCDD standard, 1 ng/yl in benzene, was subjected to MIS
analyses for determination of purity and possible interferences
for sub-ppt TCDD analyses prior to human milk studies. A TCDD
isomer was detected at 1 pg/ng 37C1-TCDD. The GLC HRMS peak
matching technique with PFK as reference was used to confirm
the elemental composition of m/e 319.8965 and 321.8936, both
corresponding to TCDD, in a concentrated solution of 37C1-TCDD
standard. The elemental compositions, the m/e 320/322 Cl ratio,
and the GLC retention time of 37C1-TCDD fortified with 2,3,7,8-
TCDD were criteria used to confirm the presence of a TCDD isomer
in 37C1-TCDD standard that satisfies the analytical criteria
for 2,3,7,8-TCDD. These results indicated that 10 ng 37C1-TCDD
foritification levels in 10 gram samples would produce 1 ppt
TCDD analyses. This was confirmed experimentally. The fortifi-
cation level was reduced from 10 to 2.5 ng 37C1-TCDD per sample
to avoid false positive results in 0 to 2 ppt TCDD analyses.
7. Human Milk Studies
EPA has initiated a study to determine the possible presence
of TCDD in human milk. Sample locations were selected based on
the aerial application of 2,4,5-T for conifer release as part of
a forestry management program. The samples were subjected to
the described acid-base extraction and cleanup procedure prior
to GLC HRMS MIS analysis.
The 60 yl human milk extracts were quantitatively concentrated
to 7-20 yl using dry nitrogen gas for sub-ppt TCDD analysis.
The MIS analysis sequence was sample, sample fortified with
-------
Revised 12/15/79 Section 9, G
Page 14
37C1-TCDD, and TCDD. A typical analysis for TCDD residues in a
QC sample of human mother's milk is shown in Figure 2. The
corrected experimental results indicated the sample contained
1.2 ppt TCDD residue. This 10 gram sample had been fortified
with 10 pg of TCDD, which corresponds to 1 ppt TCDD. The total
TCDD analyses, analytical cleanup efficiency, TCDD residue level,
and limit of detection were performed on injections of a sample
and fortified sample. Duplicate analyses were usually performed
on each sample to establish precision values.
The results of a quality assurance study incorporating human
milk fortified with 2.5 ng 37C1-TCDD and 0-5 ppt TCDD are shown
in Table 2. Evaluation of the experimental results and theoret-
ical results after completion of study indicate: (a) the analyt-
ical cleanup procedure and MIS method of analysis provided
reasonably accurate TCDD analysis in the 0.2-5 ppt concentration
range; (b) false positive results were not detected; (c) 2.5 ng
37C1-TCDD fortification levels were adequate for recovery
purposes; (d) the small amount, (2.5 pg) of TCDD, in adverse
effects in 0-5 ppt TCDD analyses, with a 0.2 ppt detection limit.
A representative number of positive results generated at this
level of detection should be confirmed with supplemental tech-
niques such as COC1 loss, peak matching analyses, etc.
Contamination was a constant problem in 0-5 ppt TCDD analysis.
A very efficient and optimized capillary column was required to
resolve TCDD from contamination, and its effectiveness could be
destroyed in the presence of high amounts of contamination. In
general, PCBs were the contaminants of major concern. The mass
resolutions 12,476 and 45,539 required to separate PCB masses
321.8677 and 327.8758 from TCDD masses 321.8935 and 327.8847
could not be used in 0-5 ppt TCDD analysis because of instrument
design and sensitivity. The PCB interference to TCDD analysis
was observed to have the following effects: (a) recovery of
37C1-TCDD became greater than 100% and (b) the TCDD m/e 320/322
chlorine isotope ratio was destroyed. Mass resolution of 5,000
to 8,000 was sufficient to resolve TCDD from other contamination
present.
8. Fish Analysis
Edible portions (2.5 to 10 grams) of fish samples were fortified
with 2.5 to 10 ng 37C1-TCDD and subjected to the described acid/
base extraction and cleanup procedures. An MIS analysis is
shown in Figure 3, for sample and fortified sample. Unusual and
high concentrations of contaminate masses were detected at m/e
320 and m/e 322 in fish collected from polluted waters. The
contamination was not detected in analyses of ocean perch, fish
from specific locations, and beef liver during the analysis
-------
Revised 12/15/79 Section 9, G
Page 15
sequence. The high concentration of co-extractable components
in fish caused serious problems (capillary column overload,
co-elution of components, and decreased MS sensitivity). To
minimize or cancel these effects, very high MS sensitivity
(4-9 pg quantification standard) and small sample size (0.5 to
3 yl from 55 yl equivalent to a 10 gram sample) were used in
analysis of fish.
A small number of highly contaminated fish extracts were
subjected to additional GLC HRMS analyses and to a "neutral"
cleanup procedure to confirm the presence of TCDD:
(a) MIS simultaneous response for the molecular ion cluster
m/e 320, m/e 322, and m/e 324 to confirm the tetrachloro
isotope ratio.
(b) MIS simultaneous response for m/e 320, m/e 322, m/e 257,
and m/e 259 to confirm the M+-COC1 loss indicative of the
TCDD structure.
(c) GLC HRMS peak matching analysis to confirm the elemental
composition of the TCDD molecular ion, m/e 319.8965.
Two exact masses corresponding to TCDD isomers were observed
eluting before and after TCDD. Contaminant masses, differing
from the exact mass of TCDD, were also observed during the time
frame of the analysis. Highly contaminated fish samples were
subjected to a "neutral" cleanup procedure described in Section
9G. Capillary column GLC HRMS MIS analysis yielded positive
37C1-TCDD and TCDD responses essentially free of contamination.
The quality assurance sample results utilized in these studies
are shown in Table 3.
9. Water and Sediment Analysis
Water and sediment samples were collected from specific areas of
the United States and subjected to the described analytical
extraction and cleanup procedures prior to MIS analysis. The
analytical results for quality assurance samples incorporated in
these studies are shown in Table 4.
Evaluation of the results shown in Table 4 indicates that the
analytical extraction and cleanup procedure and MIS technique
provided reasonably accurate analysis for 10-1000 parts per
quadrillion (lO"15) TCDD in water and 0-35 ppt TCDD in sediment.
Water extracts were very clean. Significant amounts of contam-
ination differing from the exact mass of TCDD were detected in
specific sediment samples but did not interfere with TCDD
analysis.
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Revised 12/15/79 Section 9, G
Page 16
10. TCDD Isomers
The toxicological properties of TCDD isomers are known to be
significantly different. The mass spectra of known TCDD
isomers are identical except in the low mass range, and this
minor difference would not be of significant value in ppt analy-
sis of environmental or biological extracts. Therefore, it is
extremely important that the gas chromatograph be equipped with
high resolution capillary columns to resolve TCDD isomers before
they enter the mass spectrometer. The 2,3,7,8-, 2,3,6,8-, and
1,2,3,4-TCDDs and a mixture consisting of 70% 1,3,6,8-TCDD and
30% of an unknown TCDD isomer have been separated in this
laboratory using glass capillary column GLC HRMS. The SE-30
WCOT glass capillary column resolution of TCDD isomers and
order of elution were similar to separations reported in the
literature on an OV-101 glass capillary column. The 2,3,6,8-TCDD
isomer was only partially resolved from 1,2,3,4-TCDD.
Several TCDD isomers have been detected and confirmed in environ-
mental, biological, and chemical formulation samples using the
described capillary column GLC HRMS techniques and coinjection
of specific TCDD isomers.
Preliminary studies using the acid-base cleanup procedure and
nanogram quantities of hexa-, hepta-, and octa-substituted
dioxins (analytical standards) suggest that tetrachlorodioxin
isomers are not formed from the degradation of higher chlorin-
ated dioxins by acid-base sample preparation conditions.
Nanogram quantities of 2,4,5-trichlorophenol showed no evidence
of condensation to 2,3,7,8-TCDD under the same acid-base
conditions.
XIII. ANALYTICAL QUALITY CONTROL:
The analytical cleanup laboratory should assign identification
numbers to all samples. The samples and QC samples are fortified
with 2.5-10 ng of 37C1-TCDD. The QC samples are fortified with 0 to
1, 250 pg (p to 125 ppt) of TCDD before extraction and cleanup.
A method blank is included as part of the QC sample package. All
sample extracts and quantification standards, 37C1-TCDD and TCDD, are
submitted to the GLC MS laboratory in a blind fashion, i.e., there
should be no way to distinguish QC and actual samples.
The efficiency, accuracy, precision, and validity of ppt TCDD
analyses depend on an incorporated quality assurance program. The
supplemental and conclusive GLC HRMS validation techniques involving
analyses for Mt-COCl loss and GLC HRMS peak matching analysis (real
time) can not easily be applied to 0-30 ppt TCDD analyses at this
date, using the described procedures. Based on the incorporated
-------
Revised 12/15/79 Section 9, G
Page 17
quality assurance program, analytical criteria, GLC HRMS techniques,
and multiple laboratory participation, the described methodologies
have been shown to be effective for isolation, detection, and
quantification of 0.02-100 ppt levels of TCDD in specific types of
samples. Samples containing high ppt or part per billing (ppb)
levels of TCDD can cause serious contamination problems in the sample
preparation laboratories, which result in erroneous low ppt TCDD
analysis of the sample next in the series. Extreme care and very
clean laboratory practices are mandatory for low ppt TCDD analyses.
Results of some quality assurance studies are presented in
Subsection XII.
TABLE 1. CRITERIA USED FOR CONFIRMATION OF 2,3,7,8-TCDD
RESIDUES IN HUMAN, ENVIRONMENTAL AND FISH SAMPLES
1. Capillary column GLC HRMS retention time of 2,3,7,8-TCDD.
2. Co-injection of sample fortified with 37C1-TCDD and
TCDD standard.
3. Molecular ion chlorine isotope ratio (m/e 320 and m/e 322).
4. Capillary column GLC HRMS multiple ion monitoring response
for TCDD masses (simultaneous response for elemental
composition of m/e 320, m/e 322, and m/e 328, 37C1-TCDD).
5. Response of m/e 320 and m/e 322 greater than 2.5 times the
noise level.
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Revised 12/15/79 Section 9, G
Page 18
TABLE 2. ANALYTICAL RESULTS FOR 2,3,7,8-TCDD RESIDUES IN
QUALITY ASSURANCE SAMPLES OF HUMAN MILK
Experimental Results
37C1-TCDD
% Recovery
50
72
68
64
68
84
64
51
73
52
72
100
50**
TCDD
Detection
Limit* (ppt)
0.3
0.2
0.1
0.3
0.4
0.2
0.2
0.2
0.4
0.3
0.5
0.5
0.2
TCDD
Detected*
(ppt)
1.9
0.6
0.2
ND
0.9
1.4
0.4
ND
0.6
1.4
3.0
4.0
ND
TCDD Fortification
(P9)
10
3
1
0
9
20
5
2
7.5
6.5
30
50
0
Level
(ppt)
1.0
0.3
0.1
0
0.9
2.0
0.5
0.2
0.75
0.65
3.0
5.0
0
Each 10 gram sample was fortified with 2.5 ng 37C1-TCDD,
* Corrected for recovery.
** Method blank.
ND = Not detected.
ppt = Parts per trillion.
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Revised 12/15/79 Section 9, G
Page 19
TABLE 3. SUMMARY OF ANALYTICAL RESULTS FOR QUALITY ASSURANCE
SAMPLES (OCEAN PERCH, LAKE TROUT, BEEF LIVER) GEN-
ERATED DURING ANALYSIS OF FISH FOR TCDD RESIDUES
Experimental Results
Sample
Weight
(gram)
5 (1)
5 (1)
5 (1)
5 (4)
5 (3)
5 (3)
5 (3)
5 (1)
5 (1)
10 (1)
10 (1)
10 (2)
10 (2)
10 (1)
10 (4)
10 (1)
10 (1)
37C1-TCDD
Fortifica-
tion Level
(ng)
5
5
5
5
5
5
5
5
5
10
10
10
10
10
10
5
10
37C1-TCDD
% Recovery
62
52
82
100
54
100
78
92
97
100+
100+
100+
100+
100+
67
93
84
35C1-TCDD
Detection
Limit (ppt)*
2
4
3
3
1
2
2
2
5
1
4
7
3
4
1
3
4
TCDD
Detected
(ppt)*
20
34
ND
ND
19
ND
ND
19
45
8
43
ND
ND
76
ND
56
73
TCDD Fortifica-
tion Level
(pg)
110
185
0
0
70
0
0
55
240
130
600
0
0
1250
0
650
620
(PPt)
22
35
0
0
14
0
0
11
48
13
60
0
0
125
0
65
62
* Corrected for recovery (1) ocean perch (2) lake trout (3) beef liver
(4) method blank
ND = Not detected.
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Revised 12/15/79
Section 9, G
Page 20
TABLE 4. SUMMARY OF ANALYTICAL RESULTS FOR QUALITY ASSURANCE SAMPLES
GENERATED DURING ANALYSIS OF WATER AND SEDIMENT FOR
2,3,7,8-TCDD RESIDUES
Experimental Results
Sample
Type
water
water
water
water
water
water
water
sediment
sediment
sediment
sediment
sediment
sediment
sediment
ppqd =
ppt =
*
ND =
Sample 37C1-TCDD
Weight Fortification 37C1-TCDD
(gram) Level (ng) % Recovery
1000
1000
1000
1000
1000
1000
1000
50
50
10
10
10
10
10
Parts per quadrill
Parts per trillion
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
ion (10"15).
do'12).
73
90
66
95
78
78
100+
69
100+
100+
96
100+
68
100
Detection
Limit*
15 ppqd
50 "
14 "
10 "
15 "
15 "
41 "
0.13 ppt
0.14 "
0.6 "
0.5 "
2.0
4.0 "
0.7 "
TCDD
Detected* TCDD Forti
85 ppqd
730 "
ND
50 "
116 "
28 "
422 "
1 .0 ppt
1.0 "
2.5 "
3.3 "
23.0 "
30.0 "
ND
75
1000
10
50
100
25
500
1
1
1
4
17
35
0
fication Level
ppqd
H
ii
M
H
ii
H
.0 ppt
.4 "
.6 "
.6 "
.0 "
.0 "
M
Corrected for recovery.
Not detected.
-------
Revised 12/15/79
Section 9, G
Page 21
TCDD
O m/e328
mle 322
m/e 320
20 22
TIME, (mm
26
FIGURE 1. GLC/HRMS multiple ion selection response for 1 ng 37C1-TCDD
and 2 pg <30C1-TCDD.
-------
Revised 12/15/79
Section 9, 6
Page 22
T^
."..». n:
_.JuUwW-Jv.U.i
TCDD
Q-m/e 328
0-m/e 322
0-m/e 320
I i I
16 18 20 22 24 26
18 20 22 24 26 28
TIME (min)
FIGURE 2. GLC/HRMS MIS monitoring analysis for TCDD residues in a
human milk QA sample. (A) 2 yl of sample (fortified with
1 ppt TCDD). (B) 1 ul of sample fortified with 500 pg
37C1-TCDD and 1 pg 35C1-TCDD.
-------
Revised 12/15/79
Section 9, G
Page 23
Q-m/e 328
m/e 322
i-m/e 320
12 14 16 18 20 22 24
12 14 16 18 20 22 24
TIME (min)
FIGURE 3. GLC/HRMS MIS monitoring analysis of a fish extract. (A) Sample
1 1 from 50 yl; (B) Fortified sample, 0.5 yl from 50 1 plus
quantification standard.
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Revised 6/77 Section 10, A
Page 1
THE SAMPLING AND ANALYSIS OF WATER FOR PESTICIDES
I. INTRODUCTION:
The methodology for the analysis of water described in this
section was researched by Thompson e_t al_. at the Environmental Toxi-
cology Division, Health Effects Research Laboratory, U.S. Environmental
Protection Agency, Research Triangle Park, NC (1). It is based on
modification of the multiclass, multiresidue procedure for pesticides
in air reported by Sherma and Shafik in an earlier paper (2).
Recovery studies were conducted on 42 halogenated compounds, 38 organo-
phosphorus compounds, and 7 carbamates, and the procedure proved
acceptable (>80% recovery) for 58 of the 87 compounds tested. Thir-
teen compounds yielded recoveries exceeding 60%, while the remaining
16 compounds were recovered at levels below 60%. Concentration levels
ranged from 0.09-400 ppb.
The present method provides the analyst with the means of
simultaneously monitoring water samples for a wide variety of different
pesticides, a capability not demonstrated for the few previously
published under multiclass, multiresidue analytical procedures. For
example, the method in the 1974 revision of this Manual included a
florisil cleanup column and was tested with only 16 organochlorine and
9 less-polor organophosphorus pesticides. Other published multiclass
GLC methods have employed cleanup on silica gel, Florisil, and alumina
or no column cleanup. None of these is as broadly applicable as the
following method.
REFERENCES:
1. Multiclass, Multiresidue Analytical Method for Pesticides
in Water, Thompson, J. F., Reid, S. J., and Kantor, E. J.,
Arch. Environ. Contam. Toxicol., to be published in 1977.
2. A Multiclass, Multiresidue Analytical Method for Pesticide
Residues in Air, Sherma, J., and Shafik, T. M., Arch.
Environ. Contam. Toxicol. 3_, 55 (1975). (See Section 8,
this Manual).
3. Persistence of Pesticides in River Water, Eichelberger, J. W.
and Lichtenberg, J. J., Environ. Sci. and Technol. 5(6),
541 (1971) (Table 1).
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Revised 6/77 Section 10, A
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4. Pesticide Residue Analysis in Water—Training Manual
PB-238 072, U.S. Environmental Protection Agency,
OWPO, National Training Center, Cincinnati, Ohio,
September, 1974, distributed by the National Technical
Information Service, U.S. Department of Commerce.
5. Gas Chromatographic Determination of Residues of Methyl
Carbamate Insecticides in Crops as their 2,4-Dinitrophenyl
Ether Derivatives, Holden, E. R., J. Ass. Offic. Anal. Chem.
56, 713 (1973) and 58, 562 (1975).
II. PRINCIPLE: (See Schemes I and II, Pages 15 and 16)
Compounds are extracted from water with methylene chloride, and
the extract volume is reduced at low pressure and temperature in an
evaporative concentrator. Compounds are separated into groups on a
column of deactivated silica gel by elution with solvents of increasing
polarity. Organochlorine compounds are determined by gas chromatog-
raphy with an electron capture detector, organophosphorus compounds
with a flame photometric detector, and carbamates by electron capture
GLC after conversion to 2,4-dinitrophenyl ether derivatives.
III. GRAB SAMPLE COLLECTION:
The sampling location and the method of drawing the sample will,
to a great extent, be dictated by the objectives of the sample data.
If the objective is to determine the highest pesticide pollution
present in a stream or lake, a grab sample might be drawn at the point
of highest polution introduction. If, on the other hand, the objec-
tive is an average residue profile of the entire body of water, the
final sample would preferably be a composite of a number of subsamples
taken at various locations and water depths. If samples are collected
in the area of a fish kill, a minimum of three samples are collected
in the kill area, a control sample well above the suspected source of
the pollutant, and one or two samples downstream of the kill area if
the pesticide is downstream from the area of dying fish. In the case
of a tidewater estuary, some modifications in the sampling pattern
may be indicated.
As implied by the name, a grab or dip sample would be a surface
water sample generally taken by simply filling the sample container
by immersing and allowing the bottle or jar to fill up. For sampling
at selected depths, devices such as a Precision sewage water sampler
or an Esmarch sampler may be utilized. Both devices consist of a
metal outer container with a glass bottle inside as the sample
collection vessel.
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Revised 6/77 Section 10, A
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The Precision sampler in which the interior of the collection
bottle has free access to the exterior by means of an open tube can
be used to draw a composite depth sample. As soon as the device is
immersed, collection of the sample is started. By premeasuring the
rate of lowering the device to collect a given amount of water, an
approximately uniform amount of water can be collected throughout
the entire depth sampled.
The Esmarch sampler may be manually opened and closed by means
of a chain attached to the bottle stopper. This permits a sample
or subsample to be drawn from any given depth simply by lowering the
device with the stopper closed, opening it at the proper sampling
depth to permit filling of the collection bottle, then closing the
stopper and raising the device to the surface.
IV. SAMPLE CONTAINERS AND STORAGE:
Wide mouth glass jars such as the Mason type are recommended as
suitable sample containers when the sample is to be 2 liters or less.
If the sample is of greater volume than 2 liters, the one gallon glass
bottles in which acetone, hexane or petroleum ether are normally sold
provide excellent sample containers. Furthermore, the latter require
no special precleaning before use. Other glass containers must be
scrupulously cleaned and rinsed with some of the same solvent used
for subsequent pesticide extraction. All bottle or jar caps should be
Teflon or foil lined to prevent contamination of the sample with trace
quantities of impurities which may be present in laminated paper
liners or in the composition of the material used for the seal in
Mason jar lids.
The size of sample is dictated primarily by the expected residue
levels. For example, if the sample is collected from a waterway where
pesticide levels are expectedly high (such as agricultural run-off),
a sample size of 500 to 1,000 ml may be sufficient. If the sample is
drawn in connection with a monitoring program where no especially high
residues would be expected, a sample size of 2 liters or more may be
indicated. Sample containers should be carefully labeled with the
exact site, time, date, and the name of the sampler.
Ideally, analysis of the sample should be conducted within a
matter of hours from the time of sampling. However, this is frequent-
ly impractical in terms of the distance from sampling site to labora-
tory, and/or the laboratory workload. Samples being examined solely
for organochlorine residues may be held up to a week under refrigera-
tion at 2 to 4°C. Those intended for organophosphorous or carbamate
analysis should be frozen immediately after drawing sample and should
be extracted no more than 4 days after sampling. These classes of
pesticides undergo degradation very rapidly in the aqueous medium.
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Every effort should be made to perform the solvent extraction
setep at the earliest possible time after sampling, irrespective of
the class of pesticides suspected as being present. The resulting
extracts may then be held for periods up to three or four weeks at
-15 to 20°C before conducting the adsorbent partitioning and deter-
minative portions of the analysis. The reader is referred to Table 1
at the end of Section 10,A. These data show the degradation rate of
29 pesticides in water at ambient temperature in sealed containers (3).
V. OTHER SAMPLING METHODS (Reference (4), Outline 22):
Continuous and automatic samplers of various types are appropriate
for sampling flowing rivers and streams. Samplers have been designed
to collect water samples at a rate proportional to either water flow
or time. Equipment is now available for collecting proportionalized
grab samples from gauges and instrumented streams that are proportional
to the flow of the stream. This method is particularly useful, in
fact required, to determine the total discharge load of a pesticide
from a stream. For additional details, see reference (4), outline 22.
Carbon adsorption is a standard method for continuous sampling of
water. The technique involves passage of a continuous, constantly
controlled volume of water through a column of activated carbon. The
major advantages of this method are that it takes a continuous sample
and that it yields sufficient quantities of extract for corroborative,
qualitative analyses.
The precision of the method appears to be satisfactory, but the
quantitative efficiency is open to many questions. Efficiency of
adsorption has already been found to vary dramatically, depending on
the rate of flow through the column and the total volume passed. A
broad spectrum of organics are adsorbed by the carbon, but it has been
estimated that perhaps 95% of the total organic load passes through.
Many pesticides are adsorbed by activated carbon, but little is known
at present about the efficiency of adsorption for specific pesticides.
Quantitative statements of pesticide concentration based on carbon
adsorption should be restricted to the "it is certain that no less
than (X) amount was present," variety.
Besides carbon, many other filter materials have been recommended
for continuous samplers, and continuous liquid-liquid extractors are
also available.
VI. EQUIPMENT:
1. Gas chromatograph fitted with an electron capture detector and a
flame photometric detector with a 526 nm P filter (thermionic
detection may be substituted for the FPD). GLC columns, boro-
silicate glass, 1.8 m x 4 mm i.d., packed with 1.5% OV-17/1.95%
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Revised 6/77 Section 10, A
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OV-210, and 5% OV-210, both coated on Gas-Chrom Q, 80/100 mesh,
operated with specific parameters given under Gas Chromatography,
Section IX. Criteria for high sensitivity in the GLC system are
set forth in Section 4,A,(4), Page 4 for the EC detection mode,
and in Section 4,B,(2), Page 3 for the FPD mode. These should
be carefully noted.
2. Chromaflex columns, size 22, 7 mm i.d. x 200 mm, Kontes 420100.
3. Chromaflex column, 22 mm i.d. x 300 mm, size 241, Kontes 420530.
4. Rinco evaporator, rotating, such as Scientific Glass Apparatus
Co. E-5500 or E-5500-1, with appropriate stand.
