The
IDENTIFICATION
and
MEASUREMENT
of
CHLORINATED
HYDROCARBON
PESTICIDES
in
SURFACE WATERS
U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE
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The
IDENTIFICATION
and
MEASUREMENT
of
CHLORINATED
HYDROCARBON
PESTICIDES
in
SURFACE WATERS
By A.W. BREIDENBACH
J.J. LICHTENBERG
C.F. HENKE
D.J. SMITH
J.W. EICHELBERGER, JR.
H. STIERLI
Water Quality Section
Baste Data Branch
U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE
Public Health Service
Division of Wafer Supply and Pollution Control
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PUBLIC HEALTH SERVICE PUBLICATION NO.
September 1964
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PREFACE
This manual describes the analytical methods which are
currently being used in the Public Health Service's Water
Pollution Surveillance System in identifying chlorinated
hydrocarbon pesticide levels in surface waters. It is being
issued as an informal publication, its authors recognizing that
because of today's rapid progress in methods research revisions
and additions will almost certainly be required in the future.
Organic chemicals, as a group, have presented a special
challenge to the laboratory because of the many thousands of
such chemicals in use and the many complex mixtures of wastes
produced in their manufacture. Specific identification and
measurement of one class of organics, the chlorinated hydro-
carbon pesticides, to a sensitivity of one microgram per liter
or below is of particular concern.
The carbon adsorption method, developed over a decade ago,
has been effectively employed in pesticide pollution studies.*
While it is essentially a qualitative screening and continuous
sampling technique when used on untreated surface waters, the
method provides minimum quantitative values for measurement of
specific substances. Most significant, the method has proved to
be very useful for obtaining large enough samples for corroborative
infrared and chromatographic identifications at low concentration
levels.
Chromatography and chromatographic instrumentation has made
possible the development and application of additional techniques
by the PHS Water Pollution Surveillance System laboratories. These
newer techniques, applied to carbon adsorption extracts as well as
discrete water samples, have been used to provide definitive
identification and measurement of chlorinated hydrocarbon pesticides
in surface waters• The rapid progress being made in methods research
will surely result in continued modifications, improvements, and
additions. Nevertheless, there is an urgent need for documentation
of existing methods so they are available for study by the various
agencies which have responsibilities in this field.
* This method was pioneered and developed by a team of scientists
at the Robert A. Taft Sanitary Engineering Center of the Public
Health Service: F. M. Middleton, M. Ettinger, A. Rosen, G, Walton
and H. Braus.
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Further developmental work is s^oing forward at the Robert
A. Taft Sanitary Engineering Center and at the Southeastern
Water Laboratory in Athens, Georgia. The Basic Data, Technical
Services and Basic and Applied Sciences Branches of the Division
of Water Supply and Pollution Control, as well as the Milk and
Food Research Activity of the Division of Environmental Engineering
and Food Protection, are actively engaged in the search for
additional improved procedures which will be useful for measuring
pesticides in the various parts of the aquatic environment.
Leo Weaver, P.E.
Chief, Water Quality Section
Basic Data Branch, Division of Water
Supply and Pollution Control
A Note about the Authors: Dr. Breidenbach is Chief of Technical
Operations of the Water Quality Section and in charge of the
Organic Chemistry Laboratory. Mr. Lichtenberg and Mrs. Smith
and Mr, Eichelberger are supervisor and members of the identification
and quantification activities group and Mr. Henke supervises the
laboratory's extraction and separation activities. Mr. Stierli, a
registered professional engineer, is in charge of the Equipment
Development and Instrumentation Laboratory of the Section.
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TABLE OF CONTENTS
PAGE
PREFACE iii
I. INTRODUCTION
A-Background — Public Health Service Water Pollution
Surveillance System — Organic Pollution
B-Carbon Adsorption Sampling
C-Bottled Samples
II. SAMPLE COLLECTION 7
A-The Carbon Adsorption Method — Preparation of the Carbon
Adsorption Cartridge — Precautions Necessary to Prevent
Accidental Contamination of Carbon
B-Discrete Bottled Samples — Preparation of the Container
III. PREPARATION OF SAMPLES PRELIMINARY TO GAS
CHROMATOGRAPHIC ANALYSIS 13
A-Carbon Adsorption Samples — Treatment of Carbon —
Preliminary Separation of CCE
B-Discrete Bottled Samples — Extraction of Pesticides
From Water — Concentration of Extract — Thin Layer
Chromatography
IV. DETERMINATIVE STEPS 37
A-Gas Chromatography — Application of Electron Capture
Gas Chromatography — Application of Microcoulometric
Titration Gas Chromatography — Calculations — Column
Packings — Column Conditioning
B-Infrared Spectrophotometry
V, CONTROL OF INTERFERENCES 47
A-Solvent Interferences — Chloroform — Hexane — Carbon
Tetrachloride and Acetone
B-Carbon Interferences — Carbon Blank
C-Other Sources of Interference
D-Interpretation
VI. SENSITIVITY AND SPECIFICITY 51
A-Sensitivity — Carbon Adsorption Extracts Examined by
Electron Capture Gas Chromatography — Carbon Adsorption
Extract Examined by Microcoulometric Titration Gas
Chromatography — Bottled Sample Extracts Examined by
Electron Capture Gas Chromatography — Bottled Sample
Extracts Examined by Microcoulometric Titration Gas
Chromatography
B-Specificity — Carbon Adsorption Samples — Bottled
Samples
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TABLE OF CONTENTS
(Continued)
PAGE
APPENDIX ONE 55
Engineering Aspects of Sampling
by the Carbon Adsorption Method
APPENDIX TWO 73
Chromatograms| Sample Calibration Curves,
Infrared Spectra, and Structural Formulae
APPENDIX THREE 95
Equipment, Solvents and Reagents
APPENDIX FOUR . 99
General Composition of Carbon Chloroform
and Carbon Alcohol Extracts
APPENDIX FIVE 103
Glossary
REFERENCES 105
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LIST OF ILLUSTRATIONS
figure title PAGE
1 PHS Water Pollution Surveillance System Sampling Stations 2
2 Screw-cap (Teflon-lined), Glass Sample Containers and
Expanded Polystyrene Cartons 11
3 Laboratory Data Card 15
Carbon Drying Oven 16
5 Soxhlet Extractors 17
6 Removal of Residual Chloroform from Carbon 20
7 Flow Scheme for Solubility Separation of CCE 22
8 Flow Scheme for Chromatographic Separation of Neutrals 24
9 Infrared Spectrum of Aromatic Fraction of CCE Sample
Supporting Chromatographic Identification of DDT 26
10 Infrared Spectrum of Aromatic Fraction of CCE Sample Supporting
Chromatographic Identification of Dieldrin 27
11 Spotting of TLC Plate 31
12 Diagram of Designation of Sections in the Cleanup and
Separation of CCE-Aromatics on Silica Gel Layers 33
13 Silica Gel Collection Assembly 34
Calculation of Peak Area 40
15 Carbon Adsorption Column and Shipping Container 57
16 Details of Sand Prefilter 59
17 Schematic Diagram of an Installation With Manual Backwash 60
18 Carbon Adsorption Unit Model HjO-MIC With Sand Prefilter
and Automatic Backwash 61
19 Carbon Adsorption Unit Model H20-M2C With Presettling Tank 63
and Auxiliary Equipment in Shelter
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20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
64
66
68
69
71
74
75
76
77
78
79
80
81
82
83
84
85
86
TITLE
Schematic Flow Diagram With Sampling Procedure for Organic
Sampler Models f^O-MlC and H2O-M2C
Equipment Installed in Field Test Station for Field
Evaluation of Low Flow Rate Samplers in Comparison
With Conventional Sampling Apparatus
Prototype Low Flow Rate Organics Sampler, Model LF-1
Prototype Low Flow Rate Organics Sampler, Model LF-2
Carbon Filter Data Sheet
EC Gas Chromatogram of Standard Pesticides in TLC Section II
(dieldrin, endrin)
EC Gas Chromatogram of Standard Pesticides in TLC Section III
(lindane, heptachlor epoxide, DDD)
EC Gas Chromatogram of Standard Pesticides in TLC Section IV
(heptachlor, aldrin, DDE, DDT)
MCT Gas Chromatogram of Standard Pesticides in TLC Section II
(dieldrin, endrin)
MCT Gas Chromatogram of Standard Pesticides in TLC Section III
(lindane, heptachlor epoxide, DDD)
MCT Gas Chromatogram of Standard Pesticides in TLC Section IV
(heptachlor, aldrin, DDE, DDT)
Sample Calibration Curve for Dieldrin (ECGC)
Sample Calibration Curve for Dieldrin (MCTGC)
IR Spectrum of Standard Dieldrin in Mineral Oil Mull
IR Spectrum of Standard Endrin in Mineral Oil Mull
IR Spectrum of Standard Lindane in Mineral Oil Mull
IR Spectrum of Standard DDD in Mineral Oil Mull
IR Spectrum of Standard Heptachlor in Mineral Oil Mull
• ¦ •
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FIGURE TITLE PAGE
38 IR Spectrum of Standard Aldrin in Mineral Oil Mull 87
39 IR Spectrum of Standard DDE in Mineral Oil Mull 88
HO IR Spectrum of Standard DDT in Mineral Oil Mull 89
HI Structural Formulae of Nine Chlorinated Hydrocarbon 90
Pesticides
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LIST OF TABLES
PAGE
Table 1 91
Electron Capture Retention Data
Table 2 92
Microcoulometric Titration Retention Data
Table 3 93
Some Column Packings Used for Gas Chromatographic Analysis
of Chlorinated Hydrocarbon Pesticides
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I.
