ANALYSIS OF PESTICIDE RESIDUES IN
HUMAN AND ENVIRONMENTAL SAMPLES
A COMPILATION OF METHODS
SELECTED FOR USE IN
PESTICIDE MONITORING PROGRAMS
PREPARED BY
PESTICIDES & TOXIC SUBSTANCES EFFECTS LABORATORY
NATIONAL ENVIRONMENTAL RESEARCH CENTER
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK.N.C.
EDITED BY
J. F. THOMPSON
EDITORIAL COMMITTEE
M.T. SHAFIK
R.F. MOSEMAN
REVISED IN DECEMBER, 1974
J.B.MANN
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erflata
1. Section 4,A,(6), Table 1 under 5% OV-210 column heading,
operating temp, should be changed from 200°C to 180°C.
2. Section 12,D, (2), subsection VI, 2., page 4, change
10 minutes to 45 minutes.
3. Section 12,D,(2), subsection VI, 4., page 5, under NOTE,
change 0.64 to 0.86.
4. Appendices VII and VIII, address at end of each section
change to read
Quality Assurance Section, Anal. Chem. Branch
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Parle, NC 27711 (MD-69)
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Revised 12/2/74 Page 1
TABLE OF CONTEOTS*
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 - adipose and blood
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) Figures and Tables
*Analytical methods designated by a star have been subjected to
interlaboratory study.
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4.(Cont.) B. Flame Photometric Detection
(1) Description of component modules
(2) Columns
(3) Detector
(4) Quantitation and interpretation
(5) Figures and Tables
5. Chlorinated hydrocarbon pesticides and metabolites
A. In human tissues and excreta
^(1) Adipose tissue - Modification of Mills,
Onley, Gaither method.
(2) Micro method
(a) Liver, Kidney, Bone Marrow, Adrenal, Gonads
(b) Brain and human milk
(3) Blood or Urine
^ (a) Multiple chlorinated pesticides
(b) Pentachlorophenol
(4) Urine or water
(a) Pentachlorophenol
Cb) DDA
(c) 2,4-D and 2,4,5-T
6. Organophosphorus pesticides and metabolites
A. In human tissues and excreta
(1) General comments
(2) Urine
(a) Determination of metabolites or hydrolysis
products of organophosphorus pesticides.
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(b) Paranitrophenol
(3) Blood
(a) Cholinesterase activity
7. Carbamate pesticides and metabolites
A. 1-Naphthol in urine
8. The sampling and analysis of air for pesticides
A. Sampling (withdrawn)
"&B. Analysis by GLC
C. Evaluation for purity of Ethylene Glycol
9. The poly chlorinated biphenyls
A. Introduction
B. General Comments (withdrawn)
C. Separation of PCB's from organochlorine
pesticides
D. TLC method for semi-quantitative estimation
of PCB's in adipose tissue
E. Typical chromatograms of the PCB's
F. Tables of relative retention and response ratios
10. The sampling and analysis of water for pesticides
I. Introduction
II. Sample collection
III. Sample containers and storage
IV - VIII. Analytical procedure
11. The analysis of soils, housedust and sediment
A. Soils and housedust
B. Bottom sediment
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12. Confirmatory procedures
A. Microcoulometry (withdrawn)
B. Thin layer chromatography
C. p-values
D. Derivatization techniques
(1) Microscale alkali treatment for confirmation
and sample cleanup.
(2) Rapid determination and confirmation of HCB
in fatty tissues
E. Infrared spectroscopy
F. Polarography
13. Analysis for Metals and Other Elements
A. Determination of methyl mercury in fish,
blood and urine.
B. Determination of total mercury in water.
APPENDIX
Section Subject
I. Maintenance and repair of instruments.
II. A. Analytical Quality Control
VI. Block diagram of tentative tissue, excreta and method
selection for abnormal pesticide exposure cases.
VTI. Pesticide standards repository.
VIII. Future manual revisions - very important
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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 human population.
One of the primary objectives of the Community Studies and Monitoring
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 environment 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 Pesticides and Toxic Substances Effects
Laboratory 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 environ-
mental media.
The analytical methodology compiled herein consists of bcth 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,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.
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 appropriate section identification and revision date.
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Section 1
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The cooperation of scientists using this manual is solicited in
helping to improve and up-date 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
Pesticides and Toxic Substances Effects Laboratory
EPA, National Environmental Research Center
Research Triangle Park, NC 27711
The mention of specific items of equipment and materials by brand name
or the use of manufacturer's or supplier's names and addresses does not
constitute endorsement of a product or source by the United States
Government.
EDITORIAL COMMITTEE
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Page 1
COLLECTION, PRESERVATION AND STORAGE OF SAMPLES
I. GENERAL COMMENTS:
In the procurement, storage, and transportation of samples intended
for analysis for pesticide residues, the personnel involved should be
aware of some basic considerations to ensure delivery to the analytical
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 purposes,
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 by 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, 2-1/6" high x 1-14" diam., approx.
1 oz., Owens-Illinois mold number AM-6764. Available from many
wholesale glass container distributors. There 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 exceeding
about 25 grams.
*New containers should be cleaned as described in Section 3, A.
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Section 2
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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 shouldibe avoided because of the possibility of contamination
from impurities in the rubber. The same warning applies to cork.
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 inch) 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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Section 2
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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 preparation 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.
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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Section 3, A
Page 1
MISCELLANEOUS INFORMATION
A. CLEANING OF LABORATORY GLASSWARE
In the pesticide laboratory involved in the analysis of samples
containing residues in the parts per billion range, the preparation of
scrupulously clean glassware is mandatory. Failure to do so can lead to a
myriad of problems in the interpretation of the final chromatograms due to
the presence of extraneous peaks resulting 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 contaminating substance, resulting in extraneous chromatographic
peaks that, in extreme cases, may completely overlap and mask out 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
serve to contaminate all other glassware placed therein. Many
instances of widespread laboratory contamination with a given
pesticide are traceable to the glassware washing sink.
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Section 3, A
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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
background 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
Na2S04 to the hexane extract and shake 1 minute.
Transfer extract to a Kuderna-Danish assembly
fitted with a 10 ml evaporative concentrator
tube containing one 3 mm glass bead. Reduce
extract volume to ca 3 ml in a hot water bath.
Cool, rinse down S joint and sides of tube with
hexane, diluting extract to exactly 5 ml.
Stopper tube and shake on Vortex mixer 1 minute.
Chromatograph by GLC, E.C. and evaluate
chromatogram for contaminant peaks.
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Section 3, A
- 3 -
3. No comments required.
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 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 solution. 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
attack 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
importance 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 circumstance 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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Section 3, A
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Pipet Washing
The efficient washing of pipets offers some special problems.
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 occasional exception of the chromic
sulfuric acid soak. If pipets are properly cleaned this step may be
safely eliminated for all pipets except those which are to be used in
a method involving concentration of the extract by evaporation. In this
case, the additional insurance factor of the oxidizing action is recommended
Hand washing performed by soaking in a pan or sink and then rinsing
by holding under running water is highly unsatisfactory, particularly
as applied to transfer or volumetric pipets. One simply cannot be sure
of obtaining a complete rinse of all surfaces inside the bulb. Therefore,
an automatic or semi-automatic washing system is strongly recommended.
A modular system can be assembled to operate semi-automatically at a
nominal cost. Equipment required for the assembly is as follows:
1. Electric timer w/automatic cut-off.
Fisher #6-656.
2. Electric heater, immersion, 66" length, 500 watts.
Fisher #ll-463-5B.
3. Pipet washer, stainless steel.
Fisher #15-350-5.
4. Jars, cylindrical, 6" dia. x 18" height, Corning #6942,
1-1/2 gal. (2 required).
5. Pipet baskets (2) stainless steel, fabricated by local tin shop
per sketch in Fig. 1. These are not available commercially.
One of the bell jars (Item 4) is filled with distilled water in which
is dissolved a very small amount of synthetic detergent to function
primarily as a wetting agent. After acetone rinsing the interior of the
vised pipets, they are placed in a stainless steel basket and immersed in
the weak detergent solution. They will remain here until enough are
collected to run through the complete -wash. The second jar is filled
with a detergent solution. The same detergent used in regular glassware
washing is not recommended in this application. Instead, a powdered
detergent intended for use in automatic dishwashers is recommended.
Many of these are available at wholesale or retail.
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Section 3 J A
!
- 5 -
NOTE: Conduct the same background check as previously
outlined for contaminants in the detergent^
Because the detergent is much more effective in a hot solution, it
is desirable to heat the detergent solution. The heat source is the
500-watt immersion element. A new element can be readily bent in helical
fashion by using the stainless steel pipet basket as a form. After
bending, the element will sit in the jar with the center core of adequate
diameter to accommodate insertion of the basket. Fig. 2 illustrates the
appearance of the assembly.
A dual wire (No. 16 stranded) goes to the electric timejr, which is
activated by rotating the timer arm to the number of minutes that heating
is required. Temperature should be raised to 65°C; this requires ca
50 minutes with the 500-watt element. With the timer arm set at 50
minutes, the timer unit switches off the current at the end of this period
and an alarm buzzer is activated to advise the operator._
The basket of pipets is withdrawn from the hot detergent solution,
allowed to drain about a minute, then transferred to the stainless steel
washer where hot rinse water (tap) is run through the washer at the rate
of ca 3 minutes per discharge for approximately one hour. If piped
distilled water is available, 7 or 8 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
overhead 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.
For the storage of clean pipets up to 10 ml in size, stainless steel
tubes such as Fisher No. 3-465 provide a dust-free and convenient means
of storage.
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1/4/71 Section 3,A.
Page 6
FIGURE 1. PIPET BASKET
Perrine Primate Researcn branch .
P.O. Box 490
Perrine, Florida 33157
Bale handle of 1/81
s.s. rod
Sidewalls may be of 1/8
or 1/4" s.s. mesh or
perforated s.s. sheet.
Solder used to be
95/5
Reinforced bands
of about 22 ga. s.s
¦Bottom of 1/8" mesh
s.s. screen
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1/4/71
Section 3., A
Page 7
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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Section 3, B
Page 1
MISCELLANEOUS INFORMATION
B. PREPARATION, STORAGE, AND USE OF PESTICIDE ANALYTICAL STANDARDS FOR GLC
EQUIPMENT § SOLVENTS:
1. Analytical balance capable of an accuracy of +0.05 mg.
2. Flasks, volumetric, 25, 50, and 100 ml.
3. Spatula, stainless steel.
4. Stirring rods, glass, 100 x 6 mm.
5. Bottles, inverted 1 stopper, 30 ml, Corning 1560.
6. Bottles, prescription, 1/2-oz., 1-oz. and 2-oz., with plastic screw
caps. Available from many wholesale pharmacy supply firms.
7. 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 laboratory 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.
8. Vials, screw cap, 15 x 45 mm, 1 dram, Kimble number 60910.
9. Cap liners, Teflon, size 13, 15, 18, and 22 mm, Arthur H. Thomas
2390-H22, H32, H42, and H62.
10. Primary pesticide standards. Available from the Repository at
Research Triangle Park, NC in 1/2-oz. bottles with Teflon-lined
molded screw caps.
NOTE: The organophosphorus compounds are subject to a wide
variety of oxidations, rearrangements 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 dessicator to
minimize moisture absorption and toxic vapors or cross-
contamination. ALL HANDLING OF THESE STANDARDS SHOULD
BE DONE WITH RUBBER GLOVES. SKIN CONTACT BY HIGH
CUNCENTKATES CAN KILL. Samples of organophosphates and
metabolites should be equilibrated to room temperature
in a dessicator to avoid condensation and the possibility
of long-term hydrolysis.
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Section 3, B
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11. Benzene and isooctane (2,2,4-trimethylpentane) or hexane, pesticide
quality, distilled in glass.
NOTE: (1) A 10 yl injection of each solvent should result
in a chromatogram with zero background when examined
by electron capture GLC with system sensitivity
adjusted to concur with the criteria outlined in
Section 4, A, (4).
(2) Isooctane or hexane are both suitable for
standard dilutions. Isooctane, while more expensive,
offers the advantage of a 100°C boiling point and
is therefore less subject to evaporation from repeated
opening of bottles, particularly of working standard
mixtures.
FORMULATION PROCEDURE
1. Preparation of concentrated stock standard solutions.
Except for concentrates for special purposes, a concentration
of 200 nanograms per microliter is suitable for the common
chlorinated pesticides. Ten milligrams of the primary standard,
corrected to a 100% purity basis, diluted to 50 ml will provide
this concentration.
Either benzene or hexane are suitable as the solvents for most of
the primary standards. 3-BHC, however, dissolves in hexane with
great difficulty, but readily in benzene, with stirring and a slight
application of heat from the hot water bath. Benzene has the slight
disadvantage of solidifying at freezer temperatures, but the expansion
has never proved sufficient to cause bottle breakage in this
laboratory.
The concentrated standards of chlorinated compounds should maintain
uniform strength for a 6-month period at -10° to -15°C. The
organophosphate standards are far less stable than the organochlorines.
It is recommended that the concentrated stock be held no longer than
4 months at -15°C.
NOTE: 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 quantitations
of samples will be similarly incorrect.
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Section 3, B
- 3 -
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 may be pesticide quality
isooctane or hexane. Isooctane, with a boiling point approximately
30° higher than hexane, evaporates less rapidly upon repeated opening
of the bottle, thus reducing the possibility of error from this source.
The intermediate concentration standards of the chlorinated compounds,
if stored in the freezer at -10° to 15°C, should be stable for a
4-month period. Except for the occurrence of repeated opening and
warming for making working standards, these standards could be held
much longer. Considering this factor, however, a safer time limit
is 4 months.
The organophosphorous intermediate standards should be similarly
stored in the freezer. The time limit on these standards should
not exceed 2 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 possibility of 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 stronger concentrates
required to result in given concentrations of the diluted
standards.
The use of standard mixture's of varying concentrations
is a necessity for reliable' quantitation of unknowns.
The degree of peak height variation between sample and
standard ideally should not exceed 10% although variations
up to 251 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 laboratories conducting analysis
on environmental samples may wish to make alterations in
the compound content, but the multi-concentration concept
should be applicable. (Miscellaneous NOTE 5).
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Section 3, B
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.
(A) After the working standard mixtures are made up in
volumetric flasks, they are transferred to prescription
bottles of 1/2-ox. or 1-oz. capacity with Teflon-lined
screw caps. These mixtures should be stored in the
refrigerator at all times when not in actual use. The
organochlorine and the organophosphorous working standards
should be renewed monthly and semi-monthly, respectively.
(B) The working standard mxitures are transferred
from the volumetric flasks in which they are made up
into 1 dram vials. The vials are closed with molded
plastic screw caps, each cap fitted with a No. 13
Teflon liner (See Items 8 and 9 under equipment). The
set of standards needed for immediate use may be con-
veniently fitted in a small block of polystyrene into
which holes have been cut with a rat tail file to a
size that will snugly accomodate the vials. The
remainder of the filled vials will be stored at -10
to -15°C.
Working standards of organochlorine compounds may be
held in the block at room temperature continuously for
one week during use, and then replaced with fresh vials
from the deep freeze. Vials of organophosphorous
standards in the block should be stored in the refrigerator
at all times when not in use and also replaced at the
end-of each week with fresh vials from the deep freeze.
Option (B) offers the dual advantages of (1) less frequent
formulation of working standards, and (2) reduced possibility
of errors arising from solvent evaporation resulting from
repetitive opening of the working standard containers.
III. 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 the 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 chromatographer can now make a number of tentative
peak identifications by calculating relative retention values.
-------
Revised 12/2/74
- 5 -
Section 3, B
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 concentrations that one which he
estimates will produce matching peak heights.
In many cases, it will be found that even the highest concentration
mixture will be insufficient to properly quantitate p,p'-DDE and,
sometimes, p,p'-DDT. In this case, the sample extract should be
quantitatively diluted to a degree which 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
¦^H detector when volumes of 5 to 6 yl are injected.
The range of the ^%i detector is much more restricted, and each
detector must be checked for its linearity performance.
IV. MISCELLANEOUS NOTES:
1. In addition to the diluted working standard mixtures, each laboratory
should maintain a standard of pure p,p'-DDT, made up to 60 pg
per microliter (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'-DDD and/or to DDE). In case a breakdown peak greater than 31
of the p,p'-DDT peak is noted, the glass wool plug at the column
inlet and the Vykor glass injection insert should be changed. 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 your laboratory has occasion to analyze for endrin, a similar check
with pure standard endrin should be made weekly. The concentration
should be ca 100 yg per microliter. 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 in the
general area slightly later than p,p'-DDT, the other and largest
peak, eluting extremely late, around the methoxychlor retention area
on the OV-17/QF-1 column. If this is observed, disconnect column
exit from detector and venting it into the oven, adjust column
parameters to normal operation levels, and make consecutive injections
of *Silyl 8 of 25 yl each, spaced about 1/2 hour apart. Allow
Available in 1-and 25 ml septum cap bottles from Pierce Chemical Company
P. 0. Box 117, Rockford, Illinois 61105.
-------
Revised 12/2/74
Section 3, B
- 6 -
column to purge at least 3 hours, reconnect to detector, and, after
equilibration, repeat pure endrin injection. In most cases, this
should rejuvenate the endrin response. In case it does not, discard
the column.
3. In no case should any attempt be made to scale down standard
concentrations 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 blips against blips. The optimum
range of peak heights for quantitation lies between 20 to 70$
full scale recorder deflection, provided, of course, that the com-
pound 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 overemphasized. One means of ensuring
this is to operate at a relatively high sensitivity. For example, an
attenuation of 10 x 16 on the Tracor MT-220 instrument equipped with
the Model 636800 electrometer will practically ensure this. A
fairly comparable setting on the dual channel, solid state electro-
meter, Model SS, would be 10 x 16.
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 his
attenuator linearity. The use of multi-concentration 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 standard
and sample extract. A 5-yl injection provides a convenient volume.
If alternate volumes are used, they should be restricted to the
range of 4 to 8 microliters, and each operator should make certain
that he can obtain linear response by so injecting.
-------
Revised 12/2/74
- 7 -
Section 3, B
Table 1. Suggested concentrations of the intermediate standards of
some common pesticidal compounds used in electron capture
GLC.
CHLORINATED
ng/yl
ORGANOPHOSPHOROUS
ng/yl
a-BHC
1
Mevinphos
50
6-BHC
2
Phorate
50
Lindane
1
Dimethoate
40
Heptachlor
1
Diazinon
30
Aldrin
1
Methyl Parathion
10
Hept. Epoxide
1
Ethyl Parathion
10
o,p'-DDE
1
Malathion
20
p,p'-DDE
2
Ethion
20
Endosulfan
4
Carbophenothion
10
DDA (Methyl Ester)
a
Azinphos methyl
a
Dieldrin
2
o,p'-DDD
2
Endrin
4
Perthane
a
p,p'-DDD
4
o ,p' -DDT
4
Dilan
10
Methoxychlor
10
Tetradifon
20
Mirex
10
Chlordane
10
Toxaphene
a
a Final working standard made up directly from the 200 ng/yl concentrate.
-------
Page 8
Table. I. Commonly u.',e
-------
Revised 12/2/74
- 9 -
Section 3, B
TABLE 3
SUGGESTED MIXTURES FOR QUANTITATION OF CCMMON CHLORINATED COMPOUNDS IN TISSUES
A SERIES
Concentration
in picograms per
microliter
Compound
*1
A2
A3
Lindane
5
10
20
Aldrin
5
10
20
Dieldrin
10
20
40
o,p1-DDT
15
30
60
p,p1-DDT
15
30
60
B SERIES
B1
B2
B3
B-BHC
15
30
60
Aldrin
5
10
20
Heptachlor Epoxide
10
20
40
p,p'-DDE
10
20
40
p,p'-DDD
15
30
60
C SERIES*
C1
C2
C3
a-BHC
5
10
20
d-BHC
5
10
20
Aldrin
5
10
20
o,p'-DDE
10
20
40
o,p'-DDD
15
30
60
* This series contains only those compounds which are rarely found in
tissues.
-------
1/4/71
Section 3, C
Page 1
MISCELLANEOUS INFORMATION
C. GENERAL PURITY TESTS FOR SOLVENTS AND REAGENTS
In general, the solvents used in pesticide residue analysis 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 slipped by 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 Kudema-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 pi of this concentrate into the gas chrcmatograph and allow
enough time for elution of any peak equalling 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% f.s.d. elute at the retention sites of one
or more of the pesticides of interest, the solvent would create problems
in identification and quantitation and would not be acceptable. The
electrometer attenuation should be that currently in use for sample
analysis, presumably 10 x 16, or 10 x 8 on the E 2 electrometer.
It may be possible to remove the contaminants by distillation through
an all-glass still. However, there is no certainty of this as seme
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 carbophenthion.
-------
Revised 12/2/74
Section 3, C
- 2 -
III. REAGENTS:
A. Acetonitrile - Some lots of reagent grade acetonitrile are
impure and require redistillation. Vapors from impure
(JLCN 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.
C. Sodium Sulfate, Sodium Chloride and Glass Wool - These materials
used in the cleanup procedure, even of reagent quality,
frequently 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 12/2/74
Section 3, D
Page 1
MISCELLANEOUS INFORMATION
D. 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 50-lbs.
The grade designated as "PR" 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 characteristics for the pesti-
cides of interest in the user's laboratory.
If the material is purchased in fiber drums lined "with polyethylene,
the evaluation sample may be drawn from the drum(s) in accordance with
the following quidelines. Immediately following the evaluation, the
material should be transferred from tne drum(s) to glass containers
¦with foil-lined lids to avoid the possibility of contamination of
the Florisil {jy trace quantities of organic contaminants in the
polyethylene drum liner.
II. SAMPLING:
A drum is sampled by taking six plugs from top to bottom with a
36" x 1" grain trier. The plug pattern is approximately as shown
in the following sketch:
The trier plugs from all drums are placed in a single container,
mixed thoroughly, and a quantity transferred to a 500-ml Erlenmeyer
flask. At the same time, three elution columns are prepared as
described in Section 5, A, (1). The flask and the prepared columns
are placed in a 130°C oven and held overnight or longer.
-------
Revised 12/2/74
- 2 -
Section 3, D
NOTE: If the laboratory's normal procedure is to pack the
columns immediately before use, the prepacking of columns
for overnight activation may be voided, but the flask of
Florisil should be held in the 130° oven at least 24 hours
before use.
III. STANDARD MIXTURES:
Prepare the two following standard mixtures with the concentration
shown as picograms per microliter:
Florisil Check No. 1 Florisil Check No. 2
a-BHC 20
Y-BHC 20
Heptachlor 20
Aldrin 20
Dieldrin 40
Endrin 100
o,p'-DDT 50
p ,p1 -DDT 80
Diazinon 300
M. Parathion 250
Malathion 400
Trithion 240
g-BHC 40
p,p'-DDE 60
Hept. Epox. 40
Ronnel 100
E. Parathion 250
IV. FLORISIL ELUTION:
1. Remove the prepacked columns from the oven so they may cool down
before use.
2. Place a beaker or flask under each column and prewet the packing
with 50 ml of pet ether.
NOTE: From this point and throughout the following
elution process, the solvent level should not be
allowed to go below the top of the ^£50^ layer.
3. Read and record the % relative humidity in the room.
4. With 5-ml volumetric pipets, transfer 5 ml each of mixtures 1 and
2 onto separate columns and 5 ml of hexane onto the third column
as a control.
5. Place 150-ml beakers under each column and commence elution "with
100 ml of 6% diethyl ether/pet ether, the elution rate to be 5-ml
per minute. The 100 ml portion of elution solvent is measured
in a graduate and applied to the column when the prewetting
liquid level just reaches the top of the Na-SO, layer. At the
instant the liquid level of the first 100 ml of eluting solvent
just reaches the top of the Na?S04 layer, place a second beaker
under the column and quickly add another 100 ml of eluting solvent.
Allow this second 100 ml of solvent to pass through the column.
-------
Revised 12/2/74
- 3 -
Section 3, D
6. Continue elution with 200 ml of 15$ diethyl ether/pet ether
into a succession of two more beakers identified as 200-300 and
300-400, adding the eluting solvent in 100-ml portions as previ-
ously described.
7. Then continue the elution with 50% diethyl ether/peit ether,
following the same procedure of collecting the two 100-ml
increments which are designated as 400-500 and 500-600 ml.
8. Place the 18 beakers containing the 100-ml eluate increments
on a 35 to 40° water bath and evaporate under a nitrogen stream
to ca 2-5 ml.
9. Add a pinch of anhydrous to each beaker and transfer
extracts to 10 ml grad. concentration tubes, rinsing beakers
with ca 5 ml of hexane delivered by a syringe or 5-ml Mohr pipet.
Evaporate under nitrogen stream to ca 3 ml, remove from bath,
allow to cool and dilute with hexane to exactly 5 ml.
10. Stopper evap. conc. tubes and mix on Vortex mixer 1 minute.
V. GAS CHROMATOGRAPHY:
1. With a column of 1.5% OV-17/1.95% QF-1 installed in the instrument,
prime column as described in Section 4, A, and equilibrate
instrument.
NOTE: Use only the column designated,as the compounds
in the respective mixtures will produce minimal
peak overlaps.
2. Make 5-yl injections of the two original standard mixtures to
obtain peak height data for calculations of recoveries.
3. Make a 5-^1 injection of each of the concentrated eluate incre-
ments. In case of off-scale peaks or peaks of less than 10%
f.s.d., make appropriate attenuation adjustment for both standard
and eluates. The entire series should preferably be run at one
attenuation, however.
VI. CALCULATIONS AND RECORDATION:
1. Measure all peak heights from original standards and eluate
increments.
2. For each compound, and based on peak height measurement, calculate
the percentage of the compound appearing in each 100-ml increment
and in the original standard.
-------
Revised 12/2/74
Section 3, D
- 4 -
Example: Lindane, 0-100 eluate, peak ht. 30 mm.
100-200 eluate, peak ht. 60 mm.
Original standard -98 mm.
Percentages — In 0-100 30 x 100 = 33%
30+60
In 100-200 = 60 x 100 = 67%
30+60
3. Compute the elution recovery by dividing the sum of the combined
eluate peak heights by the peak height of the original standard.
Working from the same data given above, we would have:
30 + 60 x 100 =92% Recovery
98
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 OGP compounds may not
yield high recoveries. For example, trithion may yield no
higher than 40% recovery under certain conditions as outlined
in the MISC. NOTES of Section 5, A, 1.
4. Recordation of results may be made on a form to appear comparable
to Table 2. The decision of acceptance or rejection of each
lot is based on a consideration of the elution pattern and on
the recovery efficiency of the pesticides of interest in the
program.
VII. STORAGE OF FLORISIL:
It is imperative that the Florisil be transferred from the
shipping drums into glass jars as soon as possible after the lot is
evaluated and judged acceptable. The drums are lined with poly-
ethylene which may contribute unwanted contamination over a period
of time.
Glass jars which 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, then rinsed
with distilled water and acetone. After thorough drying of the jars,
the Florisil may be transferred with a 2-lb. aluminum sugar scoop
previously washed and acetone rinsed. The net contents of each jar
when filled to 1/2-inch from the rim with Florisil is ca 2 lbs. A
square of aluminum foil is crimped over the rim of the jar and the
cap is screwed on tight. ' Each jar is labeled with the lot number
and now ready for storage.
-------
Revised 12/2/74
Section 3, D
- 5 -
VIII. NOTES:
1. Factors influencing the recovery efficiency, particularly of
certain organophosphorous compounds, include (1) presence of
impurities in the pet ether, and (2) presence of peroxides in
the ethyl ether. This is discussed in more detail in the MISC.
NOTES of Section 5, A, 1.
2. Polarity of the elution solvents exerts a profound effect on the
selective elution of a number of compounds. The ethyl ether must
contain 2% v/v of ethanol to obtain compound elution patterns
comparable to those shown in Table 1. The following chart
demonstrates the effects resulting from altering the amounts of
ethanol in the ethyl ether.
The effects of polarity variation of eluting solvent in Florisil
partitioning of 7 pesticides. Absolute ethyl ether mixed with
0, 2 ,and 4% absolute ethanol.
Elution Fraction*
Hept. Epoxide
Dieldrin
Endrin
Diazinon
Methyl Poroth ion
Ethyl Pa roth ion
Mo lath ion
No EthanoL
I
n
ni
100
87
13
100
too
100
16
84
'Eluting mixtures:
Proct. I - 6% Et,0 m pet. ether
Froct. II -15% « " " "
Froct III-50% " * " «
2"/e Ethanol
I
n
EI
100
100
100
100
100
100
100
4% Ethanol
I
H
HI
100
7
93
16
84
3
87
2
98
3
97
100
Elution Froct ion'
Hept. Epoxide
Dieldrin
Endrin
Diazinon
Methyl Farothion
Ethyl Pa rath ion
Malothion
3. If at all possible, the oven used for Florisil should be used
only for adsorbents and not for general laboratory use. 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.
-------
Revised 6/1/72
- 6 -
Section 3,D
TABLE 1, ELUTION PATTERNS AND RECOVERY DATA FOR FLORISIL, LOT H 2851i ,
BY METHOD SECTION 5,A,(l)(MANUAL OF ANALYTICAL METHODS)
FLORISIL—COLUMN PREPACKED AND HELD IN 130°C OVEN AT LEAST 2k HRS BEFORE USE
RELATIVE HUMIDITY IN LABORATORY 65 %
ELUTION INCREMENTS (ml)
6% Fraction
15$ Fraction
50% Fraction
Compound
0 - 100
100 - 200
200 - 300
300 - U00
o
0
LA
1
O
o
500 -1 600
Recover?^, %
ct-BHC
100
97
3-BHC
100
95
Lindane
100
96
Heptachlor
100
91
Aldrin
100
100
Hept. Epox.
78 | 22
105
Dieldrin !
85
15
96
Endrin
89
11
99.6
p,p'-DDE
100
97
1 1
o,p'-DDT 11 100
99.6
p, p 1-DDT li 100 :
90
Ronnel j | 100
93
Methyl I j
Parathion !;
1
U7
53
103
Maiathion j
-
I '
>
100
99
Ethyl
Parathion
1
I
!
i
!
.
78
22
96
Diazinon
i
100
,—
83
Trithion
100
U3
Numerical values represent the percentage of each compound eluting in the given cut.
-------
Revised 12/2/74
Section 3, E
Page 1
MISCELLANEOUS INFORMATION
E. 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 the establishment of
such limits so that data from all laboratories would be reported in a
comparable manner.
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
significance, 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:
Cone, in ppb
Compound
Adipose
Serum
a-BHC
10
10
20
10
10
10
20
10
10
20
20
20
20
Lindane
B-BHC
Aldrin
Heptachlor
Heptachlor Epoxide
o,p'-DDE
p,p'-DDE
Dieldrin
Endrin
2
2
2
2
o,p'-DDT
p ,p'-DDD
p,p'-DDT
-------
Revised 12/2/74
Section 4, A, (1)
Page 1
GAS CHRCMATOGRAPHY-ELECTRON CAPTURE
INSTRUMENT
In this section, operating instructions of a specific nature are
intended to apply to the model MT-220 gas chromatograph manufactured
by Tracor, Inc., Austin, TX. This instrument may be equipped with a
detector of Nickel 63 or 130 mc tritium. 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 H detector.
The *^Ni detector will be even more seriously affected. Leaks can be
detected by the application of "Snoop" at all connections or by
spraying the connection with Freon MS-180 with the instrument
operating and observing for recorder response. Spray short squirts
close to the connection. Do 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)
- 2 -
Check the "maximum" polarizing voltage available. -If at least
-130 vdc is not available on the rear panel, it is quite possible that
the power printed circuit board (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 oven temperature should
not show appreciable deviation. If the temperature fluctuation is
excessive, baselines will cycle and, in all probability, retention
measurements will be erratic.
In an emergency situation, a 10 amp variable transformer (Variac or
Powerstat) may be used as a temporary measure. Constant use of this
device is not advised as it does not operate on temperature demand, but
simply supplies a fixed voltage to the heating elements. Therefore, oven
temperature will vary with any changes in line voltage and room tempera-
ture.
V. 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 gnused 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 plain black (or gray) silicone rubber to the
sophisticated "sandwich" type selling at a significantly higher
price.
-------
Revised 12/2/74
Section 4, A, (1)
- 3 -
Excellent results have been reported on the blue silicone rubber
material marketed by Applied Science Laboratories as their
series.
The 13 mm precut septums are available in lots of 100 under catalog
number W-13. The same material listed at 'Type W" is available
in sheets of 12" x 12". About 400 13-ml septums can be cut from
this sheet with a No. 9 cork borer making the price per hundred
septums about half that of the precut septums.
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 chromatography
accessaries. 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 D.C. mode of operation.
This is piped to the instrument through a filter drier of molecular
sieve, 1/16" pellets, Linde type 13X. When the filter drier is charged
with fresh molecular sieve, the interior of the drier should be
acetone rinsed and the drier unit placed in a 130°C oven for at
least an hour. The bronze frit should be acetone rinsed 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.
-------
Revised 12/2/74
Section 4, A, (2)
Page 1
GAS Q^RQMATOGRAPHY-ELECTRON CAPTURE
COLUMNS
I. SPECIFICATIONS:
Column material shall be of borosilicate glass, 6 feet long,
1/4 inch o.d., 5/32 inch i.d. As off-cblumn injection will be used,
one side of the column shall be 1 inch longer than the other. The
Swagelok nut, ferrule and silicone "0" ring are assembled as in
Fig. 4. Complete column specifications for the Tracor 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 others which 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 alternate. 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 Micro-Tek Model 220, one column leg
should be 1 inch shorter than the other.
With a china marking pencil, place a mark on the long column
leg 2 inches from the end. Place a similar mark 1-1/8 inch from the
end of the short leg.
Add the packing to the column through a small funnel, ca 6 inches
at a time, and bounce the column repeatedly on a semihard surface.
Rapid tapping up and down the column with a wooden pencil will
promote settling of the packing. The packing is added until it reached
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
-------
Revised 12^2/74
Section 4, A, (2)
- 2 -
further settling is possible by manual vibration. The
use of mechanical vibrators is not advised as the packing
can be packed too densely, thus introducing the possibility
of an excessive pressure drop when carrier gas is applied.
Pack silanized glass wool into both ends of the column just
tightly enough to prevent dislodging when carrier flow is applied.
The glass wool should completely fill the space from the top of the
packing to the end of the column.
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 con-
tamination of the glass wool.
IV. COLUMN CONDITIONING:
The column is conditioned, or made ready for use, in two
operations, (1) by heat curing, and (2) by silylation treatment.
1. Heat Curing
A Swagelok fitting is attached to the inlet port at the top
of the oven. This is comprised of a 1/4-inch Swagelok to AN adapter,
part number 400-A-4ANF, connected to a 1/4-inch male union, part
number 400-6.
Before assembling, the bore of the union must be drilled out
with a 1/4-inch drill and burnished with a rat-tailed file so
that it will accept the 1/4-inch o.d. column glass.
The short column leg is attached to the above fitting, with
the end of the long leg venting inside the oven. The nut,
ferrule and "0" ring is assembled as shown in Fig. 4. Make sure
the nut is tight, as the "0" ring will shrink during the curing
period, thus allowing carrier gas to excape.
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-inch Swagelok nut on a short piece of 6-mm
glass rod with ferrule andM0M ring.
2. Silylating Treatment
Treatment with a silylating compound such as Silyl 8 serves
to block active adsorption sites, particularly prevalent in a
new column, thereby somewhat improving efficiency and resolution
characteristics. The most drastic effect is in the improvement
-------
Revised 12/2/74
Section 4, A, (2)
- 3 -
of endrin response and the near elimination of on-column break-
down 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 in-
jection 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 insure gas-tight septum
installation. Raise oven temperature and carrier gas flow to the
exact values given in Table 1 for the appropriate column. The oven
temperature must be monitored by some means other than the built-in
pyrometer, either with a precalibrated dial face thermometer with the
stem inserted through the oven door, or with a mercury thermometer
pushed down through an unused injection port. DO NOT RELY WHOLLY ON
THE INSTRUMENT PYROMETER.
Check the carrier gas flow rate using the sideaim 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.
-------
Revised 12/2/74
- 4 -
Section 4, A, (2)
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 para-
meters 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 riot reliable with an older,
partially fouled detector. A more reliable method is to run a polariz-
ing voltage/response curve as described in Subsection 4,A, (3) OPTIMUM
RESPONSE VOLTAGE. A polarizing voltage/response curve is shown in
Fig. 6.
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
10
Hept. Epoxide
30
o,p'-DDD
80
0- BHC
40
p,p'-DDE
40
p,p'-DDD
80
Lindane
10
Dieldrin
50
o ,p1-DDT
90
Heptachlor
10
Endrin
80
p,p'-DDT
100
Aldrin
20
-------
Revised 12/2/74
Section 4, A, (2)
- 5 -
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 quanti-
tation. 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. The column efficiency can be determined from computation
based on the p,p'-DDT peak. The equation is given on Page 6.
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.
2. Compute the relative retention value for p,p'-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.
3. Compute the absolute retention in minutes for p,p'-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, 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.
-------
Revised 12/2/74
- 6 -
Section 4, A
Peak A
(Aldrin)
Peak
Injection Point
N = f)1
R^ = (At 1/4-in/min chart speed)
r = (At 1/3-in/min chart speed)
¦ ^2^7 (At 1/2-in/min chart speed)
R
x
"x^ ~ 16.76
(At 2/3-in/min chart speed)
r^ = (At 1-in/min chart speed)
""a'-f
Where N
RXpX2> etc.
rrta
x.y.z
= column efficiency in total theoretical plates.
= absolute retention, in minutes, for peak B.
= retention ratio, relative to aldrin, for peak B.
= measurements in millimeters.
-------
Revised 12/2/74
Section 4, A, (2]
- 7 -
Based on the chromatograms of the evaluation mixture, a decision
generally can be made as to the potential quality of the column. If,
after making slight adjustments in the carrier gas flow rate, character-
istics of efficiency, absolute retention and peak resolution do not com-
pare reasonably well with the chromatograms 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 col-
umn 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% f.s.d. The DDT breakdown should not
exceed 3%. The endrin response and breakdown characteristics may be
determined similarly. This breakdown should not exceed 10%.
NOTE: This breakdown percentage is calculated by adding
up the peak areas of main peak and breakdown peak(s).
This value divided into the peak area value for the
breakdown peak(s) x 100 is the breakdown percentage.
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 signifi-
cantly altered. When a sufficient amount of residue collects in this
insert, lowered efficiency, compound breakdown, peak tailing, and de-
pressed peak height response become evident. The changing of this
-------
Revised 12/2/74
Section 4, A, (2)
- 8 -
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. Indica-
tions from the survey mentioned above were that those laboratories
changing the glass insert daily could go for long periods of time
without changing the column plug. Daily compound conversion monitoring
provides a constant check on the need for changing the glass wool plug.
When the column is idle overnight or over weekends, a low carrier
flow of ca 25 ml per minute through the column is advised. Simul-
taneously, a purge flow of ca 25-30 ml through the detector is also
advised. If a column is out of the instrument longer than 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 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 characteristics
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 labo-
ratory making their 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.
-------
Revised 12/2/74
Section 4, A, (2)
- 9 -
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 4 of this section along with the required
retention values, relative to aldrin, at a given column temper-
ature. This is of particular importance for mixed liquid phase
packing to insure the proper proportion of liquid phase com-
ponents .
(2) A statement of minimum efficiency in terms of the total theo-
retical 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,p'-DDT under prescribed operating
parameters.
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 251 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 pne 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" o.d. glass rod and install in the unused out-
let port.
3. Columns shorter than 6-ft. are generally suitable for chromatography
of specific, late eluting compounds as retention time can be shortened
for greater work output. However, for multiresidue analysis on sam-
ples 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 12/2/74 Section 4, A, (3)
Page 1
GAS CHROMATOGRAPHY-ELECTRON
CAPTURE DETECTOR
Straight D.C. polarizing voltage shall be supplied to the detector
from either an outboard power supply unit or from a strip on the back
of the electrometer. Provided the column and all electronic circuits in
the various modules of the instrument are functioning properly, the degree
of sensitivity in the electron capture mode relate most probably to the
condition of the interior of the detector. As radioactivity in the foil
decreases, so does sensitivity of the system. Measurement of the back-
ground 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 influenced 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 0V-17/QF-1 in the instrument,
set input attenuator on 10 and output attenuator on 256.
NOTE: The given attenuation values apply to electrometer
model E2. If the dual channel, solid 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/min of purge gas.
4. Set OUTPUT POLARITY switch to the polarity opposite of that used in
normal operation.
5. Reduce polarizing voltage to zero by control on power supply unit
or in front of electrometer, and adjust bucking control of electro-
meter to permit zeroing of pen on chart paper.
6. Set chart speed on 1/4 inch per minute, start 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.
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% f.s.d., a replacement
-------
Revised 12/2/74
Section 4, A, (3)
- 2 -
of the foil is indicated. Fig. 5 shows a background signal profile
on a detector in constant use for 2 months. At the time of original
installation, the background signal profile produced 68% f.s.d.
II. OPTIMUM RESPONSE VOLTAGE:
It is important that a correct polarizing voltage be supplied to
the detector to achieve maximum peak response with minimal overshoot.
An incorrect voltage can result in (1) full potential sensitivity of
the detector not being utilized, or (2) a strong overshoot in the peak
downstroke which makes for difficult quantitation of peaks. The
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 opera-
tion 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 pi.
6. Repeat injection to obtain peaks from increments of 2.5 volts,
i.e., 15, 17.5, 20, etc., until two peaks show less height
than that obtained on the highest peak.
NOTE: Occasionally a new detector will require only around
7 volts, and it may be found that the 2.5-volt inter-
vals result in too much change in response. In this
case, it may be advisable to use 1-volt intervals to
set up the response curve.
-------
Revised 12/2/74
Section 4, A, (3)
- 3 -
7. Taking the exact peak height values, measured in millimeters,
plot a peak height vs voltage curve on linear graph paper
(Fig. 6). Usually the optimum polarizing voltage is the next
voltage interval higher than the voltage producing the great-
est 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 Fig. 6 indicates the voltage se-
lected in this particular case.
III. DETECTOR LINEARITY:
In making chromatograhic runs for quantitation, it is mandatory
that compound concentration be within the linearity range of the detec-
tor. 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 is far more restrictive in linearity charac-
teristics than the 3H detector. The linearity curves below illustrate
the comparative linearity of 63Ni and 3H detectors. Linearity curves
should be run frequently, and most importantly on each new detector
or on one subjected to overhaul.
O 0.4 /A /.6 2. J O J. X J *
Llmurltgr nmi for p,p'-DDD uslag and dotactorv
-------
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 quantitation may be attempted. The first
early morning standard injection will frequently show relatively poor
response. The second and third injections will usually improve the
response to a constant level. This "priming" may be done by successive
injections of a dilute working standard mixture or it may be accom-
plished by one injection of a highly concentrated mixture. One labora-
tory 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 concen-
tration values are in nanograms per microliter.
Lindane 0.5
B-BHC 1.5
Aldrin 0.5
Hept. Epox. 1.0
p,p' -DDE 1.0
Dieldrin 1.0
o ,p' -DDT 1.5
p,p'-DDD 1.5
p ,p' -DDT 1.5
Forty microliters of this mixture is injected. If this one-shot system
is used, a special syringe should be set aside and used solely for this
purpose. Under no circumstances should the same syringe be used for
routine injections.
In the early morning the priming may be conducted while the other
daily instrument checks are being made. If more than one column in the
instrument is to be used, the priming may be done simultaneously. After
the priming mixture has eluted off the columns, the carrier flow should
be carefully adjusted for the working column using the bubble device
shown in Fig. 4 (a). The chromatograph should now be ready for the first
working standard injection.
A sample extract concentration of 10 ml from a 5 gram sample contains
the tissue equivalent of 0.5 milligram per microliter. A 5 n1 injection
of this extract (2.5 milligrams of sample) into an E. C. detector of
average sensitivity should easily produce quantifiable peaks at pesticide
concentrations of at least 0.1 ppm, provided that instrumental attenua-
tion 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 than 20 ml/min on this alternate
column or, if preferred, leave carrier flow at zero on a
column of high thermal stability. Set attenuation at an
estimated appropriate sensitivity.
-------
Revised 12/2/74
Section 4, A, (4)
- 2 -
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 concen-
tration 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 in-
jecting eluate from the 151 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 identi-
fication 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
containing aldrin and p,p'-DDT. Other compounds eluting earlier
than p,p'-DDT may be included but their presence may be irrele-
vant for this mission. Calculate from the chromatogram the RRT^
of p,p'-DDT, then by scanning horizontally across the column
opposite p,p'-DDT on the table, locate the RRT^ 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 y1 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 quantitation. The possibilities for error are greatly
enhanced by low volume injections.
-------
Revised 12/2/74
Section 4, A, (4)
- 3 -
4. Calculate the RRTa values for all 'peaks appearing on the
sample chromatogram(s). By vertically scanning the appro-
priate temperature column on the table, the calculated RRT^
values may be compared -with table values to obtain tentative
peak identifications.
5. Hie 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 quantitation. 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% f.s.d. should be quantitated.
6. At this point the task of compound identification is incomplete,
and confirmation must be conducted on an alternate column of
completely different polarity (see Section 4, A, (2"), page 1).
The chromatographer must be constantly aware that artifact
peaks may be obtained with one column which may have identi-
cal RRTa 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 alternate
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)
- 4 -
3. Electrometer attenuation should be adjusted to obtain a
minimum sensitivity level of a peak of 50% f.s.d. resulting
from the injection of 100 picograms of aldrin.
4. Quantitation 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 chromato-
grams. If there is reason to suspect any peak identification
or quantitation, instrumental controls should be switched over
to the alternate column for further scrutiny. The isomers of
BHC, o,p'-DDE, and o,p'-DDT frequently pose identification
problems. If such identification problems are present and
cannot be confidently resolved by any of the three prescribed
columns, further confirmatory work is required by electrolytic
conductivity detection and/or by T.L.C.
-------
Revised 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 p,p'-DDT peak from a clean
extract, (3) Overlapping peaks where the overlap is estimated not to
obscure the peak height, (4) 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
(Fig. 7).
II. PEAK HEIGHT X WIDTH AT HALF HEIGHT:
A. Separated, symmetrical, and fairly wide peaks (Fig. 8).
III. TRIANGULATION OR INTEGRATION:
A. Separated unsymmetrical peaks, or peaks on sloping baseline
(Fig. 9). Triangulation should not be attempted on very
narrow peaks. Extreme care must be taken in the construc-
tion of the inflectional tangents and in measurements.
IV. INTERPRETATION:
Although this subject is listed last in this section devoted
to E.C., Gl£, it is far from being the least important. An excel-
lant performance in all other areas may be nullified if the chroma-
tograms are not properly interpreted.
The electron capture detector, being non-specific, responds
to any electron-capturing materials in addition to pesticides in
the final extract being chromatographed. For this reason, the
task of interpretation is one requiring careful study of the data
-------
Revised 12/2/74
Section 4, A, (5)
- 2 -
and the application of sound judgment. The presence of chroma-
tographic peaks which precisely match the absolute and relative
retention values of those of certain pesticides does not neces-
sarily 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 o,p'-DDE. Confirmation by ancillary techniques
has never supported the E. C. indications, 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
E.C. detection, the retentions of which strongly suggest certain
pesticidal compounds. If, based on experience, these particular
compounds are not likely to be present in the sample material,
some further confirmation is required. This may be done by
(1) using an alternate column via E. C. detection, (2) applying
electrolytic conductivity detection, (3) thin-layer chromato-
graphy, or (4) chemical derivatization.