5. Variac or comparable voltage control regulator.
6. Water bath for operation at 35°C.
7. Vacuum source of 125 mm Hg, optimally.
8. Kuderna-Danish evaporators, 250 ml, Kontes 570001.
9. Centrifuge tubes, conical, 15 ml, graduated, Corning No. 8082
with Teflon lined plastic screw caps, thread finish 415-15,
Corning 9998.
10. Tubes, culture, screw caps with Teflon liner, 16 x 125 mm,
Corning 9826.
11. Evaporative concentrator tubes, 10 ml, graduated from 0.1 to
10.0 ml, size 1025 with outer joint I 19/22, Kontes 570050.
12. Tube heater with aluminum block containing 18 mm (3/4 inch) holes,
Kontes 720000 (a water bath can be used as a substitute).
13. Mixer producing a tumbling action at ca 50 rpm (Fisher Roto-Rack
or equivalent).
14. Prepurified nitrogen source with 3-stage regulation to produce
a gentle stream of gas through an extruded tip of glass or
stainless steel.
15. Vortex mini-mixer.
16. Disposable Pasteur pipets, Fisher 13-678-5A or equivalent.
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Revised 6/77 Section 10, A
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VII. SOLVENTS AND REAGENTS:
1. Methylene chloride, hexane, benzene, acetonitrile, acetone, and
methanol, all of pesticide quality.
2. Silica gel, Woelm, activity grade I, activated for 48 hours at
175°C before use. Prepare final deactivated material by adding
1.0 ml of water to 5.0 g silica gel in a vial with a Teflon-
lined screw cap. Cap tightly and mix on the Roto-Rack for 2
hours at ca 50 rpm. Discard deactivated silica gel after 5 days.
NOTE: It is recommended that the amount of silica gel
activated at 175°C be restricted to the quantity
needed for immediate deactivation.
3. Sodium sulfate, granular, anhydrous. Purify by Soxhlet extract-
ing' with methylene chloride for ca 60 discharge cycles.
4. l-Fluoro-2,4-dinitrobenzene (FDNB), J. T. Baker 5-M478 or
equivalent. Prepare a 1% reagent solution in acetone.
5. Sodium borate buffer, 0.1 M solution of Na2Bi+07.10 H20, pH 9.4,
J. T. Baker 3568 or equivalent.
6. Carborundum chips, fine. These should be purified as described
for sodium sulfate in Item 3 of this section if a precheck
indicates any contamination problems.
7. Glass wool, preextracted with methanol, acetone, and methylene
chloride to remove any contaminants.
8. "Keeper" solution, 1% paraffin oil, USP grade, in hexane.
9. Eluting solutions:
Fraction I - hexane
Fraction II - benzene-hexane (60:40 v/v)
Fraction III - acetonitrile-benzene (5:95 v/v)
Fraction IV - acetone-methylene chloride (25:75 v/v)
10. Contaminant-free water. To 1500 ml of distilled water in a 2 L
separatory funnel add 100 ml methylene chloride, stopper, and
shake vigorously for 2 minutes. Allow the phases to separate,
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Revised 6/77 Section 10, A
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discard the solvent layer, and repeat the extraction with another
100-ml portion of methylene chloride. Drain the double-extracted
water into a glass stoppered bottle for storage, withdrawing
500 ml to serve as a reagent blank with each set of samples.
11. Pesticide reference standards, analytical grade.
VIII. SAMPLE EXTRACTION AND CONCENTRATION:
1. Transfer 500 ml of water to a 1 L separatory funnel and add 10 g
anhydrous sodium sulfate and 50 ml of methylene chloride. Shake
vigorously for 2 minutes and allow a sufficient length of time
for complete phase separation.
NOTES:
1. If the expected pesticide concentration is extremely low,
i.e. under .04 yg/L, it may be advisable to increase the
initial sample to 1000 to 2000 ml. In this case, the volume
of methylene chloride should be increased to 75 ml and the
separatory funnel size to 2 or 3 L.
2. To avoid troublesome caking of the sodium sulfate at the
bottom of the funnel, shaking should be conducted instantly
after adding the sodium sulfate.
3. At this point a reagent blank of 500 ml of the preextracted
water should be carried through all procedural steps in
exactly the same manner as the sample(s).
2. Place a small wad of glass wool at the bottom of a 25 x 300 mm
Chromaflex column and add a 2 in. depth of anhydrous sodium
sulfate. Position the tip of the column over a Kuderna-Danish
assembly consisting of a 250 ml K-D flask attached to a 10 ml
evaporative concentrator tube containing two or three carborundum
chips and 5 to 10 drops of keeper solution.
3. Drain the lower layer (methylene chloride phase) from the separ-
atory funnel through the sodium sulfate column, taking care to
avoid the transfer of any of the aqueous phase.
4. Add 50 ml more of methylene chloride to the aqueous phase in the
funnel. Stopper and repeat the 2-minute shaking, phase separa-
tion, and draining of the organic layer through the sodium
sulfate column into the K-D flask.
NOTE: It is not uncommon with highly polluted water samples to
encounter persistent and sometimes severe emulsion problems
at the methylene chloride-water interface. When this occurs,
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for example, in the extraction of some waste-water samples
containing high surfactant concentrations, it is
inadvisable to pass the methylene chloride phases through
the sodium sulfate because the aqueous emulsion tends to
clog the column and make filtration difficult. A good way
to cope with an emulsion is to pack a filter tube (A. H.
Thomas 4797-N15 or equivalent) with a 25 mm thick pre-
washed glass wool pad and pass the extract containing the
emulsion through this filter into a 400 ml beaker,
applying air pressure if necessary. If the emulsion
persists on the second methylene chloride extraction,
this treatment is repeated. The glass wool pad is then
rinsed with 25 ml of methylene chloride, collecting the
extract and washing on the surface of the filtrate, a
second glass wool filter is set up and the operation is
repeated.
5. Connect the K-D flask to the rotary evaporator and incline the
assembly to an angle approximately 20° from the vertical, with
the concentrator tube about half immersed in a water bath
previously adjusted to 35°C. Turn on the rotator, adjusting
speed to a slow spin. Switch off the bath heat and apply vacuum
to the evaporator at a pressure of ca 125 mm of Hg.
NOTE: The recommended adjustments of temperature, vacuum, and
the pitch of the assembly should resutl in a steady
boiling action and with no bumping. The pitch should be
such that no extract condensate collects in the lower
position of the K-D flask. (See Figure 1.)
6. Continue evaporation until the extract is condensed to ca 4 ml,
remove the assembly from the water bath, and rinse down the
walls of the flask with 4 ml of hexane delivered with a disposable
pi pet.
7. Disconnect the concentrator tube from the K-D flask, rinsing the
joint with ca 2 ml of hexane delivered with a disposable pi pet.
8. Place the tube under a gentle stream of nitrogen at ambient
temperature and concentrate the extract to ca 0.5 ml.
NOTE: Under no circumstances should air be used for the blow-
down as certain organophosphorus and carbamate compounds
(and even low concentrations of some organohalogens)
may not survive the oxidative effects.
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IX. SILICA GEL FRACTIONATION AND CLEANUP:
Before starting the following steps, place 10 drops of the
paraffin oil-hexane keeper solution in the two 15 ml centrifuge
tubes intended as the receivers for the eluates of Fractions III
and IV.
1. Prepare a silica gel column as follows:
a. Lightly plug a size 22 Chromaflex column with a small
wad of preextracted glass wool.
b. Add 1.0 g of deactivated silica gel, tapping firmly to
settle, then top with 1 in. of anhydrous sodium sulfate
and again tap firmly.
c. Pass 10 ml of hexane through the column as a prewash,
discarding the eluate.
2. When the last of the prewash hexane just reaches the top sur-
face of the sodium sulfate, quickly place a 15 ml conical
centrifuge tube under the column, and using a disposable pipet,
carefully transfer the 0.5 ml of sample extract to the column.
When this has sunk into the bed, rinse the walls of the centri-
fuge tube with 1.0 ml of hexane, and, using the same disposable
pipet, transfer this washing increment to the column. Repeat
this 1.0 ml hexane wash twice more and finally add 6.5 ml
hexane to the column. The resulting 10 ml total effluent
is Fraction I.
NOTES:
1. There must be no interruption of the procedure during
this step. Extreme care should be taken to apply the
sample to the column at the precise moment the last of
the hexane prewash reaches the top surface of the column.
2. Faultless technique is required in this stip to avoid
any losses, particularly during the transfer of the 0.5 ml
concentrated extract and the first rinse. All the
pesticide derived from the original sample is concentrated
in this very miniscule extract. The loss of one drop
may introduce a recovery error of at least 10%.
3. Immediately position another 15 ml centrifuge tube under the
column and pass through the column 15 ml of the benzene-
hexane (60:40 v/v) eluting solution. This is the Fraction II
eluate.
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4. Make a third elution with 15 ml of the acetonitri1e-benzene
solution (5:95 v/v). This eluate is Fraction III.
5. A fourth elution fraction is necessary if there is reason to
suspect the presence of crufomate, dicrotophos, dimethoate,
mevinphos, phosphamidon, or the oxygen analogs of diazinon
and malathion. The elution solution is 15 ml of acetone-
methylene chloride (25:75 v/v). This is Fraction IV.
6. Place the el mates under a gentle nitrogen stream at ambient
temperature and concentrate as follows:
a. Fractions I and II to ca 3.0 ml, rinse down the tube
sidewalls with ca 1.5 ml hexane and adjust the volume
to exactly 5.0 ml with hexane. Cap the tubes tightly
and mix on the Vortex mixer for one minute.
b. Fractions III and IV to 0.3 ml, rinse tube sidewalls with
hexane, and dilute back to exactly 5.0 ml with hexane.
NOTE: Fractions III and IV contain eluant solvents
which may interfere in the GLC determination,
whereas those solvents in Fractions I and II
would create no such problems. For this reason,
Fractions III and IV are reduced to a lower
volume to remove the original solvents.
7. Fractions II and III may contain carbamates as well as
organophosphorus compounds. Gas chromatography of organo-
phosphorus compounds by flame photometric detection is
conducted on the eluates adjusted to 5.0 ml. When this has
been completed, the tubes are placed back under a nitrogen
stream, and the eluates are concentrated to 0.1 ml preparatory
to derivatization of the carbamates which may be present.
NOTE: The principal reason for concentrating this eluate
to 0.1 ml is to reduce the volume of benzene which
could interfere in the subsequent derivatization
reaction.
X. CARBAMATE DERIVATIZATION:
1. Add 0.5 ml of the FDNB-acetone reagent solution and 5.0 ml
of sodium borate buffer solution to the tubes containing the
0.1 ml of Fractions II and III, and add the same reagents to
an empty tube to serve as a reagent blank.
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Revised 6/77 Section 10, A
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NOTE: At this point, if any specific carbamate is suspected,
prepare a solution of known concentration from a
primary reference standard. A concentration of 5 yg
per ml in acetone may be appropriate. This should be
carried through the entire procedure starting with
this step in exactly the same manner and at the same
time as the unknowns.
2. Cap the tubes tightly and heat at 70°C for one hour in the
heating block or in a water bath.
3. Cool the tubes to room temperature and add 5.0 ml hexane to
each tube. Shake vigorously for 3 minutes, either manually
or on a wrist action mechanical shaker.
4. Allow the layers to separate and carefully transfer 4 ml of
the hexane (upper) layer to a vial or test tube which can be
stoppered tightly.
XI. GAS CHROMATOGRAPHY:
For multiresidue analysis of samples with unknown pesticidal
contamination, two GLC columns yielding divergent compound elution
patterns will aid confirmation. Two such columns are 5% OV-210
and 1.5% OV-17/1.95% OV-210. For EC detection, the column oven
should be set at 200°C for the mixed column and at 180°C for 5%
OV-210 (see exception for carbamates given under XI,5). Carrier
gas flow should be set to produce an absolute retention time of
16-19 minutes for £,p_'-DDT.
Sensitivity levels for both EC and FPD detectors should be
carefully established before starting chromatographic determination.
The majority of water samples will contain extremely low pesticide
concentrations, and, therefore an insensitive GLC system will
severely handicap the analysis. See Sections 4A and 4B of this
manual for recommended criteria.
The majority of the halogenated pesticides will be found in
Fractions I and II, with a few of the more polar compounds in
Fraction III. Most of the organophosphorus compounds will be in
Fractions II and III, none in Fraction I, and a very few in
Fraction IV. Carbamates are eluted in Fractions II and III (Tables
2-4).
The analyst is referred to Section 4,A(4) of this Manual,
pages 2 and 3, 12/2/74 revisions, for a time-saving procedure
for tentative peak identification and choice of quantisation
standards.
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A number of organophosphorus compounds chromatographed with
the FPD detector require considerable column preconditioning by
repetitive injection of standards of relatively high concentration
before attempting quantisation. Failure to carefully monitor
linearity of response may result in erroneous quantitative values.
Typical gas chromatograms of silica gel column fractions are
shown in Figures 2-4. Figure 3 illustrates the electron capture gas
chromatography of chlorinated pesticides, Figure 4 the chromatography
of organophosphates with FPD detection, and Figure 2 a chromatogram
of dinitrophenyl ether derivatives of carbamates detected by electron
capture.
XII. RECOVERY AND DETECTION DATA:
Recovery data for the extraction step alone and the total
procedure including silica gel chromatography, and the concentration
levels tested are shown in Tables 2-4. Water samples of 500 ml
are suitable for detection at these concentration levels. Of the
42 halogenated compounds evaluated, reproducible recoveries of 80%
or more were obtained for 31. Gas chromatography linearity problems
were encountered with captan and folpet, and a sizable portion of
lindane was lost during silica gel fractionation.
Thirty-one of the 38 organophosphorus compounds were recovered
in the 80+% range and six between 60 and 79%. Reproducible and
satisfactory recoveries were not achieved for carbophenoxon,
disulfoton, methamidophos, monocrotophos, and oxydemeton methyl.
Of these five compounds, excellent extraction efficiency was observed
for carbophenoxon and disulfoton, but complete loss was experienced
on the silica gel column. Six compounds were partially recovered
in the 0-60% range. Of the 17 OP compounds yielding total recoveries
of less than 80%, six of these gave over 90% extraction recovery,
but losses occurred during silica gel chromatography.
Final recoveries after fractionation were acceptable for the
carbamates metalkamate, carbofuran, methiocarb, and propoxur.
Acceptable recoveries were obtained for aminocarb and carbaryl by
direct derivatization and gas chromatography of the concentrated
methylene chloride extract, by-passing silica gel fractionation which
caused losses for these two compounds. Recoveries of mexacarbate
were highly inconsistent, both for direct analysis of spiked
methylene chloride or water extracts. Silica gel fractionation of
this compound resulted in further losses.
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XIII. MISCELLANEOUS NOTES:
1. The recommended operation of the concentrator shown in
Figure 1 is unusual for pesticide analysis. Customarily,
solvent evaporation is achieved by immersing the concentrator
tube in a water bath at a higher temperature than the boiling
point of the solvent, or the flask is attached to a conventional
rotary evaporator. The system shown in Figure 1 achieves two
important objectives: the extract is exposed to a maximum
temperature of less than 35°C to minimize degradation of heat
labile compounds; and the concentrated extract is confined to
one container, thereby eliminating need for a transfer. Using
the temperature and vacuum levels specified in Section VIII,
100 ml of methylene chloride extract can be reduced to 5 ml
in ca 20 minutes in this apparatus.
2. The activity and performance of deactivated silica gel changes
in a matter of days. It is desirable to deactivate only the
amount required for a 2 or 3 day period. Continuous storage
of activated silica gel at 175°C may result in a shift of the
compound elution pattern of deactivated columns prepared from
this adsorbent. The quantity of silica gel activated should
be limited to a one week supply.
3. The derivatization procedure for carbamates is based on work
reported by Holden (5) wherein phenols were formed by hydrolysis
in a borate buffer followed by reaction with FDNB to form
2,4-dinitrophenyl ethers. This procedure is superior to
derivatization with pentafluorophropionic anydride, as used
by Sherma and Shafik (2). With the latter method, considerable
masking of derivative peaks by EC detection is observed, and,
in addition, most of the peaks elute so early and are so
poorly resolved that quantitation is difficult. The Holden
method of reaction of intact carbamates with FDNB reagent
produces peaks which elute significantly later than those
resulting from reagent impurities or other contaminants
(Figure 4).
4. Recoveries of OP pesticides were found, in general, to be far
better when methylene chloride-extracted water rather than
unextracted distilled water was used as the spiking substrate
to evaluate this procedure. Therefore, unextracted distilled
water was used for all recovery studies. As a further test,
a sample of water was obtained a few hundred yards downstream
from the outfall of a large chemical manufacturing plant and
was fortified with a mixture of pesticides and analyzed using
the extraction and silica gel fractionation steps. Although a
few extraneous peaks were observed with the electron capture
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Revised 6/77 Section 10, A
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detector, no significant interference with pesticide peaks
occurred. This indicates the applicability of the method
to real-life water samples.
5. The OV-210 GLC column oven can be operated at an elevated
temperature (e.g., 215 to 220°C) to expedite elution of the
carbamate DNFB derivatives which have high retention times.
XIV. ANALYTICAL QUALITY CONTROL:
1. It is strongly recommended that selected analytical grade
standards of known concentrations be analyzed in parallel in
each individual sample or set of samples. This will increase
confidence in qualitative and quantitative results and will
alert the analyst to any shifts in the compound elution
pattern from the silica gel column. Certain compounds may
elute in different fractions than those shown in Tables 2-4
when different lots of silica gel are used or as atmospheric
conditions, particularly relative humidity, vary.
2. Interpretation of chromatograms should be carefully made,
based on elutoin patterns from the two dissimilar GLC columns
and detectability by the EC and FPD detectors. Further
confirmation of compound identity should be made by such
techniques as TLC, microcoulometric or Coulson conductivity
detector response, p_-values, or coupled GLC MS if the latter
equipment is available. Confirmatory procedures are discussed
in Section 8 of the EPA Quality Control Manual.
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Section 10,A
Page 15
Pesticides in Water -- Thompson et al., 1976
Water
discard
500 ml Water
10 gm Na2S04
100 ml
MeClo
Concentrate
ml
Concentrate
to 0.5 ml
Extract with
2 x 50 ml MeCl,
Percolate through
granular ^280^
Add hexane
Silica Gel Column
1 gm deactivated
with 20% water
10 ml
hexane
Fraction I
16 OGC's
PCB's
15 ml
60% benzene
in hexane
Fraction II
23 OGC's
16 OGP's
2 Carbamates
(partial)
15 ml
5% CHsCN
in benzene
Fraction III
8 OGC's
12 OGP's
7 Carbamates
15 ml
25% acel
in MeCl
Fraction IV
7 OGP's
Scheme I
-------
Revised 6/77
Section 10,A
Page 16
Pesticides in Water -- Thompson et al.
Carbamate Derivatization
Fraction III
Fraction II
0.1 ml volume
Cool and
open tubes
Aqueous
(discard)
add 0.5 ml of
1% FDNB in acetone
and 5 ml of borate
buffer, cap, heat
at 70° for" 1 hr.
add 5 ml
hexane,
shake 3 min.
EC-GC for
carbamate
derivatives
Scheme II
-------
Revised 6/77
Section 10, A
Page 17
Table 1. Persistence of Compounds in River Water
in Terms of Percentage Recover/
Compound
0-tirae
Original compound found ,%
1 wE TvEc TwE 8~wlc
Organochlorine compounds
BUG
Heptachlor
Aldrin
Heptachlor
epoxide
Telodrin
Endosulfan
Dieldrin
DDE
DDT
ODD
Chlordane (tech.)
Endrin
100
100
100
100
100
100
100
100
100
100
100
100
100
25
100
100
25
30
100
100
100
100
90
100
100
0
80
100
10
5
100
100
100
100
85
100
100
0
40
100
0
0
100
100
100
100
85
100
100
0
40
100
0
0
100
100
100
100
85
100
Organophosphorus compounds
Parathion
Nfethyl parathion
Malathion
Ethion
Trithion
Fenthion
Dimethoate
Merphos
Merphos recov.
as Def
Azodrin
Carbamate compounds
Sevin
Zectran
Matacil
Mesurol
Baygon
Monuron
Fenuron
100
80
100
100
90
100
100
0
100
100
90
100
100
90
100
80
80
50
25
25
90
25
50
100
0
50
100
5
15
60
0
50
40
60
30
10
10
75
10
10
85
0
30
100
0
0
10
0
30
30
20
<5
0
0
50
0
0
75
0
10
100
0
0
0
0
10
20
0
0
0
0
50
0
0
50
0
<5
100
0
0
0
0
5
0
0
^Pesticide concentrations were 10 yg/liter. Recoveries were rounded
off to the nearest 5%.
-------
Revised 6/77
Section 10, A
Page 18
TABLE 2. RECOVERIES OF 42 ORGANOCHLORINE COMPOUNDS
Compound
Aldrin
Atrazine
a-BHC
3-BHC
Y-BHC (Lindane)
Captan3
CDEC
Chlorbenside
Chlordane
Chlordecone (Kepone)
2,4-D, butyl ester
2,4-D, butoxyethanol
ether ester
2,4,-D, isooctyl ester
2,4-D, isopropyl ester
DCPA
p_,p_'-DDD
£,£'-DDE
o_,£'-DDT
£,£'-DDT
Dichlone
Dieldrin
Dilan
Dyrene
Cone. Extractii
(ppb) Only
.20 89
66.5 101
.09 91
.47 99
.12 90
6.50 100+
.27 63
.47 91
1.54 85
3.64 72
4.08 93
8.65 109
3.28 105
3.38 75
.50 98
.80 97
.45 96
1.05 94
1.58 104
13.4 85
.72 97
2.42 97
8.70 95
Recoveries in Percent
Silica Gel Parti
pn (Elution Fraction)
I II III IV
88
99
76 4
88
16 51
100+
41
62
89 1
18 8
90
9 90
95
71
84
94
101
93
98
79
94
77
tioning
Total
88
99
80
88
67
100+
41
62
90
26
90
99
95
71
84
94
101
93
98
79
96
94
77
Non-linear response
(Continued)
-------
Revised 6/77
Section 10, A
Page 19
TABLE 2. (CONTINUED)
Cone. Extraction
Compound (ppb) Only
Endrin 1.11 105
Endosulfan (Thiodan) .53 91
Folpetb 1.5 100
Heptachlor .18 90
Heptachlor Epoxide .31 91
Hexachlorobenzene (HCB) .20 74
l-Hydroxychlordene .34 81
Methoxychlor 5.70 97
Mi rex 2.35 83
PCNB .10 87
Perthane 66.5 89
Simazine 66.5 71
2,4,5-T, butyl ester 2.00 102
2,4,5-T, butoxyethanol
ether ester 3.00 103
2,4,5-T, isooctyl ester 6.05 109
Tetradifon (Tedion) 2.99 103
Toxaphene 22.3 103
Aroclor 1254 25.6 93
Aroclor 1260 25.6 93
Recoveries in Percent
Silica Gel Partitioning
(Elution Fraction)
I II III IV Total
98 98
24 80 104
131 131
79 79
89 89
96 96
82 82
104 104
83 83
88 88
80 15 95
28 28
99 99
71 23 94
97 97
102 102
93 93
96 96
92 92
Non-linear response
-------
Revised 6/77
Section 10, A
Page 20
TABLE 3. RECOVERIES OF 38 ORGANOPHOSPHOROUS COMPOUNDS
Cone. Extraction
Compound (ppb) Only
Azinphos Methyl (Guthion) 320 78
Carbophenothion (Trithion) 48 99
Carbophenoxon 80 94
Chlorpyrifos (Dursban) 4 99
Crufomate (Ruelene) 90 80
DEF 24 102
Diazinon 20 108
Diazoxon 10 92
Dichlofenthion (VC-13) 1.6 102
Dicrotophos (Bidrin) 120 17
Dimethoate (Cygon) 24 40
Dioxathion (Delnav) 28 103
Disulfoton (Di-Syston) 2.6 92
EPN 60 99
Ethion 20 100
Ethoprop (Prophos) 2 97
Fenitrothion (Sumithion) 12 99
Fenthion (Baytex) 12 93
Fonofos (Dyfonate) 20 98
Leptophos (Phosvel) 200 107
Malaoxon 80 104
Malathion 4 100
Methamidophos (Monitor) 200 5
Mevinphos (Phosdrin) 6 69
Recoveries in Percent
Silica Gel Partitioning
(Elution Fraction)
I II III IV Total
88 88
93 93
0
87 87
58 58
90 90
104 104
72 72
102 102
15 15
60 60
72 17 89
0
96 96
94 94
96 96
84 84
76 76
78 78
91 91
50 5G
78 78
0
32 33 65
(Continued)
-------
Revised 6/77
Section 10, A
Page 21
TABLE 3. (CONTINUED)
Compound
Cone. Extraction
(ppb) Only
Recoveries in Percent
Silica Gel Partitioning
(Elution Fraction)
I II III IV Total
Monocrotophos (Azodrin) 72 0
Naled (Dibrom) 56 92
Oxydemeton Methyl
(Metasystox R) 300 67
Paraoxon ethyl 40 99
Paraoxon methyl 36 98
Parathion ethyl 16 101
Parathion methyl 16 99
Phencapthon 60 99
Phorate (Thimet) 1.3 98
Phosalone (Zolone) 400 102
Phosmet (Irrridan) 220 82
Phosphamidon (Dimecron) 80 43
Ronnel 4 100
Ronnoxon 120 94
99
93
98
56
91
96
45
90
93
85
92
43
0
45
0
90
93
99
93
98
56
91
85
43
96
92
-------
Revised 6/77
Section 10, A
Page 22
TABLE 4. RECOVERIES OF 7 CARBAMATE COMPOUNDS
Compound
Aminocarb
Bux
Carbaryl
Carbofuran
Methiocarb
Propoxur
Zectran
Cone.