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I. INTRODUCTION
A. BACKGROUND
1, Public Health Service - Water Pollution Surveillance System
The Public Health Service Water Pollution Surveillance
System was established in October 1957 to implement that
part of Public Law 660, as later amended, wherein the
Secretary of the Department of Health, Education, and
Welfare was authorized to collect and disseminate basic
data on chemical, physical, and biological water quality
insofar as such data relate to water pollution, prevention,
and control. The Surveillance System was expanded at
the rate of 25 stations per year until it reached a total
of 122 stations in September 1962, Six sampling stations were
added during the following year. Two sampling stations have
been established in 1964 in the lower Mississippi River basin:
one at New Roads, Louisiana on the Mississippi main stem and
one at Morgan City, Louisiana on the Atchafalaya, There
are now a total of 130 sampling stations in the PHS Water
Pollution Surveillance System. Figure 1 shows the location
of PHS Water Pollution Surveillance System stations.
Participants in the Public Health Service Water Pollution
Surveillance System include more than 175 State, local, and
Federal water, sewage or other public utilities, health
departments, industries, universities, State water pollu-
tion agencies, and resident engineers of Federal reservoirs.
Active local participation is important in this operation.
The State and local agencies perform most of the conven-
tional chemical analyses and collect water samples for
the more complex examinations. The Public Health Service
performs the more complex determinations at its Cincinnati
laboratories and makes the results available to the
various participants. The program as a whole is designed
to assemble, examine, and interpret the facts which
enable water pollution control agencies and others
concerned to determine the scope and character of problems to be
solved. Specifically, the objectives of the Public Health
Service Water Pollution Surveillance System may be set forth
as follows:
Formerly the National Water Quality Network
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PHS Water Pollution Surveillance System
SAMPLING STATIONS
to
\t
f.
2 Station* in Ala*ko not shown
Anchorage and Fairbanks
7-64
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a* To maintain continuous intelligence on the nature and
extent of pollution affecting water quality.
b. To determine trends in water quality as affected by
(a) water pollution control activities, (b) water
resources development activities, and (c) water use
and reuse.
c* To provide data on water quality useful in the develop-
ment of comprehensive water resources programs.
d. To provide data which will guide State, interstate
and other agencies in their water pollution control
programs, and in the selection of sites for legitimate
water uses.
e. To provide data of likely importance to epidemiological
and toxicological studies.
Sampling locations are selected with the assistance of the various
State and local agencies concerned and each sampling station satisfies
one or more of the following criteria:
- Major waterways used for public water supply, propagation of
fish and wildlife, recreational purposes, and agricultural,
industrial, and other legitimate uses*
- Interstate, coastal, and International boundary waters.
- Water on which activities of the Federal Government may have
an impact*
The analytical work of the Surveillance System is devoted to
characterization of surface water samples in six broad disciplinary areas.
These are biological, microbiological, radiological, general chemical
as well as physical properties and synthetic organic chemicals.
Frequency of collection of the various discrete samples varies from
once per week to once per month depending on the type and purpose of the
sample*
2. Organic Pollution
A very large variety of organic pollutants is known to be
present in river water. These substances, present in small
concentrations, may be carried to the stream in runoff,
in domestic sewage or in industrial wastes. They are carried
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in solution and adsorbed to suspended solids. The 5-day
biochemical oxygen demand (BOD), chemical oxygen demand
(COD), nitrogen analyses and total carbon have been used
successfully to aid in describing the degree and type of
organic pollution as well as estimating oxidizability,
in the stream environment. These tests do not, however,
serve as tools to identify and measure the specific
organic compounds which are present in polluted water.
The rapid and economical measurement of microgram and
nanogram quantities of organic pollutants in water has
been extremely difficult and impractical until very re-
cently. Indeed, such minute quantities of specific sub-
stances, intermixed with a large variety of other inter-
fering organic substances, have presented an extremely
challenging and enigmatic analytical problem. Until re-
cently, identification and measurement of most organic
compounds in water in the parts per billion sensitivity
range, has required an extremely large sample. Most
methods, insofar as they have been developed, are not
yet sufficiently sensitive to be of use with smaller
samples.
The need for larger samples for exploratory work in
characterizing organic pollutants was recognized over a
decade ago and stimulated several years of research which
ultimately produced the carbon adsorption method (1)(2)
(3)(4)(5)(6)(7).
CARBON ADSORPTION SAMPLING
This method, described in detail herein, uses the adsorptive
capacity of activated carbon to concentrate organic materials
from large water samples, measuring up to 5000 gallons. This
large sample permits corroborative identification by several
analytical methods which can provide highly defensible identifica-
tions of specific substances. It must be noted, however, that the
concentration values obtained for specific substances with this
method must be considered as minimum values. First, the efficiency
of the adsorption on, and the desorption from, carbon cannot be
expected to be 100 percent for all compounds under widely varying
physical and physico-chemical conditions in the water being
sampled. Studies have shown recently, for example, that the
adsorption of organics in streams on carbon is most efficient
at flow rates and throughput volumes less than those which have been
employed previously (8)(9). Secondly, until a means is available to
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in solution from the large sample, the determined concentra-
tion values (e.g., microgram per liter of water) must be considered
low. The first test results from a system which approaches
this objective are encouraging. The increased yields per unit
volume of water from low flow equipment are thought to result,
in part at least, from trapping of, and subsequent desorption from
some of the suspended solids. Pesticides have been identified in
carbon adsorption samples (10)(11).
BOTTLED SAMPLES
Grab samples of one liter to one gallon are useful. The grab
sample, properly taken, contains water in which organics are dis-
solved as well as suspended solids on which organics are adsorbed.