-------
Revised 12/2/74
Section 4, A, (6)
Table 1. Conditioning, Operation Parameters and Performance Expectations for 6-ft. x 1/4-inch o.d. columns
of Precoated Packings, 3% DEGS Included Solely as a Confirmatory Column, not for routine Use.
Parameters
1.5% OV-17
-—lT95ToV-210
4% SE-30
6% OV-210
5% OV-210
3% DEGS
Liquid phase(s)
Silicone OV-17
Silicone DC QF-1
(FS1265)
Silicone SE-30
Silicone DC QF-
(FS1265)
OV-210
Tr i fluorome thylpropy1
Silicone
DEGS
Stabilized
Diethylene
Glycol
Succinate
(Analabs C4)
Solid Support
Chromosorb W, H.P.
or Gas-Chrom Q
100/120 mesh
Chromosorb W, H.P.
or Gas-Chrom Q
80/100 mesh
Chromosorb W, H.P.
or Gas-Chrom Q
100/120 mesh
Gas-Chrom P
80/100 mesh
Heat Curing Temp °C
Time Hours
245
48 (minimum)
245
72 (minimum)
245
48 (minimum)
235
20 (exact)
Operating Temp °C 200 200 200 195
Detector Temp °C (tritium) 205 205 205 205
Carrier Flow ml/min. 50-70 70-90 45-60 70-90
Elution Time for p,p'-DDT
Approx (min) 16-20 16-20 16-20 16-20
Expected Minimum
Efficiency (Total theor. 3000 3000 3000 2800
plates in 6-ft. column
basis p,p'-DDT)
-------
Revised 12/2/74
Table 2(a)
Section 4,A, (6)
1.5%OV-17/1.95% QF-1
Column Temperature, °C.
I
TO 4 Ol fM 1(M 4Ai '
170
I
I
174
I
|
178
I
|
182
|
188
|
|
190
I
I
194
I
I
198
I
|
202
1
204
|
0 32
0 32
0.32
0 32
0 32
0 32
0 32
0.32
0.32
0.32
033
0 33
0.33
0.33
0.33
0.33
033
033
044
0.45
0.45
045
0.45
0.45
0 45
046
046
046
0.48
0.46
0.46
0.47
0.47
0 47
0 47
0.47
2,4-D(ME)
0.48
048
048
0.49
0 49
0.49
050
0 50
060
0.50
0.51
0.61
0.91
0.51
0.62
0 52
0 52
052
Phorote
048
0.48
0 49
049
050
0.50
0.50
0 51
061
0 52
0.62
0 52
0.53
053
044
054
054
055
a-BHC
0 54
054
054
054
0 55
0 55
0 55
055
0.55
0 55
0 55
0 55
056
056
"~0 56
0.56
056
056
CDEC
0 56
056
056
056
0.56
056
056
056
056
056
056
0.56
0.56
058
056
056
056
056
2,4-D(IPE)
0 70
0 70
0 70
0 70
0 69
0 69
0 69
0 69
0 69
068
068
068
068
0 67
0 67
067
0.67
0.67
Simazine
0 69
0.69
06B
068
066
068
068
068
068
068
067
0 67
0.67
0 67
0 67
0.67
0 67
066
Atrazine
067
0.67
066
0.66
0.66
0 66
066
0 65
0 66
0 65
0 65
0 65
0 65
0.64
064
0.64
064
064
Diazinon
066
067
0 67
0.67
0 67
0 67
0 67
068
068
068
068
0 68
068
0 69
0.69
0 69
0 69
0.69
Lindane
0 76
0 76
0 76
0.76
0 75
075
0 75
0 75
0 75
0 74
0 74
0 74
0 74
0 73
0 73
0 73
0.73
0.73
2.4,5T(ME)
0 82
0 82
0 82
0.82
0.81
081
081
081
081
081
081
0 80
0 80
0 80
080
0.60
0 80
0 80
0BHC
0 86
0 85
0 8b
084
0 84
084
0 83
0 83
0 82
0 82
081
081
0 80
0 80
0 79
079
0 78
0 78
2,4-D(BE)l
097
0 97
0 97
096
096
096
0 95
095
094
094
094
0 93
0 93
0 93
0 92
0.92
0 92
091
5-bhc
082
0 82
0 82
0 82
0 82
082
0.82
0 82
0 82
0 82
0 82
0 82
0 82
0 87
0 82
0 82
0 62
0.82
Heptachtor
094
0.94
0 93
0 92
0 92
0.91
0 90
0 90
0 89
0 88
0 88
0 87
087
0 86
0 85
0 85
084
083
2.4.6-TOPE)
1 03
1 02
1 01
1 01
1 00
0 99
0.99
0 98
0 97
096
0 96
095
0 94
0 93
0 93
0.92
091
090
2,4-D(BE)ll
1 02
1 02
1.01
1 01
1 01
1.01
1 01
1 00
1 00
1 00
1 00
1 00
099
0 99
099
099
099
098
Dichlone
1 17
1 16
1 15
1 14
1 13
1 11
1 10
1 09
1 08
1 07
1 06
1 05
1.03
1.02
1 01
1 00
0 99
098
Dimethoate
1 00
1 00
1 00
1 00
1 00
I 00
1 00
1 00
1 00
1 00
1 00
1 00
1 00
1.00
1 00
1 00
1 00
1 OO
Aidrin (REFERENCE)
1 17
1 16
1.16
1 15
1 14
1 14
1 13
1 12
1 12
1 11
1 10
1 09
1 09
1 08
1 07
> 07
1 06
1 05
Ronnel
1 41
1 40
1 39
1.38
1 36
1.35
1 34
1.33
1 32
1 31
1 30
1 29
1 28
1 27
1 26
1 25
1 24
1.23
l-Hydroxychtadena
1 71
1 69
1 67
1 66
1 64
1.62
1 60
1 59
1 57
1 55
1 53
1 52
1 50
1 48
1.47
1.45
1 43
1 41
M. Parathion
1.70
1 69
1.68
1 67
1.66
1 65
1 64
1 63
1 6?
1 6T
1 59
1 58
1.57
1 56
1 55
1 54
1 53
1 52
Heptachtor Epoxide
207
2 04
2.01
1 98
1 95
1 92
1 89
1 87
1 84
1.81
1 78
1 75
1 72
1 69
1 66
1 63
1 60
1 57
Malathton
182
1 80
1 78
1 76
1.74
1 72
1 70
1 68
1 66
« 64
1 62
1 60
1 58
1 50
1 54
1 52
1 50
1.48
D C P A
2.15
2.13
2 11
209
2 07
2.05
2 02
200
1 98
1 96
1 94
1 91
1 89
1.87
1 85
1 83
1.8!
1 79
Dyrene
2.14
2.12
209
2 07
205
2 03
201
1 98
1 96
1.94
1 92
1 90
1 B8
1.86
1 84
1 82
1 79
1 77
o.p'-DOE
2 20
2 18
2 16
2.14
2 12
2 10
2 08
2 06
2 05
2 03
2.01
1 99
1 97
1 95
Y 93
1.91
1 89
1 87
Chlorbontkf*
2 32
2 28
225
2 22
2.19
2 16
2 13
209
2.06
2 03
ZOO
197
1 93
1 90
1 87
1 84
1 81
1 78
E. Parathron
2 20
2 1B
2 16
2 15
2 13
2 11
2.10
2.06
2 06
205
2 03
2 01
2 00
1 98
1 97
1 95
1.93
1 91
Endosulfon X
2 75
2.72
268
264
2 61
2 58
2 54
251
2 47
2 43
2 40
2 37
2 33
2 30
2 27
2 23
2.20
2.17
p.p'-DDE
2 97
2.93
288
2 84
2 79
2 75
2 71
266
2 62
2 57
2 53
2 49
2 44
2 40
235
2 31
2 27
2 22
DDA(ME)
3 27
3 22
3 18
3.13
3 07
304
300
2 95
2 91
2 86
281
2 77
2 73
268
263
2 59
254
2.50
Captan
3 32
3 27
3 23
3 18
3.13
3.06
304
2.99
2 95
2 91
2 87
2.82
277
2 73
268
264
2.59
255
Folpet
280
2.77
2.75
2 72
2 69
2 67
2 64
261
2 69
256
2 53
251
246
2 45
243
2.40
2.37
2.35
Dieldrin
3.52
346
341
3 35
3.30
3 24
3 19
3 14
308
303
29a
2 92
2.87
2 82
276
2v71
2.66
2 60
Perthane
3.34
329
3.25
320
3 15
3 11
306
3 01
2 97
2.92
288
2 83
2 77
2.74
2.69
2 65
2 60
2.56
o.p'-ODD
3 98
394
388
3 83
3.77
3 71
3.66
360
3.54
3.48
3 43
338
3 32
3.27
3.21
3.16
3.10
3.04
o.p'-DDT
3 47
343
340
3 36
3 33
3.29
3.26
3.22
3 18
3.15
3 12
3.08
304
3 01
2 97
2.93
290
2 87
Endrin
326
323
3 19
3.16
3 13
309
306
3.03
3 00
296
293
290
2 87
2 83
2.80
2 77
2.74
2 70
Chlordecone
4.65
4.67
4 49
4.41
4J3
4.26
4.18
4 10
4.02
394
3 87
3 79
3.71
364
3 61
348
3.40
3.32
p.p'-DOD
4.49
439
4.34
4 28
4 23
4.17
4.11
406
399
3 94
3.88
3.82
3 76
3 71
365
3 59
3.54
348
Endosulfon 8
e i
5.B7
5.85
5.73
5.61
5 49
536
5.24
5 12
6 OO
488
4.76
4 64
4.52
4.40
4.28
4.16
4.04
Ethton
5 57
5.48
5.39
5 29
6.20
B.11
5 01
4.92
4.83
4.74
464
4 55
446
4.38
4.27
4 18
4.09
4.00
p.p'-ODT
64
62
6.1
6 99
6.88
5.76
5.64
5 52
6 40
528
5.16
5.04
4 92
4 80
4.68
4.56
444
4.32
Carbophenolhlon
8.0
7,9
77
7,5
7.4
7.2
7.1
69
6 7
8.6
6.4
6.3
6 1
595
5 80
5.62
&48
" 134
Dllan 1
7.7
79
7.5
7 3
73
7 1
7.0
6.9
88
67
66
6.6
6.4
63
6.2
6.1
60
5.85
Mcrex
12.4
12 1
11.8
11 6
11.3
11.0
107
10 4
10 1
9.8
95
9J
9.0
8.7
84
8 1
78
76
Methoxychlor
9.4"
92
B0
8 8
6.6
8.4
8.2
80
7 8
7.6
7 3
7 1
6.9
6 7
65
6.3
6.1
5.88
Ditan II
16 9
16.6
16.1
15.7
15.3
14.9 14.5
14.1
13.7
13.3
12.9
12.5
12.1
11 7
11.3
109
105
102
Tofrodiffon
24 0
234
22.6
22.2
216
21 0
20.4
19.8
19 2
18 6
18 0
174
16 8
16.2
16.6
16 0
14 4
13 8
Axlnphotmethyt
I
170
1
1
174
1
1
178
I
1
182
I
1
186
I
i
ieo
1
1
194
1
1
198
1
1
202
1
204
Retention ratios, relative to aidrin, of 48 pesticides on a column of 1.5%OV-17/1.95%QF-1 at temperatures
from 170 to 204°C; support of Chromosorb W,H.P., 100/120 mesh, electron capture detector, tritium
source, parallel plate; all absolute retentions measured from injection point. Arrow indicates optimum
oolumn operating temperature with carrier flow at 60 ml per minute.
-------
Revised 12/2/74
Table 2(b)
Section 4,A, (6)
4%SE-30/6%QF-1
Column Temperature, °C.
I
170 174 178 182 188 190 164 198 * 202 204
1 I I I I I I I I I I I I I I I I I
0 27 0 28 0 28 0 28 0.29 0?$ 0.29 0,30 0 30 0,30 0 30 0 31 0 31_ 0 31 0.32 0.32 0 32 0.33 MevfnpHot
0.39 0.39 0.39 0.40 0 40 0 40 0.41 0 41 0.42 0 42 0 42 0 43 0 43 0 43 0.44 0 44 0 44 0 46 2,4-D(ME)
0 43 0 43 0.43 0.44 0 44 0 44 0.45 0 46 0.46 0 45 0 46 0 46 0 46 0 46 047 047 0.47 0 48 Phorole
0 42 0 43 0 43 0,44 0.44 0 44 0 45 _ Q.4S 0 4fl 048 0 47 0 47 0.48 0 48 0 4ft 0.49 0 49 0.50 fl-BHC
0 44 0.44 0 45 0 43_^0.46 0 46 0 48 0.47 0 47_ 0.48 0 48 0.48 0.49 0.49 0 50 0.50 0 60 0 51 CDEC
0 54 0.54 0 64 0 64 0.B4 0 65 0 56 0.55 0.66 0 55 0 65 0 55 0 58 0 55 0 55 0 65 0.55 0.55 2,4-D(IPE)
0 52 0.52 0 52 0.53 0.63 0.53 0.63 0 63 0.53 0 63 0 53 0 54 0 64 0-54 0 64 0 54 0.64 0 64 Simazlne
0.55 0 59 0 66 0.55 0.66 0 68 0 66 0 56 0 66 0.56 0 56 0.56 0 67 0 57 0 67 0 57 0 57 0 57 Atrazine
0 60 0 60 0.60 0 60 0 59 0.59 0.59 0.59 0.59 0 69 0 69 0 58 0 68 0 58 0 68 0.58 058 0 58 DiazinOfl
054 0 54 0 65 0.6S 066 0 66 0.66 0.57 0 57 0.57 0.58 0 68 0 69 0 69 0.59 0 60 0.60 0.61 Lindane
0 66 0.86 0.68 0.66 0 65 0 65 0.65 0 66 0.66 0.66 0.65 0 65 0 64 0 64 0 64 0 64 0 64 0.64 2,4,R-T(ME)
057 Q.S7 0*7 0 58 0 58 0 58 0 59 0.69 0 69 0.69 060~ 6io" 060 0.60 0*1 ~ Vei" 061 0.61 /3 BHC
085 0.84 0.84 0 83 083 082 0.62 0J2 061 0 8t 0.60 0 8 0 0.79 0 79 0 79 0.78 0 78 0 77 2,4-D(BE)l
066 0.66 0.66 0 66 0.66 0 66 0.67 0.67 0 67 0.67 0.68 0 66 0 68 0 69 0 69 0.69 0.69 0 70 6-BHC
OHO 0 80 OBI 0 81 _0 81_ 0 81 0.81 0.82 0 82 0 82 0 82 0 8 2 0.82 0.83 0.83 0 83 0 83 0 83 Heptachlor
089 0 89 0.88 0.88 0 87 0.87 0 87 0 86 0 66 0.85 0.66 0.65 034 084 083 0 83 0 83 0.82 2,4.5-T(IPE)
098 0.97 0 96 0 96 095 0 94 0 93 0.93 0 92 0.91 091 0.90 0.89 0.88 0 68 0 87 0 86 0.88 2,4D3 1 b3 1 52 1 51 1.50 1 50 1.49 1 48 1 48 1 47 1 46 1 46 1 45 1 44 1 44 1 43 1.42 1.42 Heptachlor EpOXldt
1./0 1 68 1.66 1 64 1 63 1 61 1 59 1 57 1 55 153 151 1 49 1 48 1 4b 144 1 42 140 1.38 Mlllthion
1.64 163 1 61 1 60 1 59 1 57 1 56 1 54 1 53 1 52 1 51 1 49 1 46 1.47 1 45 1.44 1.43 1 41 DC PA
1.53 1 52 J 51 1 60 1 49 _1 46 146 1.45 1 44 1 43 1 42 1 41 1 39 1.38 1 37 138 1 38 1 34 DyftlW
187 166 1 65 1.63 1 62 1 60 1.59 1 57 1.56 1 55 1.53 1 52 1 50 1 49 1.47 1.48 1.46 1 4J O,p'-D0E
162 1 61 1 59 1 68 1 57 1.56 1 55 1 54 1 53 1 52 1 51 1 49 1.48 1 47 1.44 1 45 1 44 1.43 Chtorbftfllld*
2 09 2 07 2 05 2 02 2 00 1 98 1 96 194 191 1 89 1 87 1 85 1 83 1 80 1 78 1 78 1.74 1 72 E. Parathlon
1.99 1 98 1.97 1 06 1 94 1.93 1 91 1 90 1 89 1 87 1 86 1.85 1 83 1 82 1.80 1.79 1 78 1 76 Endottflfofl X
2 16 2 14 2 11 2 09 2 07 2 06 2.02 2 00 1 98 1 96 1 93 1 91 1 B9 1 86 1 84 1 82 1 80 1 77 P.p'-DDE
2.27 2 26 2 22 2 19 2 16 2.13 2 10 2 07 2 04 2 01 1 98 1 96 1 93 1.90 1 87 1 84 1 81 1 78 DDA(ME)
227 2.29 2J3 2.21 2.19 2 18 2.14 2 12 2 10 2 07 2 05 2.03 2 01 1.99 1 96 1.94 1.92 190 Captan
2 22 2.20 2.18 2 16 2 14 2 12 2.10 2 08 2 06 2 04 2 02 1,99 1 97 1 95 1 93 1.91 189 1 67 Fotpet
243 2.41 239 2.37 235 2 33 231 2 29 2 27 2.25 2 22 2.20 2 18 2 16 2 14 2.12 2.10 2 08 Dieldrin
2 43 2 40 2 37 2 33 2 30 2 27 2 24 2.20 2 17 2 14 2 11 2 07 2 04 2.01 1 97 1.94 1.91 1 67 Perthan*
2.34 2 31 2 29 2.27 2 24 2.22 2.19 2.17 2.16 2 12 2 10 2 07 2 06 2 03 2.00 1.98 1 96 1.93 O.p-'-DDD
3.02 2 97 2.93 2.88 2 84 2.80 2.76 2.73 2.68 2 64 2 60 2 56 2 62 2 47 2.43 2.39 2 36 2 31 O.p'-DDT
2.76 X 73 *-71_ 2*9 2*1 2 64 2.62 2 60 2.68 2 55 2 53 2 61 2 49 2.46 2 44 2 42 2 40 2.37 Endrin
297 2.94 291 2 89 2 86 2 83 280 2 78 2 76 2.72 2 69 267 2.64 2.61 2 69 2.58 2 53 2 50 Chlordecone
3 22 3 17 3.13 3 08 3.04 298 294 290 2 86 2 82 2 77 2.73 2.68 2.04 2.69 2.55 2.61 2 46 P.p'ODO
3 19 3 16 3 13 3.10 3.07 3 04 3.00 2 97 2 94 2 91 2 88 2 85 2 81 2.76 2.76 2.72 2.69 2.66 Endotulfon B
4.08 4 09 3.96 3.89 3 81 3 76 3 68 3.60 3 63 3.47 3 40 3 32 3.27 3.20 3.13 3 06 2 98 2.90 Ethion
4.04 3.98 3.92 336 3.80 3.73 3J7 3 61 3.64 3.48 3 43 346 3.30 3.24 3.18 3.12 3.06 2.96 P.p'-OOT
408
4.02
3.96
3.90
3.83
3.78
372
366
3.59
3.52
3.47
340
3J4
3.28
3.22
3.16
3.10
3.03
Corbophefiothlon
6.2
8.1
698
6J3
8.72
6.60
5.49
6.38
6 27
6 17
5.02
4.00
4.79
4.68
4.66
443
4 32
4.20
Dllan 1
6.1
6.0
6.96
6J7
6.78
5.68
6.60
6.62
5.43
633
6 24
6.19
6.06
4 97
4.88
4.79
4.70
4.62
Mlrex
67
6.6
14
63
6.1
6.98
6.84
B.70
6.67
6.42
629
6.18
6.01
4.88
4.73
4.60
446
4.32
Methoxyehlor
74
7.2
7.1
6.9
6J
6.6
6.6
63
6.2
60
687
6.71
6.67
6.41
6.28
6.11
4.98
481
Dilan II
11.6
IIJ
11.1
10J
10.6
10.3
10.1
9.8
98
93
B.O
8 J
8.6
BJ
8.0
7.8
7.6
73
Tetrodlfofi
126
12J
11.6
11.7
11.4
11.1
10 J
10 6
10.3
10.0
*9.7
94
9.2
8.9
8 A
84
6 1
7.8
As Inphoe methyl
~r>r1i1 i 1 i 1 i 1 i 1 I 1
170 174 178 102 168 190 194 198 202 204
Retention ratio*, relative to akJrln, of 48 pestlcidei on a column of 4%SE-30/6%QF-1 at temperature
from 170 to 204°C; support of Chromoaorb W,H.P„ 80/100 rrmh; electron ceptvra detector, tritium
aouroe, parallel ptate; an abioluta retentions measured from injection point. Arrow indicate! optimum
column operating temperature with carrier flow at 70 ml per minute.
-------
Revised 12/2/74
Table 2(c)
Section 4,A, (6)
5%OV-210
170
I
|
174
1
I
178
1
*
\
182
1
i
186
I
I
190
I
\
194
1
|
198
1
I
¦
202
1
204
|
066
0 66
0.66
066
066
0 66
066
066
0 66
066
066
0 66
066
066
066
066
066
066
Mvvinphot
0 69
0 69
0 69
0 69
0 69
0 69
0.69
069
0 69
0 69
0 69
0 69
0 69
0 69
0.6B
0.69
0 69
0 69
2,4-DfME)
063
0 64
084
0 84
0 64
064
084
0.65
0 65
0 65
0 65
0.65
0 65
068
066
068
0 68
066
Phorole
0 62
0 62
0 62
0 63
0 63
0 64
0.64
0 65
0 65
0.66
.0 66
0 66
0 67
067
068
0 68
0 68
0 69
a-BHC
0 69
0 69
0.69
0 69
0 69
0 69
0 69
0 69
0 69
0 70
J0 70
0 70
0 70
0 70
0.70
0 70
0 70
0 70
CDEC
088
0,87
0 87
087
0 86
066
0 86
0 85
0 85
0 85
0 85
0 84
0 84
084
0 83
0 83
0 83
0 82
2.4-DORE)
0 87
0 88
086
0 88
0 85
085
0 85
0 84
084
0 84
0 84
0 83
0 83
0 83
0 82
082
0 82
081
Sirruzine
0.88
0 88
0 87
0 87
0 86
0 86
0 86
0.85
0.86
0 64
0 84
0 84
0 83
083
0 82
082
0 81
081
Atrazine
0 76
0 76
0.75
0 76
0 75
075
0 75
0 74
0 74
0.74
0.74
0 74
0 74
0 73
073
0 73
0 73
0 73
Diazinon
0 80
0 80
080
0 80
0.80
081
081
0 81
081
0.82'
0 82
0 82
0 82
0 83
0 83
0 83
0 83
0 84
Lindane
1 08
1.08
1 07
1 07
1.06
1 05
1.06
1 04
1 03
1 03
1.02
1 01
1 01
1 00
1 00
099
0 98
098
2,4,5-T(ME)
097
0.97
096
096
0 96
096
096
0.95
0 95
0.95
0 95
0 95
0 95
' 0 94
0 94
094
094
0 94
/3bhc
130
1 29
1 28
1 27
1 26
125
1 23
1 22
1 21
1 20
1 19
1 18
1 16
1 16
1 14
1 13
1 12
1 11
2,4-D(8EII
1 07
1.07
1 06
1 08
1 05
1 05
1 05
1 04
1 04
1 03
1 03
1 03
1 02
1 02
1 01
1 01
1 01
1 00
5-bhc
0 87
0 87
0.87
0 B7
0 07
0 87
0.87
0 87
0 87
0 87
0 88
0.88
0 88
0 88
0 88
0 88
0 88
0 88
Heptachtor
1.36
1.34
1 33
1 32
1 30
1 29
1 27
1 26
1,25
1 23
1 22
1 20
1 19
1 18
1 16
1 15
1 14
1 12
2,4,5-T(IPE)
1 47
1 46
1 44
1 43
1 41
1.40
1,38
1 36
1 35
1 33
-, 1 32
1 30
1 29
1 27
1 25
1 24
1 22
1 21
2.4-D1BEHI
1 51
1 51
1 50
1 49
1 48
1 48
1 47
1 46
1 45
1 45
1 44
1 43
1 42
i 42
1 41
1 40
1 39
1 39
Oichfone
2 IB
2 IS
2 12
2 09
2 06
204
201
1 90
1 95
1 92
1 89
1 86
1 83
1 00
1 78
1 75
1 72
1 69
Dimethoate
1 00
1 00
1 00
1 00
1 00
1.00
1 00
1 00
J 00
1 00
1 00
1 00
1 00
1 00
1 00
1 00
1 00
1 00
Aldrin (REFERENCE)
1 41
1.40
1 39
1 38
1 37
1 38
1 35
1 34
1 33
1 32
1 31
1 30
1 29
1 28
1 27
1 26
1 25
1 24
Ronnel
1 43
1 42
1 41
1 40
1 39
1,38
1 38
1 37
1 36
1 35
1 34
l 33
1.33
1 32
1 31
1 30
1 29
1 28
1-Hydroxychlorderw
3 17
3 12
3 07
3 02
2.97
2 92
2 88
2 83
278
2 73
2 68
263
258
2 54
2 49
2 44
2 39
2 34
M. Parsthion
2 02
2 00
1.98
1 97
1 95
1 93
1 91
1 89
1 87
1 85
1 83
1 81
1 79
1 77
1 75
1 73
1 71
1 69
Heptachlor E pox tot
3 20
3 14
308
3 02
2 96
291
2 85
2 79
. 2.73
2 67
2 61
2 55
' 2 49
2 44
2 38
2 32
2 26
2 20
Malathton
2 84
2 80
2.75
2 71
2 67
2 63
2 59
2 54
2 50
2 46
2 41
2 37
2 33
2 29
2 24
2 20
2 16
2 12
0 C P A
206
2.04
2.02,
1 99
1 97
1 94
1 91
1 89
1.87
1 84
1 81
1 79
» 77
1 74
1 71
1 69
1 66
1.64
Oyrene
1 67
1 65
1 63
1 62
1 60
1 59
1 57
1 55
1 53
1 52
1 50
1 49
1 47
1 45
1 43
1 42
? 40
1 39
O.p'-DDE
197
1 85
1.93
1 SO
1 88
1 86
1 84
1 82
1 80
1 78
1 75
1 73
1 71
1 69
1.67
1 65
1 63
1 61
Chlorbentidt
4 24
4 17
409
4 02
3.94
3.87
3 78
3 71
3 63
3 56
3 48
3 40
3.33
3 25
3 17
3 10
3 03
295
E. Parathion
2.63
2 60
2 57
2 54
2 51
248
2 4S
2 42
2 39
2 36
2 33
2 30
2 27
2 24
2 21
2 18
2 15
2.12
Endpsuffan X
2 24
2 21
2 18
2 16
2 12
2 10
2 07
2 04
201
1 98
1 95
1 92
1 90
1 87
1 84
1 81
1 78
1 76
p.p'DDE
3 10
SOS
300
2 94
2 89
2 84
2 79
2 74
2 69
2 64
2 59
254
248
2 43
2 38
2 33
2 28
2 23
DDA(ME)
4 47
4 40
4 32
4 24
4 17
4 09
4 01
3 93
3 85
3 77
3 69
3 62
3 54
3 46
3 38
3 30
3.22
3 14
Captsn
4 04
3 98
3 92
3 85
3 78
3.72
3 65
3 59
3 53
3 46
3 39
3 33
3 26
3 19
3 13
3 06
2 99
2 93
Folpet
321
3 17
3 13
3 09
3 04
3.00
296
2 92
288
2 83
2 79
2 75
2 71
2 68
2 62
2 58
2 54
2 49
Dietdrin
2 07
204
2 01
1 9B
1 95
1 92
1 89
1 86
1 83
1 80
1 77
1 74
1 71
1 68
1 65
1 62
1 59
1 56
Per than*
2 74
2 70
2 67
2 63
2 59
255
2 51
2 47
2 43
2 39
2 35
2 31
2 27
2 23
2 19
2 15
2 11
2 07
o.p'-DDD
2 92
2 67
2 83
2 79
2 74
2 70
2 65
2 61
2.67
2 52
2 48
2 43
2 39
2 35
2 30
2 26
2 21
2 17
o.p'ODT
3 82
3 77
3 72
3 67
3.61
356
3 50
3 45
3 39
3 34
3 28
3 23
3 18
3 13
3.07
3 02
2.96
2 91
Endrin
2 76
2 73
2 70
2 87
2 64
261
2 58
2 55
2 52
2 49
2 46
2 43
2 40
2 37
2 34
231
2 28
2 25
Chlord«c6rt*
4 10
4 03
3.96
3 89
3 82
375
366
3.61
3 53
3 46
3 38
3 32
3 25
3 18
3 11
3 03
2.96
2 89
p.p'DDD
496
4.91
4 83
4.75
4 67
4 59
4 51
4 43
4 35
4 27
4 19
4.11
4.03
3 95
3 67
3 79
3 71
3 63
Endotulfon S
80
5 89
5 76
5 64
551
5 37
6.25
5.12
499
4 86
4 73
4 60
4 48
4 35
4 22
4.09
3 96
3B3
Ethton
4 47
4 39
431
4 23
4 15
4.07
3.98
3 90
3 82
3 74
3 66
3 68
3 49
3 41
3 33
325
3 17
309
p.p'DOT
6 29
6.19
S 09
4 99
4 89
4.78
4 68
4 58
4 48
4 37
4 27
4 17
4 06
3 96
3 88
3.76
3 16
305
Corbophanofhion
>2.3
12.0
11.7
11 4
11.1
108
104
10.1
98
9.5
9 2
89
86
8.2
7 9
7.6
7 3
7 0
Dilan 1
406
4.01
395
3 89
3 84
378
3 73
3.68
3.62
3 57
3.52
3 46
3 40
3 35
3 29
3.24
3.19
3 13
Mire*
74
7.2
70
68
8 7
65
63
6 1
594
5 76
5 59
6.41
5.23
5.06
4 88
4 70
4 63
4 35
Methoxychlor
T4 2
13 8
134
13.0
12.6
12.3
11 9
11 5
11 1
10 7
10 3
10 0
96
9 2
88
84
8.1
7 7
Dilan II
21.1
206
200
195
18.9
18.4
179
17 3
16 7
16.2
15 6
15 1
14.5
14 0
134
129
12 3
118
Tefrodifon
24.7
24.0
23 3
228
21 9
21 2
206
19 9
19 2
18.5
W.8
17 1
16.4
16 7
16.0
14.3
13.6
129
Azlnphosmothyl
I
170
1
I
174
I
I
179
!
1
182
I
1
186
I
190
1
I
194
1
1
198
1
202
1
204
Retention ratios, relative to aldrin. of 48 pesticides on a column of 5%0V-210 at temperatures
from 170 to 204°C; support of Chromosorb W.H P., 100/120 mesh; electron capture detector,
tritium source, parallel plate; all absolute retentions measured from injection point. Arrow
indicates optimum column operating temperature with carrier flow at 60 ml per minute.
-------
Section Ji,A»(6)
Figure 1
Figure 2
Figure 3
-------
Revised 11/1/72 Section 4, A, (6)
Fig. 4 — Column to Port Assembly-Exploded View
Silicone O-Ring
Back Ferrule
Nut
0 - Ring Retainer
mn
Fig. 4 (a) ~ Bubble Flowmete
Buret, 50 ml
Polyeth. Tubing,l/8"o.d.
Tygon_ Tubing , l/8"i.d.—
Rubber Bulb . 30 ml —
'¦ Snoop
-------
Revised 12/2/74
Section 4, A, (6)
Figure 5. Standing current profile from detector in constant
use 60 days. Instrument Tracor MT-220; electrometer
attenuation 10 x 256, 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.
25v.
30 v. 35 v.
I *
20 v.
Optimum polarizing
voltage (see Fig. 6)
15 v.
2 10 v.
5 v.
Ov.i
-------
Revised 12/2/74
Section 4, A, (6)
Figure 6. Voltage/Response curve for 50 pg of aldrin from %
detector in continual use 60 days. Instrument Tracor MT-220;
electrometer attenuation 10 x 32; column 31 OV-1, column temp.
180°C., detector temp. 200°C., nitrogen carrier flow 60 ml/min.
\
Z5v. o 10v. 12.5v.
15v. 17.5v. o 20 v. 22.5v.
I
-------
Revised 12/2/74
Section 4, A, (6)
Fig. 7
Peak Height =CD
Peak Area = CD x AB
Half Height
Fig. 9 Triangulation
Peak Area =» 4- (FG) (JH)
-------
Revised 12/2/74 Section 4, A,
Fig. 10. Copied from Pesticide Analytical Manual, Vol. 1, U.S. Food
Drug Administration. ¦
J
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- andT-BHC sloping baseline; h,a-a£-> and 7-BHC sloping baseline;
1, chlordane flat baseline; j, heptachlor and heptachlor epoxide super-
imposed on chlordane; k, chair-shaped peaks, unsyir,metrical peak; l(
p,p'-DDT superimposed on toxaphene.
-------
Revised 12/2/74
Section 4, A, (6)
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 12/2/74
Section 4,B,(1)
Page 1
GAS CHROMATOGRAPHY-FLAME PHOTOMETRIC
INSTRUMENT
I. INTRODUCTION:
Some of the instructions in this section apply specifically to
the Melpar flame photometric unit marketed by Tracor, 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"° amperes bucking current.
V. TEMPERATURE PROGRAMMER:
See Section 4,A, (1), IV.
VI. PYROMETER:
See Section 4,A,(1), V.
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. 41 SE-30/61 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.
m. 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 deteimined that a Carbowax deposition treatment will
significantly enhance the F.P.D. response of GLC columns comprised
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 Guiffrida — 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") silanized glass wool in one end
of a 3" length of 1/4" o.d. glass tubing. Pack loosely with
2 inches of 10% Carbowax and place another wad of glass wool
in the other end of the tube (Fig- 1).
b. Place Swagelok nut, ferrule and "0" ring on each end of the
packed tube. Pass one end of the tube half way through the
male union and attach Swagelok nut to union as tight as possible.
1/ 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)
- 2 -
c. Attach other end of tube to the column inlet port in the oven,
tightening Swagelok nut as much as possible. Place 1/4" nut
on inlet end of the 6-ft. GLC column (previously heat con-
ditioned) and insert into the male union until it touches
bottom end of 3" tube, then slack off very slightly to prevent
glass ends from grinding together when nut is tightened. (Fig. 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 littlejack 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-inch length of borosilicate glass tubing,
1/4" o.d. x 5/32" 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". This must
be drilled out to accommodate the 1/4" 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.
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. Proceed as /follows:
A. Set hydrogen flow from 150 to 200 ml/min.
B. Using a standard solution of parathion, maximize response by
varying oxygen flow with air flow at zero.
C. Again maximize response by varying the air flow and holding the
oxygen flow at its optimum.
-------
Revised 12/2/74 Section 4,B,(2)
- 3 -
NOTES: 1. Some detectors respond best with air flow at zero.
2. An increase in flow velocity, although enhancing
response, may also increase the baseline noise to
troublesome levels. Response criteria is best defined
in terms of signal-to-noise ratio rather than in terms
of response alone.
D. The full list of suggested operating parameters for use with the
phosphorous cell is as follows:
Temperatures, °C Flow Rates, ml/min
Column 200 —^ Purge 70-80
/Injection block 225 Carrier 70-80
— Detector 165-200 Hydrogen 150-200
7 /Transfer Line 235 Oxygen 10-30
— Switching Valve 235 Air 0-100
— Do not operate above 170°C without heat shield.
2/
— Appropriate only if using a Valco vent valve.
E. When all parameters have been adjusted to produce optimum signal-
to-noise ratio, baseline noise should not exceed 2.5% f.s.d. and
an injection of 2.5 nanograms of parathion should result in a
peak of at least 50% f.s.d.
The following mixture should produce approximately equal peak
heights of at least 10% f.s.d.
Compound
ng
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.
-------
Revised 12/2/74
Section 4,B,(2)
- 4 -
NOTES: 2. With some organophosphorous 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 impor-
tant consideration if quantitation is to be conducted.
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 drop 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 RRTp
values. Retreatment is not advised, therefore, as
the data contained in Tables 2, 3, and 4 would become
unreliable.
-------
Revised 12/2/74
Section 4,B,(3)
Page 1
GAS CHROMATOGRAPHY-FLAME PHOTOMETRIC
DETECTOR
I. OPERATING PARAMETERS:
D.C. voltage shall be supplied to the detector from either an
outboard power supply or from a strip on the back of the electrometer.
Provided the column and all electronic circuits in the various modules
of the instrument are functioning properly, the degree of sensitivity in
the flame photometric mode relate 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.D.C. Inject
enough Ethyl Parathion to give 35 to 60 f.s.d,
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 (Fig. 4).
NOTES: 1. It should be necessary to attenuate to keep on
scale. It is therefore mandatory to check the
linearity of the electrometer at different attenu-
ations .
2. Comparison with a P.M. tube of known sensitivity
will give indication of condition of P.M. tube.
III. DETECTOR LINEARITY:
The F.P.D. has a broad range of linearity. Excluding any effect
from the instrument electronics, the effective range is from 1 to 50
times the minimum acceptable level of Ethyl Parathion (0.5 ng to 25 ng).
The appropriate attenuation will be dependent upon the sensitivity of
the particular system used. It is best to operate at the minimum
detection level and dilute the sample when necessary, however.
-------
Revised 12/2/74
Section 4,B,(3)
- 2 -
IV. PHOSPHORUS MODE:
When the detector is fitted with a 526 my filter, it is
primarily selective for phosphorus but large amounts of sulfur will
give a response in this mode.
V. SULFUR NODE:
When the detector is fitted with a 394 my filter, it becomes
selective for sulfur. Sensitivity for sulfur is usually an order of
magnitude less than for phosphorus. The square root of the response
increases approximately proportionate to the concentration.
-------
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
Rormel 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 (RRTp), on a given column matched
with a standard or matched against the RRTp values given in Tables
2, 3 or 4.
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)
TABLE I. RETENTION AND RESPONSE RATIOS, RELATIVE TO ETHYL PARATHION
ON COLUMN, OF 4% SE-30/6% QF-1
COMPOUND
RRR-pi/
RPH-P-/
TEPP
0.08
5.0
Dichlorvos
.10
5.0
Demeton-Thiono
.22
2.0
Naled
.28
0.02
Phorate
. .28
4.0
Sulfotepp
.28
5.2
Diazinon
.35
2.5
Demeton-thiolo
.37
2.0
Dioxathion
.38
0.5
Disulfoton
.40
3.8
Diazinon-oxygen analog
.42
1.0
Dimethoate
.49
0.50
Monocrotophos
.54
0.08
Ronnel
.57
1.42
Ronnel-oxygen analog
.58
0.25
Chlorpyrifos
.68
1.4
Fen+hion
.72
1.6
Methyl Parathion
.75
0.71
Malathion
.81
0.71
Methyl Parathion-oxygen analog
.83
0.10
Malathion-oxygen analog
.85
0.063
Fenitrothion
.85
0.80
Ethyl Parathion (veferenoe)
I.00
I. 00
Phosphamidon
1.02
0.16
Ethyl Parathion-Oxygen analog
1.10
0.50
Merphos
1.23
0.35
DEF
1.25
0.80
Carbophenothion-oxygen analog
1.78
0.12
Ethion
1.83
0.71
Carb ophenothion
1.90
0.36
Phen"kapton
3.04
0.20
Fensulfothion
3.16
0.03
Imidan
3.91
0.02
EPN
3.95
0.134
Azinphos methyl
6.03
0.044
Coumaphos
11.84
0.20
—^ 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
4°/» SE -30/6°/o OV - 210
Column Temperature, °C.
I
170 174 178 182 186 190 194 198 * 202 204
1 i I i 1 i I i I i I i .1 i I i 1 i_
0.06 0.06 0.06 0.06 0.06 0 07 0.07 0.07 0 07 0.08 0.0B 0.0B 0.08 0.08 0.09 Cl.m O.tM <1.09 Dlchlorvos
0 04 0.04 0.04 0.04 0.05 0.05 0.05 0.05 0.06 0.06 0.06 0.06 0.06 (1.06 0.07 n.07 (1.(17 n 07 TEPP
0.14 0.14 0.14 0.14 0.15 0.15 0.15 0.15 0.16 0.16 0.16 0.17 0.17 0.17 0 17 O.lfl (I.IB n IB Mevlnphos
0.15 0.15 0.15 0.16 0.16 0.16 0.17 0.17 0.17 0.1B 0.18 0.19 0.11 0.11 0-70 0.711 Q.?<1 0 71 Demeton Thlono
1J6 0J6 0.16 0.17 0.17 0.18 0.18 0.18 0.19 0.19 0.19 0.20 0.20 0.21 0.21 0.21 0.22 0.23 Thlonazin
0.19 0.20 0.20 0.20 0.21 0.21 0.21 0.21 0.22 0.32 0.22 0.23 0.23 0.23 0.23 0.24 0.24 0.24 Ethoprop
0.20 0.21 0.21 0.21 0.22 0.22 0.23 0.23 0.23 0.24 0.24 0.24 0.24 0 25 0.25 0.25 0.26 0.26 Phorate
0.21 0.22 0.22 0 22 0.23 0.23 0.23 0.24 0.24 0.24 0.25 0.25 0.25 0.26 0.26 0.27 0.27 0.27 Sulfotepp
0.23 0.23 0.23 0.24 0.24 0.24 0.25 0.25 0.25 0.25 0.26 0.26 0.26 0.27 0 27 0.27 0.27 0.26 Naled
0.23 0.24 0.24 0.24 0 25 0.25 0 25 0 26 0.26 0.26 0 27 0.27 0 27 0.28 0.28 0.28 0.28 0.29 Oxydemeton Methyl
0.27 0.27 0.27 0 28 0 28 0.28 0 26 0.29 0 29 0.29 0.30 0.30 Q.30 0 30 0.31 0.31 0.31 0.31 Diazinon
0.30 0.30 0.30 0.31 0.31 0.31 0.32 0.32 0 32 0.33 0.33 0.33 0.34 0.34 0.34 0.35 0 35 0 35 Dfoxathlon
0.32 0.32 0.32 0.33 0.33 0.33 0 33 0.34 0.34 0.34 0.35 0.35 0 35 0.35 0.36 0.36 0.36 0.37 Demeton Thiolo
0.30 0.31 0.31 0.32 0.32 0.33 0.33 0 33 0.34 0.34 0.35 0.35 0.35 0.36 0.36 0.37 0.37 0 37 Disulfoton
0.38 0.38 0.38 0.38 0.38 0.38 0 38 0.38 0 39 0.39 0.39 0.39 0.39 0.39 0.39 0.40 0 40 0.40 Dlazoxon
0.40 0.40 0 40 0.41 0.41 0.41 0 42 0.42 0.42 0.43 0.43 0.43 0.44 0 44 0.44 0.45 0.45 0.45 Dichlofenthion
0.46 0.46 0.46 0.46 0.47 0 47 0.47 0.47 0.47 0.48 0.48 0.48 0.48 0.49 0.49 0.49 0 49 0.49 Dimethoate
0.48 0.48 0.48 0.48 0.49 0.49 0.49 0.50 0.50 0.50 0.50 0.51 0 51 0.51 0.51 0.52 0.52 0 52 Ronnel
0.51 0.51 0.51 0.51 0.52 0.52 0.52 0.52 0.53 0.53 0.53 0.54 0.54 0.54 0.54 0.55 0.55 0.55 Cyanox
0.55 0.55 0.55 0.56 0.56 0.56 0.56 0.57 0.57 0.57 0.58 0.58 0.58 0.58 0.59 0.59 0.59 0.59 Ronnoxon
0.60 0.60 0.60 0.60 0.60 0.60 0.60 0 60 0.60 0.60 0.60 0.61 0 61 0.61 0.61 0.61 0 61 0.61 Monocrotophos
0.58 0.59 0,59 0.59 0.59 0.59 0.60 0.60 0.60 0.60 0 60 0.61 0.61 0 61 0 62 0.62 0 62 0.62 Chlorpyrlfos
0.57 0.58 0.58 0.58 0.59 0.59 0.59 0.59 0.60 0 60 0.60 0.61 0 61 0.61 0 61 0 62 0 62 0.62 Zytron
0.62 0.62 0.62 0 62 0.63 0.63 0.63 0.63 0.64 0.64 0.64 0.64 0.64 0.64 0.64 0 65 0 65 0.65 FenthIon
0.67 0.67 0.67 0.68 0.68 0.68 0.68 0 68 0.68 0.69 0.69 0.69 0 69 0 69 0 70 0.70 0 70 0.70 Mataoxon
0.72 0.72 0.72 0.73 0.73 0.73 0.73 0.74 0.74 0.74 0.75 0.75 0 75 0.75 0.76 0.76 0.76 0 76 Methyl Parathion
0.81 0.81 0.8Q 0.80 0.80 0.80 0.80 0.79 0.79 0.79 0.79 0.79 0.79 0.78 0 78 0.78 0.78 0.78 Malathlon
0.86 0.86 0.85 0.85 0.85 0.85 0,84 0.84 0.84 0.84 0 85 0.85 0.85 0.85 0 86 0.86 0.86 0.86 Fenftrothfon
0.94 0.94 0.93 0.93 0.93 0.92 0 92 0.91 0.91 0 91 0.90 0 90 0.89 0 89 0.88 0 88 0.88 0.87 Bromophos
0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0 90 0.9(1' 0.90 0.90 Methyl Paraoxon
0.93 0.93 0.93 0.93 0.92 0.92 0,92 0.92 0.92 0.91 0.91 0 91 0.91 0 90 0.90 0 90 0.90 0.90 Phenthoate
0.91 0.91 0.91 0.91 0.91 0.90 0.90 0.90 0.90 0.90 0.9Q 0.90 0.91 0 91 0.91 0.91 0 91 0.91 Bromophos Ethyl
0.85 0.84 0.84 0.84 0.83 0.B3 0.83 0.82 0.82 0.83 0.84 0.86 0 B7 0.88 0.89 0 90 0.92 0 93 Schradan
0.96 0.96 0.96 0.96 0.96 0.97 0.97 0.97 0,97 0.97 0.97 0.97 0 98 0 98 0.98 0 98 0 9B 0.98 Dicapthon
1.00 1 .00 1.00 1.00 1.00 1 00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1 00 1.00 1.00 E Pa rathion(toHrtnw)
1.02 1.02 1.02 1.01 1.01 1.01 1.01 1.00 1 00 1.00 1.00 1.00 1.01 1 01 1.01 1 01 1 01 1.01 Amldlthion
1.02 1.02 1.02 1.02 1.02 1.02 1.03 1.03 1.03 1.03 1.03 1.03 1.03 1.03 1.03 1 03 1 03 1.03 Iodofenphos
1.11 1.11 1.10 1.10 1.10 1.10 1.10 1.10 1.10 1.10 1.09 1.09 1.09 1.09 1.09 109 1.09 1.08 Crufomate
1.18 1.18 1.17 1.17 1.17 1 16 1.16 1.15 1.15 1.15 1.14 1.14 1.13 1.13 1 13 1.12 1 12 1 11 DEF
1.19 1.18 1.1B 1.17 1.17 1.17 1.16 1.16 1.15 1.15 1.14 1.14 1.14 1 13 1.13 1.12 1.12 1.12 Phosphamldon
1.18 1.17 1.17 1.17 1.17 1.16 1.16 1.16 1.15 1.15 1.15 1.14 1.14 1 14 1 13 1.13 1.13 1.12 Folex
1.25 1.24 1.24 1.23 1.23 1.22 1.22 1.21 1.21 1.20 1.20 1.19 1.19 1.18 1.18 1.17 1.17 1.16 Ethyl Paraoxon
1.23 1.22 1.22 1.22 1.22 1.22 1.21 1.21 1.21 1,21 1.20 1.20 1.20 1.20 1 19 1.19 1.19 1.19 Methidathion
1.37 1.36 1.36 1.35 1.35 1.34 1.33 1.33 1.32 1.32 1.31 1.31 1.30 1.30 1.29 129 1.28 1.28 Tetrachlorvlnphos
1.87 1.85 1.84 1.83 1.82 1.81 1.80 1.78 1.77 1.76 1.75 1.74 173 1.72 1.70 1.69 168 1.67 Ethlon
1.H9 1.88 1.87 1.86 1.85 1.84 1.83 1.82 1.80 1.79 1<78 1.77 1.76 1.75 1.74 1 73 1 72 1.71 Carbophenoxon
1.89 1.88 1.B7 1.86 1.85 1.84 1.83 1.82 1.80 1.79 1.78 1.77 1.76 1.75 1.74 1.73 1 72 1.71 Carbophenothlon
3.18 3.15 3.12 3.09 3.06 3.03 3.00 2.98 2.95 2.92 2.89 2.86 2 83 2.80 2.77 2.74 2 72 2.69 Phenkapton
3.96 3.92 3.87 3.83 3.79 3 75 3.70 3.66 3.62 3.58 3.53 3.49 3.44 3 41 3.36 3.32 3.28 3.23 Fensulfothlon
4.65 4.60 4.55 4.50 4.45 4.40 4.35 4.31 4.26 4 21 4 16 4.11 4.06 4.01 3.96 3.91 3.87 3.82 Inildan
4.66 4.61 4.56 4.51 4.46 4.41 4.36 4.31 4.26 4.21 4.16 4.11 4.06 4.00 3.95 3.90 3.85 3.80 EPN
5.S7 5.63 5.59 5.55 5.51 5.47 5.42 5.38 5.34 5.30 5.26 5.21 5.17 5.13 5.09 5 05 5.01 4 96 Famphur
7 Afl 7.3B 7.77 7.17 7.06 6.96 6.85 6.75 b.64 6.54 6.43 6.33 6.22 6.12 6.01 5.91 5.80 5.70 Azlnphos Ethyl
7.53 7.42 7.32 7.21 7.10 6.99 6.89 6 78 6.67 6.57 6.46 6.35 6 24 6 14 6.03 5.92 5 81 5.71 Azlnphos Methyl
17.4 17.0 16.7 16.3 16.0 15.6 15.3 15.0 14.6 14 3 13.9 13.6 13.2 12.9 12 6 12.2 11.9 11.5 Counaphos
^^^^—¦ 1 r—i—y—i—|—i—|—i—j—i—|—i—
186 190 194 198 202 204
Retention ratios, relative to parathion, of 54 organophosphorous pesticides on a column of
4X SE-30/6I 0V-21O at 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
Section 4,B,(5)
Table 3
10 % OV-210
Column Temperature
178 183 186 190 194 198
94 198 ' 303
i i i i T
0.04 0.04 0.04 0.04 0.04 0.05 0 05 0.05 0.05 0.05 0.05 0.05 0.06 0.06 0.06 0.06 0.06 0.06 TEPP
(57155 OT755 5~05 575? 57157 {TD7 5757 5707 07W 0.08 0.08 0.08 0.08 0.08 0.09 0.09 0.09 0.09 Olchlorvos
575? 5751 5752 575? 5751 57T3 57T5 oTO 5751 0.13 0.13 0.13 0.14 0.H 0.54 0.14 6.14 ft.14 Demeton Thiono
0.14 0.14 0.15 0.15—0.15 0.15 0.16 0.16 0.10 6.16 0.17 0.17 6.17 0.1? 0.16 0.18 0.18 0.19 Hevlnphos
5754 57T4 57T4 07T5 575? 0"T5 575? 571? 571? 0.16 0.17 0.17 0.17 0.17 0.18 0.18 0.IB 0.19 Thlonazln
5755 5755 07T5 0756 OTTS 071? 0.17 0.T7 0.18 0 18 0.19 0.19 0.19, 0.26 0.50 0.20 0.20 6.51 Phorate
571?—571? 5T5 3757 5717 6717 6718 571? 57TC 0.15 6.19 0.19 0.20 0.50 0.21 0.21—6.21 0.22 Ethoprop
5T7—5757 5773—5758—5713—57TS ri!"!.!}' "0 19 6 19 0.2S-IT 2(5 0 50 6.26 0.21 6.51 5771 0775" Dlazlnon
0755 07TS 57TB 57T9 OTTS 0~15 0770 5725 5750 0.20 0.21 5771 5771 5772 0.22 0.22 0775 677T Sulfotepp
57T9—5719 5775—5720 5771 5T1 5775 5775 5775 0 53 0.23 0.24 0.54 0.24 0.55 0 25 0.25 6.26 Naled
5~54 0~04 575? 57754 0.05 ' 0 05 077J5 5705 0 05 0.06 0.06 0.06 0.06 0.06 0.67 0.07 0.07 0.07 Oxydemeton Methyl
577? 5775 5771 5771 0.23 0 24 5774 5774 577? 0.25 0 26 0.Z6 0.26 0 27 0.27 0.27 0.28 0.28 Dlsulfoton
5775 5775 5775 0"7? 0.26 0.26 5777 5777 5"78 0.28 0.28 0 29 0.29 0.29 0 30 0.36 6.30 6.31 Dioxathlon
CT79—0~Z9 IT79 0.29 0.29 0779 0730 0730 0730 0.30 0 30 0.31 0.31 0.31 0.31 0.31 0.32 0.32 Demeton thlolo
5779—0~Z9 5779 5730 0730 0730 0~31 0775 0T? 0 35 0.35 0 33 0 33 0 33 0.34 0.34 0.34 0.35 DIchlofentMon
0733—0733 5733—0733 0T4 0734 0734 0734 0~34 0 35 0.35 6 35 0.35 0 35 0.36 0 36—6.36 0.36 Dlazoxon
5734 0734 5754 5~35 0.35 0.35 0735 5735 5737 0.37 0.37 0.38 0.38 0 38 6.39 0.39 0.39 0 40 Ronnel
5775 5745 5771 5775 OT 5TI 5*15 5~43 5T? 0.42 0.42 o 42 o 43 0.43 0.43 6 43 8 43"'6' 43 Chlorpyrlfos,
07*3 0743 0743 0744 5734 5~74 5745 5745 5745 0 4 6 0 46 0 46 0.46 0.47 0 47 0.47 0.47 0 4? Zytron
-------
12/2/74
Table 4
Section 4,B,(5)
1.5% OV-17/1.95% OV-210
Column Temperature , ° C.