(ppb)
10
10
10
10
10
10
10
Spkd. MeCl2
(No Extract)
90
101
64
95
94
100
69
Recoveries i
Silica
(Elution
I II I
4
55
n percent
Gel Parti
Fraction)
II IV
59
93
68
94
57
99
58
tioning
Total
59
93
68
98
112
99
58
-------
6/77
Section 10,A
Page 23
To Vacuum
Cone. Evap. Tube -
Rinco Rotary Evaporator
Kuderna-Danish Flask
Fig. 1. • Evaporation assembly
-------
6/77
Section 10,A
Page 24
Minute*
8
12
16
20
24
Fig. 2. Six carbamates eluted in Fraction III (portion of carbofuran and
methiocarb in Fraction II). 5.0 nanograms of each compound. GC colunn
5% OV-210.
-------
6/77
Section 10,A
Page 25
Minutes
Fig. 3. Five chlorinated compounds eluted in Fraction II. GC column
1.5% OV-17/1.95% OV-210
-------
6/77
Section 10,A
Page 26
en
c
o
c
111
ffj T ^v
S'X' T/inules 8
16
24.
32
40
Fig. 4. Four organophosphorous compounds elutcd in Fraction II. GC column 1.5% OV-17/1.95% OV-210
-------
Revised 6/77 Section 10, B
Page 1
DETERMINATION OF SOME FREE ACID HERBICIDES IN WATER
I. INTRODUCTION:
With the intensive use of herbicides for agricultural noxious
weed control and direct application in certain waterways for
aquatic broad!eaf plant control, the environmental chemist is being
called upon with increasing frequency to monitor for herbicide residue
levels in water. The free acid herbicides, particularly the
chlorophenoxys, comprise a commercially important group. The
electron capture gas chromatography of these compounds requires that
the free acids be converted to derivatives such as the ethyl or
methyl esters. One common esterification approach has been through
reaction with diazo compounds, but analysts are objecting to the
use of these compounds because of their carcinogenicity and other
personal safety risks. These extreme hazards dictate the need for
very stringent safety precautions in the handling of these reagents.
The method described below requires only the normal safety practices
customarily used for the handling of compressed gasses, moderately
toxic or corrosive materials, and flammable solvents.
REFERENCES:
1. An Improved Gas Chromatographic Method for the Analysis
of 2,4-D Free Acid in Soil, Woodham, D. W., Mitchell,
W. G., Loftis, C. D., and Collier, C. W.: J. Agr.
Food Chem., 1_9 (1), 186 (1971)
2. A Multiclass, Multiresidue Analytical Method for
Pesticides in Water, Thompson, J. F., Reid, S. J., and
Kantor, E. J.: Arch. Environ. Contam. Toxicol. in-press
(1977), and this Manual, Section 10,A
II. PRINCIPLE:
The pH of the water sample is reduced to 3.0 with h^SOtt,
and extraction and solvent evaporation are carried out as described
in Section 10,A for multiresidues in water, utilizing the same
roto-vap equipment and taking to dryness under a nitrogen stream.
An esterification reagent of 10% BC13 in 2-chloroethanol is added,
and a prescribed reaction time and temperature are applied. Hexane
and NazSOit solution are added, and, after shaking and phase separa-
tion, gas chromatography (EC) is conducted on a concentrate of the
hexane layer containing the ethanolic esters. If cleanup is required,
the partially deactivated silica gel method described in Section 10,A
-------
Revised 6/77 Section 10, B
Page 2
is used, with all esterified compounds being eluted in Fraction II.
III. EQUIPMENT AND REAGENTS:
1. All equipment and glassware specified for water method in
Section 10,A.
2. GLC columns: 5% OV-210, 1.8 m (6') x 4 mm (5/32") i.e. and
1.5% OV-17/1.95% OV-210, same dimensions, both operated at
200°C.
3. Impinger air sampling (bottle only), height 165 mm, outside
diam. 41 mm, capacity 125 ml, bottle opening § 24/25, Ace
Glass, Inc. #7540.
4. Glass stoppers, 24/25, for bottles in Item 3 above.
5. Magnetic stirring rods, Teflon-coated.
6. Evaporative concentrator tubes, graduated, 25 ml.
7. Disposable pipets.
8. Water bath, 90°C, and ice bath, 0°C.
9. Balance, top loading 1 Kg capacity, accurate to +0.2 g.
10. Sulfuric acid, cone., reagent grade.
11. Boron trichloride, 1 Ib lecture bottle with suitable shut-off
valve, Matheson Gas Products.
12. 2-Chloroethanol, 99%, Aldrich Chemical #18,574-4.
NOTE: Should be redistilled in a hood just prior to use.
13. Sodium sulfate, anhydrous, reagent grade, preextracted with
MeCl2. Prepare a 7% solution in water.
14. pH Indicator paper, range 0-3.0.
15. Reference standards of analytical grade purity.
16. Preparation of 10% BC13 in 2-chloroethanol Esterification
Reagent:
a. Place a magnetic stirring rod on the bottom of a 125 ml
impinger cylinder (Item 3) and position cylinder on top
loading balance. Load beams to the tare weight of the
-------
Revised 6/77 Section 10, B
Page 3
cylinder and rod and then increase the beam weight by
90.0 grams.
b. Add 2-chloroethanol to the cylinder until the beam
indicator shows a 90-gram addition. Record this weight.
c. Place the cylinder in an ice bath and allow time for
temperature equilibration. Insert a glass gas delivery
tube into the cylinder, extending to ca 5 mm from the
bottom of the cylinder. Extend the glass tubing line from
the cylinder to the BC13 lecture bottle through a glass
trap to prevent any back suction of solution from the
cylinder to the gas bottle.
d. Place the cylinder on the magnetic stirrer plate and
increase the stirring base velocity to full speed. Open
the gas bottle valve to bubble BC13 into the chloroethanol
at a vigorous rate so that no bubbles of BC13 escape
unabsorbed from the chloroethanol. Observe the rise in
volume of the solution in the cylinder. When it appears
that the level of the solution may be approaching a 10 ml
addition, shut off the gas flow, remove the cylinder from
the bath and place on the balance for check weighing.
Continue gas addition as necessary for the final weight to
show a 10 g addition of BC13.
NOTES:
1. All reagent preparation steps should be conducted in
an exhaust hood.
2. It may be found convenient the first time the reagent
is prepared to scratch marking lines on the cylinder
with a diamond pencil at the liquid level of the pure
chloroethanol after chilling and also at the level
reached by the addition of 10 g of BC13. These will
serve as reference points for the preparation of future
lots of the reagent.
3. The air sampling impinger cylinder (Item 3), although
intended for an entirely different use, was found
ideal in type, size, and shape for this application.
4. The reagent, if kept stoppered and refrigerated, was
found to be stable up to 30 days. Although no
observations were made after this length of time, it
may well be stable for longer periods.
-------
Revised 6/77 Section 10, B
Page 4
IV. EXTRACTION:
The sample size should be gauged by the expected concentration
levels of herbicide residues in the sample. If fairly high levels
are expected, such as in a direct run-off area from a spray appli-
cation, a 500 ml sample may be appropriate. A liter, however, may
be indicated for lower concentrations, depending, in part, on the
specific compounds. MCPB, for example, is somewhat unresponsive and
will require a larger sample for detection; on the other hand, si 1 vex
is highly responsive and, therefore, far less is required in the
final extract for chromatography.
1. Add cone. h^SO^, drop by drop, to the sample until a pH of
3.0 or slightly less is observed by testing with pH indicator
paper of a 0-3.0 pH range. Generally 4-6 drops will suffice.
2. From this point onward, follow all steps and details under
SAMPLE EXTRACTION AND CONCENTRATION, Section 10,A,VIII, fin-
ishing at Step 6 with a MeCl2 concentrate of ca 4.0 ml in a
10 ml concentrator tube.
3. With a disposable pipet, transfer the contents of the 10 ml
concentrator tube to a 25 ml graduated concentrator tube,
rinse down the sides of the 10 ml tube three times with 0.5 ml
of hexane and transfer each wash to the larger tube.
4. Place the 25 ml tube under a gentle stream of nitrogen at
room temperature and evaporate just to dryness.
V. ESTERIFICATION:
1. Add 1.0 ml of the esterification reagent to the 25 ml concen-
trator tube allowing the reagent to flow down the entire inner
walls of the tube.
2. Immerse tube in a 90°C water bath for 10 minutes.
3. Remove the tube from the bath, cool to room temperature, and
add 5.0 ml of hexane and 10 ml of 7% Na2S04 solution. Stopper
the tube and mix at high speed on a Vortex mixer.
4. Allow the layers to separate and proceed with GLC on the hexane
phase.
-------
Revised 6/77 Section 10, B
Page 5
VI. GAS CHROMATOGRAPHY:
Use a column consisting of 5% OV-210 coated on Gas Chrom-Q,
80/100 mesh. At the oven temperature parameters given in Table 1,
approximately the same RRT. values should be expected from columns
of 5% QF-1 or 5% SP-2401. rt
The confirmation column of 1.5% OV-17/1.95 OV-210 finds its
counterpart in RRTn characteristics in a commercially available
column of 1.5% SP-2250/1.95% SP-2401.
If the analysis is run on a water sample of completely unknown
constituents, the analyst may obtain a clue of the constituent(s)
by calculating the RRT,, of any peaks in the sample chromatogram and
comparing the calculated RRT,, values with those given in Table 1.
However, any reagent blank peaks of comparable RRT,, values and of
significant response must be carefully considered.
Further validation should be carried out on an alternate
column of entirely different compound elution characteristics.
VII. SILICA GEL FRACTIONATION:
This step is not recommended until gas chromatography is carried
out on esterified extract. If the contaminant background is minimal,
there is no need to do this procedure. If a significant background
is evident and the peaks are obviously of the same approximate RRT,,
values as herbicide ester peaks, the fractionation step is necessary.
The fractionating procedure is conducted in the same manner
described in Section 10,A,IX, SILICA GEL FRACTIONATION AND CLEANUP,
transferring an aliquot of 4.0 ml of the esterfied hexane extract
to a 10 ml concentrator tube and reducing the extract volume to 0.5
ml under a nitrogen stream at room temperature. All esterified
compounds should elute in Fraction II.
NOTE: The analyst must not overlook the fact that the final
sample size will be only 4/5 (0.8) of the original
sample due to the 4 ml aliquot taken from the 5.0 ml
concentrate.
VIII. QUALITY CONTROL:
If the identity of the free acid herbicide in the sample is
known, an SPRM (spiked reference material) of water should be
prepared with a compound concentration estimated to be comparable to
that of the sample. This SPRM should be carried through the entire
procedure in exactly the same manner as the sample. If the recovery
from the SPRM should turn out significantly poorer than the recovery
-------
Revised 6/77
Section 10, B
Page 6
shown in Table 1 for the compound, the analyst would be well advised
to repeat the work since, in all probability, the validity of the
results from the unknown(s) will be no better than that of the SPRM.
TABLE 1. RECOVERY AND RRTn DATA FOR 11 FREE ACID HERBICIDES.
COLUMN 5% OV-210 OPERATED AT 200°C AND CARRIER FLOW
OF 60 ML/MIN., 63Ni DETECTOR AT 300°C.
Compound
MCPP
MCPA
Dichlorprop
Fenac
Naphthalene
Acetic Acid
Silvex
2,4-D
MCPB
2,4, 5-T
2,4-DB
4-(2,4,5-TB)
Cone, in
Water (ppb)
5.0
3.0
0.4
1.36
40.0
0.25
4.0
68.0
4.0
2.0
1.0
RRTA
1.14
1.35
1 .47
1 .75*
1.90
2.15
2.19*
2.66
3.44*
3.47
5.57
Recovery After
Extract, and
Esterif. (%)
109
98
92
94
97
105
90
113
88
90
95
Recovery After
Silica Gel
Fraction (%)
88
85
83
86
87
85
75
93
63
89
91
*These RRT* values were determined at a column temperature of 180°C,
-------
Revised 12/15/79 Section 11,
Page 1
ORGANOCHLORINE INSECTICIDES IN SOILS AND HOUSEDUST
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 evap-
oration in Kuderna-Danish equipment. Interfering lipid-soluble
materials are then partially removed from the extracts by successive
cleanup on aluminum oxide and Fieri si 1 columns. Extracts are adjusted
to appropriate concentration for determinative analysis by EC and FPD
confirming as needed by MC and/or TLC.
III. EQUIPMENT:
1. Soxhlet extraction apparatus, complete with 125-ml I 24/40 flask,
extraction tube with I 24/40 lower and I 34/45 upper joints and
Friedrichs condenser with f 34/45 joint. Kimble #24010 or the
equivalent for the entire assembly.
2. Soxhlet 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 in.dia. x 2 in. depth, stainless steel.
4. Chromatographic columns, 22 x 300 mm 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
component bearing the following stock numbers:
a. Flask, 250 and 500 ml, stock #K-570001.
b. Snyder column, 3 ball, stock #K-503000.
-------
Revised 11/1/72 Section 11, A
Page 2
c. Steel springs, 1/2 in., stock #K-662750.
d. Concentrator tubes, 10 ml grad., size 1025, stock #K-570050.
6. Modified micro-Snyder columns, 19/22, Kontes K-569251.
7. Glass beads, 3 mm plain, Fisher #11 - 312 or equivalent.
8. Evap. concentrator tubes, grad., 25 ml, I 19/22, Kontes #570050.
9. Water or steam bath.
10. Glas Col heating mantles with variable autotransformers, size to
match 125-ml Soxhlet flasks.
11. Filter paper, Whatman No. 1, 15 cm.
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 contaminant
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
contains 2% ethanol, added as a stabilizer, and it is therefore
unnecessary to add ethanol unless peroxides are found and removed.
NOTE: To determine the absence of peroxides in the ether, add
1 ml of ether in a clean 25 ml cylinder previously rinsed
with ether. Shake and let stand 1 minute. A yellow color
in either 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.
-------
Revised 11/1/72 Section 11, A
Page 3
6. 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.
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.
NOTE: 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.
NOTES:
1. In a high humidity room, the column may pick up enough mois-
ture during packing to influence the elution pattern. To
ensure uniformity of the Florisil fractionation, it is
recommended 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 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 laboratories may differ somewhat from that in
Perrine. Each new batch should be tested with a mixture of
3-BHC, aldrin, heptachlor epoxide, dieldrin, £,JD'-DDE,
£,£P-DDD, and £,£'-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 compounds should be in the 6% fraction.
9. Anhydrous sodium sulfate, reagent grade granular, Mallinckrodt
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 trans-
ferring 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 ml beaker and evap-
orate down to ca 5 ml. Inject 5 yl 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
-------
Revised 6/77 Section 11, A
Page 4
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.
V. SAMPLE PREPARATION:
1. Soils and vacuum clean 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 acetone/hexane extraction solvent prior
to use. This may be conveniently done by folding several sheets
of filter paper and placing in the Soxhlet 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.
VI. EXTRACTION:
1. Weigh sample (2 grams of soil or 1 gram of dust) onto a sheet of
15 cm 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 mm; 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 grams of dust
into an evaporating dish. Add sufficient hexane to make
a slurry.
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Revised 6/77 Section 11, A
Page 5
b. Prepare a standard mixture of the following compounds, the
concentration expressed in micrograms per milliliter:*
Lindane 5.0 Dieldrin 7.5
Hept. Epoxide 5.0 £,JD'-DDD —-10.0
Aldrin- 5.0 p_,p>DDT --10.0
£,£'-DDE 7.5 £,p_'-DDT 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. Mix gently with a glass rod and evaporate the solvent
at 40°C under a nitrogen stream, stirring from time to time.
d. After removal 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 extrac-
tion, cleanup, and determinative procedures.
4. Place the sample, reagent blank and spiked sample thimbles into
separate Soxhlet extractors. Fill the boiling flasks, each
containing 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
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 5-hour period.
This should be an adequate number of cycles to ensure
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 ml K-D flask
attached to a 10 ml evap. concentrator tube containing one 3 mm
glass bead.
7. Transfer the extract from the 125 ml Soxhlet flask to the K-D
flask, rinsing the Soxhlet flask with 3 portions of 5 ml each
of hexane. Attach the Snyder column and immerce evap.
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Revised 6/77 Section 11, A
Page 6
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.
VII. ALUMINA AND FLORISIL PARTITIONING:
1. Prepare an alumina column as follows:
a. Place a small wad of prerinsed glass wool at the bottom
of a 22 x 300 mm chromatographic column.
b. Add preextracted anhydrous Na2SOit to a depth of 1/2 inch.
c. Close stopcock and fill column with hexane.
d. In a 50 ml 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 with a 1 in. layer of Na2SOit. When
settling is complete, open stopcock, and allow the hexane
to elute through the column down to a point ca 1/8 inch
above the top of the upper N32SOI+ 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 sufficient column prerinse.
2. Position a second K-D flask fitted with 10 ml evap. concentrator
tube under column.
3. Transfer the 3 ml of concentrated extract from the first K-D
evaporation 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 ml of hexane to the column, open stop-
cock 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 Extrac-
tion, 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,
(1) of this manual under FLORISIL FRACTIONATION, Steps 1 and 2,
substituting hexane for pet. ether.
7. Assemble two more K-D apparatus but with 500 ml flasks and
position the flask of one assembly under the Florisil column.
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Revised 11/1/72 Section 11, A
Page 7
However, at this point use 25 ml grad. evap. concentrator tubes
instead of the 10 ml size for previous concentrations.
8. Using a 5 ml Mohr or a long disposable pipet, immediately trans-
fer 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 ml portions of hexane, 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 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 ^SO^ layer, place the second 500 ml
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 malathion
in the sample, have a third 500 ml K-D assembly 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 1 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 reisdues are generally sufficiently high
to warrant this. Furthermore, the concentration of
contaminants remaining after cleanup is hereby reduced.
VIII. GAS CHROMATOGRAPHY:
1. Inject 5 yl of each fraction extract into the gas chromatograph
(EC mode) primarily to determine whether the extracts will
require further adjustment by dilution or concentration.
2. When appropriate dilution adjustments have been made in the
extracts and column oven is set to a known temperature, the
relative retention values of the peaks on the chromatograms
should be calculated. When these values are compared with the
values in the printed table for the appropriate column, the
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Revised 11/1/72 Section 11, A
Page 8
operator should be able to make tentative compound identifica-
tions. Microcoulometry and/or TLC may be required for positive
confirmation of some of the suspect chlorinated compounds,
whereas FPD may be utilized for the organophosphate suspects.
IX. ALUMINA COLUMN ELIMINATION:
It has been reported by several field scientists analyzing house
dust that the alumina cleanup can be bypassed with no ill effects.
In view of the expenditure of extra time and material, a laboratory
conducting monitoring studies might find it advisable to make some
recovery studies eliminating this step by taking the extract mentioned
in Step 7 under EXTRACTION AND STARTING THE Florisil fractionation
with Step 6 of Subsection VII.
TYPICAL RECOVERY DATA - Soxhlet Method
-Pesticides-
Lindane Hep. Epox. p,p'-DDE Dieldrin p,p'-TDE p,p'-DDT
SOILS:
Mean fj;™^
S.D. :
n :
ry: 85'25
5.446
12
87.83
9.446
12
83.08
6.345
12
88.25
6.210
12
91.17
6.886
12
94.17
8.922
12
HOUSE DUSTS:
Mean Per"nt : 87.33 80.58 82.92 86.27 90.00 87.78
I cL«U V C i V
S.D. : 6.997 12.85 6.345 12.89 9.715 20.20
n : 12 12 12 11 12 9
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Revised 12/15/79 Section 11, B
Page 1
ORGANOCHLORINE AND OR6ANOPHOSPHORUS INSECTICIDES IN BOTTOM SEDIMENT
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 organochlorine 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 a overall profile of the pesti-
cidal contamination of a given body of water.
REFERENCES:
1. Column Extraction of Pesticides from Fish, Fish Food and
Mud, Hesselberg, R. J. and Johnson, J. L., Bull. Environ.
Contam. Toxicol. 7_(2/3), 115-120 (1972).
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% CH2C12 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 in. x 10 in. x 2-1/2 in.
2. Oven, drying.
3. Muffle furnace.
4. Desiccator.
5. Crucibles, porcelain, squat form, size 2.
6. Omni or Son/all mixer with chamber of ca 400 ml.
7. Chromatographic columns, 300 mm x 22 mm with Teflon stopcock.
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Revised 6/77 Section 11, B
Page 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
component bearing the following stock numbers:
a. Flask, 250 ml, stock #K-570001.
b. Snyder column, 3 ball, stock #K-503000.
c. Steel springs, 1/2 in., stock #K-662750.
d. Concentrator tubes, 10 ml, size 1025, stock #K-570050.
11. Pyrex glass wool - preextracted with methylene chloride in a
Soxhlet extractor.
12. , Hot water bath, temp, controllable at 80CC.
13. Sodium sulfate, anhydrous, Baker, prerinsed or Soxhlet extracted
with methylene chloride.
14. rv-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 chloride-hexane, 15% v/v.
IV. SAMPLE PREPARATION AND EXTRACTION:
1. Decant and discard the water layer over the sediment. Mix the
sediment to obtain as homogeneous a sample as possible and
transfer to a pan to partially air dry for about 3 days at
ambient temperatures.
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Revised 11/1/72 Section 11, B
Page 3
NOTE: Drying time varies considerably depending on soil type and
drying conditions. Sandy soil will be sufficiently dry in
one day, whereas muck requires at least three days. The
silt and muck sediment 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 gram of the partially dried sample into a 400-ml Omni-
Mixer chamber. Add 50 gram of anhydrous sodium sulfate and mix
well with a large spatula. Allow to stand with occasional
stirring for approximately one hour.
NOTE: 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
50 gram sample for extraction, weigh ca 5 gram of the
partially dried sediment into a tared crucible. Determine
the percent solids by drying overnight at 103°C. Allow to
cool in a disiccator for half an hour before weighing.
Determine the percent volatile solids by placing the oven-
dried sample into a muffle furnace and igniting at 550°C
for 60 minutes. Allow to cool in a desiccator before
weighing.
3. Attach the 400 ml chamber to an Omni 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-hexane at a flow rate
of 3-5 ml/min into a 400 ml beaker.
6. Concentrate the sample extract to about 100 ml under a nitrogen
stream and at a temp, no higher than 55°C. Transfer to a 500 ml
separatory funnel containing 300 ml of distilled water and 25 ml
of saturated sodium sulfate solution. Shake the separatory
funnel for two minutes.
7. Drain the water layer into a clean beaker and the hexane layer
into a clean 250 ml separatory funnel.
8. Transfer the water layer back into the 500 ml 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 ml separatory funnel.
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Revised 6/77 Section 11, B
Page 4
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
discarding the wash water.
10. Attach 10 ml evap. concentrator tube to a 250 ml Kuderna-Danish
flask and place under a filter comprised of a small wad of glass
wool and ca 1/2 inch of anhydrous Na2SOit in a 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 hexane.
12. Attach Snyder column to top joint of K-D flask, immerse tube in
80°C water bath and concentrate extract to 5 ml or to a lesser
volume if extremely low concentration levels of pesticides are
expected.
V. FLORISIL PARTITIONING:
Remove tube from water bath rinsing joint with a small volume of
hexane. The partitioning is carried out as described in Section HA,
starting at VII, Step 6.