The absolute weight of the organic material is so small in
most grab samples as to restrict the identifiable portion to non-
infrared analysis. However, if the organic substance present can
be detected it can usually be measured at very low levels.
Thus, in the microgram per liter concentration range carbon
adsorption samples provide enough material so that the potential
for corroborated identification exists. Grab samples are most
useful for rapid and highly sensitive measurement and, in addition,
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II
SAMPLE COLLECTION
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II. SAMPLE COLLECTION
Carbon adsorption samples and discrete one-liter samples
are taken for the identification and measurement of organic
substances in surface water. Only recently has it been
possible to combine these sampling approaches with sensitive thin
layer chromatographic and gas chromatographic methods for pesti-
cide analysis. The details of the sampling techniques employed
in the laboratories of the PHS Water Pollution Surveillance
System are outlined below.
A. THE CARBON ADSORPTION METHOD (CAM)
This technique, developed in 1951 (1), was applied to
raw surface waters in a pilot study in 1956 and has been in
routine use in the PHS Water Pollution Surveillance System
since 1957. Since that time the sample collection aspects
of the technique have undergone considerable refinement.
Pertinent details of the sampling equipment are included
in Appendix One.
1. Preparation of the Carbon Adsorption Cartridge (CAC)
The carbon adsorption cartridge consists of a Pyrex
glass pipe three inches in diameter and 18 inches in
length packed with two types of carbon. To pack the
vertically oriented cylinder are added successively
4,5 inches of 4-x 10-mesh carbon , nine inches of
30-mesh carbon14, and an additional 4.5 inches of 4-x
10-mesh carbon. The cylinder is packed full but not
tightly. The coarse carbon at each end of the cartridge
aids in preventing clogging by mud and silt from turbid
waters. However, cartridges employing all fine (C-190)
carbon at low flow rates and reduced throughput volumes
are being used successfully (Appendix One). The cartridge
is shipped to the field station and installed in the
appropriate sampling system.
Sample volumes of 1000 and 5000 gallons (1)(12)(13)
of water taken at rates of 0.25 and 0.5 gpm have been
used successfully; however, sampling efficiency can
r\
Although these methods are employed currently for untreated surface
water the methods are also applicable to ground water and treated water.
3Cliff Char 4 x 10 mesh (Cliffs Dow Chemical Co., Marquette, Mich.)
^Nuchar C-190 (West Virginia Pulp and Paper Co., New York, N.Y.)
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be increased by the use of smaller volumes and
lower flow rates. Efficiency is further increased with
the low flow rate system using a column containing only
30-mesh carbon. The reproducibility (1H), and effect
of the variables of total throughput and rate of flow
through the carbon (9) have been the subject of intense
study by Booth, A sampling system for lower flow rates and
lower throughput volumes was designed by Castelli and
Booth (15). Preliminary field tests indicate sampling
efficiency is greatly improved (16). See Appendix One.
After the desired quantity of water is sampled the
carbon cartridge is disconnected and returned to the
laboratory for analysis. (See Figure 15, Appendix One.)
2. Precautions Necessary to Prevent Accidental
Contamination of Carbon
The affinity of carbon for organic substances re-
quires that supplies of carbon be protected from ex-
traneous sources of contamination. For example, carbon
can adsorb organic substances such as paint vehicles
and insecticides used for pest control from the air.
Therefore, the carbon is stored and processed in an
area adequately protected from such sources of con-
tamination. As an additional precaution, the venti-
lating, heating and air conditioning systems for the
laboratories in which carbon adsorption samples are
processed are completely isolated from all other lab-
oratories. Obviously, spraying with pest control
chemicals is not permitted in these areas. Carbon
blank determinations supplement these precautions,
(See Section V.)
DISCRETE BOTTLED SAMPLES
Approximately one liter (940 ml) of water is collected in
each of two wide-mouthed glass bottles equipped with screw caps
fitted with Teflon liners. These two bottles represent one
sample. Plastic bottles (polyethylene) are not used because
traces of plasticizer are leached from the plastic by the
water and can be a source of analytical interference. Moreover,
organics from the water are adsorbed on the plastic. It has
been suggested that high grade Teflon (Nalgene) bottles may
be satisfactory for this use; however, the cost is prohibitive
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bottles because breakage in shipment frequently causes loss of
sample. This is overcome by the use of relatively inexpensive,
expanded, polystyrene foam shipping containers molded to fit
the bottle. (See Figure 2.)
1. Preparation of the Container
Bottles are rinsed successively with chromate cleaning
solution, running tap water, distilled water, and finally
several times with redistilled solvent (e.g., acetone,
hexane, petroleum ether, chloroform). Caps and liners
are washed with detergent, if necessary, rinsed with tap
water and then distilled water and solvent.
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FIGURE 2. Screw-cap (Teflon-lined), Glass Sample Containers and
Expanded Polystyrene Cartons
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Ill
PREPARATION OF SAMPLES
PRELIMINARY TO
GAS CHROMATOGRAPHIC ANALYSIS
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III. PREPARATION OF SAMPLES
PRELIMINARY TO CAS CHROMATOGRAPHIC ANALYSIS
After a carbon adsorption or grab sample is received it is
logged and all pertinent data (source, date sampled, date re-
ceived, quantity of water sampled) are recorded. (See Figure 3.)
A. CARBON ADSORPTION SAMPLES
1. Treatment of Carbon
a. Drying the Carbon
The carbon is dried by spreading it on stainless
steel trays in an oven at 40°C. for about two davs5»6-
(See Figure 4.) If there is a backlog of dried
carbon samples on hand they are sealed in solvent-
rinsed, one-gallon, wide-mouthed tin cans and held
for further treatment.
b. Extraction of the Carbon
Large scale Soxhlet extractors are used to
extract the dried carbon. The quantity of carbon used
in the sampling cartridge is accommodated by the
extractors. (See Figure 5.)
(1) Packing the extractors
To prevent carbon fines from passing into
the boiling flask, the bottom of the extractor
is packed with about three inches of pre-extracted
glass wool7. The wool is wetted with chloroform,
the dried carbon sample added and packed by tamping
so that it just fits the extractor. If carbon is
packed too tightly siphoning will be severely
hindered; frequency of siphoning is controlled at
2 cylinder volumes per hour. Siphoning is not
always automatic and application of compressed air
to the vent of the extractor is sometimes necessary.
(2) Chloroform extraction
The Soxhlet is filled with re-distilled chloro-
form and siphoned over twice. More chloroform is
c
Copper or brass trays may also be used. Galvanized metals or aluminum
react with wet carbon.
®The air circulated through the oven is prefiltered through carbon to
prevent contamination from the atmosphere.
70ily organic substances are first removed from glass wool by extraction
with chloroform.
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STANDARD ORGANIC ANALYSIS OF
CARBON ADSORPTION SAMPLES
STATION NOl
SAMPLE NO.
DATE TO.
RECEIVED
SOURCE
LOCATION.
WEIGHT OF SAMPLES IN GRAMS
CHLOROFORM EXTRACT ALCOHOL EXTRACT
.TYPE OF WATER.
.COMPOSITE.
.QUARTER.
FLOW IN GALLONS.
LITERS.
EXTRACTION DATA
EXTRACT
GRAMS
RRB
PER CENT
OATE EXTRACTED
CHLOROFORM
ALCOHOL
TOTAL
SEPARATION OF CHLOROFORM EXTRACT
SOLUBILITY SEPARATION
P. P. B.
PER CENT
ETHER INSOLUBLES
WATER SOLUBLES
NEUTRALS
ALIPHATICS
AROMATICS
OXYS
LOSS
TOTAL
WEAK ACIDS
STRONG ACIDS
BASES
LOSS
TOTAL
NEUTRALS
NEUTRALS
REMARKS:
SEC 169a
(6-il.)