J
Xj2 20i
I I I I I I I I I I I I I I I I I I
0.04 0.04 0.05 0.05 0.05 0.05 0.06 0.06 - 0.06 0.06 0.07 0.07 0.07 0.07 0.08 0.08 0.08 0 09 TEPP
0.06 0.06 0.07 0.07 0.07 0.08 0.08 0.08 0.09 0.09 0.09 0.10 0.10 0.10 0.11 0.11 0.11 0.12 Dlchlorvos
0.1? 0.13 0.13 0.14 0.14 0.15 0.15 0.16 0 16 0.17 0.17 0.18 0.18 0.19 0,19 0.20 0.21 0 21 Mevlnphos
0.16 0.16 0.17 0 17 0.17 0.18 0.18 0.18 0.19 0.19 P.l? 0.20 0.20 Q.20 0.21 0.21 0,21 0.22 Demeton thlono
0.19 0.19 0.19 0.20 0 20 0.21 0.21 0.21 0.22 0.22 0.23 0.23 0.23 0.24 0.24 0.25 0.25 0.25 Th1on«z1n
0.20 0.20 0.20 0.21 0.21 0.22 0.22 0.22 0.23 0.23 0.24 0.24 0.24 0.25 0.25 0.26 0.26 0.26 Ethoprop
0 23 0.23 0.24 0.24 0.25 0.25 0.25 0.26 0.26 0.27 0.27 0.27 0.28 0.28 0.29 0 29 0 29 0.30 Phorate
0.24 0.25 0.25 0.25 0.25 0 26 0.26 0.27 0.27 0.27 0.28 0.28 0.28 0 29 0.Z9 0.29 0.29 0.30 Sulfotepp
0.26 0.27 0.27 0.28 0.28 0.28 0.29 0.29 0.30 0.30 0.30 0.31 0.31 0.32 0.32 0.32 0 33 0.33 Oxydemeton methyl
0 32 0.32 0.33 0 33 0.33 0.33 0.34 0 34 0.34 0 34 0.35 0.35 0.35 0.35 0.36 0.36 0.36 0.37 Oiazinon
0.31 0.31 0.32 0.32 0.33 0.33 0.34 0.34 0.35 0.35 0.36 0.36 0.36 0.37 0.37 0 38 0 38 0 39 Naled
0.34 0.34 0 34 0.35 0.35 0 35 0.36 0.36 0.36 0.37 0.37 0.37 0 37 0.38 0.38 0 38 0.39 0.39 Demeton thlolo
0 36 0 36 0.37 0.37 0.38 0.38 0.38 0.39 0.39 0.40 0.40 0.40 0 11 0.41 0.42 0,42 0.42 0.43 Dlsulfoton
0 37 0.38 0.38 0 39 0.39 0.39 0 40 0.40 0.41 0.41 0.41 0.42 0.42 0.43 0.43 0.43 0.44 0.44 Dloxathlon
0.38 0.39 0 39 0.39 0.40 0.40 0.40 0.41 0.41 0.41 0.42 0.42 0.42 0.43 Q 43 0.43 0.44 0.44 Dlazoxon
0.44 0.44 0.45 0.45 0.45 0.46 0.46 0.46 0.47 0 47 0.47 0.48 0.48 0.48 0.49 0.49 0 49 0 50 Dlchlofenthfon
0.51 0 51 0 51 0 52 0.52 0.53 0.53 0.53 0.54 0.54 0.55 0.55 0.55 0.56 0.56 0.57 0.57 0 57 Cyanox
0.53 0.53 0.53 0.54 0.54 0.54 0.55 0.55 0.55 0.56 0.56 0.56 0.57 0.57 0.57 0 58 0.58 0.58 Dimethoate
0.55 0.55 0.56 0.56 0.57 0.57 0 57 0 58 0.58 0.59 0.59 0.59 0.60 0.60 0.61 0.61 0.61 0.62 Ronnel
0 57 0 57 0.57 0 58 0.58 0.59 0.59 0.59 0.60 0.60 0.61 0.61 0.61 0 62 0.62 0.63 0 63 0 63 Ronnoxon
0.78 0.77 0 76 0.76 0.75 0.74 0.73 0.73 0.72 0.72 0 71 0.70 0.69 0.68 0 68 0.67 0.66 0 65 Nonocrotophos
0.66 0.66 0.67 0 67 0.67 0.67 0.68 0.68 0.68 0.68 0.69 0.69 0.69 0.69 0.70 0.70 0.70 0.71 Zytron
0.74 0.74 0.74 0 74 0.75 0.75 0.75 0.75 0.7j 0.75 0.75 0.75 0.76 0.76 0.76 0.76 0.76 0.76 ChlorpyrifOS
0 74 0.74 0 74 0.75 0.75 0.75 0.76 0.76 0.77 0.77 0.77 0.78 0.78 0.78 0.79 0.79 0.79 0 80 Methyl Parathlon
0.79 0.79 0.79 0 79 0.79 0 79 0.79 0.80 0 80 0.80 0.80 0.80 0.80 0.80 0 81 0.81 0.81 O 81 Methyl Paraoxon
0.93 0.93 0.92 0.92 0.91 0.91 0.91 0.90 0.90 0.89 0.89 0.89 0.88 0.86 0.87 0.87 0.B7 0 86 Malaoxon
0 92 0.92 0.92 0.92 0.91 0.91 0 91 0.91 0.91 0.90 0.90 0.90 0.90 0.90 0.89 0.89 0.89 0.89 Halathion
0.85 0.85 0 86 0.86 0 86 0.86 0.87 0.87 0.87 0.87 0.88 0.88 0.88 0.88 0.89 0.89 0.69 0.90 Bromophos
0 90 0 90 0.90 0.90 0.91 0 91 0.91 0.91 0 91 0.91 0.91 0.91 0.90 0.90 0.90 0.90 0.90 0.90 Fenthlon .
0 90 0.90 0 90 0 90 0 91 0.91 0.91 0.91 0.91 0.91 0 91 0.91 0.92 0.92 0.92 0.92 0.92 0 92 Fenltrothion
1.02 1.01 1.01 1.00 1.00 1.00 0.99 0.99 0.98 0.98 0.98 0.97 0.97 0.96 0.96 0.96 0.95 0.95 Phospiiamidon
1.25 1.23 1.21 1.19 1.18 1.16 1.14 1.13 1.11 1.09 1.07 1 .06 1.04 1.02 1.00 0.99 0.97 0.95 Schradon
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.0Q 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 ParathlonU.f.rw.)
1 06 1.06 1.06 1.06 1 05 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.04 1.04 1.04 1.04 1.04 1.04 Ethyl Paraoxon
1.05 1.05 1.05 1.05 1.06 1.06 1.06 1.06 1.06 1.06 1.05 1.05 1.05^ 1.05 1.04 1.04 1.04 1 03 Dicapthon
1.12 1.12 1.12 1.11 1.11 1.11 1.11 1.11 1.11 1.1Q 1.1Q 1.1Q 1.10 l.lQ 1.09 1 09 1.09 1.09 Branophos Ethyl
1.23 1.22 1.22 1.22 1.21 1.20 1.20 1 19 1 19 1.19 1.19 1.19 1 20 1.20 1.20 1.20 1.20 1.20 AimdUhion
1 40 1.39 1.38 1.38 1.37 1.36 1.35 1.34 1.33 1.32 1.32 1.31 1.30 1.29 1.28 1.27 1.26 1.25 Crufomate
1.36 1.35 1.35 1.34 1.34 1.33 1.33 1.32 1.32 1.31 1.31 1.30 1.30 1.29 1.29 1.28 1.27 1 27 Phenthoate
1,51
1.50
1.49
1.49
1.48
1.47
1.46
1.45
1.44
1.43
1.43
1.4?
1.41
1.40
1.39
1 38
1.37
1 36
1.51
1.50
1.49
1.49
1 48
1.47
1.46
1.45
1.44
1.44
1.43
1 42
1.41
1.41
1.40
1.39
1,38
1,37
1.57
1.57
1.56
1 56
1.56
1.55
1.54
1.54
1.53
1.53
1.53
1.52
1.52
1.51
1.51
1.50
1.50
1.49
1.72
1.71
1.70
1.69
1.68
1.67
1.66
1.65
1.65
1.64
1.63
1 62
1.61
1.60
1.59
1.58
1.57
1.56
1.74
1.73
1.73
1.72
1.72
1.71
1.71
1.70
1 70
1.69
1.69
1.68
1.68
1 67
1.67
1.66
1.65
1 65
2.69
2.67
2.64
2.62
2.59
2.57
2.54
2.52
2.49
2.46
2.45
2 42
2.40
2.37
2.35
2.32
2.30
2.27
2.88
2.85
2.82
2.79
2.76
2.73
2.70
2.67
2.64
2.61
€.58
2.55
2.52
2 49
2.46
2.43
2.40
2.37
2.99
2.97
2.94
2.92
2.89
2.87
2.84
2.62
2.79
2.77
2.75
2.72
2.70
2.67
2 66
2.62
2.60
2.57
Tetrachlorvlnphos
Meth1dath1on
Carbophenoxon
Ethlon
CarbophenotMon
4.65 4.60 4.56 4.SI 4.46 4.42 4.37 4,32 4.27 4.23 4.18 4 13 4.08 4.04 3.99 3,94 3.89 3.85 Fensulfothlon
5.57 5.50 5.42 5.35 5.27 5.20 5.12 5,05 4.97 4,90 4.82 4.75 4.67 4.60 4.52 4.45 4.38 4 30 Phenkapton
6.07 5.99 5.90 5.82 5.74 5.65 5.57 5.49 5 40 5.32 5.24 5.15 5,07 4.99 4.90 4.82 4.74 4.65 Famphur
6.63 6.53 6.44 6.34 6.25 6.16 6.06 5.97 5.88 5.78 5.69v 5.59 5.50 5.41 5.31 5*22 5.12 5.03 EPN
7.95 7.84 7.72 7.61 7.50 7.38 7.27 7.15 7.04 6.93 6.81 6.70 6.58 6.50 6.36 6.24 6.13 6.01 Imldan
10.9 10.7 10.6 10.4 10.3 10.1 10.0 9.8 9.7 9.5 9.3 9.1 8.9 8.7 8.5 8.3 8.1 7.9 Azlnphos Hethyl
14.4 14.1 13.9 13.6 13.3 13.0 12 8 12.5 12.2 11.9 11.7 11.4 11.1 10.9 10.6 10.3 10.1 9.8 Azlnphos ethyl
22.2 21.8 21.3 20.9 20.4 20.0 19.5 19.1 18.6 18.2 17.2 17.3 16.8 16.4 15.9 15.5 15.0 14.6 Coumaphos
~l 1 1 1 1 1 I i I 1 1—i 1 1 1 f—I r-
n
174 178 162 166 1$0 194 198 'W 204
Retention ratios, relative to ethyl parathlon, of 54 organophosphorous pesticides on a column of
1.51 0V-17/1.952 0V-21Q at temperatures from 170 to 204®C; column support of Gas Chrom-Q, 100/120
mesh; flame photometric detector, 5260 A° filter; all absolute retentions measured from injection
point. Arrow Indicates optimum column operation temperature with carrier flow at 70 ml per minute.
-------
11/1/72
Section 4,B,(5)
FIGURE 1
Carbowax Tube Section
10°o Carbowax
t f
Glass WocH '
FIGURE 2
Cutaway View of Column with Carbowax Assembly
-------
Section 4,B,(5)
11/1/72
FIHIiRR 3
Chromatograms of a mixture of 7 organophosphorous pesticides
on an untreated column of 4% SE-30/6% QF-1 (Fig.l),and on the
same column treated with Carbowax(Fig,2)
Column:4% SE-30/6% QF-l;amps,fuil scale 0.8 xlO -8 ;voltage 850 v.
OPERATING PARAMETERS
TEMP., C.
Column 200
Inlet 225
Detector 195
Transf.line 235
Vent 235
FLOW RATES,ml/min.
Carrier
Vent
Oxygen
Hydrogen
Air
60
60
30
180
40
M
S
tfl
-------
11/1/72
Section 4,B,(5)
Figure 4. Calculation of Signal/Noise Ratio
-------
Revised 12/2/74 Section 5,A, (1)
Page 1
ANALYSIS OF 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., Qnley, J. H., and Gaither, R. A., J.A.O.A.C.
46, 186-191 1963.
H. PRINCIPLE:
A 5 g. sample is dry macerated with sand and Na2S0. 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.
2. Aluminum foil, household type.
3. Beakers, 250 ml, stainless steel or heavy duty glass.
4. Beakers, 250 ml, Griffin low fonri.
* This method, with appropriate modifications, may be used for the analysis
of other tissues if original sample size is adequate.
-------
Revised 12/2/74
- 2 -
Section 5,A, (1)
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. Kudema-Danish concentrator fitted with grad. evaporative con-
centrator tube. Available from the Kontes Glass Company, each
component bearing the following stock numbers:
a. Flask, 500 ml, stock #K-570001
b. Snyder Column, 3-ball, stock #K-503000
c. Steel springs, 1/2", stock #K-662750
d. Concentrator tubes, 10 ml, size 1025, stock #K-570050
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 stabilizer,
and it is therefore unnecessary to add ethanol unless peroxides are
found and removed.
-------
Revised 12/2/74
Section 5,A, (1)
- 3 -
NOTE: To determine the absence of peroxides in the ether,
add 1 ml of freshly prepared 10% K1 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 ether and anhydrous sodium
sulfate (10"25 g) is added to remove moisture.
4. Eluting mixture, 151 (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.
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 insure unifomity 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 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 detemme their own pesticide recovery and
elution pattern on each new lot received, as environ-
mental conditions in the various laboratories may
differ somewhat from that in 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)
- 4 -
6. Acetonitrile, reagent grade, saturated with pet. ether.
NOTE: Occasional lots of CH3CN are impure and require redis-
tillation. Generally, vapors from impure acetoni-
trile will turn litmus paper blue when the moistened
paper is held over the mouth of the bottle.
7. Anhydrous sodium sulfate, reagent grade granular, Mallinkrodt stock
#8024 or the equivalent.
NOTE: When each new bottle is opened, it should be tested
for contaminants that will produce peaks by Electron
Capture Gas Liquid Chromatography. This may be done
by transferring ca 10 grams to a 125 ml Erlenmeyer
flask, adding 50 ml pet. ether, stoppering and shaking
vigorously for 1 minute. Decant extract into a 100 ml
beaker and evaporate down to ca 5 ml. Inject 5 yl
into the Gas Liquid Chromatograph and observe chroma-
togram for contaminants. When impurities are found,
it is necessary to remove them by extraction. This
may be done by using hexane in a continuously cycling
Soxhlet extraction apparatus or by several successive
rinses with hexane in a beaker. The material is then
dried in an oven and kept in a glass-stoppered container.
8. Sodium Chloride solution, 2%, from reagent grade NaCl.
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)
- 5 -
VI. SAMPLE PREPARATION § EXTRACTION:
1. On a cupped sheet of lightweight aluminum foil, weigh 5 grams 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) providing 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 Na2S04 to give a uniform, dry granular mass.
4. Add 50 ml of pet. ether and warm carefully on a water bath with con-
tinuous stirring until solvent boils gently.
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 rinse beaker and
paper with a final 10 ml of pet. ether.
8. Place beaker on a 40°C water bath and evaporate just to dryness
under a stream of nitrogen. Check odor to be sure all solvent is
removed and allow to cool to room temperature in a dessicator.
9. Weigh beaker and record for calculation of percent fat in the sample.
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.
-------
Revised 12/2/74
Section 5,A,(1)
- 6 -
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 NaCl 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 Na2S0. in a 150 x 24
mm filter tube and position over a 500-ml K-D evaporltor 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.
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.
-------
Revised 12/2/74
Section 5,A,(1)
- 7 -
VIII. FLORISIL FRACTIONATION:
1. Prepare a chromatographic column containing 4 inches (after
settling) of activated Florisil topped with 1/2 inch of anhydrous,^
granular Na^SO^. 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 over, 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 com-
pletely cover the Na2S0^ layer).
NOTE: From this point and through the elution process, the
solvent level should never be allowed to go below
the top of the Na-SO^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 transfer
the tissue extract (ca 5 ml) from the evaporator tube onto the
column and permit it to percolate through.
4. Rinse tube with two successive 5-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 61
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 Na2SO^ layer, place a second 500-ml Kuderna-Danish
assembly under the column and continue elution with 200 ml of 151
diethyl ether in pet. ether -(Fraction II).
-------
Revised 12/2/74
Section 5,A, (1)
- 8 -
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 Kudema-Danish flask and carefully
rinse joint with a little hexane.
10. Attach modified micro-Snyder column to collection tubes, place tubes
back in water bath and concentrate extracts to 1 ml. If preferred,
this may be done at room temperature under a stream of nitrogen.
11. Remove from bath, and cool to anibient 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 chromatographic
column using vacuum to pack. Prewash with ca 40 ml pet. ether,
discard prewash and place a Kuderna-Danish receiver under column.
Transfer concentrated Florisil eluate to column using small portions
of pet. ether. Force sample and washings into the MgO-Celite
mixture by slight air pressure and elute column with 100 ml pet.
ether. Concentrate to a suitable volume and proceed with Gas
Liquid Chromatography.
NOTE: Standard Recoveries should be made through column
to ensure quantitative recoveries.
IX. ASSESSMENT OF EXTRACT CONCENTRATION:
1. Inject 5 yl of each fraction into the gas chromatograph for the
purpose of detennining 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 61 fraction.
2. If off-scale peaks are obtained in either fraction it will be
necessary to dilute volumetrically with hexane to obtain a
-------
Revised 12/2/74
Section 5,A, (1)
- 9 -
concentration that will permit quantitation 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 con-
centration.
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 appro-
priate volume in a separatory funnel and wash it twice with portions
of water equal to about 1/2 the volume of ether. The washed ether
is shaken with 50 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 sulphate and
shaken vigorously on a mechanical shaker for 15 minutes. This
treatment should not,be attempted on ether containing ethanol, as
the amount of ethanol that would remain is indeterminate.
5. If the presence of malathion is suspected, it is necessary to pass
200 ml of 50% diethyl ether in pet. ether through the Florisil
column into a third K-D evaporator assembly, concentrating the
el'uate as described for the 6% and 15% eluates.
6. Table 1 gives the elution pattern for a number of common pesticides.
On occasion it may be observed that a portion of a given compound
-------
Revised 12/2/74
Section 5,A,(1)
- 10 -
may elute into a different fraction than the one given. For
example, some operators have difficultly 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 21 (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 quantitation 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 recoveries.
Recovery efficiency should be predetermined on standard mixtures
containing the specific compounds of interest. If low recoveries
are obtained, it may be necessary to try an alternate manufacturer's
pet. ether.
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 12,D,(2) since recoveries by the method described in
this section (5,A,1) 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 con-
firmation 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 r.p.m.
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 sub-
section VII.
-------
Revised 12/2/74
Section 5,A, (1)
- 11 -
TABLE 1. ELUTION PATTERNS AND RECOVERY DATA OF A COMPILATION OF
PESTICIDES
INTRODUCTION:
The data contained in the following table was copied in part from
Volume I of the Manual of Pesticide Analytical Methods of the U.S. Food
§ Drug Administration. It's reproduction here is intended to provide the
reader with the elution characteristics and recovery potential of many
pesticidal compounds in addition to those included which are normally
found in adipose tissue.
The elution behavior and recovery data for many of these compounds
were obtained from fatty foods, but because of the similarity of the
extraction and Florisil partitioning steps, it would be expected that
final eluants would be very similar or identical in human or animal fat.
Circumstances under which the data were obtained vary 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 in many laboratories.
When complete data on the behavior of a compound are unavailable,
the available data are given and the missing information is pointed out.
Available information is presented on elution of compounds from the
Florisil column with additional eluates in cases where the 61 and 15%
ethyl ether/pet. ether eluates 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.
Approximate percent recovery expected is given in parentheses, when
known.
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.
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Revised 12/2/74
Section 5,A,(1)
- 12 -
Florisil Elution Notations:
1. Percentages in this column refer to percent ethyl ether in
pet. ether eluants in 200 ml portions. Unless otherwise indi-
cated, 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) indi-
cates that the information was obtained during testing of Florisil
elution only.
3.
Appearance of appropriate eluant alone indicates that the infor-
mation was obtained from the entire procedure.
-------
- 13 -
Method
Florisil
Method
Florisil
Compound
Recovery
Elution
Compound
Recovery
Elution
Alachlor
ND
NR,6,15
Dicapthon
ND
15
Aldrin
C
6
Dichlobenil
ND
15
Allidochlor
ND
NR.6,15
Dichlofenthion
ND
o
Anilazine(Dyrene)
P
15
Dichlone
ND
NR,6,15
Aramite
NR
P, 15
Dichloran
ND
P,15;C,15+20
Atrazine
ND
C,50
"Dicofol
P
6,15
Azinphos ethyl
ND
50
Dieldrin
C
15
Azinphos methyl
ND
NR,6,15
Dilan
P(65)
15,50
Balan
ND
6
Dinocap
ND
P,15
«-BHC
C
6
Disulfoton
ND
6
6-BHC
C
6
Diuron
ND
C,65
y-bhc
C
6
Dyfonate
ND
6
6-BHC
C
6
Endosulfan I -
C
15
Binapacryl
ND
P, 15
Endosulfan II
C
15,30
Bomyl
ND
Endosulfan sul-
Bromophos ethyl
ND
6
fate
C
50
Bulan
P (75)
15
Endrin
C
15
Captafol
ND
NR,6,15
Endrin alcohol
C
C,6,15+20 or
Captan
ND
NR.6,15
Endrin aldehyde
C
C,6,15+20 or
Carbophenothion
P(60)
6
Endrin (delta
CDEC
C
6
Keto 153)
C
25
Chlorbenside
C
6
EPN
C
15
Chlordane
C
6
Ethion
C
6
Chlordecone
P
P,15,50
Ethaprop
C
6,15,50
Chlorfenvinphos
ND
Fenitrothion
ND
15
Chlorbenzilate
P (80)
15,30
Fenthion
ND
6,15
Chloroneb
ND
6
Folpet
P
C.15+20
Chloronitropropane
ND
NR,6,15
Heptachlor
C
6
Chloropropylate
ND
15
Hept. Epoxide
C
6
Chlorothalonil
ND
NR,6,15
Hexachlorobenzene P(60)
6
Chlorpropham
C
15
Hexachlorophene
ND
NR,6,15,50
Chlorpyrifos
ND
6
Isodrin
ND
6
Coumaphos
ND
NR,6,15,30
Malathion
ND
50
2,4-D(BOEE)
P
15
Methidathion
ND
50
2,4-D(BE)
P(10)
15
Methoxychlor
C
6
2,4-D(EHE)
C
15
Methyl Carbo-
2,4-D(IBE)
C
15
phenothion
ND
15
2,4-D(I0E)
P(75)
15
Methyl Parathion
C
15
2,4-D(IPE)
P (65)
15
Mirex
P (70)
6
DCPA
P
15
Monuron
ND
C,65
DDE-o,p'
C
6
Neburon
ND
NR,6,15,30
DDE-p,p'
C
6
Nitralin
ND
P(50),50
DDD-o,p'
c
6
Nitrofen
ND
15
DDD-p,p'
c
6
Ovex
C
15
DDT-o,p'
c
6
Oxychlordane
C
6
DDT-p.p'
c
6
Parathion
C
15
DEF
ND
50
PCNB
C
6
Dialifor
ND
15
Perthane
C
6
Diazinon
C
15
Perthane olefin
C
6
-------
- 14 -
Method
Florisil
Compound
Recovery
Elution
Phencapton
ND
6
Phorate
P(80)
6
Phosalone
ND
50
Photodieldrin
C
15,trai
Polychlorinated
C
6
biphenyls
Polychlorinated
P(65)
6,15
naphthalenes
Prolan
P (25)
15
Prometryn
ND
P(67) ,50
Propachlor
ND
NR,6,15
Propanil
ND
NR,6,15
Propazine
ND
C,50
Ronnel
C
6
Simazine
ND
C,50
Strobane
C
6
Sulfotep
ND
6
Sulphenone
ND
20,25
2,4,5-T(BE)
P
15,30
2,4,5-T(BOEE)
P
15
2,4,5-T(IOE)
C
15
2,4,5-T(IPE)
C
15
Telodrin
C
6
Terbacil
ND
NR,6,15
Tetradifon
C
15
Tetrasul
ND
6
Toxaphene
C
6
T richlorobenzenes
ND
6
Trifluralin
C
6
Zytron
ND
6
-------
Revised 12/2/74
Section 5,A, (2), (a)§(b)
Page 1
MICRO METHOD FOR THE DETERMINATION OF CHLORINATED
PESTICIDES IN HUMAN OR ANIMAL TISSUE AND HUMAN MILK
I. 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.
II. PRINCIPLE:
A 0.5 gram sample of tissue is macerated in a micro tissue grinder
with acetonitrile. An aqueous solution of Na2SO. is .added, the pesti-
cides 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 overnight before using. For routine
work it is convenient to prepare a large number of columns at one
time.
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 microliters and inject 5 microliters 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.
-------
Revised 12/2/74
- 2 -
Section 5,A, (2) ,(a)§(b)
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 r.p.m.
8. EVAPORATIVE CONCENTRATOR:
Complete with modified micro Snyder column, 1 joint 19/22, Kontes Cat.
No. K-569250.
9. CONCENTRATOR TUBE:
Size 1025, Kontes Cat. No. K-570050.
10. CONCENTRATOR TUBE:
Size 2525, Special Order, Kontes Cat. No. K-570050.
11. TEST TUBE:
25 ml with 5 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.
-------
Revised 12/2/74
Section 5,A,(2),(a)§(b)
- 3 -
2. Centrifuge and pour supernatant into a 50-ml round bottom test tube.
Repeat extraction twice more, collecting supernatants 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 microliters 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 sub-
section IV, to the top of the Florisil column with the aid of a
disposable pipet fitted with a rubber bulb. Begin immediate col-
lection of eluate in a 25 ml capacity concentrator tube.
4. Rinse the 10 ml concentrator tube with 0.25 ml of hexane trans-
ferring 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, o,p'-Dnr, and p,p'-DDT.
6. Collect a second fraction by eluting with a second 12 ml portion of
1% methanol in hexane. This fraction will contain dieldrin, heptachlor
epoxide, endrin, B-BHC, Lindane, and p,pT-DDD. (See Table 1.)
NOTE: A small amount of 3-BHC, Lindane, and/or p,p'-DDD may
appear in the first fraction.
* J. Burke et al., J.A.O.A.C. 49 (5): 999-1003, 1966.
-------
Revised 12/2/74
Section 5,A, (2), (a)f|(b)
- 4 -
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
microliters, respectively, and proceed with the GLC portion as
outlined in subsection VII.
VI. ANALYSIS OF BRAIN:
Proceed with steps (1) thru (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% Na2S0^ and extract with 2 to 3 ml portions of hexane.
8. Concentrate the combined extracts to 300 pi in a 10 ml evaporative
concentrator fitted with a modified micro Snyder column using a
glass bead for a boiling chip.
9. Proceed as described under V. FLORISIL FRACTIONATION.
VII. ANALYSIS OF HUMAN MILK
The basics of this procedure have been determined by experience
in a laboratory conducting intensive surveillance to be wholly appli-
cable 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 pre-
cautionary 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.
-------
Revised 12/2/74
- 5 -
Section 5 ,A, (2), (a)§(b)
3. In countries where the use of DUT is peimitted 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 nl 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)
- 6 -
Table 1. Elution pattern of some common chlorinated and organo-
phosphorous pesticides on micro Florisil column.
Compound
12 ml hexane +
12 ml 1% methanol
in Hexane
Fraction I
Additional 12
ml 1% methanol
in Hexane
Fraction II
Aldrin
X
cc-BHC
X
X
g-BHC
X
y-BHC
X
X
6-BHC
X
DDA, methyl ester
X
o,p'-DDD
X
X
p,p'-DDD
X
X
o,p'-DDE
X
p,p'-DDE
X
o,p»-DDT
X
p.p'-DEfT
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
Paradi ch1orob enzophenone
X
Polychlorinated biphenyls
X
Ronnel
X
Toxaphene
X
X
-------
Revised 12/2/74
Section 5, A, (3),(a)
Page 1
ANALYSIS OF HUMAN BLOOD OR SERUM
INTRODUCTION:
Because of its availability and probable diagnostic value with
regard to extent of both chronic and acute exposure to chlorinated
hydrocarbon and other classes of pesticides, blood specimens present
a convenient tissue for study, providing meaningful data pertinent
to the Community Study and Monitoring laboratory program. Of several
methods available in the literature, the Dale, et al (1966) method
provided some desirable features in rapidity, simplicity and sensi-
tivity 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 deteimination
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 derivati-
zation 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 millilliters of hexane
in a round-bottom tube. The extraction is conducted for 2 hours on a
slow-speed rotating mixer. The foimation of emulsion is unlikely, but
if it should occur, centrifugation may be used to effect separation of
the layers. A 5- ml aliquot of the hexane layer is quantitatively trans-
ferred to an evaporative concentrator tube to which is affixed a modi-
fied micro-Snyder column. The extract is concentrated in a water or
steam bath, and the final volume is adjusted to correspond to the
expected concentration of the pesticide residue. A suitable aliquot is
analyzed by electron capture gas chromatography.
-------
Revised 4/8/75 Section 5,A,(3),(a)
- 2 -
III. APPARATUS AND REAGENTS:
1. A rotary mixer so designed as to accomodate 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. Recom-
mended 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 J 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 Coming 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 4/8/75
Section 5,A,(3),(a)
- 3 -
Place whole blood sample in the refrigerator for about 30 minutes
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 r.p.m. Whether or not the analysis is to be conducted
inmediately, it is desirable at this point to transfer the 2 ml
sample aliquot to the 16 x 125 mm culture tube used for extraction.
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 if to a month without undue effects on the chlorinated pesti-
cides 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 any flocculent or
sedimentary material, it is strongly recommended
that the sample be centrifuged ca 5 minutes @ 2,000
r.p.m. before pipetting the 2 ml aliquot. Failure
to observe this point may result in poor repro-
ducibility 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 r.p.m. and rotate for 2 hours.
NOTES: (1) This speed may vary from 50 to 44 r.p.m.
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 r.p.m. 4 to 5 minutes, or longer
if necessary, to effect sufficient separation
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 particular sample. When
working with general population blood of low pesticide levels,
-------
Revised 12/2/74
Section 5,A,(3),(a)
- 4 -
it may be necessary to evaporate to ca 0.5 ml.
NOTES: (1) With some experience the operator can com-
plete 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 observation of boil agitation.
(2) Up to six tubes of extract may be evaporated
simultaneously by using the special rack shown
in Fig. 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 determined by a
preliminary analysis of the 5-ml aliquot.
5. 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 minimal dilution is required after evapo-
ration, 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 pipet for total volumes up to 3 ml.
For larger volumes, use a 5 ml Mohr pipet,
carefully measuring the volume of hexane
delivered. Above the 1 ml graduation mark, the
concentrator tube calibrations are not suf-
ficiently accurate for use in this analysis.
It is also good practice to check the graduation
marks up to 1 ml for all concentrator tubes used
in this analysis.
6. 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.
7. Proceed with electron capture GLC observing the guidelines set
forth in Section 4,A,(4).
-------
Revised 12/2/74
Section 5,A, (3),(a)
- 5 -
VI. CALCULATIONS:
The following equation is applicable when all volumes specified
in the method are followed precisely, with no exceptions:
p.p.b. =
a b x
c y
x 0.6
where
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
Example: 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 pi
volume of final extract injected = 5 pi
p.p.b.
0.3 x 80 x 1000
90 x 5
x 0.6 = 32 p.p.b,
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 know, however. If such
deviation must be made, the final calculation m?o be accom-
plished by using the following basic equation:
p.p.b. =
a b e
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.
-------
Revised 12/2/74
Section 5,A,(3),(a)
- 6 -
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
Dilution factor (e) = —^ g
i 0.3 x 61.5 x 250 __
P'P'b- " 90 x-l.d = 32
0.3
61.5
mm
90
mm
1.6
5
1,000
vl
4
ml
5
yl
= 250
VII. REPORTING LIMITS - DETECTABILITY:
The Analytical Chemistry Committee has established the following
minimum reporting limits for chlorinated pesticides in serum:
g-BHC, lindane, aldrin, heptachlor, heptachlor epoxide,
o,p'-DDE, p,p'-DDE, dieldrin 1 part per billion.
Endrin, o,p'-DDr, p,p'-DDD, p,p'-Dnr 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 indicated,
the following steps are taken: -
1. Measure 50 ml of serum into a 1 L sep. funnel containing 190 ml of
CH^CN, 200 ml of aqueous 2% Na2SO^ 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-inch column of anhydrous ^^SO^
into a 500 ml Kuderna-Danish flask fitted with a 10-ml grad., evap.
concentrator tube containing one 3-ran glass bead.
-------
Revised 12/2/74
Section 5,A,(3),(a)
- 7 -
4. Add another 50-ml portion of hexane to the aqueous solution in the
second 1-L separator; stopper and shake vigorously another two
minutes. When layers have separated, draw aqueous layer back into
the first 1 L separator and percolate the hexane layer through the
Na2S0. 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 -8- Section 5,A,(3),(a)
Figure 1. ROTO-RACK^ Mixer, variable speed
Figure 2„ Evaporative concentrator tube holder, 6-place, stainless steel
%Sr* 7^£ac/C SO**/
TZAoj oj
rtf/eoAs-i!
-------
Revised 12/2/74
Section 5,A,(3),(b)
Page 1
DETERMINATION OF PENTACHLOROPHENOL (RAPID METHOD)
IN BLOOD a URINE
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. Hie
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, J. B., Gas Chromatographic Deteimination of
PCP in Human Blood and Urine, Bull, of Envir. Contam.
5 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.
H. PRINCIPLES:
A rapid method is described for the determination of PCP based upon
its conversion to a methyl ether after a 2-hour extraction of the acidi-
fied sample in benzene. GLC, E.C. is utilized for quantitation, comparing
sample peak against peaks from known standards, similarly methylated.
III. APPARATUS:
1. Gas chromatograph with E.C. detection, fitted with either or both
columns of 4% SE-30/61 QF-1 and 5% OV-210. The 1.5% OV-17/1.953 QF-1
should not be used.
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.
-------
Revised 12/2/74
Section 5,A, (3),Cb)
- 2 -
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 r.p.m.
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, conc., 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 close
adherence to manufacturers' 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 on
storage. The volume prepared should not be greatly in
excess of that required. The original hexane-diazoalkane
-------
Revised 12/2/74
Section 5,A,(3),(b)
- 3 -
generating solution should not be stored'in ground
glass stoppered containers nor in bottles with
visible interior 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 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/pl.
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/pl solution to alkylating
reagent should be maintained. The working standards, in a range
of 10 to 30 pg/pl, are prepared by diluting the derivatized stock
with isooctane.
V. SAMPLING:
Extreme care and precautionary measures should be taken to insure
freedom of the sample from 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.
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).
-------
Revised 12/2/74
Section 5,A, (3),(b)
- 4 -
2. Add 2 drops of conc. f^SO^, seal tightly with Teflon-lined screw cap,
and rotate for 2 hours at 50 r.p.m. on the "Roto-Rack".
NOTE: If, after the extraction period, the layers do not
separate completely, centrifuge 5 minutes at 2,000
r.p.m.
3. Transfer 3 ml of the benzene (upper) layer to a 10-ml vol. flask
and proceed with methylation.
Methylation
1. Add 0.3 ml of the methylating reagent (TV., 7.), stopper flask and
mix on Vortex 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 chromatogxaph of 5 ul 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, blood serum levels
in excess of 100 ppb are not uncommon (for example, the Dade County,
(Miami) framing of all dwellings).
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 point whether
general population blood in these northern areas might contain PCP
residues approaching or less than the stated minimum detectability of
this method. Should this prove to be the case in any laboratory, a
modification of the method contained in this manual for PCP in urine
[5,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. Further-
more, using hexane as the extracting solvent, it seems probable
that less extraneous materials would be extracted.
-------
Revised 12/2/74
Section 5,A,(3),(b)
- 5 -
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 QV-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 NcOH solution followed by
deionized water and acetone rinsed. Care should be taken not to
permit contact between wooden or paper materials and glassware,
as peg boards and some brands of absorbent paper products have
been found to contain PCP.
5. If the recommended volumes are used, calculations are simplified
and are as follows:
PCP(in ppb) in serum = pg/yl injected times 10.
-------
Revised 11/1/72 Section 5,A,(3),(b)
- 6 -
TABLE I. PERCENT RECOVERY OF PCP FRCM SAMPLES FORTIFIED BEFORE EXTRACTION
Sample PCP Found,ppm PCP Added,ppm PCP Recovered ,ppm Recovery %
Blood Plasma 0.19
Urine 0.01a
0.50
0.67
96
0.65
92
0.68
98
5.00
4.58
88
4.70
90
4.70
90
4.70
90
50.0
46.9
93
48.5
97
0.50
42.0
0.46
84
Mean 92b
90
0.50
98
0.48
94
5.00
4.86
97
4.70
94
4.89
98
50.0
49.0
98
49.8
100
49.8
100
Mean 96°
^Limit of detectability for blood and urine.
Standard deviation, blood plasma — t4.5%
cStandard deviation, urine — tZ.5%
-------
Revised 12/2/74
Section 5,A,(4),(a)
Page 1
THE DETERMINATION OF PENTACHLOROPHENOL IN URINE OR WATER
I. INTRODUCTION:
Pentachlorophenol and its sodium and copper salts are exten-
sively used as wood dressings, aquatic and terrestrial herbicides,
and antimicrobics. Human exposure may occur through several routes,
including inhalation of dusts, dermal adsorption of powders and
solutions as well as ingestion of residues present in food and water.
Due to the aqueous solubility of PCP salts, a likely human elimination
route is the urinary system, providing a convenient monitor of PCP
exposure. The following method describes the preparation, gas
chromatographic determination and p-value confirmation of the methyl
ether derivative of PCP isolated from human urine.
REFERENCE: Cranmer, M. F., and Freal, J. F., Gas Chromatographic
Analysis of Pentachlorophenol in Human Urine by
Formation of Alkyl Ethers. Bull. Envir. Contamin.
and Toxicology, 9, Part II, 121-128, 1970.
II. PRINCIPLES:
A sample of human urine is made alkaline and extracted with
hexane to remove basic and neutral interfering compounds. The
alkaline urine is then acidified and re-extracted with hexane. The
PCP residue contained in a portion of the hexane extract is deriva-
tized with diazomethane and the alkylated PCP determined by gas
chromatography with electron capture detection. Quantitation is made
by comparing sample PCP-ether peaks with peaks produced from standard
solutions of PCP-ether. Confiimation can be achieved by p-value
determinations as well as GLC retention values on several columns.
Several additional alkyl ether derivatives of PCP can be prepared
for further confirmation.
III. APPARATUS $ REAGENTS:
1. Gas chromatograph equipped with electron capture detector.
Recommended columns and operating parameters are given in
Section 4, A.
2. Vortex-Genie mixer, model 550-G or its equivalent.
3. Evaporative concentrator assembly, small scale concentrator
complete with modified micro Snyder column, concentrator tube
and springs. (Kontes Glass Co., Cat. No. K-569250 size 3-19
$ 19/22 ground glass stoppered).
-------
Revised 12/2/74
Section 5,A, (4),(a)
- 2 -
4. Test tubes, 1-ml and 15-ml capacity.
5. Test tubes, conical, centrifuge 5-ml and 15-ml capacity f
ground glass stoppered.
6. Pipets, 0.1-ml, 0.4-ml, and 5-ml capacity.
7. Exhaust hood with a minimum draft of 150 linear feet per minute.
8. Sodium hydroxide, reagent grade, 0.4N solution*.
9. Sodium hydroxide, reagent grade, 20% solution*.
10. Hydrochloric acid, concentrated analytical reagent.
11. N-Methyl-N1-nitro-N-nitrosoguanidine, Aldrich Chemical Co., Inc.,
Milwaukee, Wis.