VI. GAS CHROMATOGRAPHY:
Again proceed as describe in Section 11A.
VII. CALCULATIONS:
1. Percent Dry Solids
gram of dried sample x 100 = % Dry Solids
gram of sample
2. Percent Volatile Solids
gram of dried sample - gram of ignited sample = gram of volatile
solids
gram of volatile solids x 100 = % Volatile Solids
gram of sample
3. Concentration of Pesticide in Sediment
% dry solids x 5 gram - gram of dry sample extracted
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Revised 6/77 Section 11, B
Page 5
1 of sample extract injected x gram of dry sample extracted =
1 of sample extract gram of dry sample injected
ng of pesticide = ppb of pesticide
gram of dry sample injected
VIII. SULFUR INTERFERENCE:
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 chromatograms 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 con-
ditions for pesticide analysis, the sulfur interference can completely
mask the region from the solvent peak through aldrin.
This technique eliminates sulfur by the formation of copper
sulfide on the surface of the copper. There are two critical steps
that must be followed to remove all the sulfur: (1) the copper must
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 concen-
tration 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 any exploratory injec-
tion from the final extract concentrate (presumably 5 ml) into the
gas chromatograph, 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.
2. If the sulfur concentration is such that crystallization occurs,
carefully transfer, by syringe, 500 yl of the supernatant extract
(or a lesser volume if sulfur deposit is too heavy) into a glass-
stoppered, 12 ml grad., conical centrifuge tube. Add 500 yl of
iso-octone.
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Revised 11/1/72 Section 11, B
Page 6
3. Add ca 2 ug of bright copper powder, stopper and mix vigorously
1 minute on a Vortex Genie mixer.
NOTE: The copper powder as received from the supplier must be
treated for removal of surface oxides with 6N HN03.
After about 30 seconds of exposure, decant of acid, rinse
several times with dist. water and finally with acetone.
Dry under a nitrogen stream.
4. Carefully transfer 500 yl of the supernatant-treated extract into
a 10 ml 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 final
extract volume of 1.0 ml will be of equal sample concen-
tration to a 4 ml 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
Lindane
Heptachlor
Aldrin
Hept. Epoxide
£,j3'-DDE
Dieldrin
Endrin
DDT
Chi orobenzi late
Aroclor 1254
Malathion, diazinon,
Parathion, Ethion,
Trithion
81.2
75.7
39.8
95.5
69.1
92.1
79.1
90.8
79.8
7.1
97.1
0
98.1
94.8
5.4
93.3
96.6
102.9
94.9
89.3
85.1
0
104.3
0
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Revised 12/15/79
Section
Page 1
11, C
DETECTION OF CARBAMATE PESTICIDES IN SOIL
A method investigation is being conducted on the procedure
referenced below. If the procedure proves suitable for inclusion
in this Manual, an addendum will be forwarded to all current
holders of this latest revision.
REFERENCE:
Direct Gas Chromatographic Determination of Carbamate Pesticides
Using Carbowax 20M-Modified Supports and the Electrolytic
Conductivity Detector, Hall, R. C., and Harris D. E., J.
Chromatogr., 169, 245-259 (1979).
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Revised 12/2/74 Section 12
Page 1
CONFIRMATORY PROCEDURES
INTRODUCTION
Gas Chromatography is primarily a quantitative tool which also provides
broad information on the identity of organic compounds. When the gas
chromatograph is used with the nonspecific electron capture detection system,
additional evidence is often necessary to confirm the identity of resulting
peaks.
The nature of our analyses are such that interfering materials and
artifacts are often observed and matabolic and decomposition products may be
encountered. While it is necessary that low concentrations of pesticide
residues be detected and measured, it is essential that every agent reported
be correctly identified. Whenever one observes unsymmetrical peaks, or un-
expected or unexplainable results, the identity of such peaks should be
confirmed. In the absence of this identification, one cannot produce
reliable quantitative data since quantitation with electron capture gas
chromatography depends entirely on the identity of the agent, due to the
variation in response among different pesticides. In addition, it would
be impossible to interpret the relationship of pesticides to human health by
utilizing unreliable qualitative data.
Thus, in order to provide for this most important identification factor,
confirmatory methods are included in this manual. The methods discussed
include thin-layer chromatography, infrared spectroscopy, extraction p-values
and derivatization techniques.
Since the concentrations of pesticides in human tissue are low, and
rigorous cleanup is required, and since the equipment available for con-
firmation lacks sensitivity, macro sampling is necessary. As indicated
previously, however, the determination of p-values may be accomplished with
micro-samples.
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Revised 12/15/79 Section 12 A
Page 1
CONFIRMATION AND DETERMINATION OF
ORGANOCHLORINE PESTICIDES IN HUMAN TISSUE AND MILK
I. INTRODUCTION:
The method described in the section makes use of a gas chromat-
graph equipped with a Carbowax 20M column and a Hall electrolytic
conductivity detector for the determination of chlorinated pesticides
in human adipose tissue and human milk samples at concentrations as
low as 0.01 ppm. Gel permeation chromatograph is used for additional
cleanup of extracts having an adverse effect on the performance of the
Hall detector due to excessive lipid material. A high degree of cor-
relation was obtained between results of analyses made with this
procedure and those using an electron capture detector. Application
of the Hall detector for confirmation of organochlorine pesticides can
provide an inexpensive substitute for combined gas chromatography-mass
spectrometry in some situations.
REFERENCE:
Application of the Hall Detector and a Surface-Bonded Carbowax
20M Column to Analysis of Organochlorine Pesticides in Human
Biological Samples, Crist, H. L., and Moseman, R.F., J. Chromatogr.,
160, 49-58 (1978).
II. PRINCIPLE:
Human adipose tissue and human milk samples are extracted and
cleaned up by a modified Mills, Olney, Gaither procedure. Additional
cleanup of fractions is accomplished with gel permeation chromatog-
raphy prior to gas chromtographic determination on a Carbowax 20M
column with a Hall electrolytic conductivity detector.
III. APPARATUS:
1. Tracer Model 222 gas chromatograph (or equivalent) equipped with a
Tracer 700 Hall electrolytic detector (see Section 4,C)
2. GLC column 1.8 m x 4 mm i.d. borosilicate glass, packed with
surface bonded Carbowax 20M on Chromosorb W (Section 4,A,(7)).
A 2 cm section of 5% Carbowax 20M on Chromosorb W is placed on
the injection side of the Carbowax column to protect it from
buildup of lipid material.
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Revised 12/15/79 Section 12, A
Page 2
3. Go-Getter gas purifer for helium carrier gas (General Electric
Schenectady, NY; distributed by All tech, Arlington Heights, IL).
4. Autoprep Model 1001 gel permeation chromatograph (Analytical Bio-
chemistry Laboratories, Columbia, MO) equipped with a 350 mm x
25 mm i.d. glass column containing 60 grams of 200-400 mesh BioBeads
SX-3 (see Section 5,B).
5. Equipment needed for modified MOG cleanup procedure (Section 5,A,
(D.III).
IV. REAGENTS AND SOLVENTS:
1. Pesticide analytical reference standards, available from the
Quality Assurance Section (MD-69), Health Effects Research Labora-
tory, U.S. EPA, Research Triangle Park, NC 27711.
2. BioBeads SX-3, 200-400 mesh, Bio-Rad Labs, Richmond, CA.
3. Toluene and ethyl acetate of pesticide grade quality.
4. Reagents needed for modified MOG cleanup procedure (Section 5,A,
5. Glass column, 350 mm x 25 mm i.d., Kontes K-422351, and organic
solvent plunger assembly, Kontes K-422353.
V. PROCEDURE:
1. Extract and clean up human adipose tissue and human milk samples
by modified MOG procedure in Section 5,A,(1) of this Manual.
2. Carry out additional cleanup on the petroleum ether-diethyl ether
(85:15 v/v) fraction from the adipose tissue extracts and the
(94:6 v/v) fraction from the human milk extracts by gel permeation
chromatograph.
a. Follow the column preparation and operation procedure
described in Section 8,M,j,3 of the EPA Pesticide AQC
Manual.
b. Dissolve the evaporated fractions from the MOG cleanup in
toluene-ethyl acetate (1:3 v/v).
c. Inject samples equivalent to <;! gram of fat.
d. Use toluene-ethyl acetate (1:3 v/v) as the elution solvent
with a flow rate of 5 ml/minute.
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Revised 12/15/79 Section 12, A
Page 3
e. Discard the first 100 ml solvent containing the lipids, and
collect the next 95 ml containing the pesticides.
NOTE: The analyst should determine the elution pattern
of his GPC column with pesticide standards in
order to assure quantitative recovery.
3. Concentrate the elution to an appropriate volume in a Kuderna-
Danish concentrator assembly.
VI. GAS CHROMATOGRAPHY:
1. Operate the Hall detector with the following parameters:
Quartz combustion tube 18.3 cm x 2 mm i.d.
Furnace temperature 820°C
Hydrogen flow rate 20-40 ml/minute
Transfer line 270°C
Methanol flow rate through 0.3-0.5 ml/minute
detector cell
2. Operate the Carbowax column at 175°C or 185 C°with a helium flow
rate of 50 ml/minute.
3. Pesticides eluting in the respective Florisil fractions (Section
5,A,(1), Table 1), such as oxychlordane, transnonachlor, £,p_'DDE,
and £,p_'-DDT in the 6% ether fraction and dieldrin in the 15%
fraction, can be determined using the Carbowax 20M column. Extracts
can be composited for confirmatory analyses. Figure 1 shows the
chromatogram for 2»R'-DDE from milk and Figure 2 the chromatogram
for dieldrin from adipose tissue.
NOTE: A 5% OV-1 column at 200°C was used for determination of
B-HCH because of interference from heptachlor epoxide
on the Carbowax 20M column.
VII. RESULTS:
Table 1 gives the results of quantisations of psp_'-DDE in the
6% diethyl ether MOG fraction from milk and dieldrin in the 15%
diethyl ether fraction from human adipose tissue, using both the
Hall and electron capture detectors. The 6% ether MOG fractions and
from nine adipose tissue samples were analyzed by both detectors, and
graphical comparison of the data made by plotting the results (ppm)
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Revised 12/15/79 Section 12, A
Page 4
against each other. Regression lines for five different pesticides cal-
culated by the least squares method conformed to a straight line
(y = a + bx) with coefficients of correlation ranging from 0.895 to
0.984. The relatively close agreement of these data from the Hall
detector and the electron capture detector indicate the feasibility
of using the former for determining chlorinated pesticides in biological
samples at levels as low as 0.01 ppm.
63
The linearized Ni electron capture detector was operated at 275 C
with a 230°C transfer line. A 1.5% OV-17/1.95% QF-1 column at 200°C
with a methane-argon (5.95% v/v) flow rate of 60 ml/minute was used
for determinations by EC GLC.
VIII. DISCUSSION AND MISCELLANEOUS NOTES:
1. Innumerable injections of 6% diethyl ether MOG fractions (12 mg
tissue equivalent per injection) were made without observing any
deterioration in the Hall detector sensitivity or Carbowax 20M
column performance. The glass demister tubes in the injection
port of the gas chromatograph were changed daily.
2. When the 15% diethyl ether MOG fractions were analyzed on a
routine basis, sensitivity of pesticide detection, column
resolution, and peak distortion was highly dependent on the
accumulation of lipid residue in the demister tube in the
injection port. Frequent installation of the clean demister
tube was beneficial in restoring the performance of the GLC
system, but if too many injections were made, a new combustion
tube and ion exchange resin had to be installed in the Hall
detector and the conductivity cell had to be cleaned. Re-
placing the 2 cm layer of 5% Carbowax 20M and the glass wool
plug at the injection end of the column and heating at 230-
240°C overnight was also beneficial.
3. Figure 3 indicates the beneficial effect on response of additional
GPC cleanup of the 15% diethyl ether MOG fraction from human
adipose tissue extract. As many as 20 injections were made
during a day without changing the demister trap; after 40 in-
jections, no significant decrease in response or column resolution
was observed. Removal of excess lipids by GPC reduced instrument
"down-time" and service and allowed much lower levels of pesticides
to be detected and quantitated.
-------
Revised 12/15/79
Section 12, A
Page 5
TABLE 1. DETERMINATION OF DIELDRIN AND p_,p_'-DDE IN
HUMAN BIOLOGICAL EXTRACTS*
Sample
Adipose tissue
Adipose tissue
Adipose tissue
Adipose tissue
Milk
Milk
Milk
Milk
Milk
Milk
Milk
Milk
Milk
Amount found (ppm)
Electron capture
0.11
0.02
0.07
0.10
0.01
0.04
0.05
0.02
0.04
0.04
0.03
0.08
0.03
Hall detector
0.12
0.03
0.09
0.08
0.01
0.04
0.04
0.02
0.04
0.03
0.03
0.08
0.04
Difference (%)**
9
50
29
20
0
0
20
0
0
25
0
0
33
*Adipose tissue was analyzed for dieldrin; milk was analyzed for £,£'-DDE.
**Calculated using the elctron capture result as the accepted reference value.
Average difference, 14%.
-------
Revised 12/15/79
Section 12,A
Page 6
TIME.min
Fig. 1. Chromatogram of 6% fraction from human milk extract
after cleanup with GPC (44 ppb p,p'-DDE). Injection:
3 yl/ml (42 mg tissue equivalent); detector: Hall
electrolytic conductivity; oven temperature: 185°C;
carrier gas flow rate: 50 ml/minute; reaction^gas flow
rate: 20 ml/minute; furnace temperature:
820°C.
-------
Revised 12/15/79
Section 12,A
Page 7
VENT
TIME.min
Fig. 2. Chromatogram of 15% fraction from human adipose tissue
extract after cleanup with GPC (30 ppb dieldrin) ;
Injection: 5 yl/1.0 ml (13 mq tissue equivalent);
detector: Hall electrolytic conductivity; colur.Ti:
Carbowax 20M; oven temperature: 175 C; for other
instrument conditions, see Fig. 1.
-------
Revised 12/15/79
Section 12,A
Page 8
cc
ec
Fig. 3. Chromatograms of (A) pesticide mixture before injection
of sample extracts; (B) pesticide mixture after six
injections of the 15% fraction from human adipose
tissue extracts without GPC (22 mg tissue equivalent/
injection). Pesticide mixture = 600 pg oxychlordane,
heptachlor epoxide and dieldrin in order of elution.
Detector: Hall electrolytic conductivity; oven
temperature: 185 C; for other instrument conditions,
see Fig. 1.
-------
Revised 11/1/72 Section 12, B
Page 1
CONFIRMATORY PROCEDURES
THIN-LAYER CHAROMATORAPHY
I. INTRODUCTION:
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 chromatography. 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 most chlorinated pesticides and about 50 ng of most organothio-
phosphates. Consequently, a macrosample is required. A stringent
sample cleanup procedure is also required.
REFERENCES:
1. Kovacs, Martin, F., JAOAC. 46 (1963).
2. Kovacs, Martin, 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. APPARATUS:
1. 8" x 8" glass plates, double strength window glass (Pittsburg Plate
Glass).
2. 3-1/4" x 4" clinical micros!ides (Arthur H. Thomas Co.).
-------
Revised 11/1/72 Section 12, B
Page 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.
5. Chromatographic chamber, 800 ml beaker.
6. Desaga/Brinkman standard model applicator.
7. Desaga/Brinkman drying rack, holds 10, 8" x 8" plates.
8. Spotting pipettes, 1, 5, and 10 jjl, Kontes 763800.
9. Spray bottle, 8 oz., Thomas Co. #9186-R2.
10. Desaga/Brinkman glass vacuum desiccator.
11. Desk blotter paper.
12. Ultra violet light source: 4-15 watt G. E. germicide lamps,
shielded to protect operator, General Electric Co. G 15 T 8.
III. REAGENTS:
1. Aluminum oxide G. (Brinkman or Warner-Chilcott).
2. J^-heptane, chromatopgraphic 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.
11. Ethyl ether, Reagent.
12. Acetonitrile, Chromatographic grade.
-------
Revised 11/1/72 Section 12, B
Page 3
13. Dimethylformamide, Reagent
14. Preparation of reagent solutions.
A. Developing solvents.
(1) For organochlorines
(a) 2% acetone in J^-heptane (v/v) (mobile solvent).
(b) N-heptane (mobile solvent).
(2) For thio and nonthio organophosphates.
(a) Methylcyclohexane (mobile solvent).
(b) 15% or 20% dimethylformamide (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. Immediately
add 3 drops of 30% hydrogen peroxide and mix. Keep
stored in a cool dark place not longer than 1 week.
Dark solutions should be discarded.
(2) For organothiophosphates.
(a) Stock dye solution.
Dissolve 1 gm of tetrabromophenolphthalein
ethyl ester in 100 ml of acetone.
(b) Use concentration dye solution.
Dilute 10 ml of stock solution to 50 ml with
acetone.
(c) Silver nitrate solution.
Dissolve 0.5 gm AgNOs in 25 ml of distilled water
and dilute to 100 ml with acetone.
(d) Critic acid solution.
Dissolve 5 g citric acid in 50 ml of distilled water
and dilute to 100 ml with acetone.
-------
Revised 11/1/72 Section 12, B
Page 4
(3) For thio and nonthio organophosphates.
(a) 2% £-Nitrobenzyl pyridine in acetone (w/v).
(b) 10% Tetraethylenepentamine in acetone (v/v).
IV. PREPARATION OF TIC PLATES:
8" x 8" plates
1. Add 30 g aluminum 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 "X",
approximately the size of the plate to be mounted. Press each
plate snugly into position to ensure 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.
2. Examine each 3-1/3" x 4" micro slide carefully by looking down
each edge. To ensure 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. When "bowing" is noted, discard the slide, and position
another in its place.
-------
Revised 11/1/72 Section 12, B
Page 5
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 AlaOs G of MN-silica gel G-HR into a 250 ml 5 Erlenmeyer
flask. Add 50 ml distilled water to A^Os G or 60 ml to MN-silica
get G-HR, stopper flask, shake moderately 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 for 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.
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
one 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 irregulari-
ties 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 cm) 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.
-------
Revised 6/77 Section 12, B
Page 6
11. Spot samples at 1/4" intervals along the imaginary "spotting
line." Each micro plate will accommodate 10 application points
as compared to 18 on a normal 8" x 8" plate. Spot samples and
standards on the micro slide in the same manner as described
later under SPOTTING.
V. PRECOATED PLATES:
The laboratory which conducts TLC constantly would probably find
it more economical to purchase the coating equipment and prepare their
own plates. Many smaller laboratories, however, which may conduct TLC
only occasionally as a confirmation technique will probably find it
more convenient to use precoated plates from commercial suppliers.
There are a number of high quality competitive brands of precoated
plates in the marketplace. Two sources which are known to the editor
to market suitable plates are:
(1) Brinkman Instruments Inc., Westbury, N.Y. 11590
Aluminum oxide precoated TLC sheets - aluminum oxide
F-254 neutral (Type E) on aluminum, 20x20 cm, Merck-
Darmstadt, Cat. No. 68 23 050-1.
(2) Quantum Industries, 341 Kaplan Drive, Fairfield, NJ 07006
Aluminum oxide TLC plates, 20x20 cm, type Q3, Code 1023,
25 plates per package.
Plates that incorporated the AgNOs in the precoated Al^s layer
should not be used where sensitivity is a factor.
VI. SAMPLE PREPARATION:
1. The sample must be of sufficient size that when the extract from
Florisil cleanup is concentrated to an appropriate volume, a 10 yl
spotting volume will produce detectable compound spots. A serum
extract from 50 grams concentrated to 100 yl should produce a
visible spot of 2 ppb. An adipose tissue extract from 5 grams
concentrated to 500 n1 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 6/77 Section 12, B
Page 7
VII. SPOTTING 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 making 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 standards solutions on the same plate. Standard concen-
trations 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.
VIII. COMPOUND DETECTION:
1. Organochlorines
a. Immediately after drying, spray plates with the chromogenic
reagent.
b. Air 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" 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 6/77 Section 12, B
Page 8
3. Thio and nonthio organophosphates.
a. After drying, spray plate withjD-Nitrobenzyl 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 £-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 tetraethylenepentamine should not have a deep color.
If it does, decolorize and purify with charcoal.
4. Interpret results by comparing Rf values of sample spots against
those of standard spots on the same plate.
IX. GAS CHROMATOGRAPHY (EC) CONFIRMATION OF R.F. VALUES:
At times 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. When such doubt
exists, EC GLC 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 (organochlorine) of lowest con-
centration in blood or fatty tissue such as g-BHC, heptachlor epoxide
o_,jp_'-DDT and JD,£'-ODD are frequently the most difficult to identify by
customary EC GLC. 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 nanograms each of standards of the
suspect compounds. Also spot the 6% 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
AgNOs/2-phenoxyethanol reagent.
4. Develop the spots in the sprayed area in the usual manner and
compute the Rf values for the standards.
-------
Revised 6/77 Section 12, B
Page 9
5. Utilizing the standard R. values, pin-point the elution sites for
these compounds along the 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 might contribute artifact
peaks.
8. Stopper tube and shake vigorously one minute on a Vortex mixer.
9. Inject 5 yl of this extract into the gas chromatograph and observe
the chromatogram 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, pro-
vided of course, that there are any peaks.
X. MISCELLANEOUS NOTES:
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, B
Page 10
n-HEPTANE SOLVENT SYSTEM
Adsorbent
Plate Size
Front Travel
£,£'-TDE Travel
Developing Tank
Visualization
Temperature
Amount Spotted
Pesticide
Hexachi orobenzene
Aldrin
£,£'-DDE
Heptachlor
Chlordane (tech)
PCNB
Perthane olefin
£,£'-TDE olefin
TCNB
Telodrin
Toxaphene
Strobane
£,£'-DDT
o_,£'-TDE olefin
Chlorbenside
BHC (tech)
a-BHC
Pethane
Lindane
o.,£' -TDE
£,£'-TDE
Endosulfan
Ronnel
Heptachlor epoxide
Endrin
Dieldrin
Carbophenothion
Methoxychlor
S-BHC
Al 0 G (Merck), 250 ji thick, air dried
72 hr at room termperature
8"x8"
10 cm
3.9 cm
9"x9"x3.5", saturated
AgNOa, UV exposure
24-26°C
80-200 ng
RTDE
2.7
2.1
2.0
2.0
2.0
TT9
1.8
1.8
1.8
1.7
1.7
1.7
1.7
6
1.6
1.3 (grey)
1.8, 1.4, 1.2*
1.2*
1.2*
1
1.3, 1.1, 0.27, 0.10
T73
1.3
1.1
1.1
0
0.07
1
0.88.
0785-
0.71
0.71
0.52
0.42 (yellow)
0.33, 0.27
0.27
-------
11/1/72 Section 12, B
Page 11
Pesticide RTDE
Ovex 0.18
Dichlone 0.16
Dyrene 0.15 (Grey)
Tetradifon 0.11
6-BHC 0.10
Delta Keto "153" 0.09
Kelthane 0.06
Sulphenone 0.00 (large and fuzzy)
Captan 0.00 (sharp edged grey)
Chlorobenzilate 0.00 (light)
Monuron 0.00 (light and dark)
Diuron 0.00
Endrin aldehyde 0.00 (very small)
Endrin alcohol 0.00
Most intense spot underlined
*Leaves a streak with these major spots
-------
Revised 11/1/72
Section 12, B
Page 12
2% ACETONE IN P.-HEPTANE SOLVENT SYSTEM
Adsorbent
Plate Size
Front Travel
p,p'-TDE Travel
Developing Tank
Visualization
Temperature
Amount Spotted
A1203G (Merck, 250 y thick, air dried
72 hr at room temperature
8"x8"
10 cm
5.7 cm
9"x9"x3.5", saturated
AgN02, UV exposure
24-26°C
80-200 ng
Pesticide
Hexachlorobenzene
Perthane olefin
PCNB
Aldrin
£,JD'-DDE
Chlordane (tech)
p,p'-TDE olefin
Telodrin
Heptachlor
TCNB
o.,£'-TDE olefin
Toxaphene
Strobane
£,£'-DDT
p,p_'-DDT
Uhlordenside
Perthane
BHC (tech)
a-BHC
Ronnel
Endrin
Carbophenothion
Heptachlor epoxide
£,£'-TDE
£,£'-TDE
Lindane
Endosulfan
Dieldrin
Tetradifon
1.7
1.4
1.4
4
1.4
1.4.