SOLUBILITY SEPARATION
CHROMATOGRAPHIC
SEPARATION
CHLOROFORM EXTRACT
NEUTRAL FRACTION
ETHERINSOLUBLES WATER SOLUBLES
ALIPHATICS
WEAK ACIDS STRONG ACIDS
AROMATICS
BASES NEUTRALS
OXYS
rjojrt
BACK
FIGURE 3. Laboratory Data Card
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FIGURE 4, Carbon Drying Oven
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added, if necessary, and the sample extracted
for 35 hours®. After the extraction is com-
pleted, the bulk of the chloroform is siphoned
and blown over into the boiling flask. The
flask is removed from the system, the extract
concentrated to about 250 ml by distillation^,
and filtered through solvent-washed filter paper
into a 300-ml Erlenmeyer flask. The solvent
is evaporated to approximately on a steam
bath with a jet of clean, dry air10. The con-
tents of the flask are transferred to a tared
glass vial and the remaining solvent evaporated
at room temperature in a hood without a jet of
air. The carbon chloroform extract (CCE) is
judged dry when the chloroform odor can no
longer be detected11. The weight of the residue
is obtained,
(3) Ethanol extraction
This step is not used in routine pesticide
analysis. The residual chloroform is removed
from the carbon by blowing pre-cleaned warm air
through the carbon (in place in the Soxhlet) and
exhausting chloroform vapors through the hood.
In order to do this the Soxhlet is removed from
the hood and the carbon shaken loose to facili-
tate movement of air through it. The Soxhlet,
O
Longer extraction times may be used but 35 hours (2* for ethanol) is
considered optimum. Booth (m) has confirmed this point.
^Two-zone Glas-Col heating mantles are used to prevent overheating and
scorching of the sample (CCE),
^First the compressed air is cleaned and dried by directing it through
a bed containing carbon and a drying agent such as calcium chloride.
^A trace of chloroform is retained by the CCE, This is, however,
insignificant in most samples and no correction is necessary. In very
large fluid samples correction may be necessary and can be accomplished
using a procedure developed, by Hashni (17). Unfortunately, it is not
practical to do this on ar routine basis for large numbers of samples.
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still containing the carbon, is returned to the
hood with the glass plate cover removed. A hose
from a heated air manifold (approximately 60° C.)
is attached to the bottom of the siphon tube and
the air is blown up through the carbon for three
to four hours or until it is dry. (See Figure 6)
Alternate methods^ ^ 13 may be employed but this
procedure has proved much less hazardous, consumes
less time and requires less supervision. Ethanol
(95%) is added to the dried carbon and the
extraction is carried out in the manner described
for the chloroform step. Extraction is termi-
nated after 24 hours^4-.
Concentration of the carbon alcohol extract
(CAE) is begun as described for the chloroform
extract. However, the drying, started on a
steam bath with a jet of air, is continued in
an oven at 75° C. until weight change of suc-
cessive weighings at 72-hour intervals is less
than 1%.
2. Preliminary Separation of CCE (12) (18) (19) (20) (21)
The procedure described under paragraph 2a is employed
when all classes of compounds are of interest. When only
chlorinated hydrocarbon pesticide information is sought,
the procedure 2b is used.
The separation techniques and analytical procedures
described below are carried out as quantitatively as pos-
sible. Careful attention is directed at details of quanti-
tative transfer, controlled evaporation and accurate weigh-
ing. Ether extractions are carried out in a hood.
¦^The carbon is removed from the Soxhlet and dried in the oven as in
III, A, 1, a. This procedure requires about 48 hours. Adequate
ventilation is required to remove hazardous chloroform vapors.
1 3
The residual chloroform may be leached from the carbon by pouring
alcohol over it. Proceed as follows: Siphon twice and distill
until 68° C. is reached. Repeat a second and third time, distill
to 77° C. and begin extraction. Add alcohol if necessary. The
distillate (68° to 77° C.) may be used for the initial leaching of
the carbon in succeeding extractions. This procedure requires about
4 hours.
l^See Footnote 8,
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FIGURE 6. Removal of Residual Chloroform from Carbon
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a* Procedure for General Organic Analysis
(1) Solubility separation: (See Figure 7)
(a) Weigh out approximately 0.5 gram of CCE(a)
in a 50 ml beaker. As little as 0,1 g or
less can be used; however, the percent-
age error increases as the weight of the
aliquot decreases.
(b) Add about 1 ml of methanol to the sample
and stir. Dissolve sample in 30 ml of ether
and stir. If there is appreciable insoluble
material, filter through a sintered-glass
funnel under vacuum. The residue (b) is the
ETHER INSOLUBLE fraction (EI). Transfer the
residue back to the 50-ml beaker using methanol,
evaporate on a steam bath, cool and weigh,
(c) Transfer the ether solution (c) to a 125-ml
separatory funnel. (Do not use stopcock lubri-
cants - use only glass or Teflon stopcocks).
Extract three times with 15 ml portions of dis-
tilled water and combine extracts in a tared
125-ml Erlenmeyer flask. Evaporate water (d)
to dryness on a steam bath with a jet of air,
cool and weigh. This is the WATER SOLUBLE
fraction (WS).
(d) Extract the ether solution (e) remaining in the
funnel three times with 15-ml portions of dilute
HC1 (5%). Set the ether layer (f) aside and make
the HC1 extract (g) strongly basic (pH>10) with
NaOH pellets or 25% NaOH solution. After cooling,
extract three times with 15-ml portions of ether,
combine in a 125-ml Erlenmeyer flask, dry, evapo-
rate and weigh. The residue (h) is the BASIC
FRACTION (B). Discard the water layer (i) .
15 The basic water layer (i) remaining after ether extraction may contain
some amphoteric and some water-soluble substances. If these substances
are of interest a special plan for analysis should be set up (18).
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WEIGHED SAMPLE (a)
II
add ether, filter
ii
Ether Solution (c)
extract with HgO
1
Residue (b)
evaporate, weigh
I
ETHER INSOLUBLES (El)
Ether Layer(e)
extract with HCt
Water Layer (d)
evaporate, weigh
WATER SOLUBLES (WS)
1
Ether Layer (f)
extract with NaHC03
Water Layer (j)
make acid
extract with ether
I
i
Ether Layer (h)
dry, evaporate
and weigh
I
BASES (B)
Water Layer (g)
Make basic, extract
with ether
I
1
Water Layer (i)
(contains
amphoterics, etc)
I
Ether Layer (I)
dry, evaporate
and weigh
|
Water Layer (m)
Discard
STRONG ACIDS (SA)
Ether Layer (k)
extract with NaOH
——¦———————— —— i
Water Layer (n)
make acid
extract with ether
1 —
Ether Layer (q) Ether Layer (o) Water Layer (p)
dry, evaporate dry, evaporate Discard
and weigh and weigh
NEUTRALS (N) WEAK ACIDS (WA)
FIGURE 7. Flow Scheme for Solubility Separation of CCE
-------
(e) Extract the ether layer(f) three times with
15-ml portions of NaHCO^ (5%). Set the ether
layer(k) aside and make the NaHCOg extract(j)
strongly acidic (pH<2) by careful addition
of concentrated HC1. After cooling, shake
vigorously to release C02< Extract three
times with 15-ml portions of ether, combine
in a 125-ml Erlenmeyer flask, dry, evaporate,
and weigh. The residue(l) is the STRONG ACID
fraction (SA). Discard the water layer(m).