12. Liazome thane methylating reagent.
Add 5 ml of 201 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-diazo-
methane 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 close adherence to manufacturers 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. 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,
*Refer to MISCELLANEOUS NOTES, Subsection VI,3.
-------
Revised 12/2/74
Section 5,A,(4),(a)
- 3 -
nor in bottles with visible interior etching;
however, no hazard is involved in the 5-ml test
tubes containing the PCP-hexane extract plus
diazoalkane. Extended exposure to air destroys
the diazoalkane reagents.
13. Pentachlorophenol, analytical standard available from Reference
Standards Repository, EPA, Research Triangle Park, N. C.
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 yg/ml.
14. Hexane, pesticide quality.
15. Benzene, pesticide quality.
16. Methanol, pesticide quality.
17. Methanol containing 20% of water.*
18. Acetonitrile, pesticide quality.
19. Dimethyl formamide, Nanograde solvent or its equivalent.
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 particularly 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.
;r to MISCELLANEOUS NOTES in subsection V, 3.
-------
Revised 12/2/74
Section 5,A,(4),(a)
- 4 -
All bottle caps should be Teflon or foil lined. Under no
circumstances should the paper liner of a bottle cap be allowed
to come into direct contact with the sample. Paper lined caps may
be used only if a layer of foil or Teflon is inserted to isolate
sample from paper liner.
V. PROCEDURE:
NOTE: Before starting analysis, the chemist should make certain
that all glassware used in the analysis has been specially
prepared as outlined in MISCELLANEOUS NOTE 3 in subsection
VI. THIS IS MOST IMPORTANT!
Partition and Extraction
1. Transfer 5 ml of urine into a 15 ml glass stoppered centr. tube
and add 0.3 ml of 0.4 N NaOH to alkalize the solution.
NOTE: Because of the widespread prevalence of PCP,
a reagent blank consisting of 5 ml pre-extracted
distilled water (subsection V., 3., page 5)
should be carried through the entire procedure
along with the sample(s).
2. Add 5 ml hexane, stopper tube, and shake vigorously by hand for
one minute.
3. Centrifuge, allow layers to separate and remove and discard
hexane layer by means of a pipet or an S shaped glass syphon tube.
4. Acidify the remaining aqueous layer with 0.2 ml conc. HC1 and
then add exactly 2 ml of hexane (volumetric pipet). Shake on the
Vortex mixer 3 minutes and centrifuge.
Methylation of PCP
1. Pipet 0.4 ml of the hexane extract into a 5 ml glass stoppered
test tube and add 0.1 ml of the diazomethane reagent in hexane.
NOTE: Completely avoid any introduction of water into
the solution or test tube in which the alkylation
is to be carried out. A small amount of water will
quench the reaction.
2. Mix two minutes on the Vortex mixer and allow to stand 20 minutes.
3. Add 1 ml of the methanol containing 20% F^O and mix on the Vortex
-------
Revised 12/2/74
Section 5,A,(4),(a)
5
mixer for 2 minutes; After the phases separate, the hexane
layer is ready for GLC and p-value determination.
Inject a portion, preferably 5 yl, of the methylated PCP
solution into the gas chromatograph, fitted with E. C. detector
and one of the three columns prescribed for the program. Column
operating parameters should be those given in Section 4A, Table 1
except that the flow rate may be reduced to provide for a later
retention than is possible with the carrier flow prescribed for the
column, the methylated PCP peak being an early eluter. Compare
the peaks produced, within the linear range of the detector, with
those produced by injecting a comparable volume of the standard of
the PCP-methyl ether solution (sub-section III, 11).
NOTE: It would be an extreme rarity to find a sample
requiring further concentration of the extract.
Contrarily, it would be expected that many samples
would need dilution. The GLC assay of the 5 yl
injection described above will provide an indica-
tion of the amount of dilution required. This may
be carried out by mixing an aliquot of the extract
with a quantity of hexane measured with a Mohr pipet,
or, if the required dilution is sufficiently high,
a volumetric flask may be used. (See subsection
The expected relative retention values for methylated PCP on the
three program columns operated at prescribed temperatures are as
follows:
Equilibrate each of the following solvents, acetonitrile,
methanol, and dimethyl formamide at a 1:1 v/v ratio with hexane at
room temperature for 24 hours. Pipette 0.1 ml of the 0.5 ml hexane
extract solution containing the PCP-methyl ether into a 1 ml test
tube. Add by means of a pipette, 0.1 ml of the equilibrated solvent
and thoroughly mix the two phases by means of the Vortex mixer for
approximately one minute. After the two phases separate, the upper
hexane layer is ready for GLC analysis. The p-value is calculated
as the concentration of PCP-methyl ether in the hexane phase divided
by the concentration which was determined to be in the hexane prior to
the partition. Determination of p-value from standards in each
laboratory must be carried out at the same time as the unknown since
temperature and other variables affect the partition coefficients.
VI, 7).
1.5% OV-17/1.95%QF-l
4%¦ SE-30/6% QF-1
5% OV-210 --
0.47
0.63
0.56
-------
Revised 12/2/74
Section 5,A,(4),(a)
- 6 -
The expected p-values for the solvent systems given on the
previous page are:
1. Acetonitrile:hexane 0.62
2. Methanol:hexane 0.61
3. Dimethyl formamide:hexane 0.44
VI. MISCELLANEOUS NOTES:
1. Urine samples from individuals representative of the general
population and exposed individuals have been analyzed in replicate
using the described procedure. Replications varied less than +
5% from the mean over the entire range of 2.2 to 265 p.p.b of
PCP found. The Standard deviation decreased as the concentration
of PCP increased.
2. Availability and use of several alkyl ether derivatives of PCP
increases the confidence that a correct identification has been
made. The ethyl ether derivative, for example, can be prepared
by substituting N-ethyl-N'-nitro-N-nitrosoguanidine for the
diazomethane precursor. The analyst is referred to the primary
literature source for GLC retention times,' p-values, and methods
of preparation for six additional PCP-alkyl ethers, including
the ethyl, propyl, isobutyl, iso-amyl and amyl ethers (Cranmer
and Freal, 1969).
3. All reagents including the water to make up the NaOH, HC1 and
MEOH solutions, must be extracted with hexane before use as they
may be contaminated with PCP or other materials which may interfere
with respect to the absence of PCP. Glassware should be washed
with dilute NaOH solution followed by deionized water and acetone
rinses. Care should be taken not to allow contact between wooden
or paper materials and glassware since peg boards and several
brands of absorbent paper products have been found to contain PCP.
4. A single hexane extraction of acidified urine results in 901
extraction of PCP from urine making additional extractions or
extraction with a larger volume unnecessary. Five ml of urine is
the suggested convenient volume to manipulate; however, larger or
smaller volumes of urine can be analyzed if the relative proportion
of reagents to sample is maintained. Efficient extractability
coupled with the requirement of small volumes precludes the necess-
ity for bulky glassware and keeps transfers to a minimim.
-------
Revised 12/2/74
Section 5,A,(4),(a)
- 7 -
5. Urine samples are made basic and extracted in an effort to
remove interfering compounds from the urine. This is done to
insure that the chromatographic determination of PCP is as free
as possible from error and that the gas chromatographic analysis
would not be extended due to the necessity of waiting until late
eluting peaks have cleared the gas chromatographic column. A
direct extraction of acid urine would produce an extract which
contains the neutral chlorinated hydrocarbons normally found in
urine. This problem becomes increasingly important when examining
urine samples representing occupationally exposed individuals
since their urine often contains larger amounts of compounds such
as BHC, and DDT and its neutral metabolites.
6. The relationship between the volume of urine samples and the
amount of acid and base added have been selected to insure that
sufficient quantities are present to allow for urine with different
pH values while excluding the largest amount of interfering material
from the extract. It was found, as reported by Bevenue (1966),
that a more severe treatment of the urine before extraction, i.e.,
refluxing with concentrated acid, does not result in increased
extraction of PCP. If efficient and reproducible extraction of the
urine and quenching of the alkylation reaction mixture is to be
obtained, vigorous mixing in the extraction step and after the
addition of the 20% water in methanol at the quenching step is
required. The use of a Vortex-Genie or similar mixer has proved
to be more satisfactory than the wrist action shakers or agitation
by hand.
7. Dilution of solutions containing the PCP-methyl ether is often
necessary and is conveniently accomplished in the 5 ml test tube
over a five fold range. Concentration has not been necessary
since we have not found a urine sample with less than 2 p.p.b of
PCP present and this concentration results in 40 pg of PCP ether
in a 10 yl injection, which in turn, produces a quantifiable peak.
Calculation of results is facilitated if the suggested volumes are
followed since the number of picograms of PCP derivative per yl
injected divided by 2 is equal to the p.p.b of PCP present in the
urine.
8. The efficiency of the alkylating reaction should be constant over
a wide concentration range of reactant and PCP, five fold for the
diazoalkane and one thousand fold for PCP. Study of the. effect of
the time of alkylation has shown that complete reaction is obtained
at all concentrations in less than fifteen minutes. All reactions
are run at room temperature for safety and convenience. The
suggested concentration of alkylating reagents and the twenty minutes
-------
Revised 12/2/74
Section 5,A,(4),(a)
- 8 -
time of reaction are prescribed to provide the analyst with
a wide safety factor thereby insuring consistency of results.
9. The use of a 20% water/CH^OH wash effectively removes excess
diazoalkane as well as many of the interferences present due
to the alkylating reagent. No loss of PCP ether results from
this treatment and no volume adjustment is necessary as may
happen with evaporation under air or nitrogen jet.
-------
1/4/71
Section S,A,(4),(b)
Page 1
DETERMINATION OF BIS(p-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 p,p'-DDT and p,p'-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 pre-
dominant metabolite of p,p'-DDT, p,p'-DDA, is excreted in the urine.
Excretion levels of this metabolite have been established as sensitive
indicators of exposure to p,p'-DDT (Durham et. al., 196S). However, a
rapid, sensitive gas chromatographic procedure for the analysis of this
metabolite is desirablejparticularly one which gives accurate and precise
data for low levels of p,p' -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 p,p'-DDA excretion
levels.
REFERENCES: Cranmer, M. F., J. J. Carroll, and M. F. Copeland
(1969) Determination of DDT and Metabolites In-
cluding DDA in Human Urine by Gas Chromatography.
Bull. Environ. Contamin. § Toxicol. 4_, 214 (1969).
Cueto, C., A. G. Barnes, and A. M. Mattson, (1956).
Detemiination of DDA in Urine using an Ion Exchange Resin.
J. Agr. Food Chem. £, 943 (1956).
Durham, W. F., J. F. Amstrong, and G. E. Quinby
(1965). DDA Excretion Levels, Arch. Environ. Health
11, 76 (1965).
H. PRINCIPLES:
Each sample of urine is thoroughly mixed with an equal volume of 1%
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 p,p'-DDA to the methyl
ester. After heating at 50°C for 30 minutes, 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 E.C.
detection. An osmolality correction factor is employed in reporting
p,p'-DDA excretion levels.
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Revised 12/2/74
Section 5, A,(4),(b)
-2-
III. EQUIPMENT:
1. Gas chromatograph equipped with E. C. 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 $
19/22 ground glass joint (Kontes Glass Co., Cat. No. K-570050,
size 1025 (10 ml) and size 2525 (25 ml).
8. Modified micro Snyder column, $ 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 pipets, 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 $ glass stoppers, Corning No. 9810 or the
equivalent.
-------
Revised 12/2/74
Section 5,A,(4),(b)
-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 sulphate 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
commercially prepared reagent. Applied Science
laboratories markets a "BF3 METHANOL ESTER KIT"
consisting of 25 x 5 ml ampoules of 14% BF3/Methanol.
(Nano grade methanol will be substituted on request).
-------
Revised 12/2/74
Section 5,A, (4),(b)
-4-
12. Preparation of DDA-ME standard:
1. Weigh out approximately 25 mg of Bis- (p_-chlorophenyl) -
acetic acid (p,p'-DDA) into 25 x 200 mm glass-stoppered
round bottom test tube. (Item 16, subsectionlll)
2. Dissolve the p,p'-DDA, with the aid of a Vortex mixer, in
10.0 ml of BC13-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 soldium 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 dessicator and allow to equilibrate.
9. Reweigh the concentrator tube to determine the amount of
DDA-methyl ester.
°r Prrnvrr" - mg of mA(ME) X 280 inn
< Recovery - *Mg o£ D^ x 294 x 100
10. The DDA-methyl ester is then quantitatively tranferred 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
-------
Revised 12/2/74
Section 5,A,(4),(b)
-5-
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.
In those cases where known or suspected exposure to p,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. PROCEDURE:
A control sample of urine from an unexposed donor should be carried
through the entire procedure parallel with the sample(s) being tested.
Extraction
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 two 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 pipet. The conc. tube is
fitted with a modified micro Snyder column and the extract
is concentrated in a boiling water bath to ca. 2 ml.
-------
Revised 12/2/74
Section 5,A(4),(b)
-6-
b. A urine sajnple 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 beakeT, add a pinch of
anhydrous Na2SC>4 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-inch
column of anhydrous Na2S04 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 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 conc. 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 pipet, 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°C water bath and
reduced just to dryness under a dry nitrogen stream.
-------
Revised 12/2/74
Section 5,A,(4),(b)
-7-
Exterification of Extraction
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 141 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. h^O.
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. concentrator
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.
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 temperature,
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 eluated in the second
fraction and none in the first fraction, the
eluate from the first fraction can be discarded if
DDA is the compound of sole interest.
-------
Revised 12/2/74
Section 4,A,(4),(b)
-8-
2. If a laboratory is running routine determinations
as for surveillance of a special group of donors,
and is conducting gas chromatography by micro-
coulometric detection only, the Florisil cleanup
step may be eliminated. However, the cleanup is
necessary when detection is by electron capture.
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 microliter. A 10 yl
injection should produce a quantifiable peak via E.C. under normal
conditions. An exploratory injection of 25 yl for M.C. detection
will provide the operator with information suggesting a lesser
or greater injection volume to obtain a peak height response of
10% or more f.s.d.
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 p,p'-DDA
methyl ester of known concentration. Correct the observed concentration
levels of p,p'-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 = 1000
Observed Osmolality
3. The only potential pesticide interference to the DDA, methyl ester,
peak in the second fraction eluate would be from dieldrin on the
0V-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 by reason of its greater responsiveness.
The urine from high exposure donors would be expected to contain a
small amount of p,p'-DDE as compared to the DDA levels. When the
OV-17/QF-1 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,(A),(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 extensively
as selective herbicides in the control of terrestrial and aquatic broad-
leaf plants. Because of their widespread use and relatively lengthy
persistence, particularly in treated lakes and streams, potential human
exposure to these materials may occur via several routes. These include
consumption of contaminated edible plants, livestock, and water, as well
as direct exposure by agricultural spraymen and herbicide formulators.
Thus, rapid, sensitive procedures for the detection of the free acids
and chlorinated phenol degradation products in human and animal urine
assumes an important role in the toxicological and environmental
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 E.C. detector fitted with a glass column
6' x 1/4" 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.
4. Distilling column (condenser), 200 mm jacket, fitted with tight
glass stopper at top, Kontes No. 286810.
-------
Revised 12/2/74 Section 5,A,(4),(c)
- 2 -
5. Circulating water pump.
6. Vortex mini-mixer.
7. Evaporative concentrator tubes, grad., 25 ml S 19/22, Kontes
No. 570050.
8. Conical centrifuge tubes, conical, grad., 15 ml with <£ stoppers,
Corning No. 8084 or the equivalent.
9. Disposable pipets, Pasteur, 9-inch.
10. Dry nitrogen. Tank fitted with 2-stage pressure regulator.
11. Volumetric flasks, 50 and 100 ml.
12. Mohr pipets, 0.2-, 0.5- and 5- ml.
13. Transfer (vol) pipets, 1 through 5 ml.
14. An exhaust hood with a minimum draft of 150 linear feet per
minute.
15. Centrifuge capable of 2,000 r.p.m.
IV. REAGENTS:
1. BEnzene, pesticide quality.
2. Hexane, pesticide" quality.
3. Hydrochloric Acid, conc., A.R. Grade.
4. Silica gel, Woelm, Activity Grade I.
NOTE: Dry adsorbent for 48 hours at 170°C and store in a
desiccator. On day of use, deactivate the silica
gel by adding 15 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-nitrosoguanidine, Aldrich Chemical Co.
6. Distilled water. All distilled water used throughout procedure
must be benzene extracted.
-------
Revised 12/2/74
Section 5,A, (4),(c)
- 3 -
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'-nitro-
N-nitrosoguanidine in small portions at a time, mixing
contents of flask after each addition.
d. Decant the hexane layer into a bottle with a Teflon-lined
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 nitrosoguanidine
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 solu-
tions into a single 50-ml vol. flask in the following
volumes:
2,4-D 1.0 ml 2,4,5-T 0.5 ml
c. Add diazoethane dropwise with a disposable pipet until a
definite yellow color persists.
-------
Revised 12/2/74
Section 5,A,(4),(c)
- 4 -
d. Allow solution to stand 15 minutes, then bubble nitrogen
through the solution until yellow color disappears
(ca 5-10 minutes). THIS 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 in nanograms per microliter:
2,4-D 4 2,4,5-T 2
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: Thege alkylated standards should be stored at
-18 C when not in use and discarded after one
month.
V. PROCEDURE:
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. conc. tube.
NOTE: The precise volume is predicated on the expected residue
level.
2. Add dropwise a volume of conc. HC1 equal to 1/5 the volume of
urine,and mix well.
3. Fit a stoppered reflux condenser to the tube and heat in boiling
water bath for one hour, cooling the condenser with circulating
ice water.
4. Remove frcm bath, cool and rinse inside walls and condenser tip
with 3 ml benzene.
-------
Revised 12/2/74
Section 5,A,(4),(c)
- 5 -
5. Mix contents of tube for two minutes on a Vortex set at high
speed and then centrifuge at 2,000 r.p.m.
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 ethylated extract to ca 0.3 ml at room temperature
or on a 40°C water bath under a gentle stream of nitrogen.
Silica Gel Elution Pattern
The elution pattern of the ethylated compounds must be determined
before using the silica gel column for cleanup of the ethylated urine
extracts. The column preparation and elution pattern evaluation is outlined
in the following steps:
a. Place a small wad of glass wool at the bottom of a Chromaflex
column and add 1 gram of the partially deactivated silica gel.
Top this with 1/2 inch of anhydrous, granular ^2804.
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 Na?S0,,, 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 compound
present in the fraction.
-------
Revised 12/2/74
- 6 -
Section 5,A,(4),(c)
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
Silica Gel Sample Cleanup
1. 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.
2. Transfer the concentrated extract to the column, rinsing centri-
fuge 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.
3. Finally, add 10 ml of 60% benzene/hexane followed by 10 ml of
80% benzene/hexane, collecting both these fractions in a single
tube. The ethylated 2,4-D and 2,4,5-T are contained in these
fractions.
NOTE: If the individual analyst has determined that his
elution pattern differs from that given in the
author's table and he is able to obtain a consistent
altered pattern, some appropriate revision in the
elute collection instructions may be indicated.
Gas Chromatography
Inject into the gas chromatograph 5 to 10 yl 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
-------
Revised 11/1/72
Section 5,A,(4),(c)
- 7 -
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 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. Quantitation 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
Recovery Determination
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 tijne to time. Centrifuge
5 minutes at 2,000 rpm and discard benzene (upper) layer. Fortify 5 ml of
control urine with aliquots of 0.1 to 1 ml of the aqueous extract and proceed
as described in subsection V starting at Step 2.
Partitioning
NOTE: Because of differences in ambient temperature and relative
humidity from one laboratory to another, it is imperative
that each laboratory establishes silica gel elution patterns
under local conditions. Should the compounds of interest
elute in a later fraction (i.e., in 100% benzene instead of
60% or 80% benzene-hexane) the percent water added to the
silica gel must be increased by 1% increments until desired
elution pattern is established. If the compounds of interest
elute in an earlier fraction (i.e., in 20% B-H instead of
60% or 80% B-H), the amount of water initially added to silica
gel must be decreased [use spiked control urine, not standard
compounds to determine pattern).
-------
1/4/71
Section 6,A, (1)
Page 1
ORGANOPHOSPHORUS PESTICIDES AND METABOLITES IN HUMAN TISSUES AND EXCRETA
GENERAL COMMENTS
The pesticide residue chemist is sometimes called upon 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 OGP poisoning case, time is of the essence if the chemist: is
to obtain any of the original or parent compound which was ingested or cutaneously
absorbed. The OGP 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 compound, per se, may no
longer be detectable in the sample substrates.
In cases of oral ingestion, if the gastro-intestinal fluid samples are
delivered without delay, identification of the parent compound by electron capture
and flame photometric detection is a relatively simple matter generally requiring
no more than a solvent extraction of the sample and direct injection into the
chromatography
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 supplemen-
tation 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. Ihe 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 sane
selections for assessing exposure to the OGP compounds. For example, a phenolic
metabolite of methyl and ethyl parathion is para nitrophenol. The analytical
method for detection and quantitation 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 various
OGP compounds. These metabolites appear in the urine and may be assayed by the
procedure described in Section 6,A,(2),(a). The OGP ccmpounds 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 seme guidelines for selection
of methodology, not only for the OGP compounds, but for other suspected exposure
to pesticidal compounds.
-------
Revised 12/2/74
Section 6, A, (2),(a)
Page 1
METHOD FOR DETERMINATION OF METABOLITES OR HYDROLYSIS PRODUCTS OF
ORGANOPHOSPHORUS PESTICIDES IN HUMAN URINE
I. INTRODUCTION:
The metabolism and urinary hydrolysis of organophosphorus pesti-
cides in mammals results in the excretion of a variety of alkyl
phosphates. These include the salts of dimethyl or diethyl phosphate,
phosphorothioate and phosphorodithioate. The gas chromatographic
separation and quantitation of such products in urine may be of value
in estimating the extent of exposure to the parent organophosphorus
pesticide. The procedure permits the determination of six major
metabolites and hydrolysis products of organophosphorus pesticides.
In addition, the method possesses high sensitivity and allows for
elimination of interferences from inorganic phosphates.
REFERENCES:
1. Shafik, M. T. and Enos, H. F., 1969. Determination of
Metabolites and Hydrolysis Products of Organophosphorus
Pesticides in Human Blood or Urine. J. Ag. § Food Chem.
17_ (6); 1186.
2. Bowman, M. C., and Beroza, M., 1968, Gas Chromatographic
Detector for Simultaneous Sensing of Phosphorus and Sulfur-
Containing Compounds for Flame Photometry, Anal. Chem. 40;
1448.
3. Shafik, M. T., Bradway, D., Biros, F. J. and Enos, H. F.,
1970. Characterization of Alkylation Products of Diethyl
Phosphorothioate. J. Ag. § Food Chem., 18 (6); 1174.
4. Davies, J. E., Shafik, M. T., Barquet, A., Morgarde, C.,
and Danauskas, J. X., Presentation at 167th Nat. Meeting,
ACS, Los Angeles, April, 1974.
5. Shafik, M. T., and Bradway, D. E., Presentation at 167th
Nat. Meeting, ACS, Los Angeles, April, 1974.
II. PRINCIPLES:
Organophosphate metabolites or hydrolysis products in urine are
extracted with a 1:1 (v/v) mixture of acetonitrile and diethyl ether
after acidification with 6 N HC1. An aliquot of the extraction
solvent containing the dialkyl phosphates is treated with diazopentane
to convert the dialkyl phosphates to the more volatile and gas chroma-
tographable triaklyl phosphates. It is necessary to clean up the
aklyl phosphate derivatives using silica gel chromatography to
-------
Revised 12/2/74
Section 6,A, (2),(a)
- 2 -
eliminate interferences arising from inorganic phosphate and other
materials and to obtain separations of compounds producing closely
adjacent peaks. Determination of trialkyl phosphates is accomplished
by gas chromatography using flame photometric detection with a
phosphorus filter (526 my). The presence of sulfur-containing tri-
alkyl phosphates may be confirmed by use of the flame photometric
detector with a sulfur filter (394 mv).
III. APPARATUS:
1. Dual flame photometric detector, Tracor, Inc., Austin, Texas,
equipped with 526 my filters for the determination of sulfur
and phosphorus compounds, respectively. This detection system
was fitted to a Micro-Tek MT-220 gas chromatograph for the
initial development of this method, but can be adapted for use
with any gas chromatograph.
2. Gas chromatograph modifications. The following modifications are
suggested for operation of the gas chromatograph with flame photo-
metric detection and are generally applicable to any gas chroma-
tograph.
(a) Buckout control on electrometer type E-2 equipped with a
20 megohm Victoreen resistor to reduce detector signal to a
level acceptable to electrometer circuitry.
(b) Valco switching valve #CV 4 HT interfaced between gas chroma-
tographic column and flame photometric detector. The valve
is mounted in a modified block and is coupled with R 100
dead volume Swagelok unions. The heater in the block heats
both the transfer lines and the valve. The valve permits
interchange of column effluent and nitrogen purge. The purge
flow rate is adjusted to equal the flow from the GLC column
so that when an interchange of flows is made for the purpose
of venting solvent, no change is observed in the recorder
baseline. This arrangement prevents the flame from being
extinguished when injections are made.
3. Gas chromatographic column - Borosilicate glass, 6' x 1/4" o.d.
packed with 51 OV-210 on Chromosorb W, H.P., 80/100 mesh.
Prepare and condition column by Carbowax deposition treatment
as described in Section 4, B.
NOTE: An alternate column, which may be used for con-
firmation of identity of peaks, is a 6' x 1/4" o.d.
borosilicate glass column packed with 4% SE-30/6%
OV-210 on 80/100 Gaschrom Q. Condition in a similar
manner.
-------
Revised 12/2/74 Section 6,A,(2),(a)
- 3 -
4. Centrifuge tubes - 15 ml capacity, .grad., conical, with S ground
glass stoppers.
5. Pipets - Disposable glass, Pasteur type, (9" length) fitted with
rubber bulbs (Fisher Scientific Co.).
6. Column, chromatographic, Size 23, (Kontes #420100).
7. Vortex-Genie Mixer - Model K-550-G or equiv. (Scientific
Industries, Inc., Springfield, MA).
8. Evaporative concentrator tube, 25 ml, (Kontes #570050).
9. Bottles - Reagent, narrow mouth, capacity 1 oz., complete with
polyseal screw type caps. (A. H. Thomas Co., Phila., PA., Cat.
No. 2203-C, bottles; and 2849-E polyseal caps.)
10. Exhaust hood with a minimum draft of 150 linear feet per minute.
IV. REAGENTS:
1. Acetonitrile - "Pesticide Grade" solvent, distilled from all-glass
apparatus.
2. Diethyl ether - AR, or USP grade with 2% ethanol, Mallinckrodt,
Cat. No. 0850 or equivalent.
NOTE: Under no conditions should anhydrous ether be used
for the preparation of diazopentane because of the
danger of explosion.
3. Methylene chloride, dist. from all-glass apparatus.
4., Silica gel, Woelm, activity grade I (Waters Associates, Inc.),
activated at 130°C for 48 hours and stored in desiccator.
5. Potassium hydroxide, pellets, AR grade.
6. Sodium chloride, AR grade.
7. N-amyl-N'-nitro-N-nitrosoguanidine - Aldrich Chemical Co., Inc.,
Milwaukee, Wis.
8. Extracting solvent - 1:1 (v/v) mixture of acetonitrile and anhy-
drous diethyl ether.
9. Formic acid reagent - make up a 1% solution of formic acid in
benzene.
-------
Revised 12/2/74
Section 6,A,(2),(a)
- 4 -
10. Diazopentane reagent - Preparation:
a. Dissolve 2.3 grains of KOH in 2.3 ml of dist. PLO 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 (Mallinckrodt #0850), cover
flask mouth with foil, and cool in a -18°C freezer 15 minutes.
c. In a VERY HIGH DRAFr hood, add 2.1 grams of N-amy-N'-nitro-
N-nitrosoguanidine to the flask in small portions over a
period of a few minutes, swirling the glask 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 THElDIAZO-
ALKANE TO COIC IN CONTACT WITH THE SKIN.
Wear disposable vinyl gloves and safety
goggles while handling. Avoid breathing
vapors.
(2) Do not use ground glass stoppered bottles
or bottles with visible interior etching.
Avoid strong light.
13. Standards
For brevity's sake, the names of various phosphate compounds
will be abbreviated from this point on. The identities of the
abbreviations are given in Table 3. The standard solutions are
prepared as described in the following:
a. In 15-ml grad., glass stoppered centr. tubes, accurately
weigh ca 10 milligrams of each of the following dialkyl
phosphate standards:
(1) Sodium* dimethyl phosphate (NaDMP)
(2) Potassium dimethyl phosphorothioate (KTMTP)
(3) Potassium dimethyl phosphorodithioate (KDMDTP)
(4) Diethyl* phosphate (DEP)
(5) Potassium diethyl phosphorothioate (KDETP)
(6) Potassium diethyl phosphorodithioate (KDEDTP)
*For consistency, convert the weights of these compounds to the weight of
an equivalent amount of the potassium salt. For sodium dimethyl phosphate
the conversion factor is 1.109; for diethyl phosphate it is 1.247.
-------
Revised 12/2/74
Section 6,A, (2),(a)
5
NOTE: The foregoing standards are available in 100 mg
increments from the EPA Repository at Research
Triangle Park, NC.
b. The alkylation of these standards is carried out as follows:
NOTE: The procedure, from here on, must be carried through
without interruption. Solutions of dialkyl
phosphates are unstable, especially in an acidic
medium. Therefore, the length of time in contact
with HC1 must be kept to a minimum. After alky-
lation, the trialkyl derivatives are stable
indefinitely.
(1) To each tube add 2 drops of 6 N HC1.
(2) Add sufficient diazopentane to produce a persistent
yellow color (2 to 5 ml usually suffices), mix and
allow to stand for 20 minutes.
(3) Remove excess reagent by adding the formic acid/
benzene reagent dropwise until the yellow color
just disappears. Avoid an excess of this reagent
(4) Dilute the solution(s) exactly to 10 ml with benzene,
stopper and mix thoroughly. Store in the freezer in
glass stoppered containers when not in use.
c. These alkylated concentrated stock standards may be diluted
individually or more generally prepared as mixtures at an
intermediary concentration range. For the mixtures, pipet
aliquots of appropriate volumes into three volumetric flasks
to obtain the concentrations given in the following, in
nanograms per microliter:
Mixture 1 Mixture 2 Mixture 3
KEMTP 10
KDETP 10
KDMDTP 5
KDEDTP 5
DMP
DEP
10
10
-------
Revised 12/2/74
Section 6, A,(2),(a)
- 6 -
d. Working Standard Mixtures:
(1) Dilute each of the intermediary stock mixtures in a
1:50 ratio with benzene.
(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
into the gas chromatography
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 organophosphorous pesticides. If any type
of surveillance or monitoring program is to be implemented, there
must be a highly coordinated relationship between the chemist per-
forming 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 your proposed project with the senior authors of
the publications cited.
This method, as a tool for determining the exposure index of
the subject individual sampled, is considerably more sensitive to
low levels of organophosphorous exposure than the ChE method given in
Section 6,A, (3),(a) which measures the depression in blood cholines-
terase.
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 organophosphorous pesticide
under study. Generally, the highest concentration of urinary meta-
bolites 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, EXTRACTION AND ALKYLATION:
1. Store urine samples in a freezer until ready for analysis. Do not
store acidified samples. When urine sample is thawed, centrifuge
and discard solids. Pipet a 2-ml aliquot into a 15-ml centr. tube.
-------
Revised 12/2/74
Section 6,A,(2),(a)
- 7 -
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 individual known to have no contact with
organophosphorus pesticides for at least a week.
2. If necessary, pre-extract urine samples with two 5-ml portions
of diethyl ether. Centrifuge for one minute to obtain separation
of phases and discard extracts.
NOTE: Pre-extraction of urine is usually necessary only
when the urine has been contaminated with feces as
would be the case with urine collected from experi-
mental animals housed in cages. The pre-extraction
step does not remove any of the metabolites deter-
mined by the method.
3. Add 2 grams of NaCl and pipette 4.0 ml of extracting solvent (IV,8)
to the centrifuge tubes.
4. Add 1 ml of 6 N HC1, stopper tubes and mix on a Vortex mixer for
one minute. It is advisable to do samples in pairs from this
point until diazopentane is added.
5. Centrifuge at 2,000 rpm for one minute.
NOTE: No interruptions nor delays are permissible until
Step 7 has been completed.
6. With a disposable pipet, transfer 2.0 ml of the organic solvent
layer to a grad., 15 ml, glass-stoppered centr. tube.
7. Working in a high draft hood, add ca 2 ml of diazopentane. A
pronounced orange color should persist after mixing. If necessary,
add slightly more of the alkylating reagent until the color
persists. Allow to stand for 20 minutes.
8. Concentrate solution to 0.2 to 0.3 ml under a gentle stream of
nitrogen, either at room temperature or on a 40°C water bath.
Dilute to 5 ml with dist. water, add ca 5 gm of NaCl and 3 ml
of hexane. Stopper and mix vigorously on Vortex mixer for 1
minute. Allow layers to separate and centrifuge in case of
interfacial emulsion.
-------
Revised 12/2/74
Section 6,A,(2),(a)
- 8 -
9. Prepare silica gel as follows: Partially deactivate 10 gm of
silica gel by shaking 2 hours with 2.0 ml dist. water. Transfer
2.4 gm to a size 23 chromatographic column with a small wad
of glass wool at the bottom. Top the column with ca 2 gm of anhy-
drous Na2SO^ and prewash with 10 ml of hexane.
NOTE: Elution patterns may vary from one laboratory to
another depending on the temperature and relative
humidity. This dictates the need for each laboratory
to establish an elution pattern of standards and
spiked control urine samples under local conditions
before attempting to analyze samples.
10. Transfer hexane extract (Step 8 above) to the silica gel column.
Re-extract aqueous layer with 2 ml of hexane and add to column.
Extract with another 2 ml of hexane and add to column. All
eluate to this point is discarded.
11. Place a 25-ml concentrator tube under column and add 15 ml of
methylene chloride to the column. This fraction contains EMTP,
DETP, DMDTP AND DEDTP.
12. Add 15 ml of 1% acetone in methylene chloride and discard this
eluate.
NOTE: This eluate contains most of the triamyl phosphate
and other interfering substances.
13. Place another 25-ml concentrator tube under column and add 20 ml
of 3% acetone in methylene chloride. This fraction contains
IMP and DEP.
VII. GAS CHROMATOGRAPHIC DETERMINATION:
1. Inject 5-25 yl aliquots of the silica gel column fractions and
working standard mixtures into the gas chromatograph. Dilution
or concentration of the extract will be governed by chromato-
graphic response from the initial injections.
2. Suggested operating conditions:
Column temperature 165°-175°C
Injection block temperature 200°C
Detector temperature 200°C
Transfer line temperature 200°C
Switching valve temperature 200°C
-------
Revised 12/2/74
Section 6,A,(2),(a)
9
2. Suggested operating conditions (Contd.):
Nitrogen (carrier) flow rate
Nitrogen (purge) flow rate
Hydrogen flow rate
Air flow rate
Oxygen flow rate
30-40 ml/min.
30-40 ml/min.
180 ml/min.
80 ml/min.
10 ml/min.
NOTE: It may be necessary to adjust air and oxygen
flow rates to obtain optimum response.
3. Examine the two eluates by gas chromatography. Quantitate sample
alkyl phosphate peaks by mathematical comparison with peaks
obtained from working standards using the phosphorus response.
4. Confirm the presence of sulfur-containing alkyl phosphates by use
of the flame photometric detector with sulfur filter (394 my).
See Figure 1 for chromatograms of the amyl derivatives of dialkyl phosphates.
This figure was obtained using the phosphorus filter (526 my).
Table 2 lists the relative retention times, and detector sensitivity for the
amyl derivatives of dialkyl phosphates using the phosphorus filter.
Do not inject Silyl 8 on a column connected to the FPD. If column needs
reconditioning, disconnect exit end on column from the detector and condi-
tion as required.
NOTES: 1. Often after extended use of the gas chromatographic
system, extraneous peaks may appear. They arise
chiefly from the accumulation of underivatized com-
pounds 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 are present in the column, they show as
peaks following the diazopentane injection. If
this is the case, recondition the column.
2. Quantitation is based on the amyl derivatives, MAP,
DEAP, EMATP, DEATP, DMADTP, and DEADTP. Some
inorganic phosphate is extracted which is converted
to RAP.
NOTE: IMTP and DETP isomerize upon alkylation producing
thionates and thiolates. Quantitation is based
on the DMATP and DEATP.
-------
Revised 12/2/74
Section 6,A,(2),(a)
- 10 -
3. Confirmation and Specificity.
a. 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 (Bowman and Beroza 1968), greatly enhances
the specificity of this method. Suspected thiophos-
phate can be confirmed using the sulfur filter by
simply increasing the concentration of the compound
injected into the gas chromatograph by a factor
of 5 to 10.
b. 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'-nitro-N-nitrosoguanidine
is used as the diazoalkane precursor.
c. 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 eluted in the ethyl
acetate fraction.
Vm. ANALYTICAL QUALITY CONTROL:
Because of the relative complexities of the method, it is impor-
tant that routine analyses be validated by conducting simultaneous
analyses of spiked SRM's (standard reference materials). If only
occasional routine analyses are conducted, one SRM should be analyzed
right along with the unknown and in exactly the same manner. In a
monitoring situation wherein multiple routine analyses are conducted,
the ratio of SRM to routine analyses should be at least 10%. In
other words, for every nine routine analyses, at least one SRM should
be analyzed.
The relative instability of a spiked urine sample precludes the
concept of preparing a large sample of SRM ahead of time and analyzing
it periodically. Therefore, it is preferable to prepare each SRM
as it is needed. The suggested procedure is as follows:
1. Prepare aqueous standard solution mixtures at two concentrations
of each of the following compounds: IMP, DEP, IMTP, and DEPT.
This may be done by weighing 10 mg of each into a 1 liter volumet-
ric flask, make to volume with distilled water and shake thor-
oughly. This will be STANDARD MIX A. Transfer 10 ml of Mix A
to a 100 ml volumetric flask and make to volume with distilled
-------
Revised 12/2/74
- 11 -
Section 6, A,(2),(a)
HOH. This will be STANDARD MIX B. Mixes A and B will have
the respective concentrations of 1.0 and 0.1 ppm.
2. Divide both mixes into several screw cap test tubes or vials
and freeze immediately. Do not fill tubes over half full and
lay on side during freezing to reduce probability of cracking
the glass.
3. When ready to conduct an SRM analysis one tube (of each con-
centration) is thawed and a 0.2 ml aliquot is drawn to spike
2.0 ml of control urine from an unexposed donor contained in
a 15 ml centr. tube.
NOTE: Previous experience may dictate that one of the
two concentrations (0.1 or 1.0 ppm) will closely
match the expected concentration in the unknown.
In such an instance, only one concentration may be
needed. Without knowledge of the prior case his-
tory, it is advisable to use both concentrations.
4. In another centr. tube carry along a 2.0 ml unspiked urine
sample from the same control donor.
5. In a third tube pipet 0.2 ml of the standard alone.
6. Proceed with the analysis of the two urine samples (spiked
and control) as described in subsection VI starting at Step 3.
7. To the third 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 20 minutes.
8. Evaporate to ca 0.5 ml in a 35°C water bath and under a stream
of nitrogen.
9. Add 5 ml H2O, 5 ml of hexane and 5 grains of NaCl. Mix well.
10. Transfer the hexane extract to a silica- gel column and proceed
as described in subsection VI starting at Step 10.
Recoveries are calculated by comparing the chromatographic
data of the spiked samples to those of the corresponding standard.
-------
Revised 12/2/74
- 12 -
Section 6,A,(2),(a)
Table 1. Amyl derivatives of dialkyl phosphates. Concentration ranges,
in nanograms, of standards which produce linear detector
response for normal GLC operating conditions.
Phosphorus Filter (526 my)
DMAP
0.1-25
ng
DEAP
0.1-50
ng
DMATP
0.1-50
ng
DEATP
. 0.1-50
ng
DMADTP
0.1-50
-flg-
DEADTP
0.1-50
ng
Table 2. Data from isothermal gas chromatography of trialkyl phosphates
with phosphorous flame photometric detection system. Column
51 OV-210, Tem. 173°C, carrier flow 40 ml/min. of N2, noise ±1%.
Relative
Ret. Value Response Sensitivity (ng)
Compound Retention (min) (Basis DMADTP) % fsd/ng 4:1 signal/noise
EMATP
1.46
0.59
37.2
0.11
DEATP
1.81
0.73
27.5
0.14
EMAP
2.36
0.95
57.0
0.07
DMADTP
2.48
1.00
67.8
0.06
DEAP
3.03
1.22
45.3
0.09
DEADTP
3.11
1.25
58.7
0.07
DMAPTh
3.46
1.40
-
-
DEAPTh
4.37
1.76
-
-
TAP
16.6
6.69
-
-
-------
Revised 12/2/74 Section 6,A,(2),(a)
- 13 -
Table 3. Compound identifications for abbreviations used in text:
EMP
0,0-Dimethyl phosphate
DEP
0,0-Diethyl phosphate
DMTP
0,0-Dimethyl phosphorothioate
DETP
0,0-Diethyl phosphorothioate
DEDTP
0,0-Diethyl phosphorodithioate
EMDTP
0,0-Dimethyl phosphorodithioate
DMAP
0,0-Dimethy1-0-amy1 phosphate
DEAP
0,0-Diethyl-0-airyl phosphate
DMATP
0,0-Dimethyl-0- amyl phosphorothionate
DEATP
0,0-Diethyl-0-amyl phosphorothionate
EMAPTh
0,0-Dimethyl-S-amyl phosphorothiolate
DEAPTh
0,0-Diethyl-S-amyl phosphorothiolate
DMADTP
0,0-Dimethyl-S-amyl phosphorodithioate
DEADTP
0,0-Diethyl-S-amyl phosphorodithioate
TAP
0,0,0-Triamyl phosphate
NOTES: Occurence of various compounds cited and commercial availability
of reference standards.
1. IMP and DEP are the two most prevalent compounds detected.
Available from Pfaltz § Bauer, Inc., 126-04 Northern Blvd.,
Flushing, NY 11368.
The catalog numbers are: DMP-D44180 (in lOOg lots)
DEP-D24340 (in 10g lots)
-------
Revised 12/2/74 Section 6,A,(2),(a)
- 14 -
NOTES: (Contd.)
2. Whenever DETP shows up, DEP will inevitably be present.
3. Whenever EMTP shows up, DMP will inevitably be present.
4. In a high exposure situation or a poisoning case resulting
from malathion, EMP, EMTP, and EMDTP may all be found.
5. DEDTP is rarely detected.
-------
Figure 1. Chromatograms of standard solutions.
C-DMADTP-0.63 ng, DEADTP-0.54 ng.
A DMAP-Oj. 59 ng, DEAP-0.53 ng, B DMATP-1.13 ng, DEATP-1.00 ng.
<
S
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a.
t-
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UJ
Q
B
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a
<
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o
<
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<
H-
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Oi
hJ
\
\
M
U1
o
rt
H*
o
3
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o
2
4
0
P
1
4
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6 0
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Retention in minutes
-------
1/4/71
Section 6,A,(2),(b)
Page 1
DETERMINATION OF PARA-NITROPHENOL (PNP) IN URINE
I. INTRODUCTION:
Urinary PNP, the phenolic metabolite of parathion, methyl parathion,
and EPN has been measured for years as an indicator of exposure to these
organophosphorus pesticides. The Elliott spectrophotometry method is
only semi-specific, requires a minimum of 10 ug of PNP, possesses marginal
accuracy at low levels and is somewhat 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 determinative procedure.
REFERENCES:
1. Elliott, J. W., K, C. Walter, A. E. Penick,
and W. F. Durham (I960). "A Sensitive Procedure
for Urinary para-Nitrophenol Determination as a
Measure of Exposure to Parathion." J. Agr. Food
Chan. 8_, 111.
2. Cranmer, M. F., and A. Peoples (1970). "Deteimination
of p-Nitrophenol in Human Urine." Bull. Environ.
Contamin. and Toxicol., Vol. S, No. 4.
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 deteiminative 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.
2. Centrifuge tubes (distill, recvr.), grad., 12 ml, with 1 14/20 outer
joint, Kontes, #288250.
3. Stoppers, glass, flathead, 31 14/20., Kontes #850550.
-------
1/4/71
Section 6,A,(2),(b)
^ 2 ***
4. Reflux condensers, equipped for water cooling with lower Sf inner
joint the same size as test tuhes. Kontes #2822Q0.
5. Pipets, Mohr, 0.5 ml., grad. in 0.01 ml., Kimhle #37023 or the
equivalent.
6. Pipets, Mohr, 3 ml., grad. in 0.05 ml., Kimble #37023 or the equivalent.
7. Pipets, Mohr, 1Q 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,M constant temp. 100°C., or
comparable block heater with holes of appropriate size to accomodate
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-D4.
IV. REAGENTS:
1. Hydrochloric acid, conc., 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 sulphate, A. R. grade, anhydrous, granular.
7. Hexane, pesticide quality.
8. Hexamethyldisilizane reagent, 20! in hexane.
9. Hexamethyldisilizane reagent,10% in hexane.
10. p-Nitrophenol standard solution of appropriate concentration range
in hexamethyldisilizane - 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/ul.
-------
1/4/71
Section 6,A,(2),(b)
- 3 -
V. PROCEDURE:
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 conc. HC1
and reflux the mixture for 1 hour with tube inserted in l,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 conc. 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 tuhe, taking care that
no aqueous phase is included.
8. Add 0.5 grams anhydrous Na2SO,, stopper tube and shake vigorously 1 min.
to remove traces of moisture from the solvent extract.
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.
-------
1/4/71
Section 6,A,(2),(b.)
- 4 -
Gas Chromatography
Before injecting the sample extract, pre-condition the column with
several repetitive injections of the PNP/?MDS->hexane standard (sub-section IV,
10.). This serves the dual purpose of (1) providing a quantitating standard
peak, and (2) conditioning the column prior to sample injection.
NOTE: 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-IMS.
This is done by injecting I-MDS-hexane (10:9Q 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.
VI. MISCELLANEOUS NOTES:
1. The author has reported recoveries in excess of 90% for PNP levels down
to 25 ppb in urine.
-------
Revised 11/1/72
Section 6,A,(3),(a)'
Page 1
~
CHOLINESTERASE ACTIVITY IN BLOOD
I. INTRODUCTION:
The cholinesterase enzyme in blood is inhibited in varying degrees
by organophosphate pesticides and is loosely correleated with the de-
crease of acetylcholinesterase activity in the nervous system which in
turn is accompanied by an increase in the concentration of acetyl-
choline. Therefore, a scheme for measuring the level of activity of
the cholinesterase is one means of establishing possible exposure.
The technique of continuous titration of the acetic acid released
from acetylcholine by the enzyme cholinesterase overcomes many of the
undesirable features of other methods. This method does not utilize
buffers, is temperature and atmospherically controlled, 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 determination
of red blood cell and plasma cholinesterase activity. J. Lab
Clin. Med. 34; 1964 (1949).
2. Nabb, D. P. and F. Whitfield. Determination of cholinesterase
by an automated pH-stat method. Arch. Environmental Health
15:147 (1967).