1
1.3, 1.2, l.V
1.4
1.4
1.4
1.3
1.3
1.3, 1.2*
1.3, 1.2*
1.3
1.2
1.2 (fuzzy grey)
1.2
1.1, 0.92, 0.72, 0.25
1.1
1.1
1.0
1.0 (fuzzy yellow)
1.0
1.0
0.95
0.92
0.92, 0.24
0.90
0.82
-------
Revised 11/1/72 Section 12, B
Page 13
Pesticide
RTDF
Methoxychlor 0.79
Ovex 0.76
3-BHC 0.72
Dichlone 0.72. 0.00
Dyrene 0.51
Sulphenone 0.31
Kelthane 0.28
6-BHC 0.25
Delta Keto "153" 0.23 (very small)
Captan 0.09
Chlorobenzilate 0.05
Monuron 0.05
Diuron 0.00 (dark spot)
Endrin Aldehyde 0.00 (very small)
Endrin alcohol 0.00
Most intense spot underlined
*Leaves a streak with these major spots
-------
Revised 11/1/72 Section 12, B
Page 14
RfValues
Adsorbent
Mobile solvent Methylcyclohexane
Pesticide R Value
Immobile Solvent
15% DMF 20% DMF
Dimethoate 0.01 0.01
Azinphosmethyl (Guthion) 0.09 0.06
Imidan 0.09 0.07
Methyl parathion 0.17 0.11
Coumaphos 0.23 0.15
Malathion 0.34 0.22
Dioxathion 0.37 0.24
Parathion 0.41 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 0.72
Diazinon 0.86 0.78
(1) Presence of chloride in adsorbent layer reacts with AgNOs and pre-
vents coupling of dye and pesticide to form characteristic blue or
purple spot. Some aluminum oxide coatings do not have to be pre-
washed to remove chloride. If, however, maximum compound sensi-
tivities of 0.05 yg cannot be achieved with unwashed A1203 coating,
prewashing is recommended.
(2) Chromogenic spray reacts only with sulfur-containing phosphate
esters. The following compounds do not react; oxygen analog of
parathion, dichlorvos, naled, mevinphos, Phosphamidon and
trichlorfon.
(3) The following minimum amounts of sulfur-containing phosphate esters
can be detected: 0.05 yg diazinon, demeton (thino), carbophenothion,
parathion, malathion, ronnel, dioxathion, EPN, coumaphos, sulfotepp,
and ethion; 0.1 yg azinphosmethyl, methyl parathion, and demeton
(thiol). The lower limits of detectability of dimethoate, Imidan,
methyl carbophenothion, phorate, and disulfoton were not determined.
-------
Revised 11/1/72 Section 12, B
Page 15
At 0.5 yg, or greater, the thio-phosphate esters vary as to color
produced with the chromogenic reagents. Carbophenothion, parathion,
EPN, coumaphos and diazinon appear vivid blue. Ethion, azinphos-
methyl, sulfotepp, diozathion, and malathion appear purple. Ronnel
and methyl parathion appear dull blue while both thiol and thiono
demeton appear bluish purple.
-------
Revised 1/4/71 Section 12, C
Page 1
CONFIRMATORY PROCEDURES
EXTRACTION p-VALUES
I. INTRODUCTION:
The information contained in this section is taken from the FDA
PESTICIDE ANALYTICAL MANUAL, Volume 1, based on the original work of
Bowman and Beroza (JAOAC, 48_, 943, 1965). This system is described as
a method of identifying or confirming identity of pesticides at nanogram
or other levels of analysis through the use of extractions p-values.
The p-value, determined by distributing a solute between equal volumes
of two immiscible phases, is defined as the fraction of the total
solute partitioning into the upper phase. The value may be derived from
a single distribution between the phases or from a multiple distribution
as in countercurrent distribution, (2,3). As a single distribution the
p-value may be determined easily and rapidly; it is especially useful
for confirming the identity of pesticide residues at levels amenable
to quantitative analysis by electron capture gas chromatography.
The p-values for 88 pesticides and related compounds in six binary
solvent systems are listed in Table 1. These are arranged according
to generally ascending values for retention, relative to aldrin. The
RR values, where available for the prescribed GLC column are given.
II. EQUIPMENT AND REAGENTS:
1. Gas chromatograph with electron capture detector, equipped with
6' x 1/4" o.d. glass column of 1.5% OV-17/1.95% QF-1
2. Grad. centrifuge tubes, 10 ml, with I glass stoppers.
3. Solvent systems: Use pesticide quality solvents. To remove
interferences, extract distilled water with hexane; reflux hexane,
heptane, and 2,2,4-trimethylpentane over sodium hydroxide and
distill before use. Equiliberate solvent pairs overnight in a
room maintained at 25.5 +.0.5°C before use. The six solvent systems
used in this study are shown in Table 1. Make dilutions of the
lower phase with water on a volume basis.
III. PROCEDURE:
The analyses are made by electron capture gas chromatography; 88
compounds were analyzed in this manner.
-------
Revised 1/4/71 Section 12, C
Page 2
1. Pipet 5 ml of the hexane (or upper layer) extract into a 10 ml
centr. tube, and chromatograph 5 yl.
2. Pipet 5 ml of the opposing solvent (lower layer) into the centr,
tube, stopper, and shake vigorously 1 minute.
3. Allow layers to separate and chromatograph 5 ^1 of upper layer
extract.
The p-value is the ratio of the second analysis (amount in upper layer)
to the first (total amount). It is reported in hundredths except for
values below 0.10 which are reported in thousandths.
IV. SPECIFICITY:
Figure 1 depicts graphically the number of pesticides and related
compounds falling at p-value intervals of 0.02 for the six solvent
systems. If one depends solely on p-values for identification,
specificity of a given p-value will be inversely proportional to the
number of possibilities and will increase with the accuracy of the
analysis (an error of 0.03 would bring in more possibilities than one
of 0.02). Specificity can be increased by determining more p-values,
as this process imposes additional criteria on identification. It is
also apparent that the more complete the compilation of pesticide
p-values, the more reliably can one assess the specificity of a given
p-value. The accumulation of p-values at the lower end of the hexane-
acetonitrile and the 2,2,4-trimethylpentane-DMF (deimthylformamide)
scales of Fig. 1 indicates that a p-value in this range has poor
specificity (too many possibilities). Between 0.30 and 0.91 the
specificity becomes very good because the number of possibilities at
each p-value are few. Thus, by inspecting Figure 1, one can arrive
at a decision as to the degree of specificity for a given p-value in
a given solvent system. By the same reasoning, the systems heptane-
90% ethanol, and 2,2,4-trimethylpentane-80% acetone (latter below 0.72),
appear to be more generally useful for identifications than the hexane-
acetonitrile and the 2,2,4-trimethylpentane-DMF systems. However, for
a specific case, sweeping generalizations as to the best system cannot
be made.
Since three-quarters of the p-values are below 0.21 in the hexane-
acetonitrile system and the nonpolar crop interference—as from butter--
tends to accumulate at higher p-values (2), pesticides are readily
separated from such crop interferences by simple extraction. The
result illustrates graphically why this solvent system has become
popular in pesticide analysis. 2,2,4-trimethylpentane-DMF also appears
to be good for such separations, but DMF, boiling about 70° higher than
acetonitrile, is much more difficult to evaporate and accordingly less
suitable for cleanup
-------
Revised 1/4/71 Section 12, C
Page 3
V. COMMENTS:
With the p-value technique it is not usually necessary to determine
the exact amount of a substance in an analysis, but only the relative
amounts present in the original and the extracted solution. This
feature is especially welcome in gas chromatographic analysis when one
is dealing with an unknown compound, and the response for a given
amount of compound is not known. In such cases, it is desirable to
check the linearity of the system by injecting an amount necessary to
give a reasonable response and then injecting exactly half that amount.
If the second response is half the first, the linearity of the system
may be considered satisfactory. This type of linearity check was
routinely made in the present work.
In a few instances there appears to be a reaction that takes place
between the solute and the solvent system. The reaction either prog-
resses with the time of exposure to the solvent system or may result
from the reaction of solvent and solute when they are injected into
the hot injection port during gas chromatographic analysis. Some
compounds (supposedly pure) give multiple peaks indicating breakdown.
The p-values derived from these analyses must be considered less
reliable than those of comounds chromatographing without breakdown.
-------
1/4/71
Section 12, C
Page 4
30 -
20 -
10 -
0 -
HEXANE -
907. DMSO
_
HEPTANE -
907. ETHANOL
30 -
2-,2,4-TRIMETHYLPENTANE -
20 - 807. ACETONE
o-HM
HEXANE -
ACETONITRILE
.1 i \i
20 -
o -
2,2,4-TRIMETHYLPENTANE
DMF
j.
2,2,4-TRIKETHYLPENTANE
857. DMF
Jp
.20 .40 .60 .80 1.00 0
P-VALU2S
• f_t-!•- I J * *j- • - •*•• "*--|» *• • i «• £ • .*.
.20 .40 .60 .80 1.00
Figure 1. Incidence of p-values in each of the six binary- solvent systems..
-------
Revised 1/4/71
Section 12, C
Page 5
TABLE 1.
P-VALUES OF PESTICIDES AND RELATED COMPOUNDS DETERMINED BY
SINGLE DISTRIBUTIONS BETWEEN IMMISCIBLE PHASES AT 25.5 ± 0.5°C
ARRANGED ACCORDING TO ASCENDING GAS CHROMATOGRAPHIC RETENTION
TIMES ~(Rt). COLUMN OF 1.5% OV-17/1.95% QF-1 OPERATED AT 200°C.
Solvent System
Compd.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Pesticide Rt (rela-
(Or Related tive to
Compound) Rt of
Aldrin)
naled
ethyl ene di bromide
Fumazone®
Penphene®
dichlobenil
Zinophos®
barban
chloro-IPC
CDEC 0.56
phorate 0.52
Shell SD 8447
(hydr. prod.)
trifluralin
lauseto neu
lindane 0.69
PCNB
Bayer 30911
dioxathion (pri-
mary peak)
Stauffer N-2790
diazinon 0.64
dichlone 0.99
Di-Syston®
endosulfan ether
Bayer 38156
Hercules 426
heptachlor 0.82
methyl para-
thion 1.45
dioxathion (sec-
ondary peak)
butonate
Bayer 41831
malathion 1.63
Zytron®
fenson
aldrin 1.00
1-hydroxy-
chlordene 1.25
Bayer 25141
parathion 1.84
Hexane:
Aceto-
nitrile
0.12
0.29
0.23
0.76
0.11
0.058
0.019
0.19
0.22
0.26
0.18
0.23
0.023
0.12
0.41
0.23
0.068
0.21
0.28
0.073
0.16
0.29
0.22
0.50
0.55
0.022
0.11
0.013
0.036
0.042
0.12
0.048
0.73
0.068
0.82
0.044
2,2,4-
tri-
methyl-
oentane:
DMF
(a)
(a)
0.12
0.58
0.080
0.036
0.003
0.14
0.13
0.11
0.077
0.21
0.007
0.052
0.23
0.071
0.038
0.081
0.18
0.027
0.089
0.14
0.12
0.20
0.21
0.012
,0.055
b0.005
0.016
0.015
0.058
0.013
0.38
0.026
0.32
0.029
2,2,4-
tri-
methyl-
pentane:
85%
DMF
(a)
(a)
0.32
0.89
0.15
0.23
0.007
0.17
0.32
0.44
0.24
0.81
0.010
0.14
0.67
0.24
0.12
0.33
0.52
0.068
0.36
0.42
0.39
0.72
0.73
0.015
0.25
bO. 01 4
0.046
0.037
0.14
0.032
0.86
0.062
0.78
0.082
Hexane:
90%
DMSO
0.085
0.48
0.36
0.98
0.19
0.15
0.003
0.16
0.35
0.61
0.18
0.84
0.008
0.093
0.79
0.33
0.21
0.44
0.75
0.068
0.47
0.43
0.51
0.79
0.77
0.015
0.44
C0.078
0.074
0.077
0.12
0.035
0.89
0.033
0.81
0.094
Heptane:
90%
Ethanol
0.23
0.58
0.54
0.79
0.26
0.16
0.13
0.26
0.46
0.56
0.42
0.72
0.077
0.41
0.82
0.49
0.39
0.48
0.39
0.34
0.54
0.45
0.48
0.74
0.71
0.11
0.35
0.043
0.24
0.14
0.35
0.20
0.76
0.15
0.77
0.30
2,2,4-
tri-
methyl-
pentane:
80%
Acetone
0.39
0.76
0.76
0.87
0.60
0.40
0.37
0.72
0.86
0.83
0.85
0.93
0.34
0.78
0.95
0.79
0.95
0.79
0.75
0.57
0.82
0.85
0.76
0.98
0.96
0.40
0.81
0.080
0.55
0.46
0.79
0.61
0.98
0.56
0.90
0.76
(Continued)
-------
Revised 1/4/71
Section 12, C
Page 6
TABLE 1. CONTINUED
Solvent System
Compd.
No.
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
Pesticide R^ (rela-
(Or Related tive to
Compound) Rt of
Aldrin)
Dimite®
Kel thane®
dicapthon
Chlorthion®
chlorobenzilate
(secondary peak)
dicryl
Telodrin®
Bayer 37289
isodrin
Dyrene® 1.83
heptachlor
epoxide 1.54
Morestan®
folpet 2.64
Ruelene®
Y-chlordane
Genite 923®
Sulphenone®
chlorbenside 1 .91
endosul-
fan (I) 1.95
ovex
Shell SD-8447
dieldrin 2.40
£,£'-DDE 2.23
endrin 2.93
endosul-
fan (II) 3.59
Aramite®
Methyl Trithion®
Perthane® 2.71
endrin aldehyde
TDE 3.48
p_,£'-DDT
chlorobenzilate
(primary peak)
o,£'-DDT 3.16
Kepone® 2.77
Neotran® (pri-
mary peak)
ethion 4.28
Hexane
Aceto-
ni-
trile
0.25
0.15
0.031
0.026
0.22
0.040
0.48
0.54
0.60
0.041
0.29
0.34
0.066
0.031
0.40
0.08
0.023
0.24
0.39
0.068
0.051
0.33
0.56
0.35
0.13
0.13
0.075
0.26
0.082
0.17
0.45
0.14
0.47
(e)
0.47
0.079
2,2,4-
: tri-
methyl-
pentane:
DMF
0.077
0.043
0.019
0.013
0.062
0.029
0.17
0.18
0.28
(a)
0.10
0.14
O.C15
0.012
0.14
0.032
0.012
0.039
0.16
0.024
0.038
0.12
0.16
0.15
0.060
0.075
0.019
0.077
0.041
0.038
0.10
0.032
0.11
(e)
0.11
0.045
2,2,4-
tri-
methyl-
pentane:
85%
DMF
0.27
0.18
0.044
0.039
0.24
0.041
0.63
0.75
0.78
(a)
0.39
0.43
0.036
0.013
0.48
0.076
0.009
0.21
0.52
0.061
0.051
0.46
0.65
0.51
0.14
0.23
0.075
0.44
0.083
0.15
0.42
0.12
0.51
(e)
0.59
0.20
Hexane:
90%
DSMO
0.37
0.029
0.051
0.032
0.40
0.012
0.65
0.78
0.86
0.014
0.35
0.53
0.025
0.012
0.45
0.093
0.013
0.29
0.55
0.053
0.044
0.45
0.73
0.52
0.093
0.30
0.081
0.46
0.053
0.081
0.53
0.14
0.66
(e)
0.73
0.38
: Heptane
90%
Ethanol
0.47
0.32
0.25
0.16
0.38
0.066
0.73
0.72
0.76
0.17
0.57
0.54
0.23
0.11
0.56
0.30
0.087
0.52
0.64
0.28
0.093
0.54
0.76
0.59
0.34
0.43
0.42
0.70
0.15
0.46
0.62
0.28
0.68
0.16
0.77
0.41
2,2,4-
tri-
methyl -
pentane:
80%
Acetone
0.81
0.84
0.61
0.56
0.93
0.31
0.94
0.88
0.97
0.61
0.89
0.65
0.51
0.21
0.95
0.67
0.32
0.86
0.93
0.69
0.47
0.88
0.96
0.92
0.82
0.85
0.82
0.93
0.79
0.89
0.91
0.76
0.96
0.43
0.93
0.83
(Continued)
-------
Revised 1/4/71
Section 12, C
Page 7
TABLE 1. CONTINUED
Solvent System
Compd.
No.
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
Pesticide R^ (rela-
(Or Related tive to
Compound) R+. of
Aldrin)
Prolan®
endosulfan
sulfate
Rhodia R.P.
11783
carbopheno-
thion 4.56
p_,p_'-DDT 4.18
Bui an®
endrin A-keto
compound
Geigy G-28029
EPN
dinocap (pri-
mary peak)'
methoxychlor 8.1
mi rex 6.1
tetradifon 10.9
Guthion® 15.0
dinocap (sec-
ondary peak)
coumaphos
Hexane:
Aceto-
nitrile
0.050
0.035
0.019
0.21
0.38
0.10
0.10
0.29
0.38
0.092
0.069
0.91
0.10
0.008
0.082
0.006
2,2,4-
tri-
methyl-
pentane
r\mir~
DMF
0.017
0.015
0.006
0.037
0.084
0.024
0.052
0.065
0.011
0.049
0.023
0.33
0.041
0.002
0.041
0.002
2,2,4-
tri-
methyl-
pentane:
: 85%
DMF
0.048
0.023
0.012
0.27
0.36
0.10
0.077
0.43
0.033
0.27
0.092
0.98
0.13
0.003
0.22
0.010
Hexane:
90%
DMSO
0.029
0.010
0.01
0.35
0.40
0.072
0.062
0.43
0.046
0.54
0.12
0.93
0.13
0.003
0.50
0.013
Heptane:
90%
Ethanol
0.25
0.16
0.16
0.56
0.64
0.36
0.21
0.64
0.24
0.50
0.44
0.88
0.40
0.14
0.48
0.083
2,2,4-
tri-
methyl-
pentane:
80%
Acetone
0.75
0.68
0.38
0.90
0.93
0.86
0.76
0.91
0.71
0.98
0.74
0.99
0.78
0.18
0.94
0.59
a Solvent interferes with GLC zones under these conditions.
b Reduced initial response (reaction with system?). Dyrene response continues
to diminish on standing.
c Converted to substance Rt = 0.70.
Rt changes after equilibration (reaction with system?).
e p-Values differ at different concentrations of analysis.
Actually two zones emerging as one.
-------
Revised 6/77 Section 12, D, (1)
Page 1
MICRO SCALE ALKALI TREATMENT FOR USE IN PESTICIDE RESIDUE
CONFIRMATION AND SAMPLE CLEANUP
(Reproduction of original manuscript
subsequently published in the Bull. Envir.
Contam. & Toxic., 7,2/3, 160, 1972)
Procedures involving alkali treatment for dehydrochlorination 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_ a\_, (2), studied rates of dehydrochlorination of DDT.
Milles (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 alcohol NaOH to dehydrochlorinate £,£'- and
£,£'-DDT, £,£'-TDE, and Perthane prior to gas chromatographic separation of
the respective olefins. These investigators called attention to several
earlier uses of alkali dehydrochlorination in pesticide residue chemistry.
Recent literature contains numerous references to application of this
treatment in pesticide residue analyses, including an adaption 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
derivatives, 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 appli-
cation in multi-residue analysis.
The purpose of this work was (1) to arrive at optimum and convenient
parameters of the alkali treatment in order to obtain rapid dehydrochlorin-
ation resulting in product solutions suitable for analysis by GLC; (2) to
obtain yield and recovery of olefins from a number of bis(phenyl) chloro-
ethane pesticides; (3) to determine 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.
REFERENCES:
1. Brand, K. and Busse-Sunderman, A., Berichte. 75 B 1819 (1942).
2. Soloway, S. B., Schechter, M. S., and Jones, H. A., Soap and
Sanitary Chemicals, 1946 Blue Book, 18th Ed., 215 (1946).
3. Mills, P. A., J. Ass. Offie, Anal. Chem. 42, 734-740 (1959).
-------
Revised 11/1/72 Section 12, D, (1)
Page 2
4. Klein, A. K. and Watts, J. 0., J. Ass. Offie, Anal. Chem 47,
311-316 (1964)
5. Miller, G. A. and Wells, C. E., J. Ass. Offic. Anal. Chem, 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, D.C., May 1970.
8. Greve, P. A. and Wit, S. H.., J. Agr. Food Chem. 19, 372-374 (1971).
METHOD
Reagents and Apparatus
(a) Potassium hydroxide - Anhydrous pellets.
(b) Ethanol - USP 95%
(c) Hexane - Suitable for use with electron capture gas chromatography
(Burdick and Jackson Laboratories, Inc. 1953 S. Harvey St.,
Muskegon, Michigan 49442).
(d) Micro condenser - 19/22 I; K-569250 (Kontes Glass Company, Vineland
N.J. 08360).
(e) Concentrator tube - Mills type, 19/22 f with stopper, 10 ml
graduated in 0.1 ml up to 1.0 ml; K-570050 (Kontes Glass Company).
(f) Alkali dehydrochlorination 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 chromatograph - Equipped with electron capture detector and
6' 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(HP),
Operating conditions: N2 flow 120 ml/min; temperatures, column
and detector 200"C, injector 225°C; concentric design electron
capture detector operated at DC voltage to cause 1/2 scale recorder
deflection for 1 ng heptachlor epoxide when full scale deflection
is 1 x 10 amp.
Procedure
Accurately pipet, into a 10-ml Mills tube, 2 ml of a petroleum ether
solution of sample extract (6% of 15% Florisil eluate (6)) containing
concentrations of pesticides suitable for subsequent GLC analysis. Add 1 ml
-------
Revised 11/1/72 Section 12, D, (1)
Page 3
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 manner that gentle boiling occurs. When the volume has been reduced
to about 1 ml, insert the tube completely 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-H20 (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 microliter
syringe, carefully withdraw aliquot of opper layer for determination by GLC.
NOTE: Separation of phases should be sharp so that solution withdrawn
for GLC analysis will be free from alkali.
DISCUSSION
Development of Method
Initial experimentation was performed to establish the reaction con-
ditions which would give complete and rapid dehydrochlorination of £,£'-DDT,
£,£'-DDT, £,J3'-TDE, methoxychlor, and Perthane and which would permit com-
plete recovery of the 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 discussed. 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 KOH and NaOH
have been used to the satisfaction 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) chloroethane pesti-
cides ranging from a few nanograms to 100 yg. In order to provide suffi-
cient reflux time and temperature, it was necessary that the initial volume
of ethanol be in excess of 0.5 ml. When smaller volumes were used, de-
hydrochlori nation was usually incomplete. Emulsions often occurred during
extraction of the olefin into hexane after saponification of fatty substances
-------
Revised 11/1/72 Section 12, D, (1)
Page 4
The use of ehtanol + water (1+1) instead of water as the diluent 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. Experiments
in which varying volumes of 2% ethanolic 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 dehydrochlorination of Perthane (40 yg) and methoxychlor
(4.0 ug) was not obtained. However, £,£'-DDT (8.0 yg) was completely de-
hydrochlorinated in the presence of 100-120 mg of fat. Additional experi-
mentation showed that complete dehydrochlorination of methoxychlor and
Perthane did not occur until the fat was completely saponified; these were
the bis(phenyl) chloroethanes most resistent to dehydrochlorination.
Quantities of Perthane and £,£'-DDT up to 100 yg in the presence of not more
than 50 mg butterfat were readily dehydrochlorinated with 1 ml of 2% KOH at
steam bath temperature; dehydrochlorination of larger amounts of pesticide
was not attempted. Hexane, because of its higher boiling point and greater
ease of drawing into amlcrosyringe, was used instead of petroleum ether, to
extract the olefin after reaction in order to avoid possible errors in
quantisation.
Effect on Selected Chemicals
The dehydrohalogenation reaction, as described under "Method", was
applied to £,£'-DDT (0.8 yg), £,p'-DDT (0.8 yg), £,£'-TDE (2.0 yg),
p_,£'-TDE (0.4 yg), methoxychlor (4.0 yg), and Perthane (40 yg). Quantities
shown in parentheses were chosen for ease in GLC determination. The pest-
icides were treated individually 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 fatty substances extracted
from butter by partitioning (6) between petroleum ether and acetonitrile.
Gas chromatography with electron capture detection, operated as described
under "Method", 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 compound/mol. wt x
wt. olefin compound/mol. wt.
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Revised 11/1/72 Section 12, D, (1)
Page 5
Recoveries of olefins approximated 100% and ranged from 86% for
jD,p_'-DDE and £,£'-TDE olefin in petroleum ether of 110% for £,£'-DDE in the
extract from butter. Two 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 common organochlorine pesticides and polychlorinated
biphenyls (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.