(f) Extract the ether solution (k) three times
with 15-ml portions of NaOH (5%) and once
with distilled water. Caution: Emulsions
may form during this step. Set the ether
layer(q) aside and make the NaOH extract(n)
strongly acidic with concentrated HC1. After
cooling, extract three times with 15-ml portions
of ether, combine in a 125-ml Erlenmeyer flask,
dry evaporate and weigh. The residue(o) is
the WEAK ACID fraction (WA). Discard the
water layer(p).
(g) The ether solution(q) contains the NEUTRAL
fraction (N). Place in a 125-ml Erlen-
meyer flask, dry, evaporate and weigh.
Ether solutions are dried by pouring
over a two-inch column of anhydrous
sodium sulfate followed by ether rinses or by
adding sodium sulfate (10 g) to the flask and
filtering off sulfate after standing over-
night.
(2) Chromatographic separation of the neutral fraction
(See Figure 8)
(a) Pack activated silica gel^® in a Pyrex glass
column 20 mm in diameter to a height of 10 cm.
(b) Weigh"'"' the neutral sample in a 10-ml beaker
and dissolve in a minimum amount of ether.
Add sufficient silica gel to adsorb the sample.
Evaporate the ether gently.
16Davison Code 950-08-08-226 (60 to 200-mesh), Davison Chemical Co.,
Baltimore 3, Maryland.
17It is convenient to retain for future reference about 5 mg of the
neutral fraction.
-------
I
a
I
Elute with
Iso - octane
NEUTRALS
Adsorb on Silica gel Column
Elute with
Benzene
i
c
I
Elute with
Chloroform/Methanol
(I'D
AUPHATICS (AL) AROMATICS (AR) OXYGENATED COMPOUNDS
(OXY)
FIGURE 8. Flow Scheme for Chromatographic Separation of Neutrals
-------
(c) Wet the column with about 20 ml of iso-octane
and add the sample when the last of the 20
ml reaches the surface of the adsorbent.
Rinse the beaker with iso-octane and add the
rinsings to the column. The beaker should
be rinsed several times with iso-octane and
each succeeding eluent when the eluent is
added to the column.
(d) Elute the ALIPHATIC fraction (AL) with 85 ml
of iso-octane (a) and collect in a tared 150
ml beaker. The eluent should be added care-
fully with a medicine dropper so as to disturb
the surface of the adsorbent as little as
possible. A slow, dropwise, elution rate is
desirable. It may be necessary to apply mild
pressure to obtain a satisfactory rate.
(e) After the level of the iso-octane has reached
the surface of the adsorbent, replace the re-
ceiving beaker with another tared 150 ml
beaker. Elute the AROMATIC fraction (AR)
with 85 ml of benzene (b) and collect.
(f) After the liquid level of the benzene has
reached the surface of the adsorbent, replace
the receiving beaker with another tared 150 ml
beaker. Elute the OXYGENATED fraction (OXY)
with 85 ml of, a 1:1 mixture of methanol and
chloroform (c), and collect.
(g) Carefully evaporate the three fractions on a
steam bath with a jet of dry clean air, cool,
and weigh* The beakers should be removed from
heat and air before the solvent is completely
evaporated.
Infrared spectra
(a) Chloroform extracts - infrared spectra are
obtained on selected fractions that have un-
usual physical characteristics or odors. In
analyses directed at chlorinated hydrocarbons,
the IR spectra of the aromatic fraction is run.
See Appendix Three and Figures 9 and 10.
-------
4000 3000
100
CMi
1000 900
mmm
7 8 9 10 11 12 13 14 15
WAVELENGTH (MICRONS)
SPFCTRUAA NO.
npiniw
1 Fr,FMn
PFAAARKS \
WASHINGTON
1
C..C.R.
PI IPITY
V
AROMATIC FRACTICH
PHARF
r>ATF
THICKNFSS
OPERATOR
£
FIGURE 9, Infrared Spectrum of Aromatic Fraction of CCE Sample
Supporting Chromatographic Identification of DDT
-------
4000'|i
1000 900
CM-i
1500
800
700
2000
m
2 60|
40
gp£?yj||p
WAVELENGTH (MICRONS)
SPECTRUM NO.
ORIfilKI
IFOFND
RFMAPKS
sample savannah mvbr.
—SSIXH CAROLINA
1.
C.C.B.
PURITY
0
aromatic fracticn
PHASF
fiATF
_
THICKNFSS
OPERATOR
FIGURE 10. Infrared Spectrum of Aromatic Fraction of CCE
Sample Supporting Chromatographic Identification
of Dieldrin
-------
(b) Ethanol extracts - infrared spectra, only,
are obtained on the alcohol extracts.
The percent recovery of the CCE and con-
centration (ug/1) are calculated and
recorded. The percent and concentration of
the various fractions, obtained through
the separation of the CCE are calculated and
recorded.
b. Column Chromatographic Separation of CCE (Alternate Procedure)
This procedure may be employed to separate the aro-
matic fraction more rapidly from the CCE when this frac-
tion is of interest. Since most chlorinated hydrocarbon
pesticides, including lindane, DDT, DDD, DDE,
dieldrin, endrin, aldrin, heptachlor and heptachlor
epoxide, are found in the aromatic fraction, this step
is employed when pesticide data are needed and other sub-
stances are of lesser interest. However, laboratory tests
indicate that two other pesticides, methoxychlor and
parathion (organophosphorus compounds) may be found in
the oxy fraction as might be expected from their
chemical structure.
The use of this alternate procedure does not
preclude the isolation of the other classes of organics,
because the longer, generally applicable solubility
separation can be employed with the third (chloroform-
methanol) eluate if necessary.
This chromatographic separation technique for the
CCE is identical to that used in separation of the
neutral fraction. In this case, however, 0.5 gram of
the CCE (or less) is weighed in a 50-ml beaker, dis-
solved in a minimum quantity of chloroform and added
to just enough silica gel to adsorb the dissolved
sample. The chloroform is gently evaporated and the
sample, adsorbed on the silica gel is added to the
column as described in Section A.2.a.(2) above. The
aliphatic fraction, the aromatic fraction, and a
complex residue, eluted with chloroform-methanol, are
obtained.
c« Thin Layer Chromatographic (TLC) Separation of Pesticides
From Aromatic Fraction of CCE (22).
(1) Preparation of plates
Layers of silica gel 0.25 mm thick are pre-
pared on 200- x 200-mm glass plates. A thin
slurry is prepared of 30 g of silica gel G in
-------
about 60 ml of water and spread over five plates
with the aid of a variable thickness spreading
device. The plates are allowed to stand five
minutes, then dried in an oven for 30 minutes
at 130°C., and stored in a desiccator for future
i n *
(2) Preparing the solvent system
The developing solvent, carbon tetrachloride,
is added to the chamber to a depth of 10 mm
(approximately 200 ml). Two filter paper wicks,
one on each side of the chamber, are placed so
that one end contacts the solvent. After the
lid is in place, the chamber is allowed to equili-
brate for one hour.
(3) Spotting of plates
Marks are made near the edge of each
plate at distances of 1.5 and 11,5 cm
above the bottom edge to define the spotting line
and the point at which the solvent front has moved
to 10 cm.
Layers of varying thickness have been investigated. Layers thicker
than 0.25 mm produced no better separation and thinner layers were
consistently less uniform. Aluminum oxide plates 0,25- and
0.50-mm thick were also prepared. The range of Rf values for the
pesticides spotted was considerably smaller than those observed
with silica plates using the same solvent system. Thus separ-
ation was not as good. However, alumina plates may have special
applications, for example: pesticides that show a tendency to
streak on silica gel (chlordane, toxaphene) produce single spots on
alumina. Also, it is possible to separate dieldrin and endrin, which
°n silica gel normally have approximately equal R^ values.