3. Pearson, J. R. and G. F. Walker. Conversion of acetylcholin-
esterase activity values from the Michel to the pH-stat scales.
Arch. Environmental Health, 16:809 (1968).
II. PRINCIPLES:
The whole blood sample is separated into the plasma and RBC (red
blood cells). Each fraction is placed in the reaction vessel of a
pH-stat and mixed with an excess of acetylcholine iodide. The cholin-
esterase present in the blood fraction reacts with the AChI releasing
acetic acid as illustrated in the following:
C
C+-N-C-C-0-C-C
C
C
C+-N-C-C-0H + C-C-OH
C
Acethycholine
(ACh)
Choline Acetic Acid
HA
-------
Revised 11/1/72
Section 6,A,(3),(a)
- 2 -
A standardized solution of dilute NaOH is used as the titrant for
the released HA.. The automatic titrator records the amount of titrant
delivered to the reaction in a given time period. A feedback signal
from the pH measuring electrodes controls the titrant delivery rate.
A high ChE activity produces a large hydrogen ion release in a fixed
time period, calling for a faster titrant delivery rate than will a
lower ChE activity.
III. EQUIPMENT:
1. *pH-stat, recording, complete with micro glass reference combination
electrode, 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 r.p.m.
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.
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.
*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.
-------
Revised 11/1/72
Section 6,A,(3),(a)
- 3 -
Standard Solution - Dry standard P.A.T. in 105°C oven at least 4
hours before use. Store dried salt in desiccator. Weigh exactly
0.20423 grams and transfer to a 1-liter vol. flask, dissolving and
making to volume in freshly boiled dist. water.
2. Titrant standard solutions prepared from sodium hydroxide, reag.
grade, Fisher S-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.
3. 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 R1J
(recorder units). It is not mandatory that the titrant working
solution be exactly 0.01 N as long as 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 neutrali-
zation 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 instrument operating
temperature to 37 C.
b. Set titrant delivery to appropriate position for 2.5 ml de-
livered full scale, set pH to 7.0, position pen at zero and
turn on function switch.
c. The end point is reached when pen levels off at a certain RIJ
level. Record this level and compute the titrant noimality
as follows:
XT 2.0 ml x 0.001 meq/ml _ 0.4 /m1
N ° M x 0.005 ml/RU jju-meq/nO.
-------
Revised 11/1/72
Section 6,A,(3),(a)
- 4 -
NOTE: Total syringe delivery in the assumed system is
0.50 ml, indicated by 100 RU, or 100% of full
syringe travel. It is not convenient to use the
entire syringe volume for a titrant standardiza-
tion or assay. For other syringe delivery
capacities, such as 0.25 or 1.0 ml, adjust the
RU value accordingly.
4. Acetylcholine iodide available from Calbiochem Company, P.O. 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 acetyl-
choline are hygroscopic and weight will change rapidly.
V. SAMPLE HANDLING AND PREPARATION:
The sample preparation and analysis of blood should be carried out
as soon as possible after drawing sample. If a few hours delay is un-
avoidable, keep samples refrigerated. If the delay will be overnight or
longer, blood should be centrifuged and plasma separated from RB cells
and the latter taken through the following steps (a through f) before
storage.
a. Place blood tube in centrifuge and spin for 20 minutes at 2000 r.p.m.
b. Pipet plasma into clean tubes for storage.
NOTE: If a part of this sample will eventually be analyzed
by GLC, E.C., the tube cap should be ground glass or
screw cap with Teflon or foil liner.
c. Using vacuum pipet, remove and discard fluffy layer.
d. Resuspend red cell mass in an equal volume of 0.9% NaCl solution.
This is done by gently inverting tube.
e. Centrifuge again for 10 minutes and remove supernatant NaCl solution
by vacuum pipet.
f. Repeat steps d. and e. The RBC mass is now ready for assay.
-------
Revised 11/1/72
Section 6,A,(3),(a)
- 5 -
VI. PREPARATION OF RBC HB10LYSATE:
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 cells.
NOTE: Care must be taken to wipe the tip of the pipet with
tissue while still retaining the full 0.2 ml contents.
This may require some practice.
2. With a rubber bulb attached to the pipet, draw the hemolysate solu-
tion up into the bore of the pipet repeatedly until all adhering
cells are washed into the water.
3. Stopper tube 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 manufacturer's
instructions. This is best done by using two buffers, one belgw
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.
5. Add substrate to mixture in titration vessel:
a. If plasma assay, add 0.6 ml of acetylcholine iodide solution.
b. If RBC assay, add 0.10 ml of AChI solution.
6. Start recorder and titrant flow and allow titration to proceed until
recorder describes a line with constant slope, then mark off the
start and end of a 3-minute period of titration. See Figure 1.
-------
Revised 11/1/72
Section 6, A, (3),(a)
- 6 -
NOTE: None of the line included should be nonlinear. It is
good practice to allow titration to proceed for a
minute or so before counting the RU.
VIII.SAMPLE CALCULATIONS:
Refer to Figure 1:
1. Standardization of titrant.
Average of three replicate titrations: 54.0 RLI
54.0
53.9
3)161.9 = 53.961 RU
From formula (1), N - 3^ = 5^ = 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 pM/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 pM/min/ml (only one replicate shown)
B. Alternative method, without using factors:
Substituted:
ni , RU x 0.005 ml/RU , , . . 46.5 x 0.005 n n7„.
PlasnB 1: Minutes = ml/minute = % 0.0775
ml/min.
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
pM/meq
= 0.853 uM/minute
yiM/minute = = 0^853 = 5i6g7
sample volume 0.15
-------
Revised 11/1/72
Section 6,A,(3),(a)
- 7 -
DC. MISCELLANEOUS NOTES:
1. Because of the variety of instruments available, operating instruc-
tions are not given for pH-stat equipment. Detailed instructions
furnished by the manufacturer will govern operating details. A step-
by-step outline of sample preparation, reagent preparation and stan-
dardization, plus the scheme for performing calculations are outlined.
2. Heparin is preferable to other anticoagulants, because oxalate,
citrate and EDTA will sequester calcium and magnesium, which are
required co-factors for ChE. In an emergency, these anticoagulants
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 pM/min/ml - Plasma
8.0 yM/min/ml - Red Cells
An individual laboratory should, of course, establish its own normal
ranges based on experience with the method.
7. Assay results obtained by the pH-stat method are expressed as:
micromoles (acetic acid liberated)/minute/ml sample (either packed
red cells or plasma). Abbreviated: yM/min/ml.
8. Standard sources of enzyme are available from Sigma and are useful
in intralaboratory quality control.
-------
Revised 11/1/72 Section 6,A,(3),(a)
-8-
Figure 1. Sample of strip chart record of pH-stat assay of cholinesterase
activity.
30.5 Recorder units(RU)
One minute
Fen direction
Standardization of
0«01 N NaOH against
potassium acid
phthalate, three
replications
53.9 RU
-------
Revised 11/1/72
Section 6,A,(3),(a)
- 9 -
Table 1. Factors for Three Minute Titrations
Normality NaOH
Solution Factor Plasma ChE Factor
0. 0. 0.
0095
3167
1055
0096
3200
1067
0097
3233
1078
0098
3267
1089
0099
3300
1100
0100
3333
1111
0101
3367
1122
0102
3400
1133
0103
3433
1144
0104
3467
1156
0105
3500
1167
These factors are derived as follows:
Red cell factor = ml/min x meq/ml x 1000 yM/meq
0705
Plasma factor = ml/min x meq/ml x 1000 uM/meq
TT15
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
-------
Revised 11/1/72
DETERMINATION OF 1-NAPHIHOL IN URINE
Section 7, A
Page 1
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 deter-
mination of 1-naphthol in human urine has been generally accomplished
using a colorimetric procedure. This method lacks both the sensitivity
and specificity necessary for determining the relatively small amounts
of 1-naphthol excreted in the urine of agricultural workers exposed to
low levels of this insecticide.
The 1-naphthol resulting from the hydrolysis of carbaryl has been
used as an indirect measure of the residue level of the parent insecti-
cide on a variety of agricultural crops. Argauer has described a
procedure for chloroacetylating phenols and 1-naphthol for subsequent
detection by electron capture gas chromatography. In a recent publica-
tion this procedure was utilized to determine a number of carbamate
insecticides. However, it was indicated that further 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 sensitivity down
to 20 ppb of 1-naphthol.
REFERENCES:
1. Shafik, M. T., Sullivan, H. C., Enos, H. F., Bull, of
Envir. 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 chloro-
acetate anhydride solution. After silica gel cleanup, the resulting
1-naphthyl chloroacetate is quantitatively determined by E.C., GLC,
comparing sample peaks against peaks obtained from pure 1-naphthol
standard, similarly derivatized.
III. APPARATUS:
1. Gas chromatograph equipped with E.C. detector and fitted with a
6' x 1/4" o.d. column of 1.5% OV-17/1.95% QF-1. Operating para-
meters for the column are those prescribed in Section 4,A of this
manual.
-------
Revised 11/1/72
Section 7,A
- 2 -
III. APPARATUS (Continued)
2. Chromatographic column (Chromaflex), size 22, Kontes #420100.
3. Evap. concentrator tubes grad., glass stoppered, 25 ml, f 19/22,
Kontes #570050.
4. Distilling column (condensor), 200 mm jacket, Kontes #286810,
fitted with tight glass stopper at top.
5. Centrifuge capable of 2000 r.p.m. that will accept metal shield,
I.E. Company No. 367.
6. Volumetric flasks, 10 and 100 ml.
7. Pipets, Mohr, 0.2, 0.5 and 10 ml, Corning 7064 or the equivalent.
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 S glass stoppers,
Corning 8084 or the equivalent.
12. Circulating water pump.
13. Boiling water or steam bath.
-------
Revised 11/1/72
Section 7, A
- 3 -
IV. SOLVENTS AND REAGENTS:
1. Benzene, pesticide quality.
2. Hexane, pesticide quality.
3. Pyridine, Spectro Grade, Eastman #13098.
4. Chloroacetic anhydride, Eastman #335 - prepare a 2% solution in
benzene and hold no longer than one week.
NOTE: The chloroacetic anhydride must be dry and
therefore should be stored in a desiccator as it
is extremely hygroscopic.
5. Hydrochloric Acid, reag., conc.
6. Sodium sulphate, anhydrous, granular - preextract on a Soxhlet
with benzene forQabout 50 discharge cycles, reirtove excess solvent
and store in 130 C oven.
7. Sodium sulphate, 31 solution in dist. water. Use preextracted Na^SO^.
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 1.51 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.
-------
Revised 12/2/74
Section 7, A
- 4 -
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
conc. stock of 200 ng/yl and may be held several months at -18°C.
b. Stock Standard II. With a 0.5-ml Mohr pipet, 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 21 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 r.p.m.
and remove any final traces of water from bottom of tubes.
Dilute to exactly 10 ml with benzene and mix thoroughly.
The three tubes will contain concentrations of 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 concentration of 1-naphthyl chloro-
acetate, dilutions may be made and used to check the derivatized
working standards finalized in Step c, (5) above.
-------
1/4/71
Section 7, A
- 5 -
NOTE: The derivatized standards are relatively stable.
Assuming no solvent losses from repeated opening
of the storage bottles, it is probable that the
standards could be held for 6 months, storing in
the refrigerator when not in use.
V. PROCEDURE:
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. Pipet 5 ml of urine into a 25 ml evap. concentrator tube, add
1 ml of conc. HC1, stopper and mix on the Vortex mixer 2 minutes.
2. Fit concentrator tube with a glass stoppered condenser (distilling
column) and reflux mixture in a hot water or steam bath 90 minutes,
cooling the condenser with circulating ice water.
NOTE: The top of the condenser should 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 conc. tube in centrifuge and spin for 10 minutes at 2,000 r.p.m.
6. Carefully transfer as much of the benzene (upper) layer as possible
to a clean 25 ml concentrator tube, using a disposable pipet fitted
with a rubber bulb.
7. Add 3 ml more of benzene and repeat Steps 4, 5 and 6.
NOTE: Extreme care should be taken to prevent water
from being transferred as this would seriously
affect derivatization efficiency.
-------
1/4/71
Section 7, A
- 6 -
8. Wash benzene extract with two 3 ml portions of 3% Na-SO, solution,
centrifuging and discarding each successive aqueous layer.
9. To the combined benzene extract add 2 ml of 2% chloroacetic anhydride
solution and 0.2 ml of pyridine. Stopper tuhe, 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.
Silica Gel Cleanup
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 coluim with 10 ml of hexane, discarding the eluate.
3. When the surface level of the hexane reaches a point on the column
ca 2 cm from the top of the Na2SO, transfer the concentrated benzene
extract to the column with a disposable pipet and rinse tube with two
portions of 0.5 ml of 20/80 benzene/hexane solvent applied with
another disposable pipet, directing stream so as to wash down walls
of tube. Follow this with 8.5 ml of 20/80 benzene/hexane solvent,
discarding all eluates up to this point.
4. Place a 15 ml grad. conical centrifuge tube under column and add
10 ml of 60/40 benzene/hexane solvent, collecting this fraction
which contains the 1-naphthyl chloroacetate derivative. Finally,
adjust volume of extract to exactly 10 ml with benzene.
NOTE: 1. The foregoing instructions are based on the
premise that the operator obtains an elution pattern
identical to that obtained by the authors. However,
this should not be assumed until the operator has
confirmed it by actual trial. This may be readily
done by applying Q.5 ml of the derivatized 200 ng/ul
stock standard to a silica gel column in the manner
described in Steps 1, 2 and 3 but collecting the
10 ml of hexane prewash and the 20/80 benzene/hexane
eluates instead of discarding them. In addition to
this, 10 ml each of benzene/hexane mixtures of 40/60
60/40, 80/20 and 100% benzene should be eluted
through the column, collecting each of the cuts for
-------
Revised 11/1/72
Section 7,A
- 7 -
examination by GLC to determine whether any of the
derivatized compound is eluting out of place. If so,
it may prove necessary for the individual operator to
make appropriate revisions in the eluate collection
instructions.
2. At no time during the elution should the liquid
level in the column be allowed to go below the top of
the Na2S0^ layer.
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 back-
ground 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.
VI. MISCELLANEOUS NOTES:
1. Elution patterns may vary from one laboratory to another depending
on the temperature and relative humidity. This emphasizes the
need for establishing an elution pattern of standards and spiked
control urine samples under local conditions before attempting
to analyze samples. The procedure for spiked control urine samples
is as follows:
a. In a 15-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 2,000 r.p.m.
c. Pipet aliquots of 0.1, 0.25 and 0.5 from the aqueous layer
(sodium naphthoxide solution) into separate 25-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 is obtained by chromatographing these extracts
against the derivatized working standards.
-------
Revised 12/2/74
Section 8, B
Page 1
SAMPLING AND ANALYSIS OF PESTICIDES IN AIR
I. INTRODUCTION:
Up to the date of this revision no wholly acceptable method
has been devised for the sampling of ambient air for multi-pesticides
at extremely low concentration levels. A sampling device developed
in the early 1960's by Miles, et al (1) utilized the Greenburg-Smith
micro impingers and consisted of drawing air by a vacuum pump through
a trapping medium of ethylene glycol. Pesticides were then extracted
from the EG solution and assayed via gas-liquid chromatography.
During the late 1960 's Stanley, et al (2) developed equipment utilizing
the Miles principles but capable of trapping a larger volume of air
per unit time, the latter system was implemented for a nationwide
monitoring program, but after a year the program was terminated
resulting from controversy concerning the reliability of the data.
Unfortunately, no alternative plan existed for the resumption of the
monitoring program.
The analytical method described presupposes the collection
of the sample by the Stanley, et al equipment. A customary sample
consists of 400 ml of ethylene glycol representing the contents of
four impingers of 100 ml each, two of which are operated simultaneously
for 12 hours, and two more for another 12 hours. The total air
volume sampled is generally ca 80 cubic meters.
REFERENCES:
1. Collection and Determination of Trace Quantities of Pesticides
in Air, Miles, J. W., et al, Envir. Sci, § Tech. 4_, 420 (1970).
2. Measurement of Atmospheric Levels of Pesticides, Stanley,
et al, Envir. Science § Tech., _5, 431 (1971).
3. Determination of Pesticide Residues in Air, Enos, H. F.,
et al, 163rd Annual ACS Meeting, Boston, MA (1972).
4. Levels of Selected Pesticides in Ambient Air of the United
States, Yobs, A. R., et al, 163rd Annual ACS Meeting, Boston,
Mass (1972).
II. PRINCIPLE:
The ethylene glycol contents of four 100-ml impingers are trans-
ferred to a large separatory funnel and diluted with water. Pesticides
are extracted with hexane and transferred to a Kuderna-Danish evaporator.
-------
Revised 12/2/74 Section 8, B
- 2 -
After concentration to a suitable volume, a preliminary gas chromato-
graphic evaluation is made using electron capture detection for organo-
chlorine compounds and flame photometric detection for the organophos-
phates.
The extract is then subjected to column cleanup with Florisil,
partitioning into two or three eluate fractions, each of which is
reconcentrated to a suitable volume and subjected to final gas chroma-
tographic analysis.
III. EQUIPMENT:
1. Chromatographic columns 22 mm i.d. x 300 mm length, Teflon stop-
cocks, without filter frits.
2. Separatory funnel - 1 and 2-liter capacity, Teflon stopcocks.
3. Hot water bath - 95-100°C.
4. Glass beads 3 mm plain, Fisher #11-312 or equivalent.
5. Glass wool - angel hair, fine.
6. Kuderna-Danish concentrator.
Evaporative concentrator flask - Kontes Catalog No. K-570001, 500
and 1,000 ml, lower joint 19/22 I, upper joint 24/40 $.
Snyder column - 3 ball, lower joint 19/22 S, upper joint 24/40 ?,
Kontes Cat. No. K-503000.
Tube - 10 ml grad., 19/22 ? joint, Kontes No. K-570050, Size 1025.
7. Gas Chromatographic Columns.
1.5% OV-17/1.95% QF-1, 4% Se-30/6% QF-1 and 10% OV-210, preparation
conditioning and operating parameters as outlined in Section 4, A.
IV. REAGENTS:
1. Ethylene Glycol, pesticide quality. Must meet the criteria
described in Section 8, C.
2. Hexane, redistilled in glass. Must be evaluated for background
' interference by gas chromatography, electron capture, of a 1-ml
concentrate from 180 ml of original solvent.
3. Petroleum ether - pesticide quality, redistilled in glass, B.P.
30-60°C.
-------
Revised 12/2/7-4
- 3 -
Section 8, B
IV- REAGENTS (CONTD.)
4. Ethyl ether - AR grade, peroxide free.
The above solvents should be free of materials that give a
response to the electron capture detector. Preparation of
peroxide-free ethyl ether is given in Section 5, A, (1).
5. Distilled water - check for interferences. If necessary,
extract with benzene and pet. ether as described in Section 8, C
for glycol extraction.
6. Florisil, P. R. grade. See MISCELLANEOUS NOTE 4, Page 7.
7. Anhydrous sodium sulfate.*
8. Eluting mixture (6+94 ether in pet. ether). (Used for Fraction I.)*
9. Eluting mixture (15+85 ethyl ether in pet. ether). (Used for
Fraction II.)*
10. Eluting mixture (50+50 ethyl ether in pet. ether). Prepare same
as other ether mixtures. (Used for Fraction III.)*
V. PROCEDURE:
All glassware to be used must be scrupulously cleaned and given a
final hexane rinse immediately before use.
1. Combine the impinger contents representing the collection of
81 of air (presumably 4-100 ml increments of glycol).
2. Transfer the 400 ml of E.G. to a 2-liter sep. funnel (No. 1). Wash
the sample bottle with a measured amount of dist. water and add
washings to sep. funnel. Dilute with a total of 1400 ml of water.
NOTE: All procedural instructions from here on assume the
extraction of 400 ml of E.G. Should a smaller volume
be extracted, the glassware sizes and reagent volumes
should be scaled down proportionately.
3. Transfer the prefilter glass cloths to a 400-ml beaker with a clean
pair of forceps. Add 240 ml of hexane, stopper and shake funnel
vigorously for 2 minutes. If emulsions are formed at this point,
add 10 ml of a saturated NaCl solution to the separatory funnel
(NaCl and distilled FLO should be free of interferences - preextract
if necessary).
* Instructions and comments re purity of these materials may be found
in Section 5, A, (1).
-------
Revised 11/1/72
Section 8,B
- 4 -
V. PROCEDURE (CONTD.)
4. Transfer the aqueous layer to a second 2-L sep. funnel (No. 2), and
the hexane layer to a 1-L sep. funnel (No. 3). Add another 240-ml
portion of hexane to funnel No. 2, stopper and shake vigorously for
2 minutes. Allow a few minutes for layer separation and transfer
aqueous layer back into funnel No. 1. Combine the hexane layer
with that already in funnel No. 3. Add a final 240-ml portion of
hexane to funnel No. 1 and repeat shaking and settling. Discard
aqueous layer and transfer hexane layer to funnel No. 3.
5. Add 200 ml of dist. water to S.F. No. 3, stopper and shake vigorously
1 minute. Discard aqueous layer and repeat extraction with more of
the 200-ml portion of dist. water, again discarding aqueous layer.
6. Place a glass wool retaining plug in the bottom of a 300-mm chromato-
graphic column, add 2 inches of Na„SO. and pass the combined hexane
extract through the column, collecting eluate in a 1,000-ml K-D
flask which has been fitted to a 10-ml evaporative concentrator tube
containing one 3-mm glass bead.
7. When extract has eluted from the column, rinse the sep. funnel with
three consecutive portions of 10 ml each of hexane, washing down
the walls of the column. Allow each rinse to elute off the column
before adding the next. Finally, rinse the column walls with two
more 10-ml portions of hexane.
8. Place K-D assembly in a boiling water bath and concentrate extract
to ca 5 ml. Remove from bath, cool, disconnect tube from K-D flask,
rinsing joint with a little hexane. Place tube under a nitrogen
stream at room temp., and further concentrate extract to ca 0.5 ml.
Rinse down tube sidewalls with hexane delivered from a 100-pl
syringe, diluting extract back up to exactly 1.0 ml. Stopper and
mix on Vortex 30 seconds.
9. Make exploratory injections into the gas chromatograph via FPD
detection. In the event that no peaks appear with relative reten-
tion values suggestive of organophosphorus compounds, it may be
concluded that the sample contains none of these compounds, or that
any compounds present are below the minimum detection levels. (See
Section 4,B for sensitivity guidelines.) If peaks of greater than
101 f.s.d. appear by FPD, and their identities can be determined
as organophosphorous pesticides, quantitations should be conducted
at this point.
-------
Revised 11/1/72
Section 8,B
- 5 -
V. PROCEDURE (CONTD.)
NOTE: It is probable that at this extract concentration
ratio (400:1), background interference would make
electron capture appraisal difficult or impossible.
However, if the detectors are so arranged in one
instrument that an E.C. chromatogram can be obtained
simultaneously with the FPD chromatogram, some useful,
preliminary information may be obtained.
Florisil Partitioning
1. Dilute extract up to ca 5 ml with hexane and transfer to a Florisil
column. [Prepare column as described in Section 5,A, (1).] Begin
eluate collection immediately on adding hexane to column and collect
the eluate in a 500-ml Kiderna-Danish concentrator fitted with a
10-ml grad. concentrator tube containing one glass bead. When
hexane reaches the Na^SO. layer, wash the tube with 3- to 5-ml
portions of pet. ether, rinsing the column walls each time, then
wash column walls with 1- to 10-ml portion of pet. ether. When
final wash reaches Na-SO. level, elute column with 200 ml of the 6%
mixed ethers and collect until ethers reach ^£50^ layer (Fraction I).
2. Change Kuderna-Danish concentrators and elute with 200 ml of the
15% mixed ethers in the same mariner (Fraction II).
3. Change Kuderna-Danish concentrators and elute with 200 ml of the
50% mixed ethers (Fraction III).
4. Attach Snyder columns on the K-D flasks, immerse tubes in hot water
bath and concentrate eluates to slightly less than 5 ml. Cool and
detach tubes from K-D flasks, rinsing joints with a little hexane.
5. Adjust the volume of the Fraction I and II extracts to exactly 5.0
ml and analyze by electron capture detection.
NOTE: If the 1-ml concentrate of the unpartitioned extract
was examined via electron capture, it may be possible
to estimate the degree of dilution necessary to bring
peaks of chlorinated pesticides into quantifiable
range by electron capture or electrolytic conductivity
detection.
-------
Revised 12/2/74
Section 8, B
- 6 -
V. PROCEDURE (CONTD.)
6. After completing GLC examination of Fractions I and II, continue
extract concentration, along with Fraction III, down to ca 0.5 ml
under a nitrogen stream at room temperature. Rinse down sides of
tube with hexane delivered from a 100-yl syringe, diluting extract
to exactly 1.0 ml. Stopper and shake on Vortex 30 seconds.
NOTES: (1) Do not use air for blow down and do not permit
evaporation to dryness. Organophosphate com-
pounds may be seriously affected by either of
these practices.
(2) If predetermined Florisil elution patterns have
indicated that any chlorinated pesticides are
eluting in Fraction III (endrin or dieldrin are
sometimes troublesome), the volume of this
fraction should also be adjusted to exactly 5 ml,
and examination by electron capture conducted.
7. Analyze the 1-ml extract concentrates of all three fractions by FPD
carefully observing sensitivity guidelines set forth in Section 4, B.
NOTE: The FPD analysis on the partitioned extract is run
for assurance of identifications obtained on unparti-
tioned extract. There will probably be no need for
requantitating these O.G.P. compounds except in cases
of distorted or overlapped peaks in the original
chromatograms.
VI. MISCELLANEOUS NOTES:
1. Use the 0V-17/QF-1 and SE-30/QF-1 columns for electron capture GLC,
and the SE-30/QF-1, Carbowax-treated, for all FPD work. An alter-
nate column that may prove useful is the OV-210. Expected response
data for a number of organophosphorous compounds are given in Table
1. Retention values, relative to parathion (RRTp) are given in
the tables in Section 4, B for 54 O.G.P. compounds on three diff-
erent GLC columns at temperatures ranging from 170 to 204°C.
2. The pesticides of interest may be in any of the three fractions and
can be identified on the electron capture or flame photometric
detector systems as needed, (i.e., Fraction I contains organo-
chlorine pesticides; Fraction II contains organochlorine, chloro-
phenoxy acid esters and organophosphate pesticides; and Fraction III
-------
Revised 12/2/74-
Section 8, B
- 7 -
VI. MISCELLANEOUS NOTES (CONTD.)
contains malathion.) Information on any other organochlorine or
organophosphate pesticide of particular interest may be character-
ized by monitoring the appropriate fraction with either the
electron capture or flame photometric detector.
3. As a reiteration, the miscellaneous notes discussed in the Section
5, A, (1), under the "Analysis of Adipose Tissue", are very perti-
nent to this methodology and should be thoroughly understood.
4. The analyst must know from predetermined tests of recoveries and
compound elution patterns which compounds will appear in each
Florisil fraction eluate of the particular lot of Florisil being
used. Certain laboratories operating under contract with EPA
are supplied with standardized Florisil of known adsorptive
characteristics. It may prove necessary to use more or less than
the 4 inches of Florisil specified to achieve the partitioning
indicated in Table 1, this section or in Table 1 of Section
5, A, (1).
5. Occasions arise when positive compound identification by electron
capture is not possible. This situation is not unusual with
certain of the organochlorine compounds at low concentration levels.
One confirmation technique involving minimum effort is TLC. This
is described in Section 12, B, pp 7 and 8. Another relatively
rapid technique is the p-value procedure described in Section 12, C.
Those laboratories equipped with electrolytic conductivity or
microcoulometric detectors can utilize this equipment for both
qualitation and quantitation.
-------
Revised 11/1/72
Section 8,B
" 8 "
TABLE 1. FLORISIL ELUTION PATTERNS, RECOVERY EFFICIENCY,
AND LOWER LIMITS OF DETECTION OF SOME COMMON
PESTICIDES EXTRACTED FROM ETHYLENE GLYCOL BY
PROCEDURE OUTLINED IN SUBSECTION III.
3
Elution Lower Detection Limits (ng/m )
Compound Fraction 4% SE-30/6% QF-1 1.5% OV-17/1.95% QF-1
a-BHC
I
0.2
0.2
Diazinon
II
.4
-
3-BHC
I
.8
1.2
Lindane
I
.4
.4
6-BHC
I
.4
.4
Heptachlor
I
.4
.4
Ronnel
I
.6
-
Aldrin
I
.6
.6
Heptachlor Epoxide
I
.8
.8
Dieldrin
II
1.2
1.8
Endrin
II
2.6
4.0
o,p'-DDE
I
1.4
2.0
Chlorobenside
I
1.6
1.2
p,p'-DDE
I
1.0
1.2
Ihiodan I
II
-
o,p'-DDD
I
2.0
2.8
*Perthane
I
167.
167.
p,p'-DDD
I
1.6
2.0
o,p'-DDT
I
2.0
2.6
Ihiodan II
III
-
-
p,p'-DDT
I
2.0
1.5
Tedion
II
25.
Ethyl Parathion
II
1.3
Methyl Parathion
II
.9
Malathion
III
1.3
Ethion
I
1.5
Chlordane
I
-
Carbophenothion
I
3.1
Aroclor 1254
I
-
2,4-D esters II
*ME
*BE 13.6 10.2
*IOE 50. 44.
IPE 6.2 9.
*BOEE 43. 22.
*PGBEE 19. 15.
-------
Revised 11/1/72
Section 8,B
TABLE 1 (CONTD.)
Compound
2,4,5-T esters
*IOE
*IPE
*n-Butyl
- 9 "
3
Elution Lower Detection Limits (ng/m )
Fraction 4% SE-30/6% QF-1 1.5% 0V-17/1.95% QF-1
II
18.
1.4
7.6
1.6
Lower detection limits of organophosphorous compounds determined by flame
photometric detection; all other compounds by electron capture.
^Recovery efficiencies not established for these compounds. Recoveries of
85% or better determined on all other compounds listed.
-------
Revised 11/1/72
Section 8.B
" 10 -
TABLE 2. ELLTTION AND RECOVERY OF ORGANOPHOSPHOROUS
PESTICIDES BY MOG METHOD
Copied from F.D.A. Release
Elution from Florisil
Pesticide Name % Recovery 61 15% 50%
Abate
-
Azinphosethyl
50%
-
-
-
Az inphosmethy 1
0
-
-
-
Azodrin
0
-
-
-
Bensulide
-
Bidrin
0
-
-
-
Bomyl
0
-
-
-
Bromophos ethyl
102%
+
-
-
Carbophenothion Oxygen Analog
0
-
-
-
Ciodrin
0
-
-
-
Compound 4072
0
-
-
-
Coumaphos
0
-
-
-
Def
91%
-
+
+
Demeton
0
-
-
-
Dicapthan
76%
-
+
+
Dichlorvos
-
-
-
-
Dimethioate Oxygen Analog
0
-
-
-
Dimethioate
0
-
-
-
Dioxathion
0
-
-
-
Disulfoton
51%
+
-
-
Disulfoton Oxygen Analog
0
-
-
-
Disulfoton Sulfone
0
-
-
-
Dyfonate
Falone
91%
+
Famphur
0
-
-
-
Fenthion
45%
+
+
-
Fensulfathion
0
-
-
-
Fensulfathion Oxygen Analog
0
-
-
-
Fensulfathion Sulfone
0
-
-
-
Gardona
0
-
-
-
Gophacide
-
-
-
-
Hercules 14503
95%
-
+
-
Imidan
0
-
-
-
Malathion Oxygen Analog
0
-
-
-
Maretina
v,
-
-
-
Merphos
133%°
-
+
-
Methyl Parathion Oxygen Analog
0
-
-
-
Methyl Trithion
82%
+
-
-
Mevinphos
0
-
-
-
Mocap
55%
-
-
+
-------
Revised 11/1/72
Section 8,B
- 11 -
TABLE 2 (CONTD.)
Elution from Florisil
Pesticide Name % Recovery 6% 151 501
Naled
0
-
-
-
Nemacide
110%
+
-
-
OMPA
0
-
-
-
Parathion Oxygen Analog
0
-
-
-
Phenkaptan
95%
+
-
-
Phorate Sulfone Oxygen Analog
0
-
-
-
Phosalone
83%
-
-
+
Phosphamidon
0
-
-
-
Phostex
67%
+
-
-
Ronnel Oxygen Analog
0
-
-
-
Ruelene
0
-
-
-
SD 7438
98%
-
+
-
Sulfatepp
85%
+
-
-
Sumithion
81%
-
+
-
Supracide
33%
-
-
+
Trichlorfon
0
-
-
-
Zinophos
59%
-
+
-
Zytron
100%
+
-
-
Recovery of pesticide not detemined because the compound was not
satisfactorily chromatographed using the GLC conditions of this experiment.
(6 ft. x 4 mm 10% DC 200 on 80/100 Gas Chrom Q; C.T. = 200°C, D.T. = 205 C,
Inlet = 225 C; N2 flow = 120 ml/min; KC1TD detector.)
recovery determined by averaging results of 3 determinations. The
majority of the pesticide was obtained in the 15%. elution; however, a
portion was obtained in the 50% fraction in one determination and in the
6% fraction in two of the determinations.
S/alue listed for SD 7438 is the result of a single determination. Except for
this pesticide and for Merphos, all recovery values which are listed are the
average of two determinations.
-------
Revised 11/1/72
" 12 "
Section. 8,B
Figure 1. Flew Sheet Diagram of Analysis of Air Sample
ELUTE WITH 15%
MIXED ETHERS
(FRACTION, II)
ELUTE WITH 50%
MIXED ETHERS
(FRACTION III)
INJECT G.C.
WITH E.C.D.
INJECT G.C.
WITH F.P.D.
INJECT G.C.
WITH F.P.D.
FILTER CLOTH
INJECT G.C.
WITH F.P.D.
EVAPORATE TO
EVAPORATE TO
5.0 ml
EVAPORATE TO
5.0 ml-
INJECT G.C.
WITH E.C.D.
ETHYLENE GLYCOL
INJECT G.C.
WITH E.C.D.
EVAPORATE TO
COLLECT IN K-D
FLASK
COLLECT IN K-D
FLASK
COLLECT IN K-D
FLASK
EVAPORATE TO
1.0 ml
DILUTE WITH H~0
EVAPORATE FURTHER
TO 1.0 ml
WASH COMBINED
HEXANE WITH H„0
PASS THRU
Na-SO. COLUMN
INJECT G.C.
WITH F.P.D.
EXTRACT WITH SAME
HEXANE
TRANSFER TO BEAKER
COMBINE HEXANE IN
SEPARATORY FUNNEL
ELUTE WITH 6%
MIXED ETHERS
(FRACTION I)
COLLECT IN K-D
FLASKS AND
EVAPORATE TO
1 ml
DILUTE UP TO 5 ml
AND PASS THRU
FLORISIL COLUMN
EXTRACT 3 TIMES WITH HEXANE
TRANSFER TO SEPARATORY FUNNEL
COMBINE SAMPLES
-------
Revised 11/1/72
Section 8,C
Page 1
SAMPLING AND ANALYSIS OF PESTICIDES IN AIR
C. EVALUATION OF ETHYLENE GLYCOL
I. INTRODUCTION:
Ethylene Glycol (hereinafter referred to as E.G.) is purchased in
4-kg glass bottles. Each lot received must be sampled and subjected to
analysis by GLC, E.C. and FPD, to insure that the material does not
contain contaminants which will result in chromatographic peaks that
interfere with peaks resulting from pesticides. The following procedure
is designed to provide this information.
II. APPARATUS § REAGENTS:
1. Erlenmeyer flask, 500 ml with $ glass stopper.
2. Grad. cylinders, 50 and 500 ml.
3. Separatory funnels, with Teflon stopcocks, 250 and 500 ml.
4. Chromatographic columns, 300 mm column length x 22 mm i.d. (no frit).
Kontes #420530, size 241, or the equivalent.
5. Evaporative concentrator tubes, 10 ml, Kontes #570050, size 1025,
or the equivalent.
6. Kuderna-Danish flasks, 500 ml, Kontes #570001 or equivalent.
7. Snyder columns, size 121, Kontes #503000 or Kjeldahl bulbs, 51 24/40,
Ace Glass #5230-1.
8. Hexane, pesticide grade, distilled in glass, prechecked for zero
background by GLC, E.C. at a concentration of 180:1.
9. Distilled water, prechecked for contaminants by GLC, E.C. (See
NOTE in III., 4, next page).
10. Sodium Sulphate, anhydrous, A.R. grade. Prechecked for con-
taminants and, if found, extracted as described in Section 5,A,(1).
11. Glass wool, pre-extracted with hexane.
-------
Revised 11/1/72
Section 8,C
- 2 -
III. PROCEDURE:
All glassware used must be scrupulously cleaned and given a final
hexane rinse immediately before use.
1. The E.G. will be contained in 4-kg or 1-gal. amber glass bottles
with Teflon-lined screw caps. One composite sample of each manu-
facturer's lot will be taken for evaluation. The number of
bottles to be sampled depends on the size of the lot. If the lot
consists of less than a dozen bottles, the composite should be
comprised of some fluid from each individual bottle. If the lot
is 12 to 24 bottles, half the bottles should be sampled. From 24
to 48, a quarter of the total bottles in the lot should be sampled.
2. Shake each bottle by inverting end for end about 15 times and pour
25 ml into a grad. cylinder. Transfer each 25 ml into a 500-ml
Erl. flask. Stopper flask and shake ca 50 times to insure complete
mixture of composite.
3. Carefully measure 80 ml of the E.G. composite into a 100-ml grad.
cylinder and retransfer this to a 500-ml sep. funnel.
4. Measure 350 ml of dist. water into a 500-ml cylinder and transfer to
the sep. funnel containing the E.G., rinsing the 100-ml cylinder
used to measure the E.G. composite with several portions of the
water.
NOTE: At this point, prepare a 350-ml water blank
and carry through all following steps.
5. Add 60 ml of hexane, stopper and shake vigorously two minutes.
6. Allow layers to separate, draw off lower aqueous layer into a
second 500-ml sep. funnel (No. 2), and the upper hexane layer into
a 250-ml sep. funnel (No. 3).
7. Add 60 ml of hexane to the aqueous solution in Funnel 2 and again
shake vigorously two minutes.
8. Draw off lower aqueous layer into Funnel 1 and hexane layer into
Funnel 3.
9. Repeat the extraction a third time, discarding the final aqueous
layer and combining ail three hexane extracts in Funnel 3.
-------
Revised 11/1/72
Section 8,C
- 3 -
III. PROCEDURE (CONTD.)
10. Add 50 ml of H-0 to Funnel 3, stopper and shake 1 minute, discard
aqueous layer and repeat scrubbing with another 50-ml portion of
water also discarding the final aqueous layer.
11. Place a glass wool retaining plug in the bottom of a 300-mm
chromatographic column, add 2 inches of Na^SO. and pass the
combined hexane extract through the column, collecting eluate in
a 500-ml K-D flask which has been fitted to a 10-ml evaporative
concentrator tube containing one 3-mm glass bead.
12. When extract has eluted from the column, rinse the sep. funnel
with three consecutive portions of 10 ml each of hexane, washing
down the walls of the column. Allow each rinse to elute off the
column before adding the next. Finally, rinse the column walls
with two more 10-ml portions of hexane.
13. Place K-D assembly in a boiling water bath and evaporate to 5 ml.
Remove the tube from K-D flask rinsing joint with a little hexane.
Concentrate to exactly 1 ml under a nitrogen stream at room temp.
14. Conduct GLC, E.C. with the 1.5% OV-17/1.95% QF-1 column, following
instructions given in Section 4,A of this manual.
15. At room temperature and under a nitrogen stream, evaporate extract
to exactly 0.2 ml and conduct GLC, FPD using the 4% SE-30/6% QF-1,
Carbowax-treated column (See Section 4,B).
IV. INTERPRETATION OF CHROMATOGRAPHIC DATA:
1. It is necessary that the contaminant peaks contributed by the water
be subtracted from the peak structure obtained in the sample
chromatogram. If the water blank yields troublesome contaminant
peaks, it may be necessary to purify the water by multiple extrac-
tions with benzene and hexane.
2. Generally, if the contaminant peaks from the E.G. do not exceed
10% f.s.d. and whose R.R. ratios do not indicate an overlap with
any pesticide peaks, the E.G. would be considered acceptable. If
such peaks match the R.R. ratios of any of the organophosphate
compounds via E.C. but are not detected by flame photometric, the
E.G. would be considered acceptable.
If contaminant peaks are produced of less than 101 f.s.d. which
match the R.R. ratios of any of the common chlorinated pesticides,
-------
Revised 11/1/72
Section 8,C
- 4 -
IV. INTERPRETATION OF CHROMATOGRAPHIC DATA (CONTD.)
the SE-30/QF-1 column should be tried. Should this also produce
overlaps, consideration may be given to rejection of the lot
depending on the size and location of the contaminant peak(s).
3. If contaminant peaks greater than 10% f.s.d. are obtained by
E.C. but do not produce R.R. ratios overlapping any of the
pesticides, acceptance of the sample would probably be indicated.
One possible exception to this would be an extremely late eluting
peak which, although not interfering with any pesticide, might
entail excessive delays between injections.
-------
Revised 12/4/74
Section 9,A
POLYCHLORINATED BIPHENYLS
A. INTRODUCTION
All chromatographers with experience in the analysis of bio-
logical materials are only too familiar with problems involving "artifact"
peaks which, based on their retention characteristics, could be identified
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 the 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 sub-sections 9, E and 9, F.
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 12/2/74
Section 9,C
Page 1
SEPARATION OF SOME POLY CHLORINATED 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 completely
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 chromatographic patterns because
of the various components represented. These peaks appear in and beyond
the retention time region of the organochlorine pesticides. If present
in high enough concentration, relative to pesticides present, PCB can
interfere with the determination of some organochlorine pesticides. Like-
wise, the presence of certain organochlorine pesticides can interfere
with the determination 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 determination. In order to analyze
for the PCT, it is necessary to use a GLC column and
operating parameters which permit much more rapid
elution and greater sensitivity for the chloroterphenyl
components.
A number of procedures have been proposed for dealing with the
various pesticide PCB combinations encountered. These include the silicic
acid column chromatographic separation technique presented in detail
here and several other published approaches, some of which are noted in
Subsec-tion VII. The residue analyst must make judicious use of the
available techniques in order to obtain accurate results. The proper
course of action in the determination of residues of PCB and pesticides
found together depends on the suspected identity of each and on the
estimated amounts of each. Some combinations will permit quantitation
of both pesticides and PCB without their separation from one another on
the silicic acid column. Other combinations of PCB and pesticides must
-------
Revised 11/1/72
Section 9,C
- 2 -
be separated before quantitation; still other combinations cannot be
separated by silicic acid, yet cannot be determined in the presence of
one another. The relative amounts of residues of pesticides and PCB
may also influence the decision on whether or not to perform silicic
acid separation prior to quantitation. Even when the residues will not
be completely separated by this technique, its use may be the best
means of achieving quantitative 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 be-
tween p,p'-DDE and Aroclor 1254. The interfering PCB are eluted with
petroleum ether from a column of the standardized silicic acid. The DDT
compounds, most other organochlorine pesticides, and some other PCB are
then eluted from the column with a mixture of hexane, methylene chloride,
and acetonitrile. See Table I for list of chemicals eluting in each of
the two eluates.
The method is applicable to the 6% Florisil eluate [Sect. 5,A,(1)]
obtained in the analysis of fatty tissues or to extracts cleaned up by
other methods for gas chromatography. Extracts in polar solvents must
be transferred to nonpolar solvents prior to separation.
III. 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.
-------
Revised 11/1/72
Section 9,C
- 3 -
3. Kuderna-Danish Assembly as follows:
Evaporative concentrator flask - Kontes Catalog No. K-570000,
500-ml capacity, lower joint 19/22 S, upper joint 24/40 Snyder
column - 3 ball, lower joint 19/22 S, upper joint 24/40 !; Tube -
10 or 15 ml capacity, 19/22 f upper joint.
4. Air pressure regulator - for pressure reduction to deliver ca 1 lb.
psig; air must be clean and dry.
5. Separatory funnel - Used for column eluant reservoir, 250 ml, with
Teflon stopcock, 24/40 $ joint at top, Kontes No. K-633030, or the
equivalent.
6. Hot water bath adjustable to temp, of 90-100°C.
IV. REAGENTS:
1. Petroleum ether, acetonitrile, hexane, and methylene chloride, all
pesticide quality.
2. Celite 545, Johns-Manvilie.
3. Silicic acid, Mallinckrodt, 100 mesh powder; "specially prepared for
chromatographic analysis by the method of Ramsey and Patterson,"
Analyt. Reagent No. 2847.
V. PREPARATION OF SPECIAL REAGENTS:
1. Celite 545 - must be dry and free of electron capturing substances.
If electron capturing substances are extracted by petroleum ether,
treat as follows: Slurry Celite with 1+1 hydrochloric acid -
H-0 while heating on steambath; wash with FLO until neutral; wash
successively with several portions each of methanol and acetone (to
remove H~0); 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.
-------
Revised 11/1/72
Section 9,C
- 4 -
3. Silicic acid - Place silicic acid to depth of about 1 inch in open
beaker and hegt 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% H~0 by pipette
(97 g silicic acid + 3 ml H-0 = 3% H^O). Stopper bottle tightly
and seal with tape to insure that container is air tight. Shake
well until all H?0 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 ug p,p'-DDE in hexane.
Elute as described and determine recoveries in each eluate. Inade-
quate separation of PCB from p,p-DDE will require that further test-
ing be done with other heated batches of silicic acid, treated with
different amounts of f-LO as needed to achieve the desired separation.
Increments of 0.25% or 0.5% more or less FLO are recommended for the
testing. More H^O is required when the initial test results show
PCB eluting in tne polar solvent with the p,p'-DDE; less H~0 when
p,p'-DDE elutes in the petr ether fraction.
This testing and standardization is required for each new lot of
silicic acid obtained from the manufacturer.
Once a batch of standardized silicic acid is prepared, it should be
stored in a desiccator between uses. Desired activity remains for
about 5 days.
VI. PROCEDURE:
SEPARATION OF PCB 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.
-------
Revised 11/1/72
Section 9,C
- 5 -
NOTE: Apply air pressure to top of column as much as
necessary to force enough petr ether from column
to allow space for all silicic acid - Celite.
3. Stir material in column with long glass rod to remove air bubbles,
applying air 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.
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 required for
determination. The weight of sample equivalent
placed on the column may also affect separation by
causing p,p'-DDE to appear in the petr ether eluate.
Should this occur, an amount of extract equivalent to
a smaller weight of sample should be used. In
analysis of samples by this procedure, it is suggested
that no more than 0.3-0.4 g fat equivalent be placed
on the silicic acid column.
5. 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.
-------
Revised 11/1/72
Section 9,C
- 6 -
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 CH^CN-hexane-CFLCl- eluant
to upper reservoir. Open stopcock and slowly reapply air pressure,
continuing elution until all of eluant passes through column into
the K-D concentrator.
11. Place Snyder columns on both K-D assemblies, place in a hot water
.bath and reduce eluate volumes to 5 ml in preparation for explora-
tory GLC analyses.
NOTE: The first (petr ether) eluate should contain the PCB's
and the combined solvent eluate should contain the
chlorinated pesticides.