Polychlorinated biphenyls ranging from 21 to 60% average chlorine con-
tent were stable to this treatment. Complete recoveries were obtained for
the commercial PCB mixtures, Aroclors 1221 (16 yg), 1232 (16 yg), 1242 (16 yg),
1254 (8 yg), and 1260 (8 yg).
Recoveries of unchanged aldrin (0.4 yg), dieldrin (0.4 yg), and Endrin
(0.4 yg) ranged from 70 to 90%. No alteration products were detected.
Heptachlor (0.4 yg) and heptachlor epoxide (0.4 yg) 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 re-
lative to aldrin of 1.63 after treatment of heptachlor epoxide and 0.59 and
0.93 after treatment of heptachlor.
The alkali treatment completely eliminated lindane (0.2 yg) and the
alpha (0.2 yg), beta (0.2 yg), and delta (0.2 yg) isomers of BHC. Following
the reaction, only small early eluting gas chromatographic peaks, presumably
from trichlorobenzenes, were observed.
About 40% of mirex (4.0 yg) 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/
15% QF-1 column. The peak height of the alteration product was approximately
one-tenth the peak height of the parent 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 yg) was completely eliminated, but only 65% of the major
alteration product, 4,4'-dichlorobenzophenone, was recovered. A minor
peak was observed in the chromatogram at a retention time relative to aldrin
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Revised 11/1/72 Section 12, D, (1)
Page 6
of 1.71 on the 10% DC-200 column. The 4,4'-dichlorobensophenone (2.0 ug)
was not affected by treatment with alkali.
The products resulting from alkali treatment of toxaphene (10.0 yg) gave
a multicomponent chromatogram but consisting of components with earlier re-
tention times than toxaphene itself.
The electron capture GLC responses to sulfur (20 yg), frequently en-
countered 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 most 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 confirmation of a residue of the parent pesticide. For example,
a residue of £,£'-DDT can be quantitated before alkali treatment and
verified by quantisation 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
substances. We have found the alkali treatment especially useful in connec-
tion with the real or suspected presence of residues of PCB. In this case
the characteristic olefin derivatives of £,£'-DDT, £,£'-DDT, and £,£'-TDE
can be formed and the GLC retention time region underlying 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 identity of this complex residue. The high recovery of
unreacted dieldrin, endrin, and aldrin following alkali treatment likewise
can be of value in the confirmation of identity of these pesticides.
Cleaned-up extracts of some samples may contain non-pesticidal sub-
stances 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 partic-
ularly true of the 15% ethyl ether/petroleum ether Florisil column elutate
(6) for non-fatty samples such as carrots and fatty samples such as some
fish. Electron capturing 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 micro-
coulometric 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.
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Revised 12/15/79 Section 12, D, (2)
Page 1
PERCHLORINATION OF PCBs
Subsection 9,B,(2), IX describes a method for confirmation of
polychlorinated biphenyls (PCBs) based on perchlorination of all
compounds to yield a single derivative, decachlorobiphenyl,
followed by electron capture gas chromatography. Although the
method was developed for mother's milk and is described in detail
only for this substrate, perchlorination undoubtedly has much
wider applicability to other types of samples. Suitability must
be verified in each particular situation, however, by recovery
studies on fortified check samples.
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Revised 1/4/71 Section 12, E
Page 1
INFRARED SPECTROSCOPY
I. INTRODUCTION:
Infrared spectroscopy is the most powerful single technique available
for the identification of organic compounds and is almost without equal
as empirical proof of identity. The disadvantages of infrared are low
sensitivity, requirement of a relatively pure sample, and the training
and experience necessary to interpret spectra. The low sensitivity
requires that gram quantities of sample be processed. The requirement
of a relatively pure sample dictates the use of a stringent sample
cleanup procedure plus additional cleanup of the extract either by gas
chromatographic separation or thin-layer chromatography.
Infrared is sensitive to atleastl yg and has been utilized at the
0.1 yg level. Thus, it is considerably less sensitive than either gas
or thin layer chromatography.
A number of techniques have been developed for infrared; however,
the technique presented here, potassium bromide pellets, is the most
sensitive and dependable. Considerable experience is necessary in the
preparation of pellets to minimize contamination, which is the princi-
pal problem inherent in this technique.
REFERENCES:
1. Blinn, Roger C., J.A.O.A.C., 46 (1963)
2. Blinn, Roger, C., Personal Communication (1968).
3. Boyle, H. W., Burttschell, R. H., and Rosen, A. A.
Organic Pesticides in the Environment 60, Advances in
Chemistry Series, A.C.S., Washington, D.C. (1966).
4. Curry, A. S., Read, 0. F., Brown, C., and Jenkins, R. R.,
J. Chromatography, 38:200-8 (1968).
5. Garner, H. R., and Packer, H., Applied Spectroscopy,
22: 122-3 (1968).
6. Keiser, W. E., Personal Communication (1968).
7. Kovacs, M. F., Jr., J.A.O.A.C., 47 (1964).
8. Kovacs, M. F., Jr., Personal Communication (1968).
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Revised 1/4/71 Section 12, E
Page 2
9. Perkin-Elmer Corp., Operating Manual, I.R., 337.
10. Robbins, J. D., Bakke, J.E., and Fjelstul, C. E. Practical
Micro-KBr Disk Techniques, Presented at the Am. Chem. Soc.,
Meeting, Minneapolis, Minn., April 1969.
II. EQUIPMENT:
A. Perkin-Elmer IR-337, or equivalent, equipped with a 6X beam con-
denser and holder for 1.5 mm KBr discs.
B. Micro KBr equipment, Perkin-Elmer ultra micro dye assembly.
C. Press, capable of exerting 200 pounds of pressure.
D. Micro mortar and pestle.
E. Fine tipped forceps.
F. 50 yl syringe
G. White glove liners.
H. Vacuum pump.
I. Oven at 60-70°C
J. Micro Spatula.
K. Thin-layer equipment.
L. Stream splitters: 1-100 and 1:1000.
M. Capillary Tubing, borosilicate glass, 2mm ID
N. Pipe cleaners, dyed to various colors.
0. Desiccator, vacuum
P. Evaporator, vacuum, rotary.
III. REAGENTS:
A. Standard pesticides
B. Hexane, reagent, redistilled*
C. Potassium bromide, infrared grade.
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Revised 1/4/71 Section 12, E
Page 3
D. Aluminum oxide G.
E. Ethyl ether, anhydrous, reagent grade.
F. Ethanol, 95%
G. Rhodamine B dye.
H. Silica gel G.
I. Acetone, reagent, redistilled*.
J. Methanol, reagent.
K. Chloroform, reagent.
L. Buffer, pH 6.0.
M. Methylene chloride, reagent grade, redistilled.*
N. Palladium chloride.
0. Hydrochloric acid.
P. Potassium bromide wick-sticks, Harshaw Scientific Co.
IV. INSTRUMENT CALIBRATION:
A. Gain Adjustment
1. Decrease gain until there is a sluggish response when the
sample beam is blocked and unblocked.
2. Increase gain until the correct response is obtained.
a. Partially block the sample beam with your thumb to
obtain 10% downscale deflection.
b. Rapidly, remove your thumb and note the overshoot.
c. Adjust until you obtain 1-2% overshoot.
B. Balance Adjustment
1. Partially block the sample beam with your thumb.
2. Change balance control to bring the pen to about 50%
*Redistilled in all-glass apparatus.
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Revised 1/4/71 Section 12, E
Page 4
3. Simultaneously block both beams and adjust control to no down-
scale or upscale drift.
4. Slight upscale drift can be tolerated, however, downscale cannot.
C. Zero Adjustment
1. Partially block the sample beam until the pen reads about 5%.
2. Very slowly continue blocking the beam until it is completely
blocked.
3. If pen does not read zero:
a. Remove pen tower cover.
b. Loosen the screw holding the pen carriage on the slide
wi re.
c. Set pen to zero.
d. Tighten screw.
4. Repeat procedure (1 through 3) until zero is properly adjusted.
D. 100% adjustment
1. Be sure both beams are not blocked
2. With the 100% adjustment, set the pen to 99-100%.
E. General
1. Always make the adjustments in the order in which they have
been presented.
2. Always check these adjustments before sample analysis.
3. Remember that a change in the zero adjustment will necessitate
a change in the 100% adjustment.
V. SAMPLE PREPARATION:
A. Use enough sample to provide a sufficiently large concentration of
the compounds under analysis to allow infrared observation.
B. Use a cleanup procedure which will provide relatively pure
pesticide compounds.
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Revised 1/4/71 Section 12, E
Page 5
1. Trapping of gas chromatograph effluent-capillary procedure.
a. Attach splitter to the inlet to the EC detector (split
ratio, 1/100-1000).
b. Collect effluent from splitter on KBr in cooled tube,
prepared as follows:
(1) Use a 3" length of capillary tubing (2mm I.D.).
(2) Place a pipe cleaner in the tube as a reagent support.
(3) Pack the tube with about 10 mg of dry KBr.
(4) Hold the packed tube at 150°C.
(5) Just prior to use, cool the tube in a dessicator
just below room temperature.
c. Collect center fraction of peak desired, by providing
intimate contact between packed capillary and outlet
of splitter arrangement.
d. Force KBr out of tube into micro dye, using pipe cleaner.
e. Prepare KBr pellet.
2. Wick-Stick trapping procedure
a. Collect desired peak by holding wick-stick to exit of
splitter.
b. Concentrate pesticide at tip of wick-stick by procedure
described in the wick-stick kit.
c. Break off tip of stick and prepare pellet therefrom.
3. Thin-layer cleanup and separation
a. Chlorinates pesticides
(1) Prepare plates using the mechanics presented under
"Thin-layer chromatography."
(a) Aluminum oxide G.
(b) Activate in an oven at 155°C for 2.5 hours.
(c) Store over Drierite until used.
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Revised 1/4/71 Section 12, E
Page 6
(2) Concentrate extract to 1-0.1 ml, depending on con-
centration of agent in the extract.
(3) Spot a sufficient amount of the extract, and the
appropriate standards, on the plate.
(4) Develop chromatograms with 1% ethyl ether in hexane.
(5) Spray lightly with 0.01% rhodamine B in 95% ethanol.
(6) Remove each spot desired, by vacuum, using a glass
medicine dropper plugged with glass wool.
(7) Elute the pesticide from the adsorbent with 5 ml of
4:1 hexane-ethyl ether mixture.
(8) Concentrate the eluate to about 0.1 ml.
(9) Mix sample and KBr.
(a) Weigh out 7 mg dry infrared quality KBr
into a warm micro-mortar and lightly tamp
into a small cake.
(b) Add concentrated eluate to the KBr to 2 yl
increments, allowing time for solvent evaporation
between each addition. Put eluate on KBr, not
the mortar.
b. Organothiophosophate pesticides.
(1) Prepare plates using the mechanics presented under
"Thin-layer chromatography."
(a) Mix 30 g silica gel G with pH 6 buffer in a
250 ml Erlenmeyer flask.
(b) Shake vigorously for 1 minute.
(c) Apply as a 250 micron layer.
(d) Let plates air dry overnight.
(e) Wash plates twice by letting acetone migrate
up plates for 20 cm.
(f) Air dry.
(2) Concentrate sample to 0.3 ml in methylene chloride.
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Revised 1/4/71 Section 12, E
Page 7
(3) Using a micropipette, apply sample to plate, in increments,
drying between applications.
(4) Develop with 1.75% methanol in chloroform.
(5) Air dry.
(6) Spray the plate with 5 ml of a 5% solution of palladium
chloride and 1 ml of HCL, diluted to 100 ml in 95%
ethanol .
(7) Allow plate to dry for 30 minutes at room temperature (may
be necessary to hold plate overnight before all
thiophosphates are discernible).
(8) Locate spots of interest and remove each by the method
given for chlorinated pesticides.
(9) Extract the adsorbent with five 1 ml portions of hot
acetone into a 25 ml microflask.
(10) Evaporate to dryness under vacuum in a rotating evaporator.
(11) Add 1ml of CClit, rinse the walls of the flask and re-
evaporate.
(12) Take up the residue in CCl^ and concentrate to about 0.1 ml
(13) Place 5-7 mg of dry infrared KBr in a warm mortar and
add the concentrated residue as instructed for chlorinated
pesticides.
VI. ALTERNATE METHODS OF MIXING CONCENTRATED SAMPLE EXTRACT AND KBR:
A. Wick-stick procedure
1. Place concentrated extract in vial of wick-stick kit.
2. Place wick-stick in vial and allow solvent to evaporate,
concentrating the pesticide on the tip of the stick.
3. Break off tip and use to make pellet.
B. New procedure of Blinn
1. Prepare 13 mm pellet without pesticides.
2. Using this pellet as a micro-mortar, put lightly heated KBr
powder thereon and add solution dropwise.
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Revised 1/4/71 Section 12, E
Page 8
3. Remove loose KBr into micro-dye and scrape the surface of the
13 mm pellet into the micro-dye.
4. Prepare 1.5 mm micro-pellet.
C. Syringe procedure
1. Place sample extract into a syringe equipped with a discharge
controller.
2. Eject a drop of the solution to the syringe tip, dip into KBr
powder and suspend on the needle.
3. Continue ejecting solution onto KBr, drying between injections.
4. Place KBr in micro-dye and prepare pellet.
VII. PREPARATION OF KBR PELLET:
A. Transfer the KBr sample to the micro-dye.
B. Assemble the dye.
C. Assure that the sample is spread evenly by rotating the top ram
under slight hand pressure.
D. Press and evacuate the dye.
E. Remove the pellet and analyze.
F. Clean dye immediately after use.
VIII. ANALYSIS AND INTERPRETATION:
A. Turn on instrument.
B. Place pellet in holder on instrument.
C. Recheck
1. Gain
2. Balance
3. Zero
4. 100% adjustment
D. place paper on drum.
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Revised 1/4/71 Section 12, E
Page 9
E. With scan control in stop, place range switch in proper range to
correspond with chart paper.
F. Scan sample.
G. Turn scan switch to stop.
H. Change range switch.
I. Change chart paper.
J. Replace drum in well.
K. Turn scan control to "reset."
L. Turn drum to beginning of range.
M. Put pen on paper.
N. Scan second range.
0. Interpret spectrum by comparison with spectra from standard
pesticides.
IX. MISCELLANEOUS NOTES:
A. Do not turn instrument on and off during the day it is being used.
B. Do not leave instrument stand with pen above 100%.
C. Set gain, balance, and 100% in that order and with sample in beam.
Check gain at about 4.5 microns.
D. Turn scan control to stop before changing range switch.
E. Eliminate all possible contamination when making micro KBr pellets.
F. When using thin-layer cleanup, remember "Thin-layer, Notes on
Procedure."
G. When trapping from gas chromatograph:
1. Minimize contamination from substrate, column, and previous
instrument use.
2. Work with as much sample as can be collected by taking a center
cut of the peak.
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Revised 12/2/74 Section 12, F
Page 1
Reproduced from Pesticide Analytical Manual, Volume I,
U. S. Food & Drug ADMINISTRATION (Revision 7/1/70).
POLAROGRAPHY
640.1 Introduction. The development of oscillopolarographic instrumenta-
tion and techniques has caused a renewed interest in the use of polarography
for residue determination. This technique is rapid and specific. Its
sensitivity is comparable to colorimetry.
640.2 Recommended Literature References
(1) Gajan, R. J., Residue Reviews, Vol. 6, pp 75-86, and Vol. 5,
pp 80-99, Springer Verlag, New York, 1964.
Gajan discusses the applications of polarography for the detection and
determination of pesticides and their residues. He shows 12 single sweep
polarograms with comparison of derivative and regular wave, result of
degradation, and nitro derivatives. He lists 28 references.
(2) Martens, P. H., and Nangniot, P., Residue Reviews, Vol. 2,
pp 26-50, Springer Verlag, New York, 1963.
Martens and Nangiot review polarographic applications for determining:
copper, mercury, arsenic, tin and sulfur compounds; natural organic products
such as nicotine, rotenone, and pyrethins; and synthetic organic compounds.
They list 163 references.
(3) Gajan, R. 0., JAOAC 48, 1028-1037 (1965).
Gajan discusses the practical application of polarographic techniques to
the determination of pesticide residues. He lists 46 references.
641
POLAROGRAPHIC PROCEDURE FOR PESTICIDE RESIDUES
641.01 References. Official Methods of Analysis of the Association of
Official Analytical Chemists llth Edition, Sections 29.034-29.038. Included
are official AOAC methods for parathion, methyl parathion, diazinon, and
malathion. These methods are indicated by (AOAC) at the respective num-
bered paragraphs. Paragraphs describing methods not covered by the AOAC
methods include in reference to the basic literature.
Study leading to AOAC official status:
Gajan, R. J., JAOAC 52., 811-817 (1969).
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Revised 12/2/74 Section 12, F
Page 2
641.02 Application. This procedure will enable the residue analyst to
check results obtained with various multiple residue systems, using a
portion of the same cleaned up sample extract used for multiple residue
determination.
641.1 Apparatus
Cathode-ray Polarotrace K1000 or_
Davis Differential Cathode-ray Polarotrace A1660
Silver wire electrode. A No. 20 or N. 22 silver wire on which a
very thin coating of silver chloride has been deposited.
Polarographic cells (at least 6)
Capillaries (at least 3 extra)
Stop watch
641.2 Reagents
Acetic acid. Glacial, ACS grade
Acetone. Redistilled 56.5°C. Add 1 g KMnO^ per 4 L acetone
being distilled
Alcohol. 95% USP
Ethyl acetate. Reagent grade, redistilled, 77°C ± 1°C
Hydrochloric acid. 37-38%, ACS grade
Lithium chloride
Mercury. Purified
Methanol
Nitrogen. Prepurified, water pumped.
Potassium chloride. ACS grade
Potassium hydroxide. ACS grade
Potassium permanganate. ACS grade
Sodium acetate. (NaOAc-3H20). ACS grade
Sodium chloride. ACS grade
Sodium nitrite. ACS grade
Tetramethylammonium bromide. Eastman white label No. 670
Standard pesticide solutions. Prepare standard solutions contain-
ing 1 mg of pesticide per ml of ethyl acetate; store at 0°C.
641.3 General Method. Transfer suitable aliquot (1.0 ml) of cleaned up
extract to 50 ml erlenmeyer flask and evaporate just to dryness under
gentle jet of dry air at room temperature. Extracts must be in peroxide-
free solvents.
Dissolve this residue in a definite volume of solvent as directed by
specific procedures (641.42) for the various pesticides and add the
required amount of supporting electrolyte, mix well, and transfer 5.0 ml
or less of the mixture to a polarographic cell. (Since good polarotraces
may be obtained using only 0.5 ml of solution in a polarographic cell a
minimum of 0.25 ml solvent could be used to dissolve residue.) Bubble
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Revised 12/2/74 Section 12, F
Page 3
nitrogen through cell solution for 5 min and polarograph at 25°C ± 1°C.
over designated voltage scan.
Measure any waves appearing within ±0.10 volt of peak potential of pesti-
cide being determined. Peak potential of pesticide being polarographed
is determined by polarographing a standard of the pesticide dissolved in
the same solvent and electrolyte as the sample, immediately before or after
sample. The standard solution should contain approximately the same
amount of pesticide as the sample. When uncleaned solutions are polaro-
graphed the peak potential may shift slightly due to density of the cell
solution.
Estimate amount of pesticide in the solution by comparing wave height of
sample solution with that of standard solution.
A valuable check on qualitative determination of the pesticide, if in
doubt, is the standard addition technique, i.e., add a known amount of
pesticide standard solution to cell containing the sample solution and note
any increase in wave height. The peak potential of the standard pesticide
should be the same as that of the pesticide in the sample if they are the
same compound. The amount of total pesticide in the cell can be calculated
after correcting for volume change.
641.4 Application to Specific Residues
641.41 Suitability for Mixtures. By the judicious choice of supporting
electrolyte one can determine any admixture of the pesticides in 641.42 in
approximately 20 minutes. Polarographic interference between compounds
noted in 641.42 are sometimes avoided by the separations effected in the
extraction and cleanup procedures. The analyst should be aware of which
compounds may possibly be present in a sample solution.
The extraction and cleanup procedures now being used for the various
multiple detection procedures are adequate for the polarographic procedures
described here provided the same precautions as to purity of reagents and
solvents are maintained.
641.42 Specific Residues
641.42a (AOAC) Parathion and/or methyl parathion
Limitations. Limit of quantitative detection is 0.01 ppm based on 1 g
crop sample in 1 ml cell solution.
Parathion, methyl parathion, and paraoxon give similar polarographic
responses. Therefore, report all results as total of the three unless the
specific analog has previously been identified or unless the prior analyt-
ical method permits only certain of the compounds to be in the sample
solution.
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Revised 12/2/74 Section 12, F
Page 4
Electrolyte solution. Dissolve 2.72 g NaOAc-3H20 and 1.17 g Nad in 100 ml
redistilled H20 and adjust pH to 4.8 with glacial HOAc.
Polarographic determination. Dissolve residue from evaporation in a
definite volume of acetone and add an equal volume of electrolyte solution.
Proceed as directed in the general method, 641.3, starting with "...transfer
5.0 ml or less of the mixture to a polarographic cell..."
The peak potential for parathion is -0.68 ± 0.05 volt vs. mercury pool
reference electrode and -0.70 ± 0.05 volt vs. silver wire reference
electrode.
Prepare working standard solutions by diluting appropriate amounts of
stock solution with acetone.
641.42b Guthion
Reference. Bates, J. A. R., Analyst 87_, 786-790 (1962).
Limitations. Limit of quantitative detection is 0.01 ppm based on 1 g
crop sample in 1 ml cell solution.
Guthion and its oxygen analog give similar polarographic responses. There-
fore, report all results as total of the two unless the specific analog has
been previously identified.
Electrolyte solution. Prepare aqueous solution which is 0.5M HOAc and
0.2M KC1.
Polarographic determination. Dissolve residue from evaporation in a
definite volume of acetone and add an equal volume of electrolyte solution.
Proceed as directed in the general method, 641.3, starting with "...transfer
5.0 ml or less to a polarographic cell..."
The peak potential for Guthion is -0.70 ± 0.05 volt vs. either mercury
pool reference electrode or silver wire reference electrode.
641.42c (AOAC) Diazinon
Limitations. Limit of quantitative detection is 0.2 ppm based on 1 g
crop sample in 1 ml cell solution.
Diazinon and its oxygen analog give similar polarographic responses.
Therefore, report all results as total of the two unless the specific
analog has been previously identified.
Electrolyte solution. Dissolve 7.7 g tetramethylammonium bromide in 300 ml
H20 (0.1M). Add 115 ml HOAc and dilute to 500 ml with H20.
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Revised 12/2/74 Section 12, F
Page 5
Polarographic determination. Dissolve residue from evaporation in a
suitable amount of electrolyte solution and proceed as directed in the
general method, 641.3, starting with "...transfer 5.0 ml or less to a
polarographic cell..."
The peak potential of diazinon is -0.90 ± 0.05 volt vs. either mercury pool
reference electrode or silver wire reference electrode.
Prepare working standard solutions by diluting appropriate amounts of
stock solution with petroleum ether. Evaporate carefully to dryness and
proceed as with sample determination.
641.42d (AOAC) Malathion
Limitations. Limit of quantitative detection is 0.3 ppm based on 1 g
crop sample in 1 ml cell solution.
Malathion and its oxygen analog give similar polarographic responses.
Therefore, report all results as total of the two unless the specific
analog has been previously identified.
Electrolyte solution. Dissolve 15.4 g tetramethylammonium bromide in
300 ml H20 (0.2M). Add 0.2 g lithium chloride and 4.1 ml concentrated
HC1, and dilute to 500 ml with H20.
Polarographic determination. Dissolve residue from evaporation in a defi-
nite volume of methanol. Add 1/2 as much 0.1N KOH. Let stand for 3 min
and add an amount of electrolyte solution equal to the amount of methanol
used. Let stand 5 min. Proceed as directed in the general method, 641.3,
starting with "...transfer 5.0 ml or less to a polarographic cell..."
The peak potential for malathion is -0.85 ± 0.05 volt vs. a mercury pool
electrode and -0.82 ± 0.05 volt vs. a silver wire electrode.
Prepare a working standard solutions by diluting appropriate amounts of
stock solution with methanol.
Notes. Diazinon interferes with malathion since reduction is at the same
peak potential. However, malathion does not interfere with diazinon in
procedure 641.42c. If diazinon is suspected check for diazinon according
to 641.42c. If diazinon is found present, the amount of malathion in the
sample can be estimated with an accuracy of ±10% by subtracting the amount
of diazinon found by procedure 641.42c from the total amount of pesticide
found by procedure 641.42d when calculated as malathion. The same amounts
of diazinon and malathion give approximately the same polarographic wave
heights when polarographed using the electrolyte system described in
641.42d.