Many different developing systems, both multi-component and single
component, have been investigated. In general, the single component
systems showed much more consistent results than did the
multi-component systems. The single component system which
showed the best separation of all pesticides investigated was
carbon tetrachloride. The range of R. values of pesticides was the
best of all systems investigated.
-------
The entire aromatic fraction of the CCE is
dissolved in acetone in a 15-ml centrifuge tube
and made up to 0.5 ml. A 50-ul aliquot or one-tent"
of the aromatic fraction in acetone is spotted.
(See Figure 11.)
It is necessary to direct a stream of clean
air on the point of spotting to keep the diameter
of the spot less than 1.0 cm. A typical
plate for pesticide analysis may contain three to
six samples, each spotted in duplicate; one for el"'
tion and gas chromatographic analysis and one for
possible corroborative visual identification
on the sprayed portion of the plate. In addition,
pesticide standards are spotted on the plate.
A 1-ul volume of a dye mixture is also spotted for
use as an internal standard. (See Appendix Three'
(if) Development
The spotted plate is placed in the chamber so
that the bottom edge is in contact with the solvent'
and the lid replaced. When the solvent reaches
the upper reference line (10 cm), the plate is
removed and the solvent allowed to evaporate.
The aromatic fraction of the CCE is generally 5 to 15 mg, or approxi-
mately 1 to 5% of the CCE. The average pesticide content of
most river waters sampled by the carbon adsorption method has
been estimated to be about 2% of the aromatic fraction. Fifty
microliters or 10% of the aromatic fraction yields enough material
for good pesticide peaks in gas chromatographic analysis, when
separated by thin layer chromatography,
21
The Rf of each pesticide will vary, since temperature and humidity
conditions are not controlled. However, with the aid of the dye
mixture which has been previously standardized against pesticide
spots, the sections may be adjusted to compensate for these devia-
tions.
-------
FIGURE 11, Spotting of TLC Plate
LIBRARY ' Tr— ^
PACIFIC NORTHWEST WATER LABORATORY
-------
The vertical zone of travel for each spot
arid its duplicate is divided into five horizontal
sections (identif ie^with Roman numerals) as
shown in Figure 12. The silica gel in each
section, representing only one of the duplicates,
is scraped loose from the plate with a spatula.
With the aid of vacuum, the silica gel is drawn
into an eye dropper which is plugged at the tip
with glass wool (Figure 13). The substances ad-
sorbed on the silica gel in each eye dropper are
eluted quantitatively into a graduated 15-ml centri-
fuge tube with 3 to 4 ml of acetone, and subjected
to gas chromatographic analysis.
The remaining duplicate run, still on the glass
plate, is sprayed with silver nitrate, dried
and exposed to UV light until spots appear.
Chlorinated pesticides appear as brown to black
spots. At this point, using appropriate standards
for comparison initial identification and estimates
of concentration are made.
Chlorinated and nonchlorinated pesticides
may be detected by exposing the untreated,
dry plate to bromine vapor for 30 seconds,
drying for 30 seconds, and spraying with a
fluorescein solution and finally with silver
nitrate (23). Exposure to UV light for 4 to
7 minutes causes chlorinated pesticides to
appear as brown to black spots and other pesti-
cides as yellow to white spots on the tan
background,
22 Repetitive testing of standard pesticides and the subsequent resolution
of nine chlorinated hydrocarbon pesticides resulted in the designa-
tion of the illustrated sections.
The eluate from Section V was repeatedly analyzed by gas chroma-
tography to determine if chlorinated pesticides occurred in it.
None of the pesticides studied occurs in this Section. Hence,
it is not analyzed on a routine basis.
-------
1 , ,—J—l—I , , , , [
iO.OCM
8.0 CM
6.0CM
3.0 CM
1.0 CM
SPOTTING 0
LINE „
§1
ZONE FOR:
SPOT) SF0T2 SPOT3
SECTION
3E
SECTION
TSL
SECTION
HI
SECTION
xr
SECTION
_X_
1
¦SOLVENT LINE
7.8 ALDRIN
7.5 DDE
7.3 DDT
-6.5 HEPTACHLOR
¦5.5 HEPTACHLOR EPOXIDE
¦5.2 DDD
• 4.2 LINDANE
.2.4 ENDRIN, DIELDRIN
J L
SPOT I 2
1.0 CM
8
10 II 12
FIGURE 12. Diagram of Designation of Sections in the Cleanup and
Separation of CCE-Aromatics on Silica Gel Layers
-------
AIR FLOW
EYE DROPPER
VACUUM
HOSE
GLASS WOOL
SILICA GEL COLLECTION ASSEMBLY
FIGURE 13. Silica Gel Collection Assembly
-------
B. DISCRETE BOTTLED SAMPLES
1. Extraction of Pesticides From Water
The entire measured water sample (approximately 1 liter)
is drained into a 2-liter separatory funnel equioped with a
Teflon stopcock. The sample is acidified by addition of 24
drops of concentrated HC1 and extracted successively with
100, 50, 50, 50, and 50 ml of redistilled hexane^.
The drained sample container is rinsed with three 50-ml
aliquots of hexane. The first two rinse volumes serve as
the first extraction volume (100 ml) for the sample. The
third rinse serves as the second (50 ml) extraction volume.
The sample is shaken moderately for four minutes.
(Vigorous shaking may cause severe emulsions, particularly
in waters of high organic content and/or high turbidity.)
The extracts are combined in a 300-ml Erlenmeyer flask and
dried by pouring through a two-inch column of anhydrous
sodium sulfate. The column is rinsed three times with
approximately 5 ml of hexane and the rinsings added to the
extract
2. Concentration of Extract
The hexane extract is transferred (approximately 300
ml) to a Kuderna-Danish evaporator and concentrated to
approximately 0.2 ml.
3. Thin Layer Chromatography
The entire concentrated extract is spotted on TLC
plates and developed in the same manner as the CCE aromatic
fractions. Spraying with chromogenic reagent is omitted
unless the amounts suspected are large and the extract can
be spotted in two aliquots. The four sections, obtained
in acetone solution, are subjected to gas chromatographic
analysis. Average recoveries of spotted standards range
from 85 to 98%.
23
Other solvents such as carbon tetrachloride, chloroform, and ethyl
ether-petroleum ether (1:1) may be used (2*0 (25) (26).
24
Reported values for efficiency of extraction under these conditions
range from 85 to 90% for the pesticides (24) (25) (27). It is
recommended that each analyst repeatedly check on extraction
efficiency.
-------
IV.
DETERMINATIVE STEPS
-------
IV. DETERMINATIVE STEPS
The identification and measurement of chlorinated hydro-
carbons in surface waters require extremely sensitive techniques.
In small samples the low concentrations which require identification
and measurement often provide only a few nanograms (10-9 gram) of a
pesticide. Carbon adsorption samples usually contain larger amounts
for analysis. Gas chromatography, thin layer chromatography and
infrared spectroscopy are employed as corroborative, determinative
steps.
A. GAS CHROMATOGRAPHY
Electron capture gas chromatography (ECGC) (28) is used for
identification and measurement because of its sensitivity.
Microcoulometric titration gas chromatography (MCTCG) (29),
although less sensitive, is specific for halogenated substances.
The use of both systems combines the advantages of specificity
and sensitivity. Sample gas chromatographic traces for the
ECGC and MCTGC systems are included in Appendix Two.