DETERMINATION OF POLYCHLORINATED BIPHENYLS
Determine quantity of PCB in the sample by electron capture GLC or
by halogen specific microcoulometric or electrolytic conductivity GLC.
Compare the total area of response for the residue to the total area of
response for a known weight of the Aroclor(s) reference with most similar
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 conducti-
vity detection is often preferable as means of quantitation. This type
of detection system also provides confimiatory evidence for the identifi-
cation of the residue as PCB.
-------
Revised 11/1/72
Section 9,C
- 7 -
VII. REFERENCES TO ADDITIONAL PROCEDURES:
The discussion under INTRODUCTION indicates the complexity involved
in analysis of samples containing combinations of PCB and pesticides,
including combinations of more than one Aroclor. The following procedures
can be used to provide additional information on the identification and
quantitation of residues found by the GLC determination. In certain cases,
use of one or more of these techniques may be the only way to determine
the residues. Knowledge of the behavior of compounds of interest in each
of these procedures is necessary in making a decision on the best approach
to a particular analysis.
The papers cited here do not always contain such complete information,
so it may be necessary for the residue analyst to generate additional
recovery data before using a method.
1. Alkaline hydrolysis. Young, S. J. V., and Burke, J. A., Micro Scale
Alkali Treatment for Use in Pesticide Residue Confirmation and Sample
Cleanup, Bull. Environ. Contam. Toxicol., in press. Vol. 7, No. 1
(1972); Krause, R. T., Quantitative Dehydrochlorination of Perthane
Residues and Effect of Alcoholic Potassium Hydroxide on Other
Pesticides, JAOAC, in press.
The stability of PCB to alkali permits the use of alkaline hydrol-
ysis as a test for confirming the identity of PCB residues. At the
same time, the conversion of DDT to DDE by alkali treatment provides
a means of removing some interferences from the quantitative measure-
ment of PCB. Alkaline hydrolysis also provides an additional cleanup
for many sample types. An appropriate sequence of determination
step(s) and alkaline hydrolysis can be chosen to permit quantitation
and confirmation of both PCB and the DDT compounds.
2. Oxidative treatment, (a) Mulhern, B. M., Cromartie, E., Reichel,
W. L., and Belisle, A. A., Semiquantitative Determination of Poly-
chlorinated Biphenyls in Tissue Samples by Thin Layer Chromatography,
JAOAC, 54, 548-550 (1970).
Cleaned up sample extracts are reacted with a chromic acid-acetic
acid mixture to convert DDE to 4,4'-dichlorobenzophenone. The
authors follow this reaction with ILC in which the PCB migrate as
a single spot and are not separated from DDE, but are separated from
the benzophenone. Sequential use of the alkaline hydrolysis and
oxidation on a sample containing PCB, DDE, and DDT would result in
the conversion of both o,p'- and p,p'-DDT and o,p'- and p,p'-DDE to
the respective chlorobenzophenone while leaving the PCB unchanged.
-------
Revised 11/1/72
Section 9,C
- 8 -
(b) This manual, Section 9,D, Semi-Quantita-
tive Estimation of Polychlorinated Biphenyls in Adipose Tissue.
3. Two-dimensional TLC. Fehringer, N. V., and Westall, J. E., Separa-
tion and Identification of DDT analogs in the Presence of Polychlori-
nated Biphenyl Compounds by Two-Diinensional Thin-Layer Chromatography,
J. Chromatography 57, 397-405 (1971).
Thin-layer chromatography is used in a manner designed to separate
PCB from DDT and its analogs. After an initial development in
n-heptane, the TLC place is rotated, spotted with additional
standards and developed with 2% acetone in n-heptane in a direction
perpendicular to that of the first development. The pattern of
spots indicates the identity of residues in the sample.
4. Reversed-phase partition TLC. de Vos, R. H., and Peet, E. W., Thin-
Layer Chromatography of Polychlorinated Biphenyls, Bulletin Environ.
Contam. § Toxicol. 6_, 164-170 (1971).
A stationary phase of liquid paraffin on Kieselguhr G adsorbent is
utilized in a TLC system which involves three successive developments
of the spotted plate with a mixture of acetonitrile, acetone, methanol,
and water. Visualization is by the usual silver nitrate spray and
UV irradiation. These parameters permit separation by TLC or DDE,
DDT and several other organochlorine pesticides from the components
of Aroclor 1260, and cause the PCB to appear as a number of distinct
spots rather than a long streak as sometimes occurs with other TLC
systems.
[Editor's Note: The ability of this TLC system to separate Aroclor
1260 into several spots suggests the possibility
that further work would show the system capable
of distinguishing among the different Aroclors.]
-------
Revised 11/1/72 Section 9,C
- 9 -
Table 1. Pesticides and Other Chemicals Recovered Through Silicic Acid
Column Chromatographic Separation of Some Polychlorinated
Biphenyls (PCB) from Certain Organochlorine Pesticides.3
Petroleum Ether Eluate
Acetonitrile, Methylene Chloride,
Hexane Eluate
Aldrin ,
Aroclor 1221,
Aroclor 1242,
Aroclor 1248
Aroclor 1254
Aroclor 1260
Aroclor 1262
Aroclor 4465 ,
Aroclor 5460c,a
hexachlorben zene
mirex
octachloro-dibenzo-p-dioxin ^
polychlorinated naphthalenes
2,3,7,8-tetrachloro-dibenzo-p-dioxins
Aroclor 1221,
Aroclor 1242,
Aroclor 1248,
Aroclor 5442 ,
Aroclor 5460c'
BHC (all isomers)
chlordane (technical)
p,p'-DDE
o,p'-DDT
p,p'-DDT
dieldrin
endrin
heptachlor
heptachlor epoxide
lindane
Perthane^
p,p'-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.
cDivides 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 N^/min. (Wieneke, W., private com-
munication, Jan. 1972).
^irex may be separated from Aroclors 1260 and 1254 by collecting the first
100 ml petr ether separately. This fraction will contain the mirex. (Gaul,
J., private communication, July 13, 1971).
£
Method tested with commercial polychlorinated naphthalenes: Halowaxes 1014,
1099 (Armour, J., and Burke, J., JAOAC 5£, 175-177 (1971).
%rause, R. T., in press. JAOAC.
-------
1/4/71
Section 9, D
Page 1
SEMI-QUANTITATIVE ESTIMATION OF POLY CHLORINATED BIPHENYLS IN ADIPOSE TISSUE
I. INTRODUCTION:
The incidence of certain of the polychlorinated biphenyls (PCB's)
in human adipose tissue has become quite canmon 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 a modification of the method
developed by Mulhorn et. al., (1), provides a convenient means of separating
these compounds from the common chlorinated pesticides, confirming
identification, and approximating 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-
quantitation 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 Belisle
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
2. Private communication from Monsanto
Tentative Procedure for the Determination of Airborn
Polychlorinated Biphenyls.
-------
1/4/71
Section 9, D
- 2 -
3. Pioiike, 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, 9£,
pp 900-903o
4. W. W. Sans - Multiple Insecticide Residue Determination
Using Column Chromatography, Chemical Conversion, and Gas
Liquid Chromatography J. Ag. Food Chem 15 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 DDD 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,p'-dichlorobenzophenone which has an Rf value different from the PCB's.
The PCB's are then deteimined 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, S 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 ul, Kontes #763800,,
8. All equipment specified in Section 5, A,(1) of this manual for the
extraction and cleanup of adipose tissue.
9. Equipment specified in Section 12, B of this manual to conduct thin
layer chromatography.
-------
Section 9,
- 3 -
10. A bath of white mineral oilQand 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.
. REAGENTS AND SOLVENTS;
1. Hexane, pesticide quality.
2. Benzene, pesticide quality.
30 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 KQH, 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 nig)is dissolved in 3 ml of
ethanol. Five minutes of vigorous mixing should suffice to complete
solution.
12. Oxidizing solution - 1.5 grams of CrO, is added to 1 ml of distilled
water. Finally add 59 ml of glacial "TiAC. This solution should be
suitable for a month's use,
13. TLC plate coating - Dissolve 1 gram of AgNO, in 4 ml of distilled
water and add 56 ml of methanol. Mix this with 30 grams of Al^O., -G
and prepare 8" TLC plates as described in Section 12, B of this
manual.
-------
1/4/71
Section 9, D
- 4 -
14. Analytical reference standards of the series of Aroclor compounds
available from Perrine Repository.
15. a. Stock Standard solution, Aroclor 126Q. Weigh 50 mg, dissolve
in benzene and dilute to 50 ml. Concentration is 1 mg/ml.
b0 From the stock standard, prepare four working standards of
25,50,100 and 400 ng/ul, using hexane as the diluent.
V. PROCEDURE:
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 61 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.
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 pipet, 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„
-------
1/4/71
Section 9, D
- 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 evapc concentrator tube
containing the remaining 901 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 tube
under a nitrogen stream and ..evaporate jtodryness at room temperature.
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 dichlorobenzophenone 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 vigorously
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.
70 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
-------
1/4/71
Section 9, D
- 6 -
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- yuL of extract-is 25.5 mg per microliter.
Thin Layer Chromatography
1. On one 8-inch T.L. plate, spot 10 ul each of the four working
standards of Aroclor 1260 and also 10 ul 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, seme quantitative
dilution of the sample extract is required to reduce
the spot intensity to a level comparable with ttie
standards.
VI. MISCELLANEOUS NOTES:
1. Any p,p'-DDT present in the sample may be measured by GLC
quantitation of the p,p'-DDE peak before and after dehydrochlorination.
Also, any o,p'-DDT present in the sample may be quantitated by
measurement of the o,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 p.p.m. has been tentatively established for
this procedure.
3. Recovery studies have indicated a precision of +_ 50% for this
procedure when using Aroclor 1260 as the reference standard.
-------
Revised 11/1/72
4%SE-30/6%OV-210
Chromatograms of three ARCCLORS on column of
h% SE-30 / 6% OV-210. Column temp. 200°C.,
carrier flow 60 ml/min., detector, electron,
attenuation on an E-2 10 x l6j dotted line a
mixture of chlorinated pesticides, identity and
injection concentration given below:
' 1. Diazinon — 1.5 ng 7. o,p'-DDT — 0«2li ng
2. Heptechlor — 0.03 8. p,p'-DDD — .2U
3, Aldrin — .01*5 9. p,p'-DDT — .30
h. Hept.Epox. — .09 10. Dilan — .75
5. p,p'-DDE — .0? 11. Methoxychlor .60
6.. Dieldrin — .12
AROCLOR 1232
7*0 bike Hon
-------
Revised 11/1/72
4%SE-30/6%OV-210
Chromato^rams of three AROCLORS on column of
h% SE-30 / 6$ OV-210. Column temp. 200°C.,
carrier flow 60 ml/min., detector, electrom0
attenuation on an E-2 10 x 16; dotted line a
mixture of chlorinated pesticides, identity and
injection concentration given below:
1.
Diazinon
~ 1.5 ng
2.
Heptachlor
— 0.03
3.
Aldrin
—• 0oU5
ii.
Hept.Epox.
— .09
5.
p,p'-i;de
— .0?
6.
Dieldrin
— .12
8, p,p*-DDD — a2h
9. p,p'-DDT — .30
10. Dilan — .75
11. Hethoxychlor .60
AROCIOR 1254
li<( Wmi!«
Section 9, E
AROCLOR 1248
6 ng injection
©
I
\V
8
4
U
16
AROCLOR 1260
4 ng injection
-------
Revised 11/1/72 - 3 -
hromatograins of three AROCLORS on column of
•$% OV-17 / 1.95^ QF-1. Column tenp. 200°C.,
arrier flow 60 nl/roin., detector, electrometer
tten. 10 x 16 on an E-2; dotted line, a mixture
C chlorinated pesticide compounds, identity and
ijection concentration given below:
Section 9, E
Diazinon
Heptachlor
Aldrin
Hept.Epox.
p,p'-DDE
Dieldrin
1.5 ng 7* o,P'-DCT — 0.2U ng
0.03 8. p,p '-DDD — »2k
#0U5 9. p,p'-CDT ' — »30
.09 10. Dilan — .75
.09 11. Methoxychlor .60
.12
AROCLOR 1232
7ng injection
1.5% OV-17/1.95% QF-1
AROCLOR 1221
6 ng injection
AROCLOR 1242
5 ng Injection
-------
Revised 11/1/72
1.5% OV-17/1.95% QF-1
Chroma togra.ns of three AROCLORS on column of
1.52 OV-17 / 1.952 QF-1. Column temp. 200°C.,
carrier flow 60 nl/min., 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 — 1.5 ng 7. o,p'-DDT •— 0.2U ng
2» Heptachlor — 0.03 8. p,p*-DDD — »2U
3. Aldrin — .OiiS 9. p^p'-DDT — .30
ii, Hept.Kpox. — ,'09. ,10. Dilan — .75
5. p,p '-DDE — .09 11. Methoxychlor .60
6. Dieldrin — .12
AROCIOR 13S4
3 — Mwilw
- 4 - Section 9, E
AROCLOR 1248
4 ng injection
AROCLOR 1260
3 ng injection
-------
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 , 6-ft., 4 mm. i.d., 1.5% OV-17/1.954 QF-1,
200 C Column Temp. Carrier flow 60 ml/min.
Misc.1 ^
Pesticides''7
#1221
#1232
I I
<11242
1 1
01248
r 1
tflp.5
1
4
#1260
3 1
RRR |
rphj-
RRR 1
RPH
RRR 1
RPH
RRR
RPH
RRR 1
RPH
RRR
1 RPH
¦RRR*
0.27 1
0.19
0.27 ]
0.17
1
|
1
|
1
1
|
Phosdrin
0.32
134 |
.03
.33 1
.03
1
1
1
1
.40 I
.05
.40 |
.07
1
|
1
|-
1
|
1
|
".43 }
.27
.43 1
.27
r
1
1
1
Thimet
.50
.49 1
1.00
•«!
1.00
0.48 ]
0.34
0.48
0.08
1
|
1
1
Diazinon
.63
.62 |
.05
.61 1
.38
.63.1
.41
.63
.34
1
1
*
•«!
.20
.74 {
.22
.74
.15
1
|
1
I
i'eptachloi
*
.83
.82 !
.04
".80 1
.85
.82 1
l.QO'
.82
1.00
1
1
(J-BHC
.92
.91 1.
.02
'.90 J'
• .33
.90 |
.38
.92
.31
I
1
"N
.95 1
.26
.97 1
.32
.98
.24
1
Aldrin
1.00
I
1-03 |
.13
1.05 |
".23
1.06
.68
1.04 J
0.23
1
I
w
1
i
1.22 1
.20
1.24 1
.23
1.26
.62
1.24 1
.14
1
1
1.30 |
.19
1.33 [
.21
1.34
.56
1.34 |
.08
1
1
^ , •
Farathion
1 A 7
X .TJ
1
1.44 1
.13
1.49 !
.15
1.50
AO
• it
1.48 1
.07
1
Hept.
1.54
1
1.54 !
,27
1.57 !
.29
1.58
.96
i
1.56 i
.29
1
Malathion
l.«
1
i
1
1
1.62 1
.33
1.61
'0.09
S I
P&rathion
5
1.81
1
1.83 !
.19
1.88 !
.21
1.88
.65
1.80 J
.58
1.78
| .16
1
i
. 1
1
1
1.97 1
.13
p,p'-DDE
2.23
1
2.26 j
.03
i
2.21
.13
2.20 !
.19
i
I
1
I
1
2.32
.17
2.29 1
.36
Dieldrin
2.40
1
2.43 |
.05
2.52 J
.03
2."50
• 24.
2.47 ;
.68-
2.53
[ .39
Endrin
2.93
i
I
2.78 1
.07
2.85 1
.04
2.85
¦ .32
2.82 L
1.00
2.81
1 .42
Ojp'-DDT
3-. 17
1
3.13 J
.02
3.18
.24
3.16 .[
.53
3.20
j 1.00
p.p'-DDD
A
3.49
i
3.57 1
.04
3.67 1
.02
3.66
.16'
3.60 1
.50
3.50
\'.S2
p,p'-DDT
4.18
1
4.04 |
.03
l
4.12
.05
4.08 |
.66
4.10
! *74
Ethion
4.44
1
I
¦ •
) 1
1
.08
4.38
1 .58
1
1
l
l
5.0
| .07
1
1
1
1
1
5.28 1
.11
5.4
1 .38
Dilan I
5.7
1
i
1
1
|-
1
1
5.7
! .18
1
1
1
1
1
5.9? 1
.09
1
Dilan II
6.4
1
1
1
1
r
1
6.4 J
.09
6.4
, .90
8.3
1
1
I
1
8.4 1
.Of
8.4
1 .43
1
1
1
1
i i
1
1
1 ^
I
!
1
1
1
— i
¦
1
i
1
l
1
l
1
Relative retention ratios given for 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 peak shown in
italyoa.
^Indicates chlordane peaks eluting in the appropriate area.
-------
1/4/71
Section 9, F
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 nm. i.d., 4% SE-30/6% QF-1, 200°C Column temp.
Carrier flow 70 ml/min.
Detector: Electron capture, ^H, parallel plate, 210°C.
Misc.'
Pesticides
<71221
1
171,232
1
51242
i i
01248
1 1
171254 --
1
#1260
i ' 1
XRR | RPH
RRR 1 RPH
RRR 1 RPH
RRR 1 RPH
RRR 1 RPH
RRR 1 RPH
¦RRR1
0.24 1 0.37
0.25 [ 0.26
1
|
|
i
|
1
|
Phosdrin
0.32
.32 J .07
.32 1 .07
1
1
1
1
*
.34 1 .13
.35 | .13
0.34 [ 0.04
1
|
1
|
1
1
.39 ~| .40
.39 I .33
.38 | .08
1
1
1
2,4-D(ME)
.44
.44 1 1.00
.43 J 1.00
.43 | .52
0.44 | 0.14
" 'I
|
1
!
Thimet
.47
1
|
1
.47 | .03
1
1
1
Simazine
.54
.55 1 .04
.55 J .37
.53 ] .54
.55 ] .42
1
1
I
1
Lindane
.60
.60 j .05
.62 | .28
.61 | .38
.621 .25
1
1
a-BHC
.69
.73 | .06
.72 | -.70
.72 1 1.00
i
; 73 1 .99
i
1
1
1
2,4-D.(BE)
K
.78
1
i
.79 1 .31
.77 I .43
.80| .34
1
1
Heptachlor
.83
1
.82 J .27
.81 ] .36
.83] v36
1
1
1
1
Ronnell
.91
1
1
.90.1 .14
.89 | .18
.90| .61
,0.90 | 0.38
1
Aldrin
1.00
1
1.02 J .16
1.00 [ .22
1.03] .66
1.021 .25
i
1
I
1
i
1.09 I ,20
1.07 | .27
1.101 .68
1.081 .14
1
1
1.16 ' n,z
1.15 1 .02
1 - 38 J .10
1
1
1
M
Parathion
1.34
1
I
1.31 1 .24
1.30 1 .30
1.321 I.00
1.291 .79
1.30 10.10
Hept.
Fpnyi rip
*1.43
1
1.48 | .19
1.47 | .23
l.So] .78
1.48] .95
1.52 ] .17'
p,p'-DDE
1.82
1
I
1.77 1 .03
1.77 1 .03
1.801 .19
1.751 .53
1.80 I .07
Captan
1.94
1
1.96 | .04
1.90 | .03
1.92] .25
1.87] .83
1.93 j .08
0,p'-DDD
1.98
1
i
1
2.02 1 .03
1
2.00| .09
2.02..I .36
Dieldrin
2.12
1
1
i
1
i
1 '
i
2.14] .28
2.18 1 .40
i
1
1
2.241 .07
2.2l\ 1.00
1
o.p'-DDT
2.39
1
2.27 | .05
2.27 J .02
2.30] .27
1
1
1
Endrin
2.42
I
1
1
2.51| .78
p,p'-DDD
2.55
1
1
I
i
I
1
1
2.62' .38
2.S9 h.00
Thiodan .11
'2.72
1
2.69 I .03
2.73| .18
1
1
1
1
I
1
I
,1
1
1
1
2.81 1 .35
p.p-.Dur
3.12
1
1
1
1
3.121 .06
2.991 .91
3.10 1 .67
Trithion
' 3.2C
1
1
1
I
1
I
1
1
3.39 ! .59
1
1
1
1
1
3.551 .12
3.71 I .06
3.95 J .4.1
1
1
1
1
1
1
1
, 1
1
Dilan I
4.4C
1
I
1
1
1
4.201 .10
Methoxy-
chlor
",6
1
1
1
1
1
4.66| .12
4.83 .9L
Dilan II
5.1
1
1
1
I 1
1
5.541 .05
5.77 1 .30
1
1
1
1 ;
... |
1
.. 1 . ..
1
6.2 J .20
Relative retention ratios given for Some common pesticides for comparison purposes.
2
RRR - Means the retention relative to al-drin.
^RPH - Means the peak height response relative to that of the tallest pegJc shown in
italics\
'Indicates chlgrdane peaks eluting in the approximate area.
-------
11/1/72
- 3 -
Section 9,F
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., 5/32" i.d., 5% OV-210, 200°C column temp., carrier flow 45 ml/miji.
Deteetor: Electron capture, ^H, parallel plate, 205°C.
Miscellaneous'''
Pesticides
#1221
#1232
#1242
#1248
#1254
. #1260
2rrr| 3rph
RRR [ RPH
RRR | RPH
RRR RPH
RRR J RPH
RRR RPH
"RRR
0.33 i 0.27
0.33 '0.19
1
T '
1
1
\
.411 .10
.42 , .07
t
1
1
I
.46 ¦ .31
.46 ' .24
0.47 i0.01
r- ¦
1
1
1
.51 , 1.00
.52 '1.00
.53 1 .43
0.52 1 0.08
1
.61i .02
.62 1 .30
.62 1 .50
.60! ». 23
1
1
Diazinon
0.73
.72| .06
i
: O
0
.71 1 .22
.69 J .11
1
t
I
1"
O
OO
.80 1 .84
¦ 81 {1.00
.80 | .58
1
1
.88] .03
.88 1 .13
.88 [ .16
.86 J .05
1
1
g-BHC
.94
.92; .03
.93 , .59
.94 1 .66
.92' .68
0.91 ' 0.25
1
1.12 ' .29
1
1.12 ; .29
I.O81 .54
1.081 .13
1
1
1-Hydroxychlordene
1.3C
1.29 1 .27
1.29 \ .17
1.281 .49
1.27; .38
1.28 1 0.05
¦ ¦
1.41 ' .54
1.41 1 .52
1.37' 1.00
1.391 .26
1
- -
I
1
1.471 .06
1.46 1 1.00
1
1.49 .20
1.62 ' .15
1.62 1 .17
1.591 .24
I
1.
I
1
Hep I. Epoxiile
1 "7"
1
\
t
1.771 .10
1.77; .41
1.72' .04
|
1
1
1
1
1
1
Dimethoate
1.7S
l
1
1.83 | .03
" \
1
1
1
1.86 .44
1
p,p'-DDE
1.81
1
1
1.96 1 .07
1.97 » .04
1.9 2 J .22
' 1.921 .75
1
o,p'-DDT
2.26
1
t
1
j
1
1
1
2.06,' .48
Malathion
2.32
|
|
2.32 1 .13
1
2.32 ' .08
1
2.28 L .32
2.28 j .77
1
M. Parathion
2.44
|
1
1
1
2.401 .57
2.44' .89
1
1
1
1
1
1
2.63i .11
2.66 , .22
1 _ '
1
2.82 | .05
2.86 , .04
2.78' .19
2.79; .20
1
p,p'-DDT
3.25
|
|
2.99 , .07
1
2.96i .08
2.931 .80
2.98 | 1.00
l
1
1
I
3.601 .02
3.63, .2]
3.58 t .43
|
1
1
1
4.32! .02
4.291 .13
1
Methoxychlor
4.5:
|
I
i
1
1
1
1
1
4.41, .76
|
J
1
1
1
1
5.32| .06
5.38' .34
1
1
1
»
t
1
6.5 ; .05
Dilan II
8.4
1
1
1
1
¦¦ t
1
1
8.0 1 .10
|
1
1
1
1
¦
1
l
1
1
1
t
r
1
4
RRA
' .014
1.0097
' .02
1 .02
] .025
1.027 j
1
i
1
!
1
I
1
•
1
1
1
1
.L.
I
!
Relative retention ratios given for some connon 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.
4 I
Indicates the peak height response of the tallest peak relative to that' obtained from an
- equivalent amount of aldrin.
-------
Revised 12/2/74
Section 10, A
Page 1
THE SAMPLING AND ANALYSIS OF WATER FOR PESTICIDES
I. INTRODUCTION:
The methodology for the sampling and analysis of water con-
tained in this section incorporates the basic principles and the
majority of the specific details of the method appearing in the
FEDERAL REGISTER, Volume 38, No. 125, 29 June 1973, Part II,
entitled ANALYSIS OF POLLUTANTS, Proposed Test Procedures.
This method, as described, has been determined potentially
acceptable for those compounds listed in Table 2 showing recoveries
of 80 percent or more. However, it is known that the method falls
short of acceptability for a sizable number of additional pesticides
in widespread agricultural use. For this reason, we present the
method provisionally and with reservations as to its acceptability
as an ideal multiresidue procedure. However, at the present time
it appears to offer fewer drawbacks than other published methodo-
logy. Analytical research is ongoing for a method offering a more
versatile approach to the multiresidue concept.
Minimum detection levels by this procedure as written vary
considerably for different compounds. The data in Table 2 indicated
a lower limit range of 0.03 to 4.44 ppb for the organochlorine
compounds and 0.25 to 23.0 ppb for the organophosphorous series.
REFERENCES:
1. FEDERAL REGISTER, Vol. 38, No. 125, 29 June 1973, Part II.
2. Manual of "Method for Organic Pesticides In Water and
Wastewater," 1971, EPA, NERC, Cincinnati, Ohio.
3. Persistence of Pesticides in River Water, Eichelberger,
J. W., and Lichtenberg, J. J., Envir. Sci. § Technol., 5_,
No. 6, June 1971 (Table 1).
II. 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 pollution .introduction. If, on the other hand,
the objective 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.
-------
Revised 12/2/74
Section 10, A
- 2 -
As implied by the name, a grab 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.
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.
III. 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. (See Section 3,A)
All bottle or jar caps should be Teflon or foil lined to prevent contami-
nation 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.
-------
Revised 12/2/74
Section 10, A
- 3 -
Ideally, analysis of the sample should be conducted within a
matter of hours from the time of sampling. However, this is
frequently impractical in terms of the distance from sampling site
to laboratory, and/or the laboratory workload. Samples being exam-
ined solely for organochlorine residues may be held up to a week
under refrigeration at 2 to 4°C. Those intended for organophos-
phorous 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.
Every effort should be made to perform the solvent extraction
step 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
determinative 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.
IV. EQUIPMENT:
1. Gas Chromatograph fitted with electron capture, flame photometric,
and electrolytic conductivity detectors. GLC columns to be two
of the three specified in Section 4, A, (2) with all operating
parameters as specified in Sections 4, A and 4, B.
2. Water bath capable of maintaining 95 to 100°C.
3. Separatory funnels, 2 L. with Teflon stopcocks.
4. Chromatographic columns, 22 mm. i.d. x 300 mm length, with Teflon
stopcock, without frits.
5. Filter tubes, 150 x 24 mm, Corning 9480 or the equivalent.
6. Kuderna-Danish concentrator fitted with graduated evaporative
concentrator tube. These are available from the Kontes Glass
Company, each component bearing the following stock numbers.
a. Flask, 500 ml, stock #K-570001
b. Snyder column 3-ball, stock #K-503000
c. Steel springs, 1/2" stock #K-662750
d. Concentrator tubes, 10 ml, Size 1025, stock #K-570050.
-------
Revised 12/2/74 Section 10, A
- 4 -
7. Modified micro Snyder Columns, 19/22, Kontes stock #K-569251.
8. Glass beads, 3 mm plain, Fisher #11-312 or equivalent.
9. Modified micro-Snyder column, 19/22 f joint, Kontes #K-569251.
10. Pipet, vol., 4 ml.
V. REAGENTS:
1. Hexane, pesticide quality, distilled in glass. Must meet the
purity criteria outlined in Section 3, C of this manual.
2. Isooctane, Pesticide quality.
3. 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 it is found necessary to remove peroxides.
NOTE: To determine the absence of peroxides, test in
accordance with method outlined in Section 5, A,
(1), page 2.
4. Petroleum Ether, pesticide quality, redist. in glass, b.p. 30-60°C.
Refer to Sect. 5, A, (1), page 9, NOTE 7.
5. Methylene chloride, pesticide quality.
6. Methylene chloride/hexane, 15% v/v.
7. Eluting mixture, 6% (6+94)-60 ml of diethyl ether is diluted to
1000 ml with pet. ether and ca 15 grams of anhydrous Na2SO^ is
added to insure freedom from moisture.
8. Eluting mixture, 151 (15+85)-150 ml of diethyl ether is diluted
to 1000 ml with pet. ether and ca 15 grams of anhydrous Na2S0^
is added.
9. Eluting mixture, 50% (50+50)-500 ml of diethyl ether is diluted
to 1000 ml with pet. ether and ca 15 grams anhydrous Na-SO. is
added.
/
NOTE: None of the eluting mixtures should be held
longer than 24 hours after mixing..
-------
Revised 12/2/74
Section 10, A
- 5 -
10. Anhydrous sodium sulfate, reagent grade, granular, Mallirikrodt~
stock #8024, or equivalent.
NOTE: The purity of this material should be tested as
outlined in Section 5, A, (1), page 3, except
that 15% methylene chloride/hexane should be
substituted for pet. ether.
11. Florisilj 60/100 mesh, PR grade.
NOTE: Observe comments on Florisil given.in Section 5, A,
(1), page 2 of this manual.
VI. PROCEDURE:
Extraction
It is assumed that final TLC and electrolytic conductivity
confirmation may be applied to supplement the information obtained
by electron capture detection. For this reason a larger sample is
used than would be necessary for E. C. alone. Dilution of an aliquot
of the final extract for E.C., GLC requires far less time than the
extraction of another sample for confirmatory purposes.
1. Transfer 2 L. of sample (or a lesser volume, if indicated) to a
4 L. sep. funnel and add 120 ml of 15% methylene chloride/hexane.
NOTES: 1. If, on the basis of prior analysis.of a given
waterway, the residue levels may be expected to
run high, a sample of^SUD" ml or 1 L. may be in-
dicated. In this event, the size of the sep.
funnel should be 2-liters and the extraction
solvent volumes given as 120 ml should be reduced
to 100 ml.
2. A 500 ml graduated cylinder is a suitable
measuring device for the initial sample.
Any measuring discrepancy up to 5.0 ml would
result in an error no greater than 1.0%.
2. Stopper funnel and shake vigorously 2 minutes. Allow layers to
separate and draw off aqueous layer into a second 2 L. sep. funnel.
3.. Add another 120 ml of 15% MC/hexane to the aqueous phase in sep.
funnel #2, stopper .and shake vigorously another 2 minutes.
4. Prepare a 2-inch column of anhydrous, granular Na2S0. i.n a 150
x 24 mm filter tube with a small wad of pre-extractea glass wool
at the* bottom. Position this over a 500 ml K-D flask to which
is attached a 10 ml concentrator tube with one" -3-mm glass bead
in bottom.
-------
Revised 12/2/74
Section 10,A
- 6 -
5. Filter the MC/hexane extract in sep. funnel #1 through the
Na2SO^ column into the flask.
6. Draw off the aqueous layer in sep. funnel #2 into empty sep.
funnel #1.
7. Add 120 ml of straight hexane to the aqueous solution in sep.
funnel #1, stopper and shake again for 2 minutes. "Draw off and
discard the aqueous layer.
8. Filter the solvent extracts in both sep. funnels through the
Na^SO. into the flask, rinsing down filter tube with three 10 ml
portions of hexane.
9. Attach a 3-ball Snyder column to the K-D flask, place assembly
in a boiling water bath and concentrate extract to ca 5 ml.
10. Remove K-D assembly from bath, cool and rinse 1 joint between
tube and flask with a small volume of hexane, also rinse down
walls of tube. Rinse should be delivered with 2 ml Mohr pipet
and should not exceed 3 ml.
11. Place tube under a slow nitrogen stream at ambient temperature
and reduce extract volume to ca 0.5 ml. Using a disposable
pipet, carefully add hexane to adjust volume to exactly 1.0 ml
in the tube tip. Then, with a 4 ml vol. pipet, add 4 ml of
hexane. DO NOT rely on the accuracy of the tube graduation
at the 5 mT mark.
12. Stopper concentrator tube and mix vigorously on Vortex mixer
for 1 minute.
GAS CHROMATOGRAPHY
The chromatographic approach should be made in accordance with
the guidelines presented in Section 4, A, (4), Steps 1 through 6,
and applying the instrumental operating parameters specified for the
respective GLC columns listed. By following the given protocol, it
should be possible to make some tentative compound identifications
upon computation of relative retention times (RRT^) of peaks in the
preliminary chromatograms via electron capture.
Full reliance should not be placed on the chromatographic data
obtained from one column. An alternate column of completely different
compound elution characteristics should be used to (1) confirm a number
of compounds tentatively identified on the first column, and (2)
isolate and tentatively identify any corrpound pairs which may have
eluted as single peaks on the first column. Subsequent quantitations
are conducted as outlined in Section 4, A, (4).
-------
Revised 12/2/74
Section 10, A
- 7 -
NOTE: If the initial chromatogram indicates the presence of
a sufficient amount of interfering materials, it may-
prove necessary to conduct a Florisil cleanup on the
extract. Based on the general experience of water
chemists, this is rarely necessary on most surface water
samples. If it should prove necessary, the cleanup
should be carried out as prescribed in Section 5, A (1),
pages 6-9. After the cleanup, another exploratory
injection, is.made followed by peak identifications and
quantitation.
If the E.C. data indicates the probable presence of one or more
chlorinated pesticide compounds, the chromatographer would be well
advised to conduct confirmation via electrolytic conductivity detection
in the reductive mode even though positive identifications were made
on two columns via electron capture. This extra step provides needed
validation, particularly when compounds are tentatively identified
which appear to be out of place in light of known supplemental data
concerning the waterway sampled.
It is improbable that parent compounds in the organophosphorous
class will be detected in an average water sample. Compound degra-
dation is rather rapid in the aqueous medium. However, if the
waterway receives heavy run-off from nearby agricultural land under-
going current spray programs, the presence of these residuals is
possible.
In general, many of the O.G.P. compounds are far less responsive
to electron capture detection than compounds in the O.G.C. glass.
This factor, combined with their highly diluted concentration in a
waterway, makes E.C. detection a dubious matter. Therefore, specific
detection is preferable for identification and quantitation of these
compounds or their metabolites. Flame photometric detection provides
a suitable mode for this mission. Guidelines for the use of this
detector are provided in Section 4, B. It will undoubtedly be necessary
to, use the original 5 ml extract (undiluted) because of the expectedly
low concentrations. In fact, it may prove necessary to concentrate
the extract even further if a preliminary chromatogram via FPD gives
any indication of peaks that may he of insufficient size to meet
the response criteria given in Section 4, B.
VIII. MISCELLANEOUS NOTES:
i ¦
1. This method will not detect the acid form of herbicides such
as 2,4-D or 2,4,5-T, but should be suitable for certain of the
esters of these compounds which are used commercially. However,¦
-------
Revised 12/2/74
Section 10, A
- 8 -
as these compounds are only about one tenth as responsive to
electron capture as a number of the common chlorianted pesticides,
it appears somewhat remote that they would be detected in an
average water sample by this procedure.
2. In a laboratory study conducted on river water in the Water
Quality Laboratory of EPA in Cincinnati, the degradation pattern
shown in Table 1 was reported on a 20-gallon sample of water
held in the laboratory under sunlight and fluorescent light.
These data are presented for supplemental information.
3. Table 2 provides recovery data on 25 compounds, 14 of them
organochlorine, 9 organophosphorous and two of the more common
PCB's. The column to the far right gives estimated minimum
detection values in ppb assuming that (1) the method and
suggested final extract volume is followed precisely, (2) an
initial sample value of 500 ml is used, and (3) instrumental sensi-
tivity criteria provided in Sections 4A and 4B are maintained.
-------
12/2/74
- 9 -
Section 10, A
Tabic 1.
Persistence of
in Terms of l'cr
Com] lot iritis in River Water
centage Recover)'
Ori f
Compound
0-time
1 fck
2 wk
4 wk
8 wk
Orgajiochlorinc compounds
B1IC
100
100
100
100
100
Heptachlor
100
25
0
0
0
Aldrin
100
100
80
40
40
Heptachlor
epoxide
100
100
100
100
100
Telodrin
100
25
10
0
0
Endosulfan
100
30
5
0
0
Dieldrin
100
100
100
100
100
DDE
100
100
100
100
100
DDT
100
100
100
100
100
DDD
100
100
100
100
100
Chlordane ftech.)
100
90
85
85
85
Endrin
100
100
100
100
100
Organophosphorus compounds
Parathion
100
50
30
<5
0
Methyl parathion
80
25
10
0
0
Malathion
100
25
10
0
0
Ethion
100
90
75
50
50
Trithion
90
25
10
0
0
Fen th ion
100
50
10
0
0
Dimethoate
100
100
85
75
50
Merphos
0
0
0
0
0
Merphos recov.
as Def
100
SO
30
10
<5
Azodrin
100
100
100
100
100
Carbamate compounds
Sevin
90
5
0
0
0
Zectran
100
15
0
0
0
Matacil
100
60
10
0
0
Mesurol
90
0
0
0
0
Baygon
100
50
30
10
5
Monuron
80
40
30
20
0
Fenuron
80
60
20
0
0
aPesticide concentrations were 10 ug/liter. Recoveries were rounded
off to the nearest 5%.
-------
12/2/74
" 10 -
Section 10, A
Table 2. Recoveries and minimum detection levels of various pesticidal
compounds in water. Chlorinated compounds by electron capture,
phosphorous compounds by flame photometric.
Compound
Recovery
(percent)
Concentration
(ppb)
Est. Minimum
Detect. Level
(PPh)
Aldrin
91
0.25
0.04
b-bhc
98
0.55
0.09
•y-BHC
96
0.14
0.03
p,p'-DDE
110
0.55
0.09
p,p'-DDT
100
1.40
0.24
Dieldrin
104
0.80
0.14
Endosulfan
106
0.65
0.11
Endrin
106
1.40
0.22
Heptachlor
101
0.20
0.04
Hept. Epoxide
102
0.35
0.06
Hexachlorobenzene ;(HCB)
93
0.25
0.08
Methoxychlor
93
7.00
1.10
Mirex
103
3.00
0.47
Toxaphene
99
26.0
4.44
Aroclor 1254
97
26.0
1.60*
Arochlor 1260
99
26.0
1.60*
i
Azinphos methyl
90
80.0
23.0
Carbophenothlon
84
16.0
2.80
Diazinon
80
2.40
0.40
Malathion
95
8.00
1.40
Parathion ethyl
89
6.00
1.00
Parathion methyl
86
8.00
1.40
Phorate
42
1.50
0.25
Ronnel
94
4.00
0.70,
Dimethoate
0
12.0
* Estimated
-------
Revised 11/1/72
Section 11,A
Page 1
SAMPLE PREPARATION AND ANALYSIS OF SOILS AND HOUSE DUST
I. INTRODUCTION:
The analytical method described below is similar in principle to
the method presented in the Analytical Manual distributed at the annual
Chemist's Meeting in Tucson in 1968. The main difference lies in the
incorporation of the standard Mills, Onley, Gaither Florisil cleanup
technique for which all laboratories have equipment and a degree of
expertise in manipulation. This is preceded by percolation through an
alumina column for further removal of contaminants.
II. PRINCIPLES:
Organochlorine pesticides, together with other lipid-soluble sub-
stances, are extracted from homogenized samples by continuous Soxhlet
extraction with acetone-hexane. Bulk of solvent is removed by evapora-
tion in Kuderna-Danish equipment. Interfering lipid-soluble materials
are then partially removed from the extracts by successive cleanup on
aluminum oxide and Florisil columns. Extracts are adjusted to appro-
priate concentration for determinative analysis by B.C. and F.P.D.,
confirming as needed by M.C. and/or T.L.C.
III. EQUIPMENT:
1. Soxhlet extraction apparatus, complete with 125-ml S 24/40 flask,
extraction tube with $ 24/40 lower and f 34/45 upper joints and
Friedrichs condenser with "S 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" dia. x 2" 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 com-
ponent 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
- 2 -
c. Steel springs, 1/2", stock #K-662750.
d. Concentrator tubes, 10 ml grad., size 1025, stock #K-570050.
6. Modified micro-Snyder columns, 19/22, Kontes K-569251.
7. Glass beads, 3 mm plain, Fisher #11 - 3l2 or equivalent.
8. Evap. concentrator tubes, grad., 25 ml, 3F 19/22, Kontes #570050.
9. Water or steam bath.
10. Glas Col heating mantles with variable autotransfoimers, 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 contam-
inant background. If any interferences are detected,
the water must be hexane extracted before use.
5. Diethyl ether - AR grade, peroxide free. The ether must contain
2% (v/v) absolute ethanol. Most of the AR grade ethyl ether con-
tains 2% ethanol, added as a stabilizer, and it is therefore un-
necessary to add ethanol unless peroxides are found and removed.
NOTE: To determine the absence of peroxides in theNether,
-------
Revised 11/1/72
Section 11, A
- 3 -
add 1 ml of ether in a clean 25-ml cylinder pre-
viously 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.
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.
NOTE: (1) In a high humidity room, the column may pick up
enough moisture during packing to influence the
elution pattern. To insure uniformity of the Florisil
fractionation, it is 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 labora-
tories may differ somewhat from that in Perrine.
Each new batch should.be tested with a mixture of
8-BHC, aldrin, heptachlor epoxide, dieldrin, p,p'-DDE,
p,p'-DDD, and p,p'-DDT, eluting the standard mixture
as described/in Section 5,A,(1) of this manuial.
Dieldrin should elute entirely in the 151 diethyl
ether fraction, whereas all other compounds should be
in the .6% fraction.
9. Anhydrous sodium sulfate, reagent grade granular, Mallinckrodt
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Revised 11/1/72
Section 11, A
- 4 -
Stock #8024 or the. equivalent.
NOTE: When each new bottle is opened, it should be tested
for contaminants that will produce peaks by Electron
Capture Gas Liquid Chromatography. This may be done
by transferring ca 10 grams to a 125-ml Erlenmeyer
flask, adding 50 ml pet. ether, stoppering and shaking
vigorously for 1 minute. Decant extract into a
100-ml beaker and evaporate down to ca 5 ml. Inject
5 pi into the Gas Liquid Chromatograph and observe
chromatogram for contaminants. When impurities are
found, it is necessary to remove them by extraction.
This may be done using hexane in a continuously
cycling Soxhlet extraction apparatus or by several
successive rinses with hexane in a beaker. The
material is then dried in an oven and kept in a
glass-stoppered container.
V. PROCEDURE:
Sample Preparation
1. Soils and vacuum cleaner bag dusts are analyzed in the air-dry state.
If a soil sample is obviously damp, it is allowed to equilibrate its
moisture content with room air before handling. Trials have shown
that house dust screenings generally contain approximately 0.11
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.
-------
Revised 11/1/72
Section 11,A
5 -
Sample 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 iji 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.
b. Prepare a standard mixture of the following compounds, the
concentration expressed in micrograms per milliliter:*
*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.Q 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 extraction,
cleanup and determinative procedures.
4. Place the sample, reagent blank and spiked sample thimbles into
separate Soxhlet extractors. Fill the boiling flasks, each con-
taining six glass beads, about half full with the 1:1 acetone/hexane
co-solvent, assemble the extraction apparatus, position in the
heating mantles and start extraction.
Lindane
Hept. Epoxide
Aldrin
p,p'-DDE
5.0
5.0
5.0
7.5
Dieldrin 7.5
p,p'-DDD 10.0
o,p' -DDT 10.0
p,p'-DDT 10.0
-------
Revised 11/1/72*
Section 11,A
- 6 -
NOTE: Each laboratory will need to determine the setting
of their voltage controller/ There should be
sufficient heat to result in 1 discharge cycle
about every 5 "minutes, or ca 60 syphon discharges
in a 5-hour period. This should be an adequate
number of cycles to insure complete extraction.
5. At the completion of the extraction period disassemble the extraction
apparatus, rinsing the joint between flask and extractor with a
few ml of hexane.
6. Assemble a Kuderna-Danish evaporator with the 250-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 immerse evap. concentrator tube about
1-1/2 inches into the boiling water bath. Evaporate extract down
to ca 3 ml, remove from bath and.cool. Extract is, now ready for
cleanup.
Alumina and Florisil Cleanup
1. Prepare an alumina column as follows:
a. Place a small wad of prerinsed glass wool at the bottom of a
22 x 300 mm chromatographic column.
b. Add preextracted anhydrous Na2SO^ 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 witl a 1-inch layer of Na?S0.. When settling is
complete, open stopcock and allow the nexane to elute through
the column down to a point ca 1/8 inch above the top of the
upper Na2SO^ 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 suffi-
cient column prerinse.
-------
Revised 11/1/72
Section 11, A
- 7 -
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 evapor-
ation to the column.- Rinse tube with three portions of 3 ml each
of hexane transferring the rinsings to the column.
4. Open stopcock and add 85 ml of hexane to the column, open stopcock
wide and elute into the K-D flask.
5. Concentration of the eluate from the alumina column is conducted
exactly the same as outlined above in Step 7 under Sample Extraction,
taking extract down to 3 ml. This extract is now ready for Florisil
partitioning.
6. Florisil column: Prepare the column as described in Section 5,A, (1)
of this-manual-under FLORISIL FRACTIONATION, Steps 1 and 2, sub-
stituting hexane for pet. ether.
7. Assemble two more K-D apparatus but with 500-ml flasks and position
the flask of one assembly under the Florisil column. 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 transfer
the extract from the evaporator tube in Step 5, above, onto the
column and permit it to percolate through. Rinse tube with two
successive 5-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 61 diethyl ether in pet. ether
(Fraction I). The elution rate should be ca 5 ml per minute. When
the last of the eluting solvent reaches a point ca 1/8 inch from
the top of the Na?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 mala-
thion in the sample, have a third 500-ml K-D assembly
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Revised 11/1/72
Section 11,A
- 8 -
ready. At the end of the 15% fraction elution,
add 200 ml of 501 diethyl ether in pet. ether
(Fraction III), evaporating the eluate in the
same manner.
10. Remove K-D assemblies from bath, cool and rinse 5 joint between tube
and flask with a little pet. ether. Finally, dilute both extracts
to exactly 25 ml and proceed with the GLC determinative step.
NOTE: A relatively high dilution is suggested as it has
been observed that residues are generally suffi-
ciently high to warrant this. Furthermore, the
concentration of contaminants remaining after
cleanup is hereby reduced.
Determinative by GLC
1. Inject 5 pi of each fraction extract into the gas chromatograph
(E.C. mode) primarily to determine whether the extracts will re-
quire 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 reten-
tion 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 operator should be able to make
tentative compound identifications. Microcoulometry and/or TLC
may be required for positive confirmation of some of the suspect
chlorinated compounds, whereas FPD may be utilized for the organophos-
phate suspects.