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Revised 12/2/74 Section 12, F
Page 6
641.42e Dimethoate
Reference. Gajan, R. J., and Gaither, R. A., unpublished method.
Limitations. Limit of quantitative detection is 0.05 ppm based on 1 g
crop sample in 1 ml cell solution.
Electrolyte solution. 0.1 N KOH in H20.
Polarographic determination. Dissolve residue from evaporation in a
definite volume of ethanol and add the required amount of electrolyte to
maintain a ratio of 3 parts electrolyte to 2 parts ethanol. Proceed as
directed in the general method, 641.3, starting with "...transfer 5.0 ml
or less of the mixture to a polarographic cell..."
The peak potential of dimethoate is -0.30 ± 0.05 volt vs. mercury pool
reference electrode and -0.55 ± 0.05 volt vs. silver reference electrode.
641.42f Carbophenothion
Reference. Nangnoit, P., Anal. Chem. Acta. 3]_, 166-174 (1964).
Limitations. Limit of quantitative detection is 0.2 ppm based on 1 g crop
sample in 1 ml cell solution.
Electrolyte solution. 50% w/v KOH in H20.
Polarographic determination. Dissolve residue from evaporation in a
definite volume of ethanol. Add an equal volume of electrolyte, mix well
and proceed as directed in the general method, 641.3, starting with
"...transfer 5.0 ml or less of the mixture to a polarographic cell..."
The peak potential for carbophenothion is 0.28 + 0.05 volt vs. a mercury
pool reference electrode and -0.43 ± 0.05 volt vs. silver wire reference
electrode.
641.42g Carbaryl.
Reference, Gajan, R. J., Benson, W. R., and Finocchiaro, J. M., JAOAC 48,
958-962 (1965).
Limitations. Limit of quantitative detection is 0.05 ppm based on 1 g
crop sample in 1 ml of cell solution.
Electrolyte solution. Mixture of glacial HOAc, 1.0 N NaN02 in H20, and
50% w/v KOH in H20, in ratio of 1:1:3.
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Revised 12/2/74 Section 12, F
Page 7
Polarographic determination. Dissolve residue from evaporation in a
definite volume of glacial acetic acid. Add an equal volume of 1.0 N
NaNO and let stand for 3 min. Add an amount of 50% KOH equal to three
times the volume of acetic acid used. Let stand for 15 min and proceed
as directed in the general method, 641.3, starting with "...transfer 5.0
ml or less of the mixture to a polarographic cell..."
The peak potential for carbaryl is -0.45 ± 0.05 volt vs. mercury pool
reference electrode and -0.68 ± 0.05 volt vs. a silver wire reference
electrode.
641.42h DDT in the Presence of Toxaphene (100X)
Reference, Gajan, R. J., and Link, J., JAOAC 47_, 1119-1124 (1964).
Limitation. Limit of detection is 0.5 ppm based on 1 g crop sample in
1 ml cell solution.
Electrolyte solution. Dissolve 7.703 g tetramethylammoniurn bromide in
250 ml distilled H20 (0.2M).
Polarographic determination. Dissolve residue from evaporation in a
definite volume of acetone and add 1.5 times as much ethanol. Add a volume
of electrolyte equal to that of the mixed solvent. Proceed as directed in
the general method, 641.3, starting with "...transfer 5.0 ml or less of the
mixture to a polarographic cell..."
The peak potential for DDT is -0.60 ± 0.05 volt vs. mercury pool reference
electrode and -0.70 ± 0.05 volt vs. silver wire reference electrode.
Notes. Parathion interferes with DDT in this procedure; however, the two
can be separated by Florisil column chromatography. DDT is found in the
6% Florisil column eluate; parathion in the 15% eluate (see 201). Other
analogs of DDT containing the trichloroethane configuration will also
interfere; however, they can also be separated from parathion by Florisil
column chromatography. Their absence should be checked for by 6LC or TLC.
-------
-------
Revised 12/15/79 Section 13
Page 1
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
See Subsection 6N through 6S of the EPA Pesticide AQC Manual
for a discussion of high performance liquid chromatography (HPLC)
including instrumentation, theory and principles, columns and
solvents, practical aspects of successful operation, and applica-
tions to pesticide analysis.
By far the most popular mode for the determination of
pesticide residues has been reverse phase HPLC on chemically
bonded C18 columns combined with ultraviolet adsorption detection.
However, the electrochemical LC detector has great promise for
residue analysis because of its subnanogram sensitivity for certain
compounds (see Determination of Halogenated Anilines and Related
Compounds by HPLC with Electrochemical and Ultraviolet Detection,
Lores, E. M., Bristol, D. W., and Moseman, R. F., J. Chromatogr.
Sci., 16, 358 (1978).
Retention and response data for pesticide standards and
detailed analytical procedures for residues will appear in future
revisions of this section as they are developed and tested in EPA
laboratories. HPLC data for 166 pesticidal compounds have been
compiled by J. F. Lawrence and D. Turton in J. Chromatogr., 159,
207 (1978). This reference lists the column packing, column
dimensions, mobile phase, elution volume, nature of the pesticide
(standard or residue from a particular substrate), UV detection
wavelength, and the literature reference.
-------
Revised 6/77 Appendix I
Page 1
GENERAL COMMENTS FOR THE
MAINTENANCE AND REPAIR OF INSTRUMENTS
The subject of instrumental servicing is obviously far too complex
to treat in a meaningful way in a manual such as this. In fact, a full
treatment would undoubtedly require an entire manual the size of this one.
The few comments which are offered here are primarily intended for
those laboratories which are a part of the U.S. Environmental Protection
Agency or which have formal contractual agreements with EPA and are
therefore eligible to obtain full benefits from the electronic repair
facility.
The Instrument Shop at Research Triangle Park, N.C. is fully equipped
to handle all repairs, modifications and calibrations on the Tracer MT-220
or MT-222 gas chromatographs and on miscellaneous brands of strip chart
recorders. The services of the shop are available to all EPA regional
laboratories and to the Epidemiologic Studies and Human Monitoring
laboratories holding contracts with EPA, these services to be supplied
on a cost-free basis. In such instances where the service required is
covered by a no-cost manufacturer's warranty, the manufacturer or dis-
tirbutor should be contacted for repairs.
The following services are available to the qualified laboratories:
1. Repair of Electron Capture Detectors including
foil replacement, rods, BNC connectors, Teflon
internal parts, and other parts as required.
2. Repairs, calibrations, modifications to: Electrometers,
Programmers, Temp-Set Controllers, E C Power Supplies,
Recorders, Microcoulometer and FPD Detector Systems.
In addition to the items above, a variety of miscellaneous repairs
are performed on blower motors, limit switches, oven heaters, thermometers,
etc., on a "one-for-one" basis.
Replacement modules, components, or recorders used with the MicroTek
GC MT-220 are available from the Instrument Shop
These include in part:
Electrometers - complete or integral components.
Programmers - complete or integral components.
-------
Revised 12/2/74 Appendix I
Page 2
Temp. Set controllers - complete or integral components.
Recorders - Westronics or Honeywell - complete or integral
components.
Flow Controllers, rotometers, damper systems, blower systems,
heaters 50 or 100 watt, limit switches adjustable and preset
oven coils, specified wiring, voltage monitor kits (for 110
V., A.C., only), accessory variacs, light duty Sola regulators,
thermometers, signal cables (BNC-BNC), pilot lamps, multidials,
10 turn potentiometers, compensator PCB, Insulation (Microfibre).
Dohrman system components, power supplies, printed circuit
boards (PCB's) and componets thereof.
A few items - Freon, Snoop, "0" rings, Septums, etc., should be
purchased by the user and only requested on an emergency basis where the
possibility of "down time" exists before normal purchase procedures can be
accomplished.
Our services may be obtained by phone or mail, depending on the
urgency. If "down time" is anticipated, a phone call should be made re-
questing the desired service or equipment. It is suggested that complete
information be written prior to a telephone service call - model numbers,
age of unit to be replaced if known, and pertinent data on the isolation
service procedures already tried. It is imperative that onedoesnot
attempt to use the "Mobile Reserve" to "Stock" their laboratory. It is
requested that all malfunctioning units be sent to the Instrument Shop for
repairs or survey.
In case of instrument breakdown requiring on-site servicing, the
appropriate area project coordinator should be contacted for discussion of
the need/cost involved.
Problems encountered with the gas chromatograph and not definitely
isolated as being electronic in nature should be channeled through Dr.
E. 0. Oswald, Chief, Chemistry Section, or discussed directly with J. F.
Thompson, Analytical Quality Assurance Chief, both at RTP.
Detailed trouble shooting instructions for all instrument modules are
far too lengthy for inclusion in this manual. Copies of such instructions
may be obtained from the RTP Instrument Shop.
Materials to be shipped to the Instrument Laboratory for service
should be placed in appropriate containers with sufficient packing to
insure against damage. Articles should not be shipped COLLECT except when
specific agreement has been reached to do so. We suggest the use of second-
class air mail when possible and that insurance in the full amount of the
units cost be purchased. Special shipping cartons and packing materials
-------
Revised 12/2/75
Appendix I
Page 3
are used to send various items - "These are not to be discarded". The
materials are expensive and should be returned or reused for future shipments
to the Instrument Shop.
Phone
Mr. Frank Wilinski, 919-549-8411, X2508
(commercial) or 629-2508 (FTS).
Mail, Truck Instrument Shop
or REA Quality Assurance Section
Shipments: Environmental Toxicology Division
EPA, Health Effects Research Laboratory
Room 113, Monsanto Bldg. (MD-69)
Research Triangle Park, NC 27711
-------
Revised 12/15/79 Appendix II, A
Page 1
ANALYTICAL QUALITY CONTROL
The term "quality control (QC)" may connote to some a system of con-
tolling the quality of a manufactured product. The phrase is wisely applied
in this role, but it also can be aptly used to indicate a "level of perfor-
mance". It is in the latter sense that the term is properly 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.
A Systematic program of quality control is of equal importance in an
analytical laboratory to any other activity performed by the laboratory.
Notwithstanding, many laboratory administrators fail to recognize its
importance, and they make no provision in time and resources for its
incorporation in the overall laboratory program. The comment has often been
heard from individual bench chemists to the effect that "we couldn't possibly
fit the type of QC program recommended into our work schedule." Unquestion-
ably, this statement has been a true one and has resulted from failure by
the administrator to recognize and provide for quality control in the
analytical program planning.
The unfortunate consequence of a lack of a systematic QC program is
the output of highly questionable analytical data of little or no value
for decision making. In the case of a regulatory laboratory, such data can
not be introduced as evidence in a court because of the danger of its dis-
creditation by the opposition. In a monitoring situation, such questionable
data could, for example, lead to false conclusions as to the pesticidal
profile of some sector of the environment.
In the preceding pages of this Manual, a number of multiresidue and
specific residue analytical procedures have been presented. A number of
these have been subjected to collaborative studies and are known to yield
acceptable inter!aboratory precision and accuracy. Yet, no method presented
should be expected to produce unquestionable data unless it is conducted
within the framework of systematic controls. Pesticide residue procedures
in general are highly complex and exacting, requiring highly sophisticated
electronic instrumentation. The lack of adequate controls is tantamount to
a ship without a compass.
In 1976 a complete and separate manual for analytical quality control
was developed, and it is now available in its 1st revised edition (1979).
The specifics of a QC program for pesticide analysis area treated in this
Manual. An outline of the EPA QC Manual is shown on the following page:
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Revised 12/15/79 Appendix II, A
Page 2
Title: Manual of Analytical Quality Control for Pesticides
and Related Compounds in Human and Environmental Media
Author: Dr. Joseph Sherma, Department of Chemistry, Lafayette
College, Easton, PA 18042
Revisions by: Dr. Joseph Sherma and Dr. Morton Beroza, The Association
of Official Analytical Chemists, Arlington, Virginia.
Editors: Randall R. Watts and Jack F. Thompson, EPA, Research
Triangle Park, NC
1. General Description of Pesticide Residue Analytical Methods.
2. Interlaboratory Quality Control.
3. Intralaboratory Quality Control.
4. Evaluation, Standardization, and Use of Materials for Pesticide
Residue Analysis.
5. Operation of the Gas Chromatograph.
6. Additional Procedures in Pesticide Analysis.
7. Multiresidue Extraction and Isolation Procedures for Pesticides
and Metabolites.
8. Confirmatory Procedures.
9. Maintenance, Troubleshooting, and Calibration of Instruments.
10. Training of Pesticide Analytical Chemists.
Persons wishing a copy of the QC Manual may write to the
following address:
Environmental Toxicology Division
EPA, HERL (MD-69)
Research Triangle Park, NC 27711
-------
1/4/71
TENTATIVE TISSUE, EXCRETA AND METHOD SELECTION FOR
ABNORMAL PESTICIDE EXPOSURE CASES; BLOCK DIAGRAM
LIVE DONORS
Suspected CHI.
Pestic. Exposure
BLOOD
5,A, (3), (a)
4 ML
URINE
5,A,(4),(b)
10 ML
Appendix VIt
Page 1
1
TISSUE BIOPSY
5,A, (2), (a)
0.5 GRAMS
Suspected PCP
Exposure
1 URINE
5, A, (4), (a)
1 5 ML
1
UR
5,A,(
2
;_
INE
3) , (b)
ML
BLOOD
5,A,(3),(b)
4 ML
Suspected 2,4-D ' URINE
or 2,4,5-T " |5,A,(4),(c)
Exposure ;
Suspected Hg Exposure
BLOOD-TISSUE
13, A
10 GRAMS
Suspected O.G.P.
Pestic.Exposure •
1
URINE
6,A,(2), (a) i
20 ML i
Suspected J
Carbaryl Exposure
URINE
7, A
5 ML
URINE
6,A, (4),(a)
5 ML
BLOOD
6,A, (3), (a)
3 iviL
AUTOPSY SAMPLES
Suspected CHL.
Pestic. ExposureJ
Suspected Hg
Exposure
URINE
5,A,(4),(b)
10 ML
i
Suspected PCP Exposure
Suspected 2,4-D or
2,4,5-T Exposure
Suspected OGP Pest-
icide Exposure
Suspected Carbaryl
Exposure
ADIPOSE TISSUE
5,A, (1)
5 GRAMS
SAME AS FOR LIVE DONORS
1
BLOOD
5,A, (3), (a)
100 GRAMS
OR
MODIFICATION
OF 5,A,(1)
Sample size given represent^ a maximun, number and letter designations refer to
method number as listed in the TABLE OF CONTENTS.
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Revised 12/2/75 Appendix VII
Page 1
PESTICIDE ANALYTICAL REFERENCE STANDARDS
The pesticide repository of the Environmental Toxicology Division was
initially established at the former Perrine, Florida, location. The
repository was created primarily to provide pesticide reference standards
for Pesticide Community Studies and other field laboratories under contract
to the United States Government to conduct pesticide monitoring programs.
It was created in the belief that a central source of analytical-grade
reference compounds would greatly assist in the assurance of accurate,
reliable analytical data.
In addition to meeting its primary responsibility to its program
laboratories, the ETD has extended this service to other nonprofit government
and university laboratories on a discretionary basis as time and resources
permit. Because of great demand from many sources, and limited supplies, the
amount of each standard sent out is restricted to no more than 100 milligrams
and the number of standards to only those necessary for limited immediate
needs. The short shelf life of many standards is one of the reasons for
restricting field pesticide inventories.
Most of the high-purity analytical-standard compounds carried in the
repository stock are difficult and expensive to prepare, and are therefore
in short supply. The reader is referred to Section 3,B of this manual for
suggested guidelines for the efficient preparation of reference standard
solutions.
The repository stock is reviewed biennially and compounds for which
there has been no demand or those which are no longer commercially produced
are removed from the stock and replaced by pesticidal compounds more recently
introduced to the marketplace. At the time of the stock overhaul a printed
index entitled ANLYTICAL REFERENCE STANDARDS AND SUPPLEMENTAL DATA FOR
PESTICIDES AND OTHER SELECTED ORGANIC COMPOUNDS is also updated, reflecting
the stock overhaul. This booklet provides a complete list of all the com-
pounds in stock along with some supplemental data such as chemical name
structure, molecular weight, use, toxicity and an innovation introduced in
the 1976 issue, literature references to residue analytical methodology
for each compound if any could be located.
In preparing requests for standards, the requester is asked to list
by code number and common name each compound needed. This assists
repository personnel in processing requests, particularly those that are
lengthy.
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Revised 12/2/75 Appendix VII
Page 2
A final note is directed to all scientists associated with university
laboratories. Requests for standards must be made on stationery bearing
the letterhead of the institution and must be signed by a university official
such as a department head. Pesticides will not be mailed to individuals
submitting requests on personal stationery.
A special word of gratitude and apprecition is extended to pesticide
manufacturers for their wholehearted cooperation in providing the repository
program with analytical-grade standard materials at no cost to the program.
All requests for the current catalog and for standards should be directed
to:
Quality Assurance Section, Analytical Chemistry Branch
EPA, Environmental Toxicology Divison (MD-69)
Health Effects Research Laboratory
Research Triangle Park, North Carolina 27711
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Revised 12/15/79 Appendix VIII
Page 1
REVISIONS TO THE MANUAL
This manual is revised biennially and all persons on the mailing list
will automatically receive copies of the revisions. The question then for
each manual holder is whether his name is in fact on the list. Consider the
following points:
1. If you received this Appendix section as part of a group of
revisions, you are definitely on the list.
2. If you received this Appendix section as part of an entire manual
you requested by mail or phone, you are definitely on the list.
3. If you received this Appendix section as a handout at some training
course, and your name and affiliation were not recorded, you are
probabaly not on the list and, therefore, will not automatically
receive revisions.
4. If you obtained your copy of the Manual from some individual not
associated with the Laboratory at Research Triangle Park, NC, you
are probably not on the list and therefore will not automatically
receive revisions.
If after, reading the foregoing, there is some doubt that you may not
be on the mailing list, please clip off the section below, complete it in
full and mail it as shown.
Date
Quality Assurance Section, Analytical Chemistry Branch
Environmental Toxicology Division
EPA, HERL (MD-69)
Research Triangle Park, NC 27711
This is to request that your record be reviewed to be certain the under-
signed is on your mailing list to recieve copies of all future analytical
manual revisions.
(Print or type name and full business address below)
-------
Revised 12/79 Index
A
Agricultural media, sample containers for, 2, p. 2
Agricultural samples, storage of, 2, p. 3
Air
chlorinated, phosphate, and carbamate pesticides and PCB
determination in, 8B, p. 1-22
Kepone determination in, 5A5a, p.7-8
sampling of, 8A, p. 1-29
Air analysis
determination of carbamate pesticides, 8B, p. 5-7
determination of chlorinated pesticides and PCBs, 8B, p.4-5
determination of OP pesticides, 8B, p. 5
extraction of sampling module for, 8B, p. 3-4
GLC in, 8B, p.8-10
recovery data in, 8B, p.10-11
Air sampler
device for calibration of, 8A, Fig. 4, p.22
for ambient air, 8A, p. 2-9; Fig. 1, p. 19
for source sampling, 8A, p. 9-14
for workplace air - personnel monitoring, 8A, p. 14-16
Air sampling
collection efficiencies of PCBs, 8B, Tables 6 and 7, p. 18
collection efficiencies of pesticides, 8B, Tables 1-5, p. 13-17
Alkaline treatment, micro method for pesticide confirmation and cleanup,
12D1, p. 1-7
Alky! phosphates
alkylation of, 6A2a, p.9
analytical quality control, 6A2a, p.15-16
cleanup for derivatives, 6A2a, p.9-10
determination of, in tissues, 6A2a, p.1-19
extraction of, 6A2a, p.7-9
GLC of, 6A2a, p. 10-12
sampling of, 6A2a, p. 7
standards, 6A2a, p. 5-7
Alumina cleanup column, 5B, Fig. 2, p. 12
Alumina for TLC, 12B, p.4-6
Analytical quality control, App. IIA, p. 1-2
alkyl phosphates, 6A2a, p. 15-16
herbicides, 10B, p. 5-6
Kepone, 5A5a, p. 17
PCBs, 9B1, p.12-13; 9B2, p. 7
water analysis, 10A, p.14
Aroclors, see PCBs
Atrazine, gas chromatograms of, with Hall detector in N and Cl modes,
4C2, Figs. 1 and 2, p.4
Autoprep 1001, schematic diagram of, 5B, Fig. 1, p. 11
Autoprep GPC system, 5A5a, p. 16
-------
Revised 12/79 Index
B
Background signal profile, of electron capture detector, determination
of, 4A3, p.1-2
Baseline construction, for gas chromatograms, 4A6, Fig. 10, p. 16
Beef liver, TCPD determination in, 9G, p.1-23
Bis (p_-chlorophenyl) acetic acid, see DDA
Blood
chlorinated pesticides, determination in, 5A3a, p.1-8
cholinesterase activity determination in, 6A3a, p.1-10
Kepone determination in, 5A5a, p.4-6
OP pesticide metabolite determination in, 6A2a, p.1-20
pentachlorophenol determination in, 5A3b, p.1-6
sample containers for, 2, p.1-2
storage of samples, 2, p.2-3
Boron trifluoride, preparation of, 5A4b, p.3
a-Bromo-2,3,4,5,6-pentafluorotoluene, carbamates derivatization with,
8B, p. 12 Table 8, p.19
Bubble flow meter, 4A6, Fig. 4a, p.11
Carbamates
derivatization of, 8B, p.5-6; 10A, p.11-12
detection in soil, 11C, p.l
determination of, as PFB ether derivatives 8B, p.8-9
determination of, by derivatization with a-bromo-2,3,4,5,6-pentafluoro-
toluene, 8B, p. 12; Table 8, p. 19
retention data and gas chromatograms, on Carbowax 20M-modified supports,
detected with the Hall detector, 4C5, p. 1-5
retention times, on Ultra Bond columns, 4C5, p.2
Carbaryl
determination of, by polarography, 12F, p. 6-7
determination of exposure to, 7A, p.1-8
Carbowax 20M columns, see support bonded Carbowax 20M columns
Carbowax 20M modified supports, retention data and gas chromatograms of
carbamate pesticides on, with detection by the Hall detector, 4C5, p.1-5
Carbowax treatment of GLC columns, apparatus for, 4B5, Figs. 1 and 2, p.5
Chlordecone, see Kepone
Chlorinated anilines, separation of, on 2% OV-101 on Chromosorb W support
bonded Carbowax 20M GLC column, 4A7, Fig. 4, p. 9
Chlorinated pesticides
cleanup for, 5Ala, p.6-8; 5A2a and b, p.3-4
confirmation and determination of, in human tissue and milk, 12A, p. 1-8
determination of,
in human and animal tissue, 5Ala, p. 1-19
in human blood or serum, 5A3a, p. 1-8
in sediment, 11B, p. 1-6
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Revised 12/79 Index
in soil and house dust, 11A, p.1-8
in tissue and milk, micro method, 5A2a and b, p.1-6
extraction of, 5Ala, p.5-6; 5A2a and b, p.2-3; 5A3a, p.3
gas chromatogram of, with Hall detector, 4C2, Fig. 4, p.5
gas chromatograms of, on various stationary phases, 4A6, Figs. l-3d, p.10-11
GLC of, 5Ala, p.8-9
PCBs separation from, 9C, p.1-7
sampling of, 5Ala, p.4
Chlorinated phenol metabolites of PCP and HCB, determination of, in urine,
5A4a, p.1-16
Cholinesterase activity, determination of, in blood, 6A3a, p.1-10
Cholinesterase assay, 6A3a, p.5-10
Cleaning of laboratory glassware, 3A, p.1-6
Cleanup
by gel permeation chromatography, 5B, p. 1-14
by micro scale alkali treatment, 12D1, p. 1-6
Coated GLC packings, procedure for preparation of, with HI-EFF Fluidizer,
4A7, p. 11-14
Collection, preservation, and storage of samples, 2, p. 1-3
Column, U-shaped, for gas chromatography, 4A6, Fig. 11, p. 17
Column packings, sources of, 4A2, p.9
Column to port assembly, for gas chromatograph, 4A6, Fig. 4, p. 12
Columns
conditioning of
for electron capture GLC, 4A2, p.2-3;4A6, Table 1, p.3
for flame photometric GLC, 4B2, p.1-2
evaluation of
for electron capture GLC, 4A2, p.3-7
for flame photometric GLC, 4B2, p.3-4
for electron capture GLC, 4A2, p.1-10
for flame photometric GLC, 4B2, p.1-5
for GLC, operation parameters and performance expectations, 4A6, Table 1,
p. 3
maintenance of
for electron capture GLC, 4A2, p.8-9
for flame photometric GLC, 4B2, p. 5
packing of, for GLC, 4A2, p.1-2
selection of
for electron capture GLC, 4A2, p. 1
for flame photometric GLC, 4B2, p.1
Concentrator, 10A, Fig. 1, p. 23
Conditioning, of GLC columns, 4A2, p.2-3;4A6, Table 1, p.3;4B2, p. 1-2
Confirmation
by IR spectroscopy, 12E, p. 1-9
by micro scale alkali treatment, 12D1, p. 1-6
by polarography, 12F, p. 1-7
by thin layer chromatography, 12B, p. 1-15
of HCB in adipose tissue, 5Alb, p. 4-5
Confirmatory procedures, general comments, 12, p. 1
-------
Revised 12/79 Index
Copper, for removal of sulfur interference, 11B, p. 5-6
2,4-D
cleanup for, 5A4c, p. 5-6
determination of, in urine, 5A4c, p. 1-7
ethylation of, 5A4c, p. 3-5
extraction of, 5A4c, p. 4-5
6LC of, 5A4c, p. 6-7
DDA
cleanup for, 5A4b, p. 7-8
determination of, in urine, 5A4b, p. 1-8
extraction of, 5A4b, p. 5-6
GLC of, 5A4b, p.8
methylation of, 5A4b, p. 3-7
sampling of, 5A4b, p. 4-5
DDE, oxidation of, in PCB analysis, 9D, p. 5-6
DDT, dehydrochlorination of» in PCB analysis, 9D, p. 4-5
Degradation of pesticides
in standard solutions, 3B, p. 2-3
in water, 10A, Table 1, p. 17
Dehydrochlori nati on
by micro scale alkali treatment, 12D1, p. 1-6
of DDT and ODD in PCB analysis, 9D, p. 4-6
Detectability limits, for chlorinated pesticides in tissue, 3E, p. 1
Detection, of OC1 and OP pesticides on thin layers, 12B, p. 7-8
Development, of thin layer plates, 12B, p.7
Diazinon, determination of, by polarography, 12F, p.4
Diazomethane, preparation of, 5A3b, p.2-3; 5A4a, p. 2-3
Diazopentane, preparation of, 6A2a, p.3-4
1 ,l-Di'chloro-2,2-bis (£-chlorophenyl) ethylene, see DDE
2,4-Dichlorophenoxyacetic acid, see 2,4-D
Dimethoate, determination of, by polarography, 12F, p. 6
Dinitrophenyl ethers, gas chromatogram of carbamate derivatives, 10A,
Fig. 2, p.24
Disulfoton, chromatogram on Carbowax 20M modified Chromosorb GLC column
coated with OV-210, 4A7, Fig. 6, p. 10
DuPont constant flow sampling pump, 8A, Fig. 8, p. 26
DuPont personal sampling pump, 8A, Fig. 11, p.29
Efficiency, of GLC columns, calculation of, 4A2, p.6
Electrolytic conductivity detector, 5A5a, p.15-16; 9B1, p.