1. Application of Electron Capture Gas Chromatography
a. Extracts of TLC Sections of CCE Aromatics
Pesticides from each sample separated by thin
layer chromatography are contained in acetone in
four 15-mlf calibrated, Pyrex centrifuge tubes. The
volume, usually three to five ml, is reduced by evapora-
tion to 0.5 ml in a water bath at H0° C. with a jet of
clean dry air. A 5-ul aliquot is withdrawn from each
tube with a 10-ul Hamilton microsyringe and injected
into the previously conditioned and stabilized column*^.
Although these conditions are adequate for the concen-
tration range of pesticides found in most samples, in
some instances the volume of the TLC extract must be
The instrument employed in developmental work was a Perkin-Elmer
154-L equipped with a parallel plate E.C. detector, pulser, D.C.
power supply, amplifier and Leeds 6 Northrup (0-5 mv) recorder.
It was operated with a Pyrex glass, 3 ft. long x 1/8 inch 0.D,
column packed with 5% Dow 11 silicone (Dow-Corning, Midland, Mich.)
on Chromosorb W (Johns-Manville, N.Y.), with the power supply in
the pulse mode and the column temperature at 175° C. The carrier
gas used was 95% argon - 5% methane at a flow rate of 50 ml/min.
(nitrogen carrier gas is used for the D.C. mode). The amplifier
was operated at an attenuation of 64.
-------
adjusted by evaporation in a 40° C. water bath or by
dilution. It is often possible to predict the need
for adjustment of injection volume or the total
volume on the basis of the TLC result.
After the chromatogram is obtained, it is exam-
ined for peaks which possess retention times and peak
geometries which match known pesticides. See Table 1.
Since the aromatic fraction has been separated by TLC,
the number of possible pesticides in a given injec-
tion is reduced. The areas under these peaks are
calculated and compared to a standard calibration
curve which is prepared by obtaining peak areas from known
quantities of the individual pesticides under identical
conditions. The peak areas are measured as illustrated
in Figure 14.
The calibration curve is obtained by plotting peak
area in square millimeters against sample size in
nanograms. (See Appendix Two.) As seen in the dis-
cussion of thin layer chromatography, only two curves
are necessary for TLC II, three curves for TLC III,
and four curves for TLC IV. From the calibration
curve, the nanograms of each pesticide per TLC section,
W, is calculated.
(ul TLC extract)(ng determined per injection) = ng/TLC section
(ul injected)
Extracts of TLC Sections from
Solvent Extract of Bottled Samples
Essentially the same procedure as outlined in IV.A.la
is employed except for the changes noted herein. The
TLC extracts of sections II, III, and IV are reduced in
volume to 0.2 ml. A 5-ul aliquot is chromatographed
with an attenuation of less than 64.
The nanograms of each pesticide determined per TLC
section, W, is calculated.
(ul TLC extract)(ng determined/injection) = ng/TLC section = W
(ul injected)
-------
B C
AREA UNDER PEAK = a x C
WHERE
c = WIDTH AT 1/2 HEIGHT
a = HEIGHT OF PEAK
A
D
CALCULATION OF PEAK AREA
FIGURE 14. Calculation of Peak Area
-------
2. Application of Microcoulometric
Titration Gas Chromatography
a. Extracts of TLC Sections of CCE-Aromatics
In many instances the entire remainder of the
extracts of sections II, III and IV must be in-
jected in order to elicit a response within the
sensitivity of the instrument. The result of the
ECGC run is used as a guide in the choice of an
injection volume. The volume selected is injected
into the microcoulometric titration gas chromato-
graphic system25. The procedure for identifying
and measuring pesticides from the gas chromatographic
traces are identical to those described for the ECGC
system. See Table 2 and Appendix Two. The nanograms
per TLC section, W, are calculated as previously.
(ul TLC extract)(ng determined/injection) = ng/TLC section
(ul injected)
b. Extracts of TLC Sections From
Solvent Extracts of Bottled Samples
Using the result of the ECGC injection as a guide,
an aliquot of extracts II, III, and IV is selected and
injected under the same conditions as described for the
CCE-aromatic-TLC extract in IV.A.2.a. The evaluation of
the chromatographic trace in terms of identification and
measurement is carried out as described.
(ul TLC extract)(ng determined/injection) = ng/TLC section
(ul injected)
26
Developmental work was on a Perkin-Elmer 800 gas chromato-
graph equipped with an S-100 furnace, Titration cell T-200 and
coulometer C-100 (Dohrman Inst., San Carlos, Calif.), and a
Minneapolis-Honeywell Brown (0-1 mv) recorder equipped with a disc
integrator. A 3 ft. x 1/4" O.D. aluminum column, packed with 5% Dow-11
silicone grease on 60/80 mesh non acid-washed Chromosorb W, was ooer-
ated at 200° C. with a carrier gas of helium at a flow rate of 120
ml/min. The injection block temoeratur© was 215° C. The coulometer
is operated at maximum sensitivity (51.2 ohms) at damping position 4.
-------
3. Calculations
a. Bottled Samples
(V„x>
= ]ig pesticide/liter
-------
b. Carbon Adsorption Samples
27 jn the light of the unknown efficiency of adsorption on and desorption
from carbon for most organic compounds, these concentrations must be
considered minimum; the actual concentration being equal to or most
likely greater than that determined.
See footnote 27.
-------
Column Packings
The glass and aluminum gas chromatographic columns de-
scribed are used for routine analysis of chlorinated pesti-
cides. These columns can be expected to give satisfactory
results for about 500 to 700 injections or for a period of one
to one and one-half months. The types of injections made
include: pesticide standards, eluates of TLC sections of CCE
aromatic fractions from raw and finished waters, and solvent
extracts of grab samples of raw and finished water. Other
column packings have been employed to a limited extent in
our laboratory. Some additional column packings used for
pesticide analysis are shown in Table 3, Appendix Two.
Column Conditioning
To obtain optimum response and peak resolution, pas
chromatographic columns must be adequately conditioned.
Conditioning requirements vary for different column packings
and for different pesticides.
Columns packed with Chromosorb W coated with 5% Dow 11
silicone are conditioned, in our laboratory, according to
the following procedure. The column is installed in the oven,
the carrier gas is adjusted to the proper flow rate, and the
column temperature increased very slowly to 200° C. (program-
med at a rate of 1° C. per min.).
It is held at 200° C. overnight and then increased to
225° C. for two to three hours and brought back to 200° C.
The column is held at this temperature continuously for about
14 days with carrier gas passing through it. During this time
50-to 100-ug quantities of standard pesticides and normal ali-
quots of actual samples are periodically injected. For some
pesticides, such as endrin, the column may require additional
conditioning.
Columns tend to lose their response and resolution abruptly
and new columns should be pre-conditioned and ready for use when
this occurs. Insertion of quartz wool ahead of the column will
tend to lengthen the life of the column by retaining the non-
volatile waxes and oils present in many of the samples. Inser-
tion of a Pyrex glass or quartz tube in the injection block is
also helpful in extending column life.
-------
B. INFRARED SPECTROPHOTOMETRY
The aromatic fraction of each carbon adsorption sample is
prepared for infrared spectroscopy (10). A portion of the
aromatic fraction is diluted with chloroform, A suitable volume
is spread evenly on a simple salt plate. The solvent is evapo-
rated under a heat lamp. A standard 12-minute scan is made on
a Perkin-Elmer 137-B Infracord^. The resulting spectrum is
examined for specific absorption bands coincident with those
appearing on standard spectra. (See Appendix Three.) When
pesticides are present in overriding concentrations^spectra are
specific enough to permit unilateral identification. In many
cases the number and position of the absorption peaks in the
spectrum will lend support to the identifications made by pre-
viously described chromatography. (See Figures 9 and 10.)