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Revised 11/1/72
Section 11,A
- 9 -
TYPICAL RECOVERY DATA,.- Soxhlet Method
-Pesticides-
SOILS:
Lindane Hep. Epox. p,p'-DDE Dieldrin p,p'-TDE p,p'-DDT
Mean recovery1 85,25 87,83 85,08 88,25 91,17 94,17
S.D. : 5.446 9.4.46 6.345 6.210 6.886 8.922
n : 12 12 12 12 12 12
HOUSE DUSTS:
Mean : 87.33 80.58 82.92 86.27 90.00 87.78
recovery
S.D. 6.997 12.85 6.345 12.89 9.715 20.20
n : 12 12 12 11 12 9
-------
11/1/72
ection 11,B
age 1
SAMPLE PREPARATION, AND ANALYSIS OF BOTTOM SEDIMENT
I. INTRODUCTION:
L
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 v
readily biodegradable. This information combined with residue data
obtained by analysis of the water and tissues from resident marine life
contribute in the development of an overall profile of the pesticidal
contamination of a given body of water.
"REFERENCES:
1. , Column Extraction of Pesticides from Fish, Fish Food and Mud,
Hesselberg, R. J. and Johnson, J. L., in press.
»
2. Sediment Extraction Procedure, Southeast Water Laboratory,
EPA, Athens, Georgia, Method Number SP-8/71.
II. PRINCIPLES:
The sediment sample is partially dried and extracted by column
elution with a mixture of 1:1 acetone/hexane. The extract is washed
with water to remove the acetone and then the pesticides are extracted
from the water with 15% CH.Clo in hexane. The extract is dehydrated,
concentrated to a suitable volume, subjected to Florisil partitioning,
desulfurized if necessary, and analyzed by gas chromatography.
III. EQUIPMENT,AND REAGENTS:
1. Pans, approximately 14" x 10" x 2-1/2".
2. Oven, drying.
3. Muffle furnace.
4. Desiccator.
5. Crucibles, porcelain, squat form, size 2.
6. Omni or Sorvall mixer with chamber of ca 400 ml.
7. Chromatographic columns, 300 mm x 22 mm with Teflon stopcock.
-------
11/1/72
Section 11, B
- 2 -
8. Separatory funnels, 500 ml and 250 ml with Teflon stopcocks.
9. Filter tube, 180 mm x 25 mm.
10. Kuderna-Danish concentrator fitted with grad. evaporative concen-
trator tube. Available, from the Kontes Glass Company, each com-
ponent bearing the following stock numbers:
a. Flask, ,250 ml, stock #K-570001.
b. Snyder column, 3 ball, stock #K-503000.
c. Steel springs, 1/2", stock #K-662750.
d. Concentrator tubes, 10 ml, size 1025, stock #K-570050.
11. Pyrex glass wool - preextracted with methylene chloride in a
Soxhlet extractor.
12. Hot water bath, temp, controllable at 80°C.
13. Sodium sulfate, anhydrous, Baker, prerinsed or Soxhlet extracted
with methylene chloride.
14. n-Hexane, pesticide quality.
15. Acetone, pesticide quality.
16. Methylene chloride, pesticide quality.
17. Ace tone-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. PROCEDURE:
1. Decant and discard the water layer over the sediment. Mix the
sediment to obtain as homogeneous a sample as possible and transfer
-------
11/1/72
Section 11, B
- 3 -
to a pan to partially air dry for about 3 days at ambient tempera-
tures.
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 gm of the partially dried sample into a 400-ml Omni-Mixer
chamber. Add 50 gm of anhydrous sodium sulfate and mix well with a
large spatula. Allow to stand with occasional stirring for approx-
imately 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 50gra sample for extraction, weigh
ca 5 gm of the partially dried sediment into a tared
crucible. Determine the percent solids by drying
overnight at 103°C. Allow to cool in a desiccator
for half an hour before weighing. Determine the
percent volatile solids by placing the oven-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 ace tone-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.
-------
11/1/72
Section 11,B
- 4 -
7. Drain the water layer into a clean beaker and the hexane layer into
a clean 2SO-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.
9. Wash the combined solvent extract by shaking with 100 ml of dis-
tilled water for 30 seconds. Discard the wash water and rewash the
extract with an additional 100 ml of distilled water, again dis-
carding the wash water.
10. Attach 10 ml evap. concentrator tube to a 250-ml Kuderna-Danish
flask and place under a filter comprised of a small wad of glass
wool and ca 1/2 inch of anhydrous 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.
13. Remove tube, rinsing joint with small volume of hexane. The sample
is now ready for Florisil partitioning. This is conducted as
described on page 3, Section 8,B of this manual.
V. CALCULATIONS:
1. Percent Dry Solids
gm of dried sample x 100 = % Dry Solids
gm of sample
2. Percent Volatile Solids
gm of dried sample - gm of ignited sample = gm of volatile solids
gm of volatile solids x 100 = % Volatile Solids
gm of sample
-------
11/1/72
Section 11,B
- 5 -
3. Concentration of Pesticide in Sediment
% dry solids x 50 gm = gm of dry sample extracted
1 of sample extract injected x gm of dry sample extracted = gm of
1 of sample extract dry sample injected
ng of pesticide = ppb of pesticide
gm of dry sample injected
VI. 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 conditions 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 63 and 151 Florisil
eluates with copper if sulfur crystallizes out upon concentration of the
6% eluate.
Certain pesticides will also be degraded by this technique, such as
the organophosphates, chlorobenzilate and heptachlor (see Table 1).
However, these pesticides are not likely to be found in routine sediment
samples because they are readily degraded in the aquatic environment.
If the presence of sulfur is indicated by an exploratory injection
from the final extract concentrate (presumably 5 ml) into the gas chroma-
tograph, proceed with removal as follows:
1. Under a nitrogen stream at ambient temp., concentrate the extract
in the concentrator tube to exactly 1.0 ml.
-------
11/1/72
Section 11,B
- 6 -
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 ul of
iso-octone.
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 HNCL. After about 30 seconds of exposure, decant
off 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 concentration 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
81.2
98.1
Lindane
75.7
94.8
Heptachlor
39.8
5.4
Aldrin
95.5
93.3
Hept. Epoxide
p,p'-DDE
69.1
96.6
92.1
102.9
Dieldrin
79.1
94.9
Endrin
90.8
89.3
DDT
79.8
85.1
Chlorobenzilate
7.1
0
Aroclor 1254
97.1
104.3
Maiathion, diazinon,
0
0
Parathion, Ethion,
Trithion
-------
Revised 12/2/74
CONFIRMATORY PROCEDURES
Section 12
Page 1
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 metabolic and decomposition pro-
ducts 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
unsyraietrical peaks, or unexpected 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 confirmation lacks sensitivity, macro sampling is necessary. As
indicated previously, however, the determination of p-values may be
accomplished with micro-samples.
-------
Revised 11/1/72
Section 12, B
Page 1
CONFIRMATORY PROCEDURES
B. THIN-LAYER CHROMATOGRAPHY
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 chroma-
tography. Thin-layer chromatography introduces a second physical
basis for separation, that of adsorption chromatography.
Additional advantages of this technique, include simplicity,
rapidity, low man-hour consumption, and its utility as a resolving,
and cleanup procedure, for use with other methods of analysis.
The method is, in general, somewhat less sensitive than micro-
coulometry, being limited to about 10 Ng for easy visual inspection
of 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.,
JAQAC. 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 (Pittsburgh
Plate Glass).
2. 3-1/4" x 4" clinical microslides (Arthur H. Thomas Co.).
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Revised 11/1/72 Section 12, B
- 2 -
3.
Developing tank, Thomas-Mitchell, 8-1/2" x
(Arthur H. Thomas Co.).
4-1/2" x 8-1/2" deep
4.
Desaga/Brinkmann standard counting board.
5.
Chromatographic chamber, 800 ml beaker.
6.
Desaga/Brinkmann standard model applicator.
7.
Desaga/Brinkmann drying rack, holds 10, 8"
x 8" plates.
8.
Spotting pipettes, 1, 5, and 10 pi, Kontes
763800.
9.
Spray bottle, 8 02., Thomas Co. #9186-R2.
10.
Desaga/Brinkmann 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. (Brinkmann or Warner-Chilcott).
2. N-heptane, chromatographic grade.
3. Methycyclohexane, practical, B.P. 100.5 - 101.5°C. (Matheson,
Coleman, and Bell).
4. Tetrabromophenolphthalein ethyl ester (Eastman Organic Chemicals
#6810).
5. Acetone, Reagent.
6. Silver Nitrate, Reagent.
7. Tetraethylenepentamine.
8. Citric Acid, granular, Reagent.
9. p-Nitrobenzyl pyridine.
10. Hydrogen peroxide, 30%, Reagent.
-------
Revised 11/1/72 Section 12, B-
- 3 -
11. Ethyl ether, Reagent.
12. Acetonitrile, chromatographic grade.
13. Dimethylformamide, Reagent.
14. Preparation of reagent solutions.
A. Developing solvents
(1) For organochlorines
(a) 2% acetone in N-heptane (v/v) (mobile solvent).
(b) N-heptane (mobile solvent).
(2) For thio and nonthio organophosphates.
(a) Methylcyclohexane (mobile solvent).
(b) 15% or 201 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.
-------
Revised 11/1/72
Section 12, B
- 4 -
(c) Silver nitrate solution.
Dissolve 0.5 AgNO^ gm in 25 ml of distilled water
and dilute to 100 ml with acetone.
(d) Citric acid solution.
Dissolve 5 gm citric acid in 50 ml of distilled
water and dilute to 100 ml with acetone.
(3) For thio and nonthio organophosphates.
(a) 2% p-Nitrobenzyl pyridine in acetone (w/v).
(b) 10% Tetraethylenepentamine in acetone (v/v).
IV. PREPARATION OF T.L.C. 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 comer 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 on the applicator board, forcing out
excess water under the plate to insure a tight fit. (Add enough
water to the applicator board to prevent the appearance of air
bubbles under the plate after it has been pressed into position).
-------
Revised 11/1/72
Section 12, B
- 5 -
2. Examine each 3-1/3" x 4" micro slide carefully by looking down
each edge. To insure flatness of plates, use only slides
that are visibly straight along all edges.
3. Mount the micro slides individually on the surface of the photo-
glass plates.with their long axis perpendicular to the direction
of layer application. With an eye dropper, place a few ml dis-
tilled water on the surface of the photoglass plate and mount
micro slides. Force out the excess water so that no large air
bubbles remain under the slide. The presence of a large air
bubble indicates slide "bowing" due to an irregularity in the
slide. When "bowing" is noted, discard the slide, and position
another in its place.
4. Repeatedly slide an empty applicator across the series of mounted
slides to force out excess water, and wipe surface of slides dry
each time with a tissue. The empty applicator must ride smoothly
and without effort across the series of slides. If not, re-
examine the uniformity and positioning of micro slides.
5. To remove any remaining water, wipe the surface with a dry tissue,
then with one soaked in 95% ethanol, and let dry.
6. Weigh 30 g Al-0 G or MN-silica gel G-HR into a 250 ml 5 Erlenmeyer
flask. Add 50 ml distilled water to Al-O, G or 60 ml to MN-silica
gel 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.
-------
Revised 11/1/72
- 6 -
Section 12, B
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
ong 8" x 8" plate and place the plates in a rack for drying at
80 C for 1 hour in a forced draft oven.
9. After heating, cool the micro plates, and examine each individually
in strong transmitted light for possible gross irregularities in
the uniformity of the coating. Discard plate if gross
irregularities are observed. Place 4 micro plates on the surface
of each 8" x 8" plate, slide into a drying rack, and store in a
desiccating storage cabinet until needed.
10. Sample spotting - Make a pencil mark at each side 1/2" above the
bottom edge of the slide. The imaginary line between these points
serves as the sample "spotting line." Draw an actual line across
the slide 2-3/8" (about 6 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.
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. 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 pi should produce a
visible spot of 2 ppb. An adipose tissue extract from 5 grams
concentrated to 500 yl should give a readable spot at 10 ppb.
These values assume the detection of chlorinated pesticides.
2. The extract from the 15% diethyl ether fraction* contains far more
lipids than are present in the 6% fraction. For this reason, some
further cleanup is required. This is conveniently accomplished by
spotting the equivalent of 5 grams of blood or 0.5 grams of fat on
a 3-1/4" x 4" micro plate, developing with acetonitrile, and
scraping off the alumina from the area at the solvent front. This
is extracted, in hexane and the resulting extract respotted on a
standard 8" x 8" TLC plate.
-------
Revised 11/1/72 Section 12, B
- 7 -
VI. 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 standard solutions on the same plate. Standard concentrations
should bracket the calculated amount of residue in sample.
5. Prepare chromatographic tank by placing 50 ml of developing solvent
in the trough and 75 ml in the bottom of the tank.
6. Seal the tank and develop to the line scribed on the plate.
7. Remove plate and air dry in the hood.
VII. 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 11/1/72 Section 12, B
- 8 -
3. Thio and nothio organophosphates
a. After drying, spray plate with p-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 p-Nitrobenzyl pyridine should be
yellow. If there is any purple color, recrystallize from
acetone. Oxidized solution will cause high background color
and will reduce sensitivity.
2. The tetraethylenepentamine should not have a deep
color. If it does, decolorize and purify with charcoal.
4. Interpret results by comparing values of sample spots against
those of standard spots on the same plate.
VIII. GAS CHROMATOGRAPHY (E.C.) CONFIRMATION OF R.F. VALUES:
At times there may be reason to question the validity of a spot
because of a slight shift i'n the position of the R. F. site or because
of spot diffusion or a very faint appearing spot. When such doubt
exists, GLC, EC examination of the material from the questionable R. F.
site can serve to either confirm or negate the presence of the
suspected compound.
Any of the pesticidal compounds (organochlorine) of lowest con-
centration in blood or fatty tissue such as 3-BHC, heptachlor epoxide,
Ojp'-DDT and p,p'-DDD are frequently the most difficult to identify by
customary GLC, EC. These compounds, if present, are generally in such
low concentration that an extract aliquot equivalent to 5.0 grams of
blood and 0.5 grams of fat is needed for spotting on the TLC plates.
1. Spot the TLC plate with 200 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 AgNO^/2-
phenoxyethanol reagent.
-------
Revised 11/1/72 Section 12, B
- 9 -
4. Develop the spots in the sprayed area in the usual manner and
compute the R£ values for the standards.
5. Utilizing the standard 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, provided
of course, that there are any peaks.
IX. 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
- 10 -
n-Heptane Solvent System
Section 12, B
Adsorbent Al,Oj G (Merck), 250 // thick, air dried 72 hr at room
temperature
Plate Size 8"x8"
Front Travel 10 cm
p,p'-TDE Travel 3.9 cm
Developing Tank 9"x9"x3.5", saturated
Visualization AgNOj, UV exposure
Temperature 24-26°C
Amount Spotted 80-200 ng
Pesticide Rtde
Hexachlorobenzene 2.7
Aldrin 2.1
p.p'-DDE 2.0
Heptachlor 2.0
Chlordane (tech) Z0, 1.8, 1.4, 1.2*
o,p'-DDT 1.9
PCNB 1.8
Perthane olefin 1.8
p,p'-TDE olefin 1.8
TCNB 1.7
Telodrin 1.7
Toxaphene 1.7, 1.2*
Strobane 1.7, 1.2*
p,p'-DDT 1.6
o,p*-TDE olefin 1.6
Chlorbenside 1.3 (grey)
BHC (tech) L3, 1.1, 0.27, 0.10
a -BHC 1.3
Perthane 1.3
Lindane 1.1
o,p'-TDE 1.1
p,p'-TDE 1.0
Endosulfan 0.88.0.07
Ronnel 0.85
Heptachlor epoxide 0.71
Endrin 0.71
Dleldrln 0.52
Carbophenothlon 0.42 (yellow)
Methoxychlor 0.33, 0.27
fi -BHC 0.27
Ovex 0.18
Dlchlone 0.16
Dyrene 0.15 (grey)
-------
11/1/72
Section 12, B
- 11 -
Pesticide
rTDE
Tetradlfon
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 (large and dark)
Diuron
0.00
Endrin aldehyde
0.00 (very small)
Endrin alcohol
0.00
Most Incense spot underlined
'Leaves a streak with these major spots
-------
11/1/72
- 12 -
Section 12, B
2% Acetone in n-Heptane Solvent System
Adsorbent AljOs G (Merck), 250 ft thick, air dried 72 hr at room
temperature.
Plate Size 8"x8"
Front Travel 10 cm
p,p'-TDE Travel 5.7 cm
Developing Tank 9"x9Mx3.5", saturated
Visualization AgNOg, UV exposure
Temperature 24-26°C
Amount Spotted 80-200 ng
Pesticide R rD)
Hexachlorobenzene I 7
Perthane olefin 1.4
PCNB 1.4
Aldrin 1.4
p,p'-DDE 1.4
Chlordane (tech) 1J, 1.3, 1.2, 1.1°
p,p'-TDE olefin 1.4
Telodrin 1.4
Heptachlor 1.4
TCNB 1.3
o,p'-TDE olefin 1.3
Toxaphene 1.3, 1.2°
Strobane 1.3, 1.2®
o.p'-DDT 1.3
P.P'-DDT 1.2
Chlorbenside 1.2 (fuzzy grey)
Perthane 1.2
BHC (tech) U, 0.92, 0.72, 0.25
a -BHC 1.1
Ronnel 1.1
Endrin 1.0
Carbophenothion 1,0 (fuzzy yellow)
Heptachlor epoxide 1.0
p.p'-TDE 1.0
o.p'-TDE 0.9S
Lindane 0.92
Endosulfan 0.92.0.24
Dieldrin 0.90
Tetradifon 0.82
Methoxychlor 0.79
Ovex 0.76
0 -BHC 0.72
Dichlone 0.72.0.00
-------
11/1/72
Section 12, B
Pesticide
Dyrene
Sulphenone
Kelthane
i-BHC
Delta Keto "153"
Capean
Chlorobenzilate
Monuron
Diuron
Endrin aldehyde
Endrln alcohol
Most Intense spot underlined
"Leaves a streak with these major spots
- 13 -
Rtde
0.51
0.31
0.28
0.25
0.23 (very small)
0.09
0.05
0.00
0.00 (dark spot)
0.00 (very small)
0.00
-------
11/1/72
- 14 -
Section 12, B
Rf Values
Adsorbent
Mobile solvent
Pesticide
AliOs G
Methylcyclohexane
Rf Value
Immobile Solvent
Dlmethoate
Azlnphosmethyl (Guthlon)
Imldan
Methyl parathion
Coumaphos
Malathlon
Dloxathion
Parathion
Demeton (thiol)
EPN
Methyl carbophenothion
Sulfotepp
Carbophenothion
Ronnel
Ethlon
Demeton (thiono)
Phorate
Dl8ulfoton
Dlazlnon
(1) Presence of chloride In adsorbent layer reacts with AgNO, and prevents coupling
of dye and pesticide to form characteristic blue or purple spot. Some aluminum oxide
coatings do not have to be prewashed to remove chloride. If, however, maximum com-
pound sensitivities of 0.05 i/g cannot be achieved with unwashed Al,Oj coating, pre-
washlng Is recommended.
15% DMF
20% DMF
0.01
0.01
0.09
0.06
0.09
0.07
0.17
0.11
0.23
0.15
0.34
0.22
0.37
0.24
0.41
0.27
0.44
0.32
0.49
0.33
0.50
0.36
0.69
0.55
0.74
0.59
0.76
0.62
0.77
0.63
0.79
0.67
0.81
0.71
0.82
0.72
0.86
0.78
(2) Chromogenic spray reacts only with sulfur-containing phosphate esters. The fol-
lowing compounds do not react; oxygen analog of parathion, dichlorvos, naled,
mevlnphos, Phoephamidon and trichlorfon.
(3) The following minimum amounts of sulfur-containing phosphate esters can be
detected: 0.05 p g dlazlnon, demeton (thiono), carbophenothion, parathion, malathlon,
ronnel, dloxathion, EPN, coumaphos, sulfotepp, and ethion; 0.1 fig azlnphosmethyl,
methyl parathion, and demeton (thiol). The lower limits of detectability of dlmethoate,
Imldan, methyl carbophenothion, phorate, and disulfoton were not determined. At 0.5
g, or greater, the thlo-phosphate esters vary as to color produced with the chromo-
genic reagents. Carbophenothion, parathion, EPN, coumaphos and dlazlnon appear
vivid blue. Ethion, azlnphosmethyl, sulfotepp, dloxathion, and malathlon appear
purple. Ronnel and methyl parathion appear dull blue while both thiol and thiono
demeton appear bluish purple.
-------
1/4/71
Section 12, C
Page 1
CONFIFMATOKC PROCEDURES
C. EXTRACTION p-VALUES
I. INTRODUCTION:
The information contained in this section is taken frcra 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 extraction 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
confiiming the identity of pesticide residues at levels amenable to
quantitative analysis by electron capture gas chromatography.
The p-values for 88 pesticides and related ccmpounds 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% 0V-17/1.95% QF-1.
2. Grad. centrifuge tubes, 10 ml, with S 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. Equilibrate 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.
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 jjI of upper layer extract.
-------
1/4/71
Section 12, C
- 2 -
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 Q.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-IMF (dimethylformamide)
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-
EMF 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 frcm 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-EMF also appears to be good for such separations, but
EMF, boiling about 70 higher than acetonitrile, is much more difficult to
evaporate and accordingly less suitable for cleanup.
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
-------
1/4/71 Section 12, C
- 3 -
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 he a reaction that takes place
between the solute and the solvent system. The reaction either progresses
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 compounds chromatographing without breakdown.
-------
1/4/71
_ 4 -
Section 12, C
30 -
20 -
10 -
0 -
HEXANE -
907. DMSO
jl'ij L|. H1111111
ili^ 11
HEPTANE -
907. ETHANOL
m
>
o.
&
o
z
30 -
20 -
10 -
0 -
2,2,4-TRIMETHYLPENTANE
807, ACETONE
|n.l| jLj-ijjJ.uij). 11|' 1111—lLl|xi ij
HEXANE -
ACETONITRILE
IiL
ill,.! ii iii| i
hi ii i (i i >i
30 _
20 -
K) -
0 -
2,2,A-TRIMETHYLPENTANE
DMF
ill
iU_Ja_
, T I 1 I 1 I ' I
.20 .AO .60 .80 1.00
2,2, A- TRINETHYLPENTANE
857. DMF
.20 .AO .60 .80 1.00
p-VALUES
Figure 1. Incidence of p-values in each or the six binary- solvent systems.
-------
- 5 -
Section 12, C
Compd.
No.
Pesticide
(Or Related
Compound)
Solvent System
Rt (rela-
tive to
Ri of
Aldrin)
Hexane;
Aceto-
nitrile
2,2,4-
tri-
methyl-
pentane:
DMF
2,2.4-
tri-
methyl-
pentane:
85%
DMF
Hexane;
90%
DMSO
Heptane;
90%
Ethanol"
2,2,4-
tri-
methyl-
pentane;
80%
Acetone
1
naled
0.12
(a)
(a)
0.085
0.23
0.39
2
ethylene dibromide
0.29
(a)
(a)
0.48
0.58
0.76
3
Fumazone®
0.23
0.12
0.32
0.36
0.54
0.76
4
Penphene®
0.76
0.58
0.89
0.98
0.79
0.87
5
dlchlobenll
0.11
0.080
0.15
0.19
0.26
0.60
6
Zinophos®
0.058
0.036
0.23
0.15
0.16
0.40
7
barban
0.019
0.003
0.007
0.003
0.13
0.37
8
chloro-IPC
0.19
0.14
0.17
0.16
0.26
0.72
9
CDEC
0.56
0.22
0.13
0.32
0.35
0.46
0.86
10
phorate
0.52
0.26
0.11
0.44
0.61
0.56
0.83
11
Shell SD 8447
(hydr. prod.)
0.18
0.077
0.24
0.18
0.42
0.85
12
trifluralin
0.23
0.21
0.81
0.84
0.72
0.93
13
lauseto neu
0.023
0.007
0.010
O.OOS
0.077
0.34
14
lindane
0.69
0.12
0.052
0.14
0.093
0.41
0.78
15
PCNB
0.41
0.23
0.67
0.79
0.82
0.95
16
Bayer 30911
0.23
0.071
0.24
0.33
0.49
0.79
17
dloxathion (pri-
mary peak)
0.068
0.038
0.12
0.21
0.39
0.95
18
Stauffer N-2790
0.21
0.0S1
0.33
0.44
. 0.48
0.79
19
diazlnon
0.64
0.28
0.18
0.52
0.75
0.39
0.75
20
dichlone
0.99
0.073
0.027
0.068
0.068
0.34
0.57
21
Di-Syston®
0.16
0.089
0.36
0.47
0.54
0.82
22
endosulfan ether
0.29
0.14
0.42
0.43
0.45
0.85
23
Bayer 38156
0.22
0.12
0.39
0.51
0.48
0.76
24
Hercules 426
0.50
0.20
0.72
0.79
0.74
0.98
25
heptachlor
0.82
0.55
0.21
0.73
0.77
0.71
0.96
26
methyl parathlon
1.45
0.022
0.012
0.015
0.015
0.11
0.40
27
dloxathion (sec-
ondary peak)
0.11
0.055
0.25
0.44
0.35
0.81
28
butonate
0.013
b0.005
b 0.014
c 0.078
0.043
0.080
29
Bayer 41831
0.036
0.016
0.046
0.074
0.24
0.55
30
malathion
1.63
0.042
0.015
0.037
0.077
0.14
0.46
31
Zytron®
0.12
0.058
0.14
0.12
0.35
0.79
32
fenson
0.043
0.013
0.032
0.035
0.20
•0.61
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.
-------
1/4/71
Section 12, C
- 6 -
(Table 1 contd.)
Solvent System
Compd.
No.
Pesticide
(Or Related •
Compound)
Rt (rela-
tive to
Rt of
Aldrin)
Hexane:
Aceto-
nltrile
2,2,4-
tri-
methyl-
pentane;
DMF
2,2,4-
tri-
methyl-
pentane:
85%
Hexane;
90%
DMSO
Heptane;
90%
Ethanol
2.2,4-
tri-
methyl-
pentane:
80%'
DMF
Acetone
33
aldriri
1.00
0.73
0.38
0.86
0.89
0.76
0.98
34
1-hydroxychlor-
dene
1.25
0.068
0.026
0.062
0.033
0.15
0.56
35
Bayer 25141
0.82
0.32
0.78
0.81
0.77
0.90
36
parathion
1.84
0.044
0.029
0.082
0.094
0.30
0.76
37
Dimite®
0.25
0.077
0.27
0.37
0.47
0.81
33
Kelthane®
0.15
0.043
0.18
0.029
0.32
0.84
39
dicapthon
0.031
0.019
0.044
0.051
0.25
0.61
40
Chlorthion®
0.026
0.013
0.039
0.032
0.16
0.56
41
chlorobenzilate
(secondary
peak)
0.22
0.062
0.24
0.40
0.38
0.93
42
dfcryl
0.040
0.029
0.041
0.012
0.066
0.31
43
Telodrin®
0.48
0.17
0.63
0.65
0.73
0.94
44
Bayer 37289
0.54
0.18
0.75
0.78
0.72
0.88
45
isodrin
0.60
0.28
0.78
0.86
0.76
0.97
46
Dyrene®
1.83^
0.041
(a)
(a)
0.014
0.17
0.61
47
heptachlor
epoxide
1.54
0.29
0.10
0.39
0.35
0.57
0.89
48
Morestan©
0.34
0.14
0.43
0.53
0.54
0.65
49
folpet
2.64
0.066
0.015
0.036
0.025
0.23
0.51
50
Ruelene®
0.031
0.012
0.013
0.012
0.11
0.21
51
Y-chlordane
0.40
0.14
0.48
0.45
0.56
0.95
52
Genlte 923®
0.08
0.032
0.076
0.093
0.30
0.67
53
Sulphenone®
0.023
0.012
0.009
0.013
0.087
0.32
54
chlorbenside
1.91
0.24
0.039
0.21
0.29
0.52
0.86
55
endosulfan (I)
1.95
0.39
0.16
0.52
0.55
0.64
0.93
56
ovex
0.068
0.024
o;o6i
0.053
0.28
0.69
57
Shell SD-8447
0.051
0.038
0.051
0.044
0.093
0.47
58
dieldrin
2.40
0.33
0.12
0.46
0.45
0.54
0.88
59
p, p'-DDE
2.23
0.56
0.16
0.65
0.73
0.76
0.96
60
endrln
2.93
0,35
0.15
0.51
0.52
0.59
0.92
61
endosulfan (II)
3.59
0.13
0.060
0.14
0.093
0.34
0.82
62
Aramlte®
0.13
0.075
0.23
0.30
0.43
0.85
63
Methyl Trithion©
0.075
0.019
0.075
0.081
0.42
0.82
64
Perthane®
2.71
0.26
0.077
0.44
0.46
0.70
0.93
65
endrln aldehyde
0.082
0.041
0.083
0.053
0.15
0.79
-------
1/4/71
(Table 1 contd.)
- 7 -
Section 12,C
Compd.
No.
Pesticide
(Or Related
Compound)
Solvent System
Rt (rela-
tive to
Rt of
Aldrin)
Hexane;
Aceto-
nitrile
2.2,4-
tri-
methyl-
pentane:
DMF
2,2,4-
tri-
methyl-
pentane:
85%
DMF
Hexane;
90%
DMSO
Heptane:
90%
Ethanol-
2,2,4-
tri-
methyi-
pentane;
80%
Acetone
66
TDE
3.48
0.17
0.038
0.15
0.081
0.46
0.89
67
o,o' -DDT
0.45
0.10
0.42
0.53
0.62
0.91
68
chlorobenzilate
(primary peak)
0.14
0.032
0.12
0.14
0.28
0.76
69
o,p'-DDT
3.16
0.47
0.11
0.51
0.66
0.68
0.96
70
Kepone®
2.77
(e)
-------
11/1/72
Section 12,D,(1)
Page 1
MICRO SCALE ALKALI TREATMENT FOR USE IN PESTICIDE RESIDUE
CONFIRMATION AND SAMPUE CLEANUP
(Reproduction of manuscript in press, 1972 by-
Susan J. V. Young and Jerry A. Burke
Division of Chemical Technology
Bureau of Foods
Food § Drug Administration)
Procedures involving alkali treatment for 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 al. (2), studied rates of dehydrochlorination of DDT. Mills
(3) used refluxing alcoholic KOH in the cleanup of fatty foods for paper
chromatographic detection of alkali-stable organochlorine pesticides. Klein
and Watts (4) used alcoholic NaOH to dehydrochlorinate o,p'- and p,p'-DDT,
p,p'-TDE, and Perthane prior to gas chromatographic separation of the respec-
tive olefins. These investigators called attention to several earlier uses of
alkali dehydrochlorination in pesticide residue chemistry. Recent literature
contains numerous references to application of this treatment in pesticide
residue analyses, including an adaptation for use in a pre-gas chromatographic
(GLC) column (5).
In spite of the knowledge of this reaction, we have observed that it is
not fully and effectively utilized by residue laboratories for forming deriva-
tives, gaining information for identity confirmation, or obtaining better
cleanup of troublesome extracts. This is probably because the procedure has
not been described in detail for simple micro scale application in multi-
residue analysis.
The purpose of this work was (1) to arrive at optimum and convenient para-
meters of the alkali treatment in order to obtain rapid dehydrochlorination
resulting in product solutions suitable for analysis by GLC; (2) to obtain
complete yield and recovery of olefins from a number of bis(phenyl) chloro-
ethane pesticides; (3) to 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.
METHOD
Reagents and Apparatus
(a) Potassium hydroxide - Anhydrous pellets.
(b) Ethanol - USP 95%.
-------
11/1/72
Section 12,D,(1)
- 2 -
(c) Hexane - Suitable for use with electron capture gas chromatography
(Burdick and Jackson Laboratories, Inc. 1953 S. Harvey St.,
Muskegon, Mich. 49442).
(d) Micro condenser - 19/22 J; K-569250 (Kontes Glass Company, Vineland,
WTJ. 08360).
(e) Concentrator tube - Mills type, 19/22 31 with stopper, 10 ml graduated
in 0.1 ml up to 1.0 ml; K-570050 (Kontes Glass Company).
(f) Alkali dehydechlorination 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 chromatography - Equipped with electron capture detector and
6' x 4 mm id glass column containing either (1) 101 DC-200 or (2)
1:1 mixture of 151 QF-1 + 101 DC-200 on 80-100 mesh Chromosorb W(HP).
Operating conditions: ^ flgw 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 full scale recorder
deflection for 1 ng heptachlor epoxide when full scale deflection
is 1 x 10~9 amp.
Procedure
Accurately pipet, into a 10-ml Mills tube, 2 ml of a petroleum ether solution
of sample extract [6% or 15% Florisil eluate (6)] containing concentrations of
pesticides suitable for subsequent GLC analysis. Add 1 ml of 2% ethanolic KOH
and a few carborundum chips and fit the tube with a micro condenser. [Note:
Avoid getting alkali on the ground glass joint: light greasing of joint with
silicone lubricant may prevent sticking.] With a test tube clamp, hold the
tube over an opening in the steam bath in such a manner that gentle boiling
occurs. When the volume has been reduced to about 1 ml, insert the tube com-
pletely into the steam bath opening and heat vigorously for 15 minutes or until
the volume reaches 0.2 ml. Remove tube from the steam. If a precipitate has
formed, as is often the case with extracts containing fatty substances, add a
few drops of 2% ethanolic KOH and warm gently in steam with swirling until the
precipitate dissolves. After the solution has cooled slightly, add about 2 ml
ethanol-H-O (1 + 1). Allow solution to reach room temperature and pipet 1 or 2
ml hexane into tube. Stopper tube with ground glass, invert, shake vigorously
for about 30 seconds, and allow solvent layer to separate sharply. With micro-
liter syringe, carefully withdraw aliquot of upper layer for determination by
GLC. [Note: Separation of phases should be sharp so that solution withdrawn
for GLC analysis will be free from alkali.]
-------
11/1/72
Section 12,D,(1)
- 3 -
Discussion
Development of Method
Initial experimentation was performed to establish the reaction conditions which
would give complete and rapid dehydrochlorination of p,p'-DDT, o,jd'-DDT,
j^jd'-TDE, o,p'-TDE, methoxychlor, and Perthane and which would permit complete
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 dis-
cussed. The steam bath was chosen as a source of heat because of its ready
availability and convenience. The Mills reaction tube was fitted with a micro
condenser to eliminate losses due to volatilization, which often occurred in
the absence of the condenser. Both KQH and NaOH have been used to the satis-
faction of previous investigators. The more frequent use of KOH by other
workers and its higher solubility in ethanol made it the choice for this work.
An alkali concentration of 2% has been widely used, and was found ideal for
treatment of aliquots of cleaned-up sample extracts containing quantities of
bis(phenyl) chloroethane pesticides ranging from a few nanograms to 100 yg.
In order to provide sufficient reflux time and temperature, it was necessary
that the initial volume of ethanol be in excess of 0.5 ml. When smaller vol-
umes were used, dehydrochlorination was usually incomplete. Emulsions often
occurred during extraction of the olefin into hexane after saponification of
fatty substances. The use of ethanol + 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 701. Less than 301 ethanol did not adequately enhance separation of the
two layers. The non-volatile fatty substance transferred to acetonitrile by
partitioning a petroleum ether solution of butterfat with acetonitrile was used
in tests to evaluate the effects of fatty substances on the reaction. Experi-
ments in which varying volumes of 2% 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 yg) was
not obtained. However, g_,g_'-DDT (8.0 yg) was completely dehydrochlorinated in
the presence of 100-120 mg of fat. Additional experimentation showed that com-
plete dehydrochlorination of methoxychlor and Perthane did not occur until the
fat was completely saponified; these were the bis(phenyl) chloroethanes most
resistent to dehydrochlorination. Quantities of Perthane and jo,p1-DDT up to
100 yg in the presence of not more than 50 mg butterfat were readily dehydro-
chlorinated 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 a microsyringe, was used
-------
11/1/72
Section 12,D,(1)
- 4 -
instead of petroleum ether, to extract the olefin after reaction in order to
avoid possible errors in quantitation.
Effect on Selected Chemicals
The dehydrohalogenation reaction, as described under "Method", was applied to
kP'-DDT (0.8 yg), o,£'-DDT (0.8 yg), ^jd'-TOE (2.0 yg), 0,^-TOE (0.4 yg),
methoxychlor (4.0 yg), and Perthane (40 yg). Quantities shown in parentheses
were chosen for ease in GLC determination. The pesticides were treated indi-
vidually in petroleum ether, in 6% ethyl ether/petroleum ether Florisil eluates
(6) containing the equivalent of 6 g of kale, and in petroleum ether containing
30-60 mg of fatty substances extracted from butter by partitioning (6) between
petroleum ether and acetonitrile. Gas chromatography with electron capture
detection, operated as described under "Method", 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
wt. parent compound represented in aliquot to GLC
wt. parent compound/mol. wt.
wt. olefin compound/mol. wt. x
Recoveries of olefins approximated 100% and ranged from 86% for £,£*-DDE and
o,£*-TDE olefin in petroleum ether to 1101 for -DDE in the extract from
Eutter. Two olefin derivatives, the cis and trans isomers, are formed from
o,]}1 -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 bi-
phenyls (PCB) were subjected to the described alkali treatment. All tests
were made with petroleum ether solutions of the chemical under study. The
quantity of each chemical was chosen for ease of determination by GLC and is
given in parentheses.
Polychlorinated biphenyls ranging from 21 to 601 average chlorine content 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.
-------
11/1/72
Section 12,D,(1)
- 5 -
Heptachlor (0.4 yg) and heptachlor epoxide (0.4 yg) were markedly affected;
recoveries of the original compound ranged from 30 to 501. Minor GLC peaks
were observed on the 10% DC-200 column at retention times relative to aldrin
of 1.63 after treatment of heptachlor epoxide and 0.59 and 0.93 after treat-
ment of 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 altera-
tion product, 4,4'-dichlorobenzophenone, was recovered. A minor peak was
observed in the chromatogram at a retention time relative to aldrin of 1.71
on the 10% DC-200 column. The 4,4'-dichlorobensophenone (2.0 yg) 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 reten-
tion times than toxaphene itself.
The electron capture GLC responses to sulfur (20 yg), frequently encountered in
residue analysis at retention times relative to aldrin of 0.23, 0.55, and 1.13
on the 10% DC-200 column, were eliminated by the alkali treatment.
Application of Method
The 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
-------
11/1/72
Section 12,D,(1)
- 6 -
confirmation of a residue of the parent pesticide. For example, a residue of
£,£' -DDT can be quantitated before alkali treatment and verified by quantita-
tion as £,£'-DDE after treatment.
In addition, the GLC retention time region of the reacted compound can be
examined for presence of pealcs from unreacted and presumably interfering sub-
stances. We have found the alkali treatment especially useful in connection
with the real or suspected presence of residues of PCB. In this case the
characteristic olefin derivatives of ^,£r-DDT,o,g_' -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 iden-
tity of this complex residue. The high recovery of unreacted dieldrin, endrin,
and aldrin following alkali treatment likewise can be of value in the con-
firmation of identity of these pesticides.
Cleaned-up extracts of some samples may contain non-pesticidal substances
which give rise to electron capture response. Other extracts may contain
fatty substances, not removed by the cleanup, which can prohibit application
of some tests, e.g., thin layer chromatography. This is particularly true of
the 15% ethyl ether/petroleum ether Florisil column eluate (6) for non-fatty
samples such as carrots and fatty samples such as some fish. Electron captur-
ing substances present in carrots must be eliminated before determination of
dieldrin and/or endrin residues. Treatment with alkali serves well for this
purpose. Extracts of fatty samples may require treatment to eliminate both
electron capturing substances and non-volatile fatty substances. In many
instances thin layer chromatography or microcoulometric GLC cannot be
accomplished prior to alkali treatment. Electron capture responses to sulfur,
often a source of annoyance to the residue chemist, are also eliminated by
this treatment.
-------
11/1/72
Section 12,D,(1)
- 7 -
1. BRAND, K. and BUSSE-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. Offic. Anal. Chem. 42, 734-740 (1959).
4. KLEIN, A. K. and WATTS, J. 0., J. Ass. Offic. 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. KRALISE, 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).
-------
12/2/74
Section 12, D, (2)
RAPID DETERMINATION AND CONFIRMATION OF
HEXACHLOROBENZENE IN FATTY TISSUES
T- 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) On chromatography by electron capture
detection, the retention characteristics of the HCB peak are
quite similar to the alpha isomer of BHC (Hexachlorocyclohexane)
on a number of GLC columns; (2) Because of the unfavorable
partition ratio of HCB in the acetonitrile/pet. ether solvent
system, low recoveries are obtained using the multiresidue method
outlined in Section 5, A, (1).
REFERENCE: Crist, H. L., Moseman, R. F., and Noneman, J. R.
(In review as of 12/74).
II. PRINCIPLE:
The fat sample is dissolved in a small volume of hexane and
applied to a prewashed Florisil column topped with anhydrous Na2S0^.
The column is eluted with a measured volume of hexane at a given
elution rate. The eluate containing the HCB is evaporated in a
Kuderna-Danish assembly and subsequently under a nitrogen stream.
This concentrated eluate is chromatographed by electron capture
for tentative identification of HCB along with heptachlor, aldrin,
p,p'-DDE and o,p'-DDT, if present. All or an aliquot of the con-
centrate extracted is then evaporated to dryness. The residual
material is treated with pyridine and KOH in propanol, heated for a
given time period, and then 2% ^£50^ and hexane are added. After
mixing, phase separation and appropriate volume adjustment, the
derivatization product, bis-isopropoxytetrachlorobenzene (BITB)
is chromatographed using electron capture, and the resulting peak is
quantitatively referenced against the peak obtained from a known
quantity of pure HCB, derivatized in the same manner.
III. APPARATUS:
1. Gas chromatograph fitted with electron capture detector and a
column of 1.51 0V-17/1.95% QF-l(or OV-210). Operating parameters:
column temp. 200°C., detector (tritium) 210°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.
-------
Revised 12/2/74 Section 12, D, (2)
- 2 -
3. Kuderna-Danish concentrator assembly, Kontes No. K-570000, fitted
with evaporative concentrator tube, 25 ml, Kontes No. Special
K-897900.
4. Micro Snyder column, Kontes No. K-569250.
5. Disposable pipets.
6. Compressed, gaseous nitrogen equipped with regulator valve for
pressure reduction to approximately S-lbs/sq. in.
7. Water bath capable of temp, of 95 to 100°C.
8. Glasswool preextracted with benzene by Soxhlet cycling.
9. Centrifuge tubes, grad., 13 ml with $ stopper, Kontes No. 410550
or the equivalent.
10. Vortex mini-mixer.
11. Mechanical Mixer/rotator, capable of 50 r.p.m.
IV. REAGENTS § SOLVENTS:
1. Hexachlorobenzene, 99 + %, analytical reference standard, available
from Repository, Pesticides § Toxic Substances Effects Laboratory,
EPA, Research Triangle Park, NC 27711.
2. Pyridine, spectograde.
3. Potassium hydroxide, reagent grade.
4. Sodium sulphate, anhydrous, granular. Soxhlet extract with benzene
and oven dry at 130°C.
5. Sodium sulphate, 2% aqueous solution prepared from preextracted
Na^SO^.
6. Florisil, PR grade, the Floridin Company, "Berkeley Springs, W.V.
7. 2-Propanol, reagent grade.
8. Keeper solution, 1% paraffin oil in hexane, pesticide quality.
9. Hexane, distilled in glass, pesticide quality.
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12/2/74
Section 12, D
- 3 -
V. PROCEDURE:
1. Weigh 2.50 g of well macerated fat on aluminum foil ca 65 mm
square.
2. Transfer fat to a 13 ml centrifuge tube and rinse foil with
hexane, bringing volume in tube to exactly 10 ml.
3. Stopper tube securely and place on mechanical mixer/rotator
adjusted for a speed of ca 50 r.p.m. Rotate for 30 minutes
to distribute fat uniformly in the hexane layer.
4. Prepare Florisil column as described in Section 5, A, (1),
subsection V. and prewet column with 40 to 50 ml of hexane or
a sufficient quantity to more than' cover the Na2SO^ lazer.
5. Prepare a Kuderna-Danish assembly by attaching a'250 ml K-D
flask to a 25 ml grad. evap. concentrator tube containing a
3 mm glass bead (or caitiorundum chip) in bottom.
6. When prewash hexane just reaches the top level of the Na^SO.
layer, position column over K-D flask and, with a volumetric
pipet, transfer 2.0 ml of the extract from the centrifuge
tube to the column (0.5 g. of fat).
7. Add 100 ml hexane to the column and elute at a carefully pre-
measured rate of 5 ml per minute.
8. Immerse the concentrator tube in a boiling water bath to about
1/3 of its depth and concentrate extract down to ca 5 ml.
9. Remove K-D assembly from bath, cool, and carefully remove
concentrator tube, rinsing joint with ca 3 ml of hexane.
10. Place tube under a gentle nitrogen stream and reduce extract
volume to ca 3 ml. Do Not Allow to go to Dryness.
11. Rinse sidewalls of tube with hexane and adjust volume to exactly
5 ml. Stopper and Vortex mix one minute.
12. Make an exploratory injection of 5 yl of the extract into the
gas chromatograph fitted and adjusted as described earlier in
subsection III, 1.
-------
12/2/74 Section 12, D, (2)
- 4 -
On the basis of observations from this
injection, it may prove necessary to
further dilute or concentrate the extract.
This 100 ml hexane fraction may contain, in
addition to any HCB present, heptachlor,
aldrin, p,p'-DDE and o,p'-DDT. The various
isomers of hexachlorocyclohexane (BHC) which
sometimes present toublesome peak overlaps with
HCB, particularly the alpha isomer, are re-
tained on the column. HCB, if present, should
produce a peak with an RRT^ value of 0.47.
No attempt should be made to quantitate any
compounds other than HCB which may appear in
this eluate as the partitioning may be incom-
plete, and therefore, expectedly low recoveries
could occur.
VI. CONFIRMATION BY DERIVATIZATION:
1. Add 5 drops of the 1% paraffin oil/hexane keeper to the extract
in the 25 ml concentrator tube and place under a gentle nitrogen
stream at room temperature. Continue evaporation just to the
complete disappearance of the hexane.
2. Add 0.2 ml pyridine and 0.5 ml 10% KOH in 2-propanol, attached
micro-Snyder column to tube and place in boiling water bath for
exactly 10 minutes.
3. Remove tube, cool under tap water and add 10 ml 2% Na SO^ and
5 ml hexane. Stopper and Vortex mix one minute. ^
NOTE: The 5 ml hexane should be delivered with a
volumetric pipet as this is a quantitative
dilution.
4. After the phases have completely separated, inject 5 yl of
the hexane extract (upper layer) in the gas chromatograph,
fitted and adjusted as described in Subsection III., 1. An
additional quantitative volume of hexane may be added, as
estimated, to bring the BITB peak on scale. In this case,
after dilution, the tube should be stoppered, reshaken, and time
allowed for layer separation. In the event the BITB peak is
visible but has a peak height less than 10% fsd, some further
concentration is required. This should be done by withdrawing
4 ml of the extract with a volumetric pipet, transferring to a
NOTES: 1.
2.