Electrometer, gas chromatograph, 4A1, p.1-2
-------
Revised 12/79 Index
Electron capture detector, 4A3, p.1-3
background signal profile, 4A3, p. 1-2
linearity of, 4A3, p.3
linearized 63Ni (Tracor), 4A3, p. 6
optimum response voltage, 4A3, p. 2-3
standing current profile, 4A6, Fig. 5, p. 13
voltage-response curve, 4A6, Fig. 6, p. 14
Electron capture GLC
instrument for, 4A1, p.1-3
test for interfering substances, 3C, p. 1
Environmental media, sample containers for, 2, p. 1-2
Environmental samples
determination of Kepone in, 5A5a, p. 1-18
storage of, 2, p. 2-3
EPA high-volume air sampler, 8A, Fig. 3, p. 21
EPN, determination of exposure to, 6A2b, p. 1-4
ERGO high volume air sampler, 8A, Fig. 7, p. 25
Evaporative concentrator tube holder, photo of, 5A3a, Fig. 2, p. 8
Fish
Kepone determination in, 5A5a, p. 8-11
TCDD determination in, 9G, p. 1-23
Flame photometric detector, 4B3, p. 3
linearity, 4B3, p. 3
operating parameters, 4B3, p. 1
optimum response voltage, 4B3, p.1
phosphorus and sulfur modes, 4B3, p. 3
Flame photometric GLC, instrument for, 4B1, p. 1
Florisil
effect of varying polarity on eluting solvent, 3D, p. 6
elution pattern and recovery data from, 5Ala, Table 1, p. 11-19
elution pattern from micro column, 5A2a and b, Table 1, p. 6
evaluation of quality, 3D, p. 1-7
fraction of 17 compounds, 3D, Table 1, p. 7
sampling of, 3D, p. 1-2
storage of, 3D, p. 5
Flow system, gas chromatograph, 4A1, p. 1
l-Fluoro-2,4-dinitrobenzene, derivatization of carbamates with,
10A, p. 10-11
Gas chromatograms
baseline construction, 4A6, Fig. 10, p. 16
of chlorinated pesticides on various stationary phases, 4A6, Figs. l-3d,
P- 10-11
-------
Revised 12/79 Index
of PCBs and OC1 pesticides, 9E, p.1-4
peak area measurement, 4A6, Figs. 7-9, p. 15
Gas chromatograph, column to port assembly, 4A6, Fig. 4, p. 12
Gas chromatography
combined with IR spectroscopy for confirmation, 12E, p.5
U-shaped column, 4A6, Fig. 11, p. 17
Gas chromatography-electron capture
chromatography of sample, 4A4, p.1-4
columns for, 4A2, p. 1-10
instrument, 4A1, p.1-3
quantisation and interpretation, 4A5, p.1-2
Gas chromatography-f1ame photometric
columns for, 4B2, p.1-5
sample quantisation and interpretation, 4B4, p.1-2
Gas chromatography-Hall electrolytic conductivity detector
columns for, 4C2, p. 1-6
detector operation, maintenance, and troubleshooting, 4C3, p.1-12
instrument for, 4C1, p.1-3
sample quantisation and interpretation, 4C4, p.1-2
Gas chromatography-nitrogen-phosphorus detector, 4D, p.1-6
Gel permeation chromatography
cleanup of extracts by, 5B, p.1-14
elution patterns and recovery data for pesticides, 5B, p. 4-8
H
Hall electrolytic conductivity detector, 4C3, p.1-12
block diagram of, 4C3, Fig. 1, p. 9
columns for, 4C2, p.1-6
confirmation and determination of chlorinated pesticides in tissue
and milk with, 12A, p.1-8
instrument for, 4C1, p. 1-3
linearity, Cl mode, 4C3, Fig. 4, p. 11
micro cell assembly, diagram of, 4C3, Fig. 2, p.9
peak shapes obtained with, 4C3, Fig. 5, p. 12
retention data and gas chromatograms of carbamate pesticides on Carbowax
20M-modified supports with, 4C5, p.1-5
sample quantisation and interpretation, 4C4, p. 1-2
selectivity compared with FPD in S-mode, 4C4, Fig. 2, p.2
selectivity comparison with N-P detector, 4C4, Fig. 1, p.2
selectivity of, 4C3, Fig. 3, p.10
HCB, determination and confirmation of, in adipose tissue, 5Alb, p.1-11
Herbicides, gas chromatogram of triazines, with Hall detector, 4C2,
Fig. 5, p.6
Hexachlorobenzene, see HCB
HI-EFF Fluidizer, preparation of coated GLC packings with, 4A7, p.11-14
High performance liquid chromatography, 13, p. 1
House dust, chlorinated pesticides determination in, 11A, p. 1-8
-------
Revised 12/79 Index
Hydrolysis products of OP pesticide metabolites, determination of,
6A2a, p.1-19
I
Infrared spectroscopy, 12E, p. 1-9
sample preparation for, 12E, p. 4-8
Instrument maintenance and repair, general comments, App. I, p. 1-3
K
KBr pellets, preparation for IR spectroscopy, 12E, p. 7-8
Depone
analytical quality control, 5A5a, p.17
confirmation-derivatization of, 5A5a, p.14-15
determination of, in blood and environmental samples, 5A5a, p. 1-18
GLC of, 5A5a, p.11-12
recovery and response of, 5A5a, p.12-14
sampling of, 5A5a, p. 2
Leptophos, determination of metabolites in tissue, 6A2a, p.1-19
Linearity
of electron capture detector, 4A3, p.3
of flame photometric detector, 4B3, p. 3
M
Maintenance, of GLC columns, 4A2, p. 8-9
Maintenance and repair of instruments, App. I, p.1-3
Malathion, determination of, by polarography, 12F, p. 5
Mass spectrometry, of Kepone, 5A5a, p. 17
Metabolites, of OP pesticides, determination of, 6A2a, p. 1-19
Methyl parathion, determination of exposure to, 6A2b, p. 1-4
Micro method, analysis of tissue and milk by, 5A2a and b, p. 1-6
Milk
chlorinated pesticides confirmation and determination in, 12A, p.1-8
chlorinated pesticides determination in, micro method, 5A2a and b,
P. 1-6
chromatogram of PCBs in, before and after perchlorination, 9B2, Figs. 1
and 2, p. g_io
PCBs determination in, macro method, 9B1, p. 1-13
PCBs determination in, micro method, 9B2, p. 1-10
TCDD determination in, 9G, p. 1-23
-------
Revised 12/79 Index
Mirex, determination of, in adipose tissue, 5Alb, p. 1-11
MOG cleanup, application to serum, 5A3a, p. 6-7
Monocrotophos, chromatogram on Carbowax 20M modified Chromosorb W GLC
column, 4A7, Fig. 5, p. 10
MSA personal sampling pump and calibration unit, 8A, Figs. 9 and 10, p.27-28
N
1-Naphthol
cleanup for, 7A, p. 6-7
derivatization of, 7A, p.5
determination of, in urine, 7A, p.1-8
extraction of, 7A, p.5
GLC of 1 naphthyl chloroacetate derivative, 7A, p.7-8
hydrolysis of, 7A, p. 5
£-Nitrobenzyl pyridine reagent, for TLC detection of OP pesticides,
12B, p. 7-8
Nitrogen-phosphorus detector, 4D, p.1-6
chromatograms of pesticides with, 40, Fig. 2, p.5; and Fig. 4, p. 6
description of, 4D, p. 2
diagram of, 4D, Fig. 1, p. 5
GLC columns for, 4D, p.4
linearity of, for malathion, 40, Fig. 3, p.6
mechanism of selectivity, 4D, p. 3
response characteristics, 4D, p. 3-4
selectivity of, 4D, Fig. 5, p. 6
]D-Nitrophenol, see PNP
0
Olefins, from bis(phenyl) chloroethane pesticides by alkali treatment,
12D1, p.1-6
Operation parameters of GLC columns, 4A6, Table 1, p.3
Organophosphorus pesticides
determination intact, in tissue and blood, 6A1, p. 2
determination of exposure to, in tussue, 6A2a, p. 1-20
exposure to, 6A1, p. 1-3
gas chromatograms on SE-30/QF-1 column, 4B5, Fig. 3, p.6
"0"-Rings, for electron capture GLC columns, 4A1, p. 3
Parathion
determination of, by polarography, 12F, p. 4
determination of exposure to, 6A2b, p. 1-4
-------
Revised 12/79 Index
PCBs
analytical quality control, 9B1, p.12-13; 9B2, p.7
cleanup for, 9B1, p.5-7; 9B2, p.2-3; 9D, p.4
determination of
by macro method, in human milk, 9B1, p.1-13
by micro methods, in human milk 9B2, p.1-10
general comments, 9A, p.1-2
in adipose tissue, 9D, p. 1-7
in air, 8B, p.1-22
extraction of, 9B1, p.4-5; 9B2,p. 2; 9D, p.4
gas chromatograms of, 9E, p.1-4
in milk, before and after perch!orination, 9B2, Figs. 1 and 2, p.9-10
GLC of, 9B1, p.8-9; 9B2, p. 3; 9C, p.6
GLC retention and response values, 9F, p.1-4
perch!orination of, 9B2, p.5-10; 12D2, p.l
sampling of, 9B1, p.2
separation from OC1 pesticides on silicic acid, 9B1, p.7-8; 9B2, p. 3-4;
9C, p.1-7
thin layer chromatography of, 9D, p.6
PCP, see pentachlorophenol
Peak area measurement, 4A6, Figs. 7-9, p.15
Peak identification, in gas chromatography, 4A4, p.2-3; 4A5, p.1-2;
4B4, p. 1-2
Pentachlorophenol
acid alumina column chromatographic cleanup of, 5A4a, p.6-7
analytical quality control, 5A4a, p.14
confirmation of, by GLC MS and p-values, 5A4a, p.12-13
detection and recovery data from urine, 5A4a, p.9
determination of, in blood, 5A3b, p. 1-6
determination of, in urine, 5A4a, p. 1-16
extraction of, 5A3b, p.3-4; 5A4a, p.5
GLC of, 5A4a, p. 7-8
methylation of, 5A3b, p.2-4; 5A4a, p.6
retention times of methyl ester, 5A3b, p.5; 5A4a, Table 1, p.8
sampling of, 5A4a, p.4-5
Pentafluoropropionic anhydride, derivatization of carbamates with,
8B, p. 8-9
Perchlorination, PCBs derivatization by, 9B2, p.5-10;12D2, p.l
Performance expectations, of GLC columns, 4A6, Table 1, p.3
Pesticide reference standards, source of, App. VII, p. 1-2
Pesticide repository, App. VII, p. 1
Pesticide residues, polarographic determination of, 12F, p.1-7
Pesticides
determination of
in air, 8B, p. 1-22
in water, 10A, p.1-26
PFB ether derivatives, for GLC determination of carbamates, 8B, p.8-9
-------
Revised 12/79 Index
Phosphonate metabolites
GLC of, 6A2a, p. 9-12
of leptophos, determination of, in tissues, 6A2a, p. 1-19
Phosvel, see leptophos
Pipet washer, diagrams of, 3A, p. 5-6
Pipets, washing of, 3A, p. 4-6
PNP
cleanup for, 6A2b, p. 3
determination of, in urine, 6A2b, p.1-4
extraction of, 6A2b, p. 3
GLC of, 6A2b, p. 4
hydrolysis of, 6A2b, p. 3
Polarography, 12F, p. 1-7
Polychlorinated biphenyls, see PCBs
Preparation, storage, and use of pesticide standards for GLC, 3B, p. 1-12
Priming, of GLC columns, 4A4, p. 1
Purity tests for solvents and reagents, 3C, p.1-2
p-Values
for PCP confirmation, 5A4a, p. 13
for pesticide confirmation, 12C, p. 1-7
Pyrometer, gas chromatograph, 4A1, p. 2
Q
Quantification, of GLC peaks, 4A4, p. 2-3; 4A5, p. 1
RBC hemolysate, preparation of, 6A3a, p. 5
Reagents, purity tests for, 3C, p. 1-2
Recovery
of PCP from blood plasma, Table 1, 5A3b, p. 6
of pesticides from water, 10A, Table 1, p. 17
Repair, of instruments, App. I, p. 1-3
Retention times
of carbamate pesticides, on Ultra-Bond columns, 4C5, p. 2
of compounds in GLC, calculation of, 4A2, p. 5-6
of PCBs, 9F, p. 1-4
on Carbowax 20M modified supports coated with OV-210, 4A7, Table 1, p.6-7
on DC-200 column, 4A6, Table 2d, p. 7
on DC-200/QF-1 column, 4A(6), Table 2e, p. 8
on OV-17/OV-210 column, 4A(6), Table 2f, p.9; 4B5, Table 4, p.4
on OV-17/QF-1 column, 4A6, Table 2a, p. 4
on OV-210 column, 4A6, Table 2c, p. 6; 4B5, Table 3, p. 3
on SE-30/OV-210 column, 4B5, Table 2, p. 2; 4A6, Table 2b, p.5
on SE-30/QF-1 column, 4B5, Table 1, p. 1
on uncoated Carbowax 20M-modified supports, 4A7, Table 2, p. 7
-------
Revised 12/79 Index
Revisions, for this Manual, availability of, App. VIII, p. 1
Rp values, of pesticides on alumina thin layers, 12B, p.10-11
Roto-Rack mixer, photo of, 5A3a, p.8
Sample containers, 2, p. 1-3
Sampling
of air, 8A, p. 1-29
of water, 10A, p. 2-5
Sediment
chlorinated pesticides, determination in, 11B, p.1-6
extraction of, 11B, p. 2-4
Kepone determination in, 5A5a, p.7-8
TCDD determination in, 96, p. 1-23
Sediment analysis, sample preparation for, 11B, p.2-4
Selection of method, for tissue and excreta analysis, App. VI, p.l
Septums, for electron capture GLC, 4A1, p. 2-3
Serum, chlorinated pesticides determination in, 5A3a, p. 1-8
Signal to noise ratio, calculation of, for FPD, 4B5, Fig. 4, p.7
Silica gel, fractionation of OC1, OP, and carbamate pesticides on, 10A,
p. 9-11
Silicic acid, separation of PCBs and OC1 pesticides on, 9B1, p. 7-8;
9B2, p. 3-4; 9C, p. 1-7
Silver nitrate reagent, for TLC detection of OC1 and OP pesticides, 12B,
p. 7
Silylation, of GLC column, 4A2, p. 2-3
Simazine, gas chromatogram of, with Hall detector, 4C2, Fig. 3, p. 5
Sludge, Kepone determination in, 5A5a, p. 7-8
Soil
chlorinated pesticides, determination in, 11 A, p. 1-8
detection of carbamate pesticides in, 11C, p. 1
Kepone determination in, 5A5a, p. 7-8
Soil and dust, extraction of, 11A, p. 4-6
Soil and dust analysis, sample preparation for, 11 A, p. 4
Soil, sediment, and dust analysis
cleanup for, 11A, p. 6-8
GLC in, 11A, p. 8
Solvents, purity tests for, 3C, p. 1-2
Spotting, of thin layer plates, 12B, p. 7
Standards for GLC
concentrations of intermediate standards for EC, 3B, Table 1, p. 9
dilution values for concentrated solutions, 3B, Table 2, p. 10-11
mixtures for quantisation of chlorinated pesticides in tissues by EC GLC,
3B, Table 3, p. 12
preparation, storage, and use, 3B, p. 1-12
Standing current profile, electron capture detector, 4A6, Fig. 5, p. 13
Stationary phases, for GLC of pesticides, 4A6, p. 1
-------
Revised 12/79
Index
Storage of samples, 2, p.2-3
Sulfur-containing insecticides, gas chromatogram of, with Hall detector,
4C2, Fig. 6, p.6
Sulfur interference, elimination of, in sediment analysis, 11B, p. 5-6
Support bonded Carbowax 20M columns, 4A7, p. 1-14
applications, gas chromatograms, and data, 4A7, p.4-10
preparation of supports, 4A7, p.2-3; 4A7, Fig. 1, p.8
SURC air sampler, 8A, Fig. 2, p. 20
2,4,5-T
cleanup for, 5A4c, p. 5-6
determination of, in urine,
ethylation of, 5A4c, p. 3-5
extraction of, 5A4c, p. 4-5
GLC of, 5A4c, p.6-7
TCDD
acid-base cleanup procedure
analytical quality control,
capillary column GLC
determination of, in
9G, p. 1-23
neutral cleanup procedure for, 9G, p. 8-9
Temperature programmer for electron capture gas chromatograph, 4A1, p. 2
2,3,7,8-Tetrachlorodibenzo-p_-dioxin, see TCDD
Thin layer chromatography
chlorinated and OP pesticides, determination by, 12B, p. 1-15
combined with GLC, 12B, p. 9-10
combined with IR spectroscopy for confirmation, 12E, p. 5-7
confirmation by, 12B, p. 1-15
PCBs determination by, 9D, p. 1-7
12B, p. 6
5A4c, p. 1-7
for, 9G, p. 5-7
9G, p. 16-17
HRMS multiple ion selection analysis of, 9G, p.10-11
human milk, beef lever, fish, water, and sediment,
determination
micro method,
in, 12A,
5A2a and
sample preparation for
Tissue
chlorinated pesticides, confirmation and
chlorinated pesticides determination in,
cleanup of adipose, by gel permeation chromatography, 5B, p. 9-10
HCB and mi rex determination in adipose, 5Alb, p. 1-11
human or animal adipose tissue analysis of, 5Ala, p. 1-19
limits of detectability of chlorinated pesticides in, 3E, p. 1
mixtures of standards by quantitation of chlorinated pesticides
EC GLC, 3B, Table 3, p. 12
OP pesticide metabolite determination in
PCPs determination in, 9D, p. 1-7
sample containers for, 2, p. 1-3
selection of method for analysis of, App
storage of samples, 2, p. 2-3
Tissue or blood, intact OP pesticides determination in, 6A1, p. 2
P.
b,
1-8
p.1-6
by
6A2a, p. 1-20
VI, p. 1
-------
Revised 12/79 Index
Titrant solution, for cholinesterase determination, preparation of, 6A3a,
p. 3-4
Triazine herbicides
gas chromatogram with N-P detector, 4D, Fig. 4, p. °
separation of, on Carbowax 20M QIC columns, 4A7, Figs. 2 and 3, p. 8-9
1,1 ,l-Trichloro-2-(p_-chlorophenyl)-2-(£-chlorophenyl) ethane, see DDT
2,4,5-Trichlorophenoxyacetic acid, see 2,4,5-T
U
Ultra Bond column, see Carbowax 20M modified supports and support bonded
Carbowax 20M columns
Urine
2,4-D and 2,4,5-T determination in, 5A4c, p. 1-7
DDA determination in, 5A4b, p. 1-8
1-naphthol determination in, 7A, p.1-8
pentachlorophenol and PCP and HCB chlorinated phenol metabolite
determination in, 5A4a, p. 1-16
PNP determination in, 6A2b, p. 1-4
selection of method for analysis of, App. VI, p.l
V
Voltage-response curve, electron capture detector, 4A6, Fig. 6, p. 14
W
Water
chlorinated, phosphate, and carbamate pesticides determination in,
10A, p. 1-26
Kepone determination in, 5A5a, p. 6-7
sample containers for, 2, p. 2-3
sampling of, IDA, p. 2-5
TCDD determination in, 9G, p. 1-23
Water analysis
analytical quality control, 10A, p. 14
cleanup for, IDA, p. 9-11
6LC in, IDA, p. 12-13
sample extraction for, 10A, p. 7-8
Wick-Stick technique, for IR confirmation, 12E, p. 5,7
App. = appendix
Fig. = figure
p. = page or pages
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/8-80-038
3. RECIPIENT'S ACCESSION'NO.
4. TITLE ANDSUBTITLE
Manual of Analytical Methods For the Analysis of
Pesticide Residues _In_ Human and Environmental Samples
5. REPORT DATE
June 1980
6. PERFORMING ORGANIZATION CODE
Revised Dec. 1979
7. AUTHOR(S)
Dr. Morton Beroza
Dr. Joseph Sherma
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
Association of Official
1111 N. 19th St.
Arlington, VA 22209
Analytical Chemists
1ED 868
11. CONTRACT/GRANT NO.
68-02-2474
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory, Office of Research
and Development, U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
6. ABSTRACT • ; ~~~~
This manual provides the pesticide chemist with methodology useful in determining
human exposure to pesticides and related industrial chemicals. Methods are also pre-
sented for measuring the extent of environmental contamination with these compounds.
This manual has been compiled and produced in an effort to promote general acceptance
and adoption of uniform chemical methodology of utmost reproducibility and accuracy
and to ensure that analytical results can be correlated and directly compared between
laboratories. Methods contained in this manual have generally been developed and/or
evaluated by this laboratory within the Environmental Toxicology Division.
The analytical methodology compiled herein consists of both multiresidue and
specific residue procedures. Included also, are miscellaneous topics treating a
number of important activities such as the cleaning of laboratory glassware, the
preparation of analytical reference standards, and the calibration and maintenance of
the gas chromatograph. Several of the methods have been subjected to collaborative
studies and have thereby been proved to produce acceptable inter!aboratory precision
and accuracy. These methods are designated by stars placed at the left of the title
in the TABLE OF CONTENTS. Other methods presented are thought to be acceptable but
have not been validated by formal interlaboratory collaboration.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COS AT I Field/Group
Quality Control
Chemical Analysis
Chemical Tests
Pesticides
Bioassy
Environmental Samples
07,B
07,6
. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport}
Unclassified
21. NO. OF PAGES
685
20. SECURITY CLASS (This page}
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
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