29
Other infrared spectrophotometric equipment may also be used.
-------
V.
CONTROL OF INTERFERENCES
-------
V. CONTROL OF INTERFERENCES
When using ultrasensitive analytical techniques, particularly
electron capture gas chromatography for pesticide analyses, nossi.ble
interferences from solvents, activated carbon, and other reagents
and materials employed throughout the procedure must be Riven con-
tinuous attention. Adequate steps must be taken to eliminate or
minimize any interferences and ensure that, if present, they are
taken into consideration in the interpretations of the gas chroma-
t op,rams.
To accomplish these objectives, all solvents, carbon and
other materials are checked routinely by subjecting them to
analyses identical to those used for samples.
A. SOLVENT INTERFERENCES
1. Chloroform
All chloroform (analytical reagent fpnade) used
for extraction of the carbon adsorption samples is
distilled before use. To determine interferences, a
volume of CHCl^ equivalent to that used for extrac-
tion of the carbon is distilled a second time. The
residue obtained, representing that attributable to
CHC13 which would have been contained in the CCE
sample is given the column chromatographic separation.
See Section III.A.2a(2). One-tenth of the aromatic
fraction thus obtained is given the usual TLC cleanup,
A volume of the eluate from each section, equivalent
to the volume of sample routinely used, is injected
into both the electron capture and the microcoulometric
gas chromatographs. The significance of interferences,
if present, is noted in terms of retention time peak
geometry and peak intensity, i.e., area and height.
Interferences, if noted under these conditions, can be
considered at their maximum effect.
2. Hexane
Analytical reagent grade hexane is distilled
before use in extraction of water grab samples. A
volume of hexane equivalent to that used in the
extraction (300 ml) is evaporated in a Kuderna-Danish
evaporator to the appropriate volume. This residue
is given the TLC cleanup and the eluates from the
four sections are injected into the gas chromatograph
as discussed previously.
-------
Other solvents (ethyl ether, petroleum ether,
chloroform, and carbon tetrachloride) used for the
extraction are checked in the same manner,
3. Carbon Tetrachloride and Acetone
A quantity of carbon tetrachloride and acetone
approximately equivalent to that used for development
of the TLC plates and elution of the pesticides from
the silica gel are also checked for interferences.
B. CARBON INTERFERENCES
As discussed in Section II entitled Sample Collection,
it is possible for small amounts of organic substances to
become adsorbed upon carbon in the period between its
activation and its use in the cartridge. Together with a
precautionary program to reduce the probability of such
contamination occurring during transport or storage, an
aliquot of the unused carbon is analyzed by a procedure
identical to that used for carbon adsorption samples.
1. Carbon Blank
The presence or absence of interferences in the
carbon blank are determined according to the procedure
for determining the CCE (Section III.A.l.). A quan-
tity of carbon equivalent to that used in a carbon
adsorption cartridge is extracted with three liters
of double distilled analytical reagent grade chloro-
form. A blank determination is made whenever a new
container (bag or barrel) is opened. All cartridges
filled with carbon from a given container are so re-
corded. The residue is subjected to the standard
column chromatographic separation. One-tenth of the
aromatic fraction is given the normal TLC cleanup and
gas chromatographic analysis as previously described.
Interferences, if noted under these conditions,
would be at maximum effect.
C. OTHER SOURCES OF INTERFERENCE
The silica gel (Davison Code 950) used for the column
chromatographic separation, silica gel C used for TLC
cleanup, and the anhydrous sodium sulfate used for drying
-------
solvent extracts are examined for interferences using
the appropriate solvents for elution and development.
The eluates are subjected to gas chromatography as
previously described. When quantities of residue permit
infrared spectra are determined for solvents, carbon,
and other reagents.
INTERPRETATION
The interpretation of all gas chromatographic
analyses are made in light of any interferences deter-
mined by the foregoing procedures. If interferences
are present and are significant enough to invalidate
specific results, either qualitatively or quantitatively
those results are discarded.
-------
VI.
SENSITIVITY AND SPECIFICITY
-------
SENSITIVITY AND SPECIFICITY
A. SENSITIVITY
In discussing sensitivity, in terms of concentration, it
must be pointed out that concentrations obtained with the car-
bon adsorption method are minimum values. See Section IV.
1. Carbon Adsorption Extracts Examined by
Electron Capture Gas Chromatography
The electron capture detector varies in its sensitivity
for the various members of the chlorinated hydrocarbon
pesticide series. However, it is, in general, capable of
detecting absolute quantities of 1 nanogram (10" g) or
less on a routine basis. Remembering that (a) only 1/10
of the aromatic fraction is subjected to TLC and
-------
2. Carbon Adsorption Extract Examined by
Microcoulometric Titration Gas Chromatography
Since 25-50 ng are required to produce a minimum
recognizable response for most chlorinated hydrocarbon
pesticides and recalling that it is usually necessary to
inject all of the TLC section extract (rather than 1/100
as in the case of ECGC) the lowest detectable concentra-
tion under these procedures has been conservatively
estimated at 0,001 yg/1. Potentially, using the entire
sample and with significant additional effort, detection
of 0.0000025 pg/1 is possible.
3. Bottled Sample Extracts Examined by
Electron Capture Gas Chromatography
The lowest measurable concentration is approximately
0.001 yg/1 in a 1-liter sample.
4. Bottled Sample Extracts Examined by
Microcoulometric Titration Gas Chromatography
The lowest measurable concentration is 0.025-0.050 yg/1
in a 1-liter sample.
B. SPECIFICITY
1. Carbon Adsorption Samples
In the examination of CCE for chlorinated hydrocarbon
pesticides by the procedure outlined, it is demonstrated that
the pesticide: 1-is adsorbed on carbon, 2-is desorbed with
chloroform, 3-is ether soluble, 4-is not water soluble,
5-is not acidic, 6-is not basic, 7-is neutral, 8-is benzene
soluble, 9-moves on TLC in the same fashion as a given
standard, 10-is eluted from ECGC at the same retention time
as, and having the same peak geometry as a given standard,
11-is identical to the same standard when chromatographed
with MCTGC in terms of its retention time, peak geometry,
and degree of chlorination, and produces an infrared spectrum
which in many cases supports the identifications made by
chromatography.
2. Bottled Samples
The examination of bottled samples by the procedure
outlined provides for three corroborative chromatographic
identifications which serve as a three-way cross check on
identification.
-------
APPENDIX ONE
ENGINEERING ASPECTS OF SAMPLING
BY THE CARBON ADSORPTION
METHOD
-------
ENGINEERING ASPECTS OF SAMPLING
BY THE CARBON ADSORPTION METHOD
The carbon adsorption method of organics sampling consists of
the passage of up to 5,000 gallons of raw water at rates up to 1/2
gallon per minute through a carbon adsorption column. Following
the sample run, the column is shipped to the Water Quality Section,
Cincinnati, Ohio, for analysis.
I. TYPES OF SAMPLING EQUIPMENT IN USE
A. GENERAL
At the present time there are three tvpes of carbon adsorp-
tion sampling apparatus used in the PHS Water Pollution Sur-
veillance System. The first and oldest of these consists of
a piping arrangement that was originally assembled and installed
at the sampling location. This device is referred to as the
manual type installation and is discussed on page 58. The second
type is a prefabricated system with automatic backwash of a sand
prefilter. Two models of this type system were developed for
extensive use in water pollution surveillance. One model is a
panel unit equipped with automatic backwash device, designed for
mounting on