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12/2/74
Section 12, D, (2)
- 5 -
10 ml grad. concentrator tube. The appropriate degree of
concentration may then be achieved by evaporation under a nitro-
gen stream. Quantitative reference is obtained by carrying a
precise amount of reference standard HCB through the derivati-
zation procedure along with the unknown.
NOTE: Using the prescribed column temperature of 200°C.,
the RRT^ of the BITB peak should be 0.64.
VH. MISCELLANEOUS NOTES:
1. The analyst may find that the 100 ml hexane eluate will yield
a clearly delineated peak of the precise value for HCB.
In this case, there is little need for pursuing the derivati-
zation route. However, for full irrefutable confirmation, the
derivatization step is advised. Early eluting compounds such
as the isomers of hexachlorocyclohexane (BHC), and heptachlor,
which could present interfering peaks, are altered and present
no interfering peaks after derivatization. Aldrin, dieldrin,
endrin, p,p'-DDE and the PCB's are not altered, but their
normal elution characteristics pose no interference problems.
2. Recovery studies by the authors over a concentration range of
0.01 to 1.9 ppm have indicated recoveries from 86 to 107% on
quantitation of the 100 ml Florisil eluate. See Table 1.
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),
it is feasible to provide ahead of time for this contingency
during the extraction step of Method 5, A, (1). See MISC. NOTES,
subsection 8. of Section 5, A, (1).
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Revised 12/2/74
- 6 -
Section 12,D,(2)
TABLE I
Recovery of HCB from Fortified Chicken Fat by Direct
Elution with 100 ml of Hexane
Fat, mg
HCB
Added, ng
HCB
Recovered, ng
HCB
Cone, yg/g
Recovery
%
403
4
4.1
0.010
102
494
8
7.0
0.016
88
477
16
14.8
0.034
92
474
25
24.8
0.053
99
583
50
51.5
0.086
103
482
48
51.2
0.100
107
530
150
143
0.283
95
497
160
160
0.322
100
446
250
254
0.561
102
555
350
301
0.631
86
528
500
477
0.947
95
525
1000
1003
1.900
100
Mean 97.4%
Range 86-1071
Std. Dev. + 6.31
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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 at least 1 ug and has been utilized at the
0.1 ug 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 principal 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, J. F., Brown, C., and Jenkins, R. R.,
J. Chromatography, 38:2Q0-8 (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).
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.
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1/4/71
Section 12, E
- 2 -
II. EQUIPMENT:
A. Perkin-Elmer IR-337, or equivalent, equipped with a 6X beam
condenser and holder for 1.5 mm KBr discs.
B. Micro KBr equipment, Perkin-Elmer ultra micro dye assemhly.
C. Press, capable of exerting 200 pounds of pressure.
D. Micro mortar and pestle.
E. Fine tipped forceps.
F. 50 ul 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.
D. Aluminum oxide G.
E. Ethyl ether, anhydrous, reagent grade.
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1/4/71
Section 12, E
- 3 -
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 5Q%.
*
Redistilled in all-glass apparatus.
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1/4/71
Section 12, E
- 4 -
3. Simultaneously block both heams 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 pan 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.
bc Loosen the screw holding the pen carriage on the slide wire.
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 1QQ% 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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1/4/71
Section 12, E
- 5 -
1. Trapping of gas chromatograph effluent-capillary procedure.
a. Attach splitter to the inlet to the E.C. 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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1/4/71
Section 12, E
- 6 -
(2) Concentrate extract to 1-0.1 ml, depending on
concentration 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 adsoibent 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 in 2 ul
increments, allowing tin© for solvent evaporation
between each addition. Put eluate on KBr, not
the mortar.
b. Organothiophosphate 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.
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1/4/71
Section 12, E
- 7 -
(2) Concentrate sample to 0.3 ml in methylene chloride.
(3) Using a micropipette, apply sample to plate, in increments,
drying between applications.
(4) Develop with 1.75% methanol in chlorofoim.
(5) Air dry.
(.6) Spray the plate with 5 ml of a 5% solution of palladium
chloride and 1 ml of HC1, diluted to 100 ml in 95% ethanol.
(7) Allow plate to dry for 30 minutes at roan 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 1 ml of CCl^, 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.
Vr. 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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1/4/71
Section 12, E
- 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 THE 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. 10Q% adjustment
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1/4/71
Section 12, E
- 9 -
D. Place paper on drum.
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 heginning 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 1001 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 possihle contamination when making micro KBr pellets.
F. When using thin-layer cleanup, rememher "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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12/2/74
Section 12,F
Page 1
Reproduced from Pesticide Analytical Manual, Volume I, U.S. Food
& Drug ADMINISTRATION
POLAROGRAPHY
640.1 Introduction. The development of oscillopolarographic instrumentation and tech-
niques 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 pes-
ticides and their residues. He shows 12 single sweep polarograms with comparison of deriv-
ative 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 Nangniot review polarographic applications for determining: copper, mercury,
arsenic, tin and sulfur compounds; natural organic products such as nicotine, rotenone, and
pyrethlns; and synthetic organic compounds. They list 163 references.
(3) Gajan, R. J., 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 11th 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 numbered 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).
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 cample extract
used for multiple residue determination.
7/1/70
1
-------
CONFIRMATORY TESTS
Section 641. 3
Pesticide Analytical Manual Vol. I
Foods and Feeds
641.1 , Apparatus
Cathode-ray Polarotrace K1000 or
Davis Differential Cathode-ray Polarotrace A1660
Silver wire electrode. A No. 20 or No. 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 KMn04 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 containing 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 tempera-
ture, 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 polaro-
traces 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 nitrogen through cell solution for
5 min and polarograph at 25 C ± 0.1 C over designated voltage scan.
Measure any waves appearing within ±0.10 volt of peak potential of pesticide being deter-
mined. 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, imme-
diately before or after sample. The standard solution should contain approximately the same
amount of pesticide as the sample. When uncleaned solutions are polarographed the peak
potential may shift slightly due to density of the cell solution.
2
7/1/70
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Pesticide Analytical Manual Vol. I
Foods ar.d Feeds
CONFIRMATORY TESTS
Section 641.42a
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 ad-
dition 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 pes-
ticide 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. Polaro-
graphic interference between compounds noted in 641.42 are sometimes avoided by the sepa-
rations 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 pro-
cedures 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 identi-
fied or unless the prior analytical method permits only certain of the compounds to be in the
sample solution.
Electrolyte solution. Dissolve 2.72 g NaOAc-3H20 and 1.17 g NaCl 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 ace-
tone 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 soludon with
acetone.
7/1/70
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CONFIRMATORY TESTS
Section 641.42d
Pesticide Analytical Manual Vol, I
Foods and Feeds
641.42b Guthlon
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. Therefore, report all re-
sults 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 KCl.
Polarographic determination. Dissolve residue from evaporation in a definite volume of ace-
tone 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 elec-
trode 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.
Polarographic determination. Dissolve residue from evaporation in a suitable amount of elec-
trolyte solution and proceed as directed in the general method, 641.3, starting with "...trans-
fer 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 a'l
results as total of the two unless the specific analog as been previously identified.
4
7/1/70
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Pesticide Analytical Manual Vol. I
Foods and Feeds
CONFIRMATORY TESTS
Section 641.42f
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 definite 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 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. How-
ever, 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 dia-
zinon 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 ap-
proximately the same polarographic wave heights when polarographed using the electrolyte
system described in 641.42d.
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 etha-
nol 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 uid
-0.55 i 0.05 volt vs. silver wire reference electrode.
641.42f Carbophenothion
Reference. Nangnoit, P., Anal. Chem. Acta. 31, 166-174 (1964).
Limitations. Limit of quantitative detection is 0.2 ppm based on 1 g crop sample in 1 rnl cell
solution.
Electrolyte solution. 50% w/v KOH in H20.
7/1/70
5
-------
CONFIRMATORY TESTS
Section 641.42g
Pesticide Anolyticq/ Manual Vol. I
Foods and Feeds
Polarographic determination. Dissolve residue from evaporation in a definite volume of etha-
nol. 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 elec-
trode 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 HO Ac, 1.0 N NaN02 in H20, and 50% w/v KOH in H20,
in ratio of 1:1:3.
Polarographic determination. Dissolve residue from evaporation in a definite volume of glacial
acetic acid. Add an equal volume of 1.0 N NaN02 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, starving with ".. .transfer 5.0 ml or less of the mix-
ture 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 tetramethylammonium bromide in 250 ml distilled H20
(0.2M).
Polarographic determination. Dissolve residue from evaporation in a definite volume of ace-
tone 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 GLC or TLC.
6
7/1 P<\
-------
Revised 12/2/74
Section 13, A
Page 1
DETERMINATION OF METHYL MERCURY IN FISH, BLOOD, BRAIN AND URINE
I. INTRODUCTION:
In past years, mercury has been found to be a frequent con-
taminant of fish in various parts of the world. Much effort has
been spent in improving or developing methods for analyzing commo-
dities for total mercury content, usually by colorimetric (1) or
atomic absorption procedures (2). However, it has been shown (3)
that most, if not all, mercury found in fish is present as methyl
mercury, a compound thought to have much greater chronic toxicity
than other forms of mercury. Therefore, appropriate methodology,
specific for methyl mercury, is desirable. (The form in which
mercury may be present in other contaminated commodities varies
and is not necessarily methyl mercury).
The most widely used method for methyl mercury analysis is that
described by Westbtt (3,4,5,6). This method has been used success-
fully for the analysis of fish, red blood cells, plasma and brain
of hamsters fed methyl mercury. The method described herein repre-
sents a modification of the procedures described by Westbb in her
papers and private communications.
REFERENCES:
1. "Official Methods of Analysis" 10th Ed., Association of
Official Agricultural Chemists, Washington, D.C., 1965,
pp.
375.
2.
W.
R. Hatch and W. L. Ott, Anal.
Chem. 40, 2065 (1968).
3.
G.
Westbb, Acta. Chem. Scand. 21,
1790-1800 (1967).
4.
G.
Westbb, Acta. Chem. Scand. 20,
2131-2137 (1966).
5.
G.
Westbb, Acta. Chem. Scand. 22,
2277-2280 (1968).
6.
G.
Westbb, private communication.
7.
Laboratory Information Bulletin,
FDA, October, 1970
DETERMINATION OF METHYL MERCURY IN FISH AND ANIMAL
TISSUES BY GAS LIQUID CHROMATOGRAPHY, Kamps, L., and
Malone, B.
-------
Revised 12/2/74
Section 13, A
- 2 -
II. PRINCIPLES:
In this procedure, methyl mercury chloride (Q-LH CI) is formed
by addition of hydrochloric acid to homogenized tissue, and the
organic salt is extracted into benzene. The methyl mercury is
extracted into benzene. The methyl mercury is extracted from the
benzene solution by partitioning it with aqueous cysteine solution,
which layer is then acidified and" the salt reextracted back into
benzene. The final benzene layer is analyzed by gas-liquid
chromatography.
III. APPARATUS:
1. Gas Chromatograph fitted with column 6' x 4 mm i.d. of 5%
HIEFF-10B on Chromosorb W, H.P., 80/100 mesh and with an electron
capture detector.
2. Sorval homogenizer with 50-ml cups. Available from Ivan Sorval
Co., Norwalk, Conn.
3. Centrifuge tubes, 35 ml, conical, Kimble #45201 or the equivalent.
4. Centrifuge bottles, 250 ml, Corning #1280, or the equivalent.
5. Centrifuge with adjustable speed up to 3,000 r.p.m. with carrier
heads appropriate for tubes and bottles listed above in 3 and 4.
6. Cylinder, grad., 100 ml.
7. Separatory funnels, 30, 60 and 250 ml with Teflon stopcock.
8. Pipets, transfer, 2,4,6 and 50 ml.
9. Pipets, measuring 1 and 2 ml, grad., in 0.01 ml.
IV. REAGENTS AND SOLVENTS:
1. Benzene, pesticide quality. Background contamination must be
checked by injecting 5 pi of a 15 to 1 concentrate. No peaks
should appear in the area shortly after the solvent front.
2. Methyl mercuric chloride*. Available from K § K Laboratories,
stock number 23308, or from the E.P.A. Repository at Research
Triangle Park, NC. Stock Standards of:
a. 1 mg/ml in benzene.
-------
Revised 12/2/74
- 3 -
Section 13, A
b. 1 mg/ml in ethanol-water (1:1).
Dilute stock solutions a. and b. as needed for recovery trials
and GLC working standards.
NOTE: The stability of CHjHgCl dilute standard solutions
is questionable. Stock solutions, 1 mg/ml
in benzene or in (1:1) alcohol/I^O should be
stable indefinitely. It is suggested that dilute
solutions in H2O be made fresh every 2 or 3
days and dilute solutions in benzene every 2
months.
*Some of the mercury standards will be available from the
EPA Repository at Research Triangle Park, NC
3. Ethyl mercuric acetate. Available from Ventron, P. 0. Box 159,
Beverly, Mass. 01915, stock #87301.
Prepare benzene solution of 1 mg/ml.
4. Phenyl mercuric acetate. Available from K § K Laboratories
stock #17412.
5. Methyl mercuric iodide. Available from K § K Laboratories
stock #2508.
Prepare benzene solution of 1 mg/ml.
6. Column conditioning mixture: Combine equal portions of foregoing
items 2a, 3, 4 and 5 and set aside for column conditioning.
7. Cysteine hydrochloride-monohydrate. Aldrich Chemical Co., Stock
#C 12, 180-0.
8. Sodium acetate .3H2O, Mallinckrodt #7368 or the equivalent.
9. Sodium chloride, reag. grade, fine crystal.
10. Sodium sulfate, reag. grade, anhydrous.
NOTE: Contaminant peaks are sometimes observed from
this material. Each batch should be checked by
injecting a benzene extract into the gas chroma-
tograph.
-------
Revised 11/1/72
Section 13,A
- 4 -
11. Cysteine Solution - Dissolve 1.00 g cysteine hydrochloride, 0.775 g
sodium acetate. (3 F^O) and 12.5 g anhydrous Na-SO, in distilled
^0 and dilute to 100 ml. Prepare fresh every 3 days.
12. Mercuric Chloride Solution - Dissolve 5.0 g HgCl? and 17.0 ml conc.
HC1 in distilled FLO and dilute to 100 ml. Shake 4 times (3 min.
each) with 50-ml portions of benzene to remove impurities.
13. Hydrochloric acid, conc., reagent grade.
14. 6.0 N Hydrochloric acid - Prepare by diluting 185 ml conc. HC1 to
1 liter with dist. water.
NOTE: It has been found that reagent grade HC1 may
occasionally be contaminated with materials
that interfere with the chromatographic deter-
mination of CHjHgCl. It is suggested that the
acid be triple washed with equal volumes of
pesticide quality benzene in a sep. funnel.
15. Column packing - 5% HIEFF-10B (phenyldiethanolamine succinate).
Available from Applied Science Laboratories, Stock #09120. Coat on
80/100 mesh Chromosorb W, H.P. After the 6-ft. column is packed, conditi
conditioning should be carried out as follows: Place in condition-
ing oven in the manner described in Section 4,A,(2), raise the
temperature to 210 C and apply a carrier flow of ca 25 ml/min.
Maintain conditioning for 4 days. During this period, the organic
mercury mixture (Subsection IV,6) is injected into the column in
three sessions during each day, each injection session consisting
of three 10 yl injections spaced one or two minutes apart. There-
fore, at the end of the 4-day period, the column should have
received a total of 36 injections of the mixture (see MISC. NOTE 1).
V. PROCEDURE:
All glassware used in the procedure shall, after washing in the
usual manner, be rinsed successively with NH.OH, distilled water and
ethanol (see MISC. NOTE 2). The following procedure, unless otherwise
noted, is intended for fish containing 0.1 ppm or more of CH^Hg:
-------
Revised 11/1/72
Section 13,A
- 5 -
Extraction and Partitioning
A. Fish, Blood § Brain
1. Weigh 10 grams of sample into 50-ml homogenization cup. Measure
55 ml of dist. water in a 100-ml grad. cylinder and add a suffi-
cient amount to just cover sample. Homogenize at medium speed
for 3 minutes.
2. Transfer sample to a 250-ml sep. funnel rinsing with the water
remaining in the cylinder. Add 14 ml of conc. HC1, 10 grams
of NaCl and mix.
NOTE: In the analysis of red blood cells, plasma or
brain, add at this point 2 ml of the impurity-
free HgC^ solution (Subsection IV,12).
3. Add 70 ml of benzene, stopper funnel and shake vigorously for
5 minutes (a reciprocating mechanical shaker is preferable if
available ).
4. Transfer sample to a 250-ml centrifuge bottle, cover with foil
and centrifuge at 2000 r.p.m. for 10 minutes.
NOTE: The initial 10-minute centrifuging may not
adequately separate the aqueous-organic phases.
The solid material at the organic-aqueous inter-
face must be quite compact to allow easy removal
of 50 ml benzene. If recentrifugation is
necessary, stir the sample vigorously with a
glass rod before centrifuging. Should the ben-
zene layer appear cloudy, centrifuge for an
additional 10 minutes.
5. With a vol. pipet, carefully transfer 50 ml of the benzene layer
into a 60-ml sep. funnel. Take care not to disturb the solids
collected at the benzene-water interface.
6. Add 6.0 ml of the cysteine solution to the 60-ml sep. funnel,
stopper and shake vigorously for 2 minutes.
NOTE: To increase the limit of detectability, the
volume of cysteine solution should be reduced
to 1.2 ml, applied with a 2-ml Mohr pipet.
Further modifications in this connection are
given in NOTE form below.
-------
Revised 11/1/72
Section 13, A
- 6 -
Allow to stand 10 minutes, then drain aqueous layer emulsion
into a 35-ml centrifuge tube. Centrifuge at 3000 r.p.m. for
20 minutes. If emulsions persist, stir vigorously with a glass
rod and repeat centrifuging as necessary.
With a volumetric pipet, transfer 2 ml of the cysteine solution
from the centrifuge tube to a 30-ml sep. funnel. Add 1.2 ml of
6N HC1 and 4.0 ml benzene, stopper and shake vigorously for 2
minutes.
NOTE: For lower detectability levels, transfer 0.80 ml
(or as much as possible to remove and measure)
of the cysteine solution, then reduce the addi-
tion volumes of 6N HC1 and benzene to 0.6 and
2.0 ml, respectively.
B. Procedure for Urine
1. Pipet 15 ml of urine into a 60-ml separatory funnel. Add conc.
HC1 until a pH of 1 is obtained, and mix slowly to avoid loss
of sample due to foaming.
2. Add 30 ml of benzene and shake vigorously for 5 minutes (a re-
ciprocating mechanical shaker is preferable if available).
3. Transfer sample to a 100-ml centrifuge bottle, cover and
centrifuge at 2000 r.p.m. for 10 minutes.
4. Volumetrically transfer 25 ml of the benzene layer to a 60-ml
sep. funnel.
5. Add 5 ml of cysteine solution to the 60-ml sep. funnel, stopper
and shake vigorously for 2 minutes.
6. Drain aqueous layer emulsion into a 35-ml centrifuge tube.
Centrifuge at 3000 r.p.m. for 10 min. If emulsions persist,
stir with a glass rod and repeat centrifugation as necessary.
7. Carefully transfer 4 ml of the cysteine solution to a 30-ml
sep. funnel. Add 1.2 ml of 6N HC1 and 5 ml of benzene, stopper
and shake vigorously for 2 minutes.
8. Allow to stand for 10 minutes, discard aqueous layer and dry
benzene by adding a pinch of anhydrous Na2SO^ prior to GLC.
7.
8.
-------
Revised 11/1/72
Section 13,A
- 7 -
Gas Chromatography
Set column temperature at 170°C and adjust carrier flow to 120 ml
per minute. Set attenuator to obtain a peak height of 60-80% f.s.d.
resulting from the injection of 1.6 ng of methyl mercuric chloride in
benzene. If column is properly prepared and conditioned, the peak
should be of reasonable symmetry with minimal tailing as illustrated in
Figure 1. Make a sufficient number of repetitive standard injections
to assure response reproducibility before making sample injections.
It has been observed that all methyl mercury salts elute at the same
retention time. These include the CI, Br and I salts of methyl mercury,
as well as Panogen (methyl mercury dicyandiamide).
VI. CALCULATIONS:
If 50 ml of benzene and 2.0 ml of cysteine are recovered during the
procedure for 10 g samples, the amount of sample per ml of final benzene
solution is 0.595 g. This calculation is as follows:
P F
g/ml = A x x ^ t F where
A = of sample analyzed.
B = ml benzene added (normally 70 ml).
C = ml benzene recovered (normally 50 ml).
D = ml cysteine solution added (normally 6.0 ml).
E = ml cysteine solution recovered (normally 2 ml).
F = ml benzene added for final partition (normally 4.0 ml).
This calculation may be applied to all variations of the CH^HgCl
method. It is readily apparent that sample size affects the limit of
detectability; however, the analysis of samples larger than 10 g is not
recommended. Results in terms of ppm CH,HgCl can be converted to ppm Hg
by multiplying by a conversion factor of 0.80.
VII. MISCELLANEOUS NOTES:
I. Obtaining and maintaining acceptable gas chromatography of methyl
mercury is difficult. Chromatography has been tried on the follow-
-------
Revised 11/1/72 Section 13,A
7 8 -
ing liquid phases: 10% Carbowax 20 m, 20% Carbowax 20 m, 15% DEGS
(stabilized), 5% HIEFF-10B [Phenyldiethanolamine Succinate (PDEAS)].
Acceptable chromatography was not obtained on a 15% DEGS column. To
date 5% HIEFF-10B has produced the best chromatography. Proper
pre-conditioning of the column is very critical. Provided that
column is properly conditioned, it should be possible to obtain a
peak of at least 50% f.s.d. resulting from the injection of 0.150
nanograms of CH^HgCl. If the sensitivity should fall short of this,
further conditioning with repeated injections of the concentrated
mixture may be indicated.
2. Glassware such as pipets which come in contact with fairly con-
centrated solutions of CHjHgCl should be rinsed with NH^OH, distilled
FLO, and C^rOH, but it is probably not necessary to rinse the rest
of the glassware with anything but distilled F^O.
3. Benzene solution of CFLHgCl cannot be concentrated without signifi-
cant loss of CH,HgCl. Limit of detectability is therefore restricted,
as discussed above in Subsection VI.
4. The analysis should not be stopped overnight at any point except when
the sample is in benzene solution. Prolonged contact with cysteine
solution results in low recoveries.
-------
l/U/71 Section 13,A
-9-
Figure 1. Typical chromatogram of 1.6 ng of CH^HgCl on 5% K-EFF-10B
at 170°C. and carrier flow of 120 ml/minute.
— Peak Height
-------
1/4/71
Section 13, B
Page 1
DETERMINATION OF TOTAL MERCURY IN WATER
FLAMELESS ATOMIC ABSORPTION
I. INTRODUCTION:
The method described was obtained from the Analytical Quality
Control Laboratory of the Federal Water Quality Administration, and is
designated as a "tentative or provisional" method.
The procedure outlined sets forth a sensitive and accurate method
for the determination of total mercury down to 0.5 ppb in solution.
Hie method as outlined is applicable to surface waters, saline waters,
wastewaters and effluents. Following appropriate pretreatment, the
method is also applicable to mud, botton sediment and fish tissue.
In addition to inorganic forms of mercury, organic mercurials
may also be present in an effluent or surface water sample. These
organo-mercury compounds will not respond to the flameless atomic
absorption technique unless they are first broken down and converted to
mercuric ions. Potassium permanganate oxidizes many of these compounds
but recent studies have shown that at least two organic mercurials,
phenyl mercuric acetate and methyl mercuric chloride, are only partially
oxidized by this method.
Potassium persulfate has been found to give approximately 100%
recovery when used as the oxidant with these compounds. Therefore, a
persulfate oxidation step following the addition of the permanganate
has been included to insure that organo-mercury compounds, if present,
will be oxidized to the mercuric ion before measurement.
REFERENCES:
1. Hatch, W. R., and Ott, W. L., "Determination of
Sub-Microgram Quantities of Mercury by Atonic
Absorption Spectrophotometry", Anal. Chem. 40,
2085 (1968).
2. Uthe, J. F., Armstrong, F. A. J. and Stainton, M. P.,
"Mercury Determination in Fish Samples by Wet Digestion
and Flameless Atomic Absorption Spectrophotometry",
Fisheries Research Board of Canada, 501 University Crescent.
Winnipeg 19, Manitoba.
3. Brandenberger, H. and Bader, H., "The Determination
of Nanogram Levels of Mercury in Solution by a Flameless
Atomic Absorption Technique", Atcmic Absorption
Newsletter, 6, 101 (1967).
-------
1/4/71
Section 13, B
- 2 -
4. Brandenberger, H. and Bader H., "The Determination
of Mercury by Flameless Atomic Absorption II. a
Static Vapor Method" Atomic Absorption Newsletter,
7, 53 (1968).
5. Tentative Method for Mercury (Flameless AA Procedure),
Analytical Quality Control Laboratory, Federal Water
Quality Administration, 1014 Broadway, Cincinnati,
Ohio 45202
II. PRINCIPLES:
The flameless AA procedure is a physical method based on the
adsorption of radiation at 253.7 nm wavelength by mercury vapor. The
mercury is reduced to the elemental state and aerated from solution in
a closed system. The mercury vapor passes through a cell positioned in
the light path of an atomic absorption spectrophotometer. Absorbance
(peak height) is measured as a function of mercury concentration and
recorded in the usual manner.
III. APPARATUS:
1. Atomic Absorption Spectrophotometer: Any commercial atomic absorption
unit having an open sample presentation area in which to mount the
absorption cell is suitable.
Instrument Settings: Lamp current - 9 ma, wavelength - 253-7°A,
slit setting - 320 y, scale - 2.5.
2. Mercury hollow cathode lamp, Westinghouse WL-22847, argon-filled, or
the equivalent.
3. Recorder, Sargent Welch, model DSRLG, or the equivalent. Recorder
settings: Logarithmic mode, scale selector - MV, range - full scale =
30 MV, chart speed 1/2 -inch per minute.
4. Absorption Cell: Constructed from plexiglass tubing, 1" O.D. x 4-1/2".
The ends are ground perpendicular to the longitudinal axis and quartz
windows (1" diameter x 1/16" thickness) are cemented in place. Gas
inlet and outlet ports (also of plexiglass but 1/4" O.D.) are
attached approximately 1/2" from each end. The cell is strapped to
a burner for support and aligned in the light beam by use of two 2"
by 2" cards. One inch diameter holes are cut in the middle of each
card; the cards are then placed over each end of the cell. The cell is
then positioned and adjusted vertically and horizontally to give the
maximum transmittance.
-------
Revised 11/1/72
Section 13,B
- 3 -
5. Masterflex Pump: With electronic speed control. Any peristaltic
pump capable of delivering 1 liter of air per minute may be used.
6. Flowmeter: Capable of measuring an air flow of 1 liter per minute.
7. Aeration Tubing: A straight glass frit having a coarse porosity.
Tygon tubing is used for passage of the mercury vapor from the sample
bottle to the absorption cell and return.
8. Drying Tube: 6" x 3/4" diameter tube containing 20 grams of magne-
sium perchlorate. The apparatus is assembled as shown in Figure 1.
NOTE: In place of the magnesium perchlorate drying tube a
small reading lamp with 60W bulb may be used to pre-
vent condensation of moisture inside the cell. The
lamp is positioned to shine on the absorption cell
maintaining the air temperature in the cell about
10 C above ambient.
9. Bottles, B.O.D., 300 ml, with "S glass stoppers. Fishers #2-926, or
the equivalent.
10. Pipets, transfer - 1, 2 and 5 ml.
11. Pipets, measuring - 1,2 and 5 ml.
IV. REAGENTS:
1. Sulfuric acid, canc., reagent grade.
2. Sulfuric acid, 1.0 N. Dilute 28.0 ml of conc. H-SO^ to 1000 ml with
distilled water.
3. Sulfuric acid, 0.5 N. Dilute 14.0 ml of conc. ^SO^ to 1000 ml with
distilled water.
4. Nitric acid, conc., reagent grade, of zero or low Hg content.
NOTE: If a high reagent blank is obtained,, it may be
necessary to distill the HNOj.
5. Stannous sulphate, reagent or purified grade. Add 25 g of the
reagent to 250 ml of 0.5 NILSO.. This mixture is a suspension
and requires continuous stirring during use.
NOTE: Stannous chloride or hydroxylamine hydrochloride may
be substituted for the stannous sulphate.
-------
Revised 11/1/72
Section 13,B
- 4 -
I
6. Sodium chloride - hydroxylamine sulfate solution. Dissolve 12 g
of each compound in distilled water and dilute to 100 ml.
7. Potassium permanganate, cryst*, reag. grade. Dissolve 5 g in 100 ml
of distilled water for a 5% w/v solution.
8. Potassium persulfate, reagent grade. Dissolve 5 g in 100 ml of
distilled water for a 51 w/v solution.
9. Mercuric chloride, reag. grade, cryst., A.C.S.
a. Stock mercury solution - dissolve 0.1354 grams of HgCl- in
100 ml of 1.0 N H2S04. 1 ml = 1 mg Hg.
b. Working mercury solution - Make two successive 1/100 dilutions
of the stock solution to obtain a final working standard con-
taining 0.1 ug per ml.
NOTE: The stock solution is relatively stable and
may be held, tightly stoppered, for periods
up to a month. The working solution should
be prepared weekly.
V. PROCEDURE:
Calibration
1. Transfer 1, 2 and 5 ml aliquots of the working Hg solution to 300-ml
BOD bottles and add distilled water to each bottle for a total
volume of 100 ml.
NOTE: In additional BOD bottle, add 100 ml of distilled
water and carry through all successive steps as
a reagent blank.
2. Stopper and shake bottles vigorously for complete mixing.
3. Add 5 ml of conc. H^SO., mix by swirling and then 2.5 ml of conc.
HNOj to each bottle ana, without stoppering, mix gently by swirling.
NOTE: Loss of Hg may occur at elevated temperatures.
However, with the specified volumes of acids, the
temperature should rise only 13 C and no Hg
losses have been observed under these conditions.
-------
1/4/71
Section 13, B
- 5 -
4. Add 1 ml of 5% KMnO^ solution to each bottle, mix by gently-
swirling, and allow to stand 15 minutes.
5. Add 2 ml of 51 potassium persulfate solution, mix by gently
swirling and allow to stand 30 minutes.
6. Add 2 ml of the sodium chloride - hydroxylamine sulfate solution
to reduce the excess permanganate.
7. Treating each bottle individually, add 5 ml of the stannous
sulfate - hLSO. solution, taking care to mix the reagent thoroughly
iimiediately oefore adding.
8. Immediately attach the bottle to the aeration apparatus (Figure 1)
forming a closed system. The circulating pump, previously adjusted
to a flow rate of 1 liter per minute, is allowed to run continuously.
9. The absorbance will increase and reach maximum within 30 seconds.
As soon as the recorder pen levels off (approximately 1 minute),
open the bypass valve (L) and continue the aeration until the
absorbance returns to its minimum value.
NOTE: Because of the tcxic nature of mercury vapor
precaution must be taken to avoid its inhalation.
Therefore, a bypass has been included in the system
to either vent the mercury vapor into an exhaust
hood or pass the vapor through some absorbing
media, such as:
a) Equal volumes of 0.1 NKMnO^ and 101 h^SO^.
b) 0.25% iodine in a 3% KI solution.
A specially treated charcoal that will adsorb mercury
vapor is also available from:
Barnebey Cheney
E. 8th Avenue and North Cassady St.
Columbus, Ohio 43219
CAT. No. 580-13 or 580-22
-------
1/4/71
Section 13, B
- 6 -
Sample Assay
1. Accurately transfer 100 ml or an aliquot diluted to 100 ml to a
300 ml BOD bottle. The sample must contain not more than 0.5 yg
of mercury.
2. Add 5 ml of cone. H~SO. and 2.5 ml of conc. HN0,, mixing after each
addition.
3. Add 1 ml of 5% KMnO^ to each sample bottle. Mix and add additional
portions of KMnO. until the purple color persists at least
15 minutes.
4. Add 2 ml of 5% potassium persulfate to each bottle and allow to stand
30 minutes.
5. Add the sodium chloride-hydroxylamine sulfate in 2 ml increments to
reduce the excess permanganate.
6. Add 5 ml of the stannous sulfate - HLjSO. suspension (the reagent
well mixed before adding) and immediately attach the bottle to the
aeration assembly.
7. Continue as previously described in Steps 8 and 9 under CALIBRATION
observing precautionary note.
VI- CALCULATIONS:
Determine peak height of the unknown from the chart and read mercury
value from the standard curve.
Calculate mercury concentration in sample by formula:
ugHg/1 = ug/Hg x 1000
in aliquot volume of aliquot
Report mercury concentrations as follows:
Below 0.5 pg/1, <0.5; between 1 and 10 ug/1, one decimal;
above 10 yg/1, whole numbers.
VII. MISCELLANEOUS NOTES:
1. Until more conclusive data are obtained, samples may be preserved by
addition of 3 ml of 1:1 HNO., per liter of sample. If only soluble
mercury is sought, the sample should be filtered immediately before
the acid is added. For total mercury the filtration is omitted.
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1/4/71
Section 13, B
- 7 -
2. The presence of sulfide interferes, with the determination, but can
be eliminated by the addition of potassium permanganate. Copper
has also been reported to interfere; however, copper concentrations
as high as 10 mg/1 had no affect on the recovery of mercury from
spiked samples.
3. a. Using spiked Ohio River samples at concentration of 1.0, 5.0,
10 and 15 yg hg/1, standard deviations were +_ 0.4, +_ 0.6, +_ 0.9
and ^ 1.1, respectively (AQC laboratory).
b. Using spiked Ohio River samples at 2.0 and 12 yg Hg/1, recoveries
were 95% and 100% respectively. (AQC laboratory).
4. The range of the method may be varied through instrument and/or
recorder expansion. A detection limit of 0.5 yg Hg/1 can be achieved;
concentrations below this level should be reported as <0.5.
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1/4/71
Section 13, B
- 8 -
Figure 1. Aeration apparatus for mercury deteimination by cold vapor AA tecnnique.
I
J
A - Sample container, approximately 300 ml (BOD bottle)
B - Drying tube, 150-200 ml capacity with Mg C10^.
C - Rotameter, = 1 liter of air/minute.
D - Cell, with quartz windows.
E - Air pump, = 1 liter of air/minute.
F - Glass tube with fritted end.
G - Hollow cathode Hg Lamp.
H - AA Detector.
J - Gas washing bottle containing 0.251 iodine in a potassium iodide solution.
K - Recorder, any compatible mode, three-way L. by-pass valve.
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1/4/71
Section 13, B
- 9 -
Figure 2. Cell for mercury measurement by cold vapor technique.
T
18 mm
J_
t
10 mm
I
«s 10 - 10 cm >
The length and 0D of the cell are not critical. The Perkin-Elmer 303 or 403
Spectrophotometers can accomodate a cell of about 18 an; the IL 153 instrument
takes a maximum length of about 11 cm. The body of the cell may be of any tubular
material but the end windows must be of quartz because of the need for UV transparency.
The length and diameter of the inlet and outlet tubes are not important, but tne
position of the side arms may be a factor in eliminating recorder noise. Tnere is
some evidence that displacement of the air inlet aim away from tne end of tiie cell
results in smoother readings. A mild pressure in the cell can bfe tolerated, but
too much pressure may cause the glued-on end windows to pop off.
Cells of this type may be purchased from various supply houses; see Fisher Catalogue
70, pp 992-993, Item 14-385-904D.
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Revised 12/2/74
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. If 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 contractural 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 Tracor 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 distributor should be contacted
for repairs.
The following services are available from the RTP shop:
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
Temp. Set Controllers - complete or integral components.
-------
Revised 12/2/74
Appendix I
-2-
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 components 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 requesting 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 one does not 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.
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Revised 12/2/74
Appendix I
-3-
Special shipping cartons and packing materials 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 919-549-2508(FTS)
Mail, Truck
or REA
Shipments:
Instrument Shop
Pesticides § Toxic Substcs. Effects Laboratory
EPA, National Environmental Research Ctr.
Research Triangle Park, N.C. 27711
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Revised 12/2/74
Appendix II, A
Page 1
ANALYTICAL QUALITY CONTROL
I. INTRODUCTION:
The term "quality control" may connote to some a system of
controlling the quality of a manufactured product. Truly, the
phrase is widely applied in this role, but it also can be aptly
applied to a "level of performance". It is in the latter sense
that the term is applied in the chemical analysis for pesticide
residues. A program of quality control is a means of assuring
the output of reliable and valid analytical data.
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 make no provision in time and
resources for its incorporation in the overall laboratory program.
The Editor has often heard the comment from individual bench chemists
to the effect that "we couldn't possibly fit the type of QC program
recommended into our work schedule." Unquestionably, this state-
ment 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 an organized 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 could not be introduced as evidence in a
court because of the danger of discreditation of the data 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 foregoing 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 interlaboratory 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 com-
plex and exacting, requiring highly sophisticated electronic
instrumentation. The lack of adequate controls is tantamount to
a ship without a compass.
Within a matter of months from the time of these manual
revisions, it is expected that a complete and separate manual
-------
Revised 12/2/74
Appendix II, A
- 2 -
dealing with Quality Control will become available for distri-
bution. For this reason our comments here will not pursue any
specifics. The manual outline is given in the following:
Tentative Outline:
Quality Control Manual
for the Analysis of Pesticides
in Human and Environmental Media
I. General Description of Pesticide Residue Analytical Methods.
II. Analytical Quality Control, its purpose and coverage in this
manual.
A. Introduction to statistics, precision, accuracy.
B. Interlaboratory Program.
a. Report forms - data
b. Sample data.
c. Relative performance ranking of laboratories.
III. Intralaboratory Program.
a. Control charts and report forms.
b. Sample data.
IV. Evaluation and Standardization of Materials Used in Pesticide
Residue Analysis.
A. Adsorbents such as Florisil, Silica gel, etc., activity
characteristics.
B. GLC column packing - discussion of importance of establish-
ing various characteristics such as efficiency, freedom
from active adsorption sites which promote compound break-
down, etc.
V. GLC Operation
A. Temperature control systems, accuracy of pyrometer readout,
method and need of monitoring to prevent such an occurence
as tritium foil vaporization due to excessive temperature
or column oven significantly higher or lower in temperature
than indicated by pyrometer.
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Revised 12/2/74
Appendix II, A
- 3 -
B. Gas Flow System
1. Filter - need for maintenance.
2. Purity of gases.
3. Effects on instrument caused by leaks and comments
on checking.
4. Carrier and purge flow velocity - inaccuracies in
many manometers and procedure for checking to guard
against completely erroneous gas velocities.
C. Detectors
1. EC
2. Thermionic
3. FDP
4. Microcoulometric
5. Coulson
6. Miscellaneous
D. Injection Port - importanc
1. On-column or off-column injection through a "demister"
tube to trap unwanted contaminants.
2. Injection techniques - various methods, preferred
volume range avoiding extremely low volumes. Cite
error potential such as 0.2 pi error in 1.0 pi shot
yields 201 error whereas the same 0.2 pi error in a
5 pi shot reduces the percentage to 4%.
3. Discussion of various septems and problems.
E. Selection of optimum columns for pesticides - advantages
of selecting'those which resolve largest number of pesti-
cidal compounds.
F. Use of at least one alternate column to avoid identification
trap of certain pesticide peaks overlapping. Alternate
column should elute pesticides in a significantly different
pattern than the main working column.
G. Sensitivity of system - For analysis of pesticides in
environmental media, concentrations of residues commonly
in ppb or ppt range. Need high sensitivity. Minimum
acceptability determined by peak ht. response resulting
from 100 pg of aldrin shall be 50% f.s.d.
H. Qualitative approach - absolute vs. relative retentions,
advantages of latter in reproducibility.
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Revised 12/2/74
Appendix II, A
- 4 -
I. Quantitative - Factors affecting accuracy
1. Linearity of detector for various pesticides -
vs. 63^^ the latter much more restrictive.
2. Ideal range of peak ht. response, 20 to 601 f.s.d.?
Minimum acceptable 10% f.s.d.
3. Discussion of comparison of relative peak heights from
standard and sample. Ideally should vary no more than
10% for highest accuracy, but should vary not more than
25%.
4. Pitfalls and superfluousness of use of a standard curve
due to response variations from day to day or even
within the working day. Need for frequent injections
of standards to monitor response.
5. Methods and pitfalls of peak measurements.
VI. Miscellaneous Procedures in Analysis of Environmental Substrates
A. Sample collection, storage and preparation - discussion
of importance of the sample container, temperature and time
lapse of storage.
1. Biological samples - tissues, etc.
2. Air
3. Water
4. Soil and stream bottom sediment
B. Control of Procedures for extraction of residues
1. Use of p-values for evaluation of extraction efficiency.
2. Various methods with discussion of pitfalls in each.
C. Control of methodology for evaporation and concentration
of sample solutions and fractional eluates.
1. Special importance of freedom of glassware from organic
contaminants.
2. Various methods - air-blow-down, nitrogen blow-down,
K-D apparatus, micro Snyder. Advantages and pitfalls
of each.
3. Technique for wash-down of evaporator tube to avoid
hang-up of pesticides' on upper glass.
4. Avoidance of boil-down to dryness - consequences.
D. Multiresidue and Individual Extraction - Cleanup-Fractiona-
tion Procedures for Pesticides and Metabolites (stress
problem areas; brief descriptions, not complete details
as in analytical manual; literature references).
-------
Revised 12/2/74 Appendix II, A
- 5 -
1. Chlorinated Insecticides.
1. Tissue - MOG.
2. Micromethod.
3. Blood.
4. Urine
2. Organophosphates.
1. Urine.
2. Blood.
3. Carbamates.
1. Fat.
2. Urine.
4. Herbicides.
5. Air Analysis.
6. Water.
7. Soil.
8. PCB - pesticide mixtures.
VII . Confirmatory Procedures.
A. Requirements for positive confirmation of pesticide identity.
1. Three or more pieces of independent evidence.
2. Data must be truly independent.
B. Relative Retention Times.
C. Selective Detectors.
D. Differential Elution from Cleanup Column.
E. Thin Layer Rp Values.
1. Preparation of TLC plates.
2. Spotting and Development of the Chromatogram.
3. Detection of Zones.
(a) Qualitative
(1) Rp Values.
(2) Co- chromatography.
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Revised 12/2/74
Appendix II, A
- 6 -
(b) Quantitative.
(1) Visual estimation.
(2) Densitometry.
F. Partition Studies (p-Values).
G. Derivatization (chemical reaction) techniques.
H. Spectrometry.
1. Visible - Fluorescence.
2. UV.
3. IR.
4. NMR.
5. MS
VIII. Maintenance, Troubleshooting, and Calibration of Instruments.
IX. Training of Pesticide Analytical Chemists
I. Dissertation on the need for practical bench training.
2. Non-availability at educational institutions.
Any recipient of these revisions wishing a copy of the Quality
Control manual is requested to write the editor of this manual. When
the manual comes off the press, a copy will be forthcoming.
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1/4/71
Appendix VI.
Page 1
TENTATIVE TISSUE, EXCRETA AND METHOD SELECTION FOR
ABNORMAL PESTICIDE EXPOSURE CASES; BLOCK DIAGRAM
LIVE DONORS
Suspected CHI.
Pestic. Exposure
BLOOD
5,A, (3), (a)
URINE
5,A,(4),(b)
10 ML
1
TISSUE BIOPSY
5,A,~(2),(a)
0.5 GRAMS
Suspected PCP
Exposure
Suspected 2,4-D_
or 2,4,5-T
Exposure
URINE
5,A,(4),(a)
URINE
5,A,(4),(c)
URINE
5,A,(3),Cb)
Suspected Hg Exposure
BLOOD
5,A,(3),(b)
BLOOD-TISSUE
13, A
10 GRAMS
Suspected O.G.P.
Pestic.ExposureJ
Suspected
Carbaryl Exposure
J
URINE
7, A
5 ML
1
URINE
6,A,(2), (a)
20 ML
URINE
6,A, (4),(a)
5 ML
BLOOD
6, A, (3), (a)
3 ML
AUTOPSY SAMPLES
Suspected CHL.
Pestic. Exposure1
Suspected Hg
Exposure
Suspected PCP Exposure —
Suspected 2,4-D or
2,4,5-T Exposure
SAME AS FOR LIVE DONORS
Suspected OGP Pest-
icide Exposure
Suspected Carbaryl
Exposure
Sample size given represents a maximum, number and letter designations refer to
URINE
5,A,(4),(b)
10 ML
ADIPOSE TISSUE
5,A, (1)
5 GRAMS
BLOOD
5, A, (3), (a)
100 GRAMS
OR
MODIFICATIOfy
OF 5,A,(1)
Sample size given represents a maximum, number and letter designations refer to
method number as listed in the TABLE OF CONTENTS.
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Revised 12/2/74
Appendix vn
Page 1
PESTICIDE ANALYTICAL REFERENCE STANDARDS
The pesticide repository of the Pesticides and Toxic Substances Effects
Laboratory (P^TSEL) 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 P^TSEL 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, we restrict the amount of each standard sent out 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 are difficult to
prepare, and are therefore expensive. It is suggested that not more than
20 mg of primary standard be weighed out and diluted to 100 ml with an
appropriate solvent. This yields a concentration of 200 ug per ml and should
provide a sufficiently high concentration for just about any gas-liquid or
thin layer chromatographic method. In fact, a solution of this concentration
should require one or more serial dilutions to provide an appropriate concen-
tration for most electron-capture GLC work.
For the user's information, supplemental data such as the use, chemical
name, molecular weight, empirical and structural formulae, and toxicity are
given for each compound. Unless otherwise stated, the toxicity is expressed
as the LDjq on the basis of oral feeding of male rats. The figure given is
the number of milligrams of the compound required per kilogram of animal
weight to produce mortality in 50 percent of the test animals. Consequently,
the lower the figure, the higher the toxicity. All users are cautioned to
exercise extreme care in the handling of any compound with an LD^g of 50 or
less. A number of these highly toxic compounds are also dermally toxic;
therefore, scrupulous care should be taken to avoid contact with the skin.
The current index lists over 400 compounds. Over'30 compounds previously
stocked have been deleted as they are no longer manufactured or too difficult
to obtain. On the other hand, 67 new compounds have been added to the stock
during the past year Each compound is listed by its common name if one has
-------
Revised 12/2/74
Appendix VII
- 2 -
been assigned. In cases where no common name has been assigned, the trade
or chemical name is given.
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.
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 appreciation 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, Chemistry Branch
EPA, Pesticides § Toxic Substances Effects Laboratory
National Environmental Research Center
Research Triangle Park, North Carolina 27711
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12/2/74
REVISIONS TO THE MANUAL
Appendix VIII
Page 1
This manual is revised biennially and all persons on the mailing
list will automatically be mailed 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
probably not on the list and therefore will not automatically receive
revis ions.
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 a 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, Chemistry Branch
Pesticides § Toxic Substances Effects Laboratory
EPA, NERC
Research Triangle Park, NC 27711
This is to request that your record be reviewed to be certain the
undersigned is on your mailing list to receive copies of all future
analytical manual revisions.
(Print or type name and full business address)
U.S. GOVERNMENT PRINTING OFFICE: 1975 - 640-920/2503 - Region 4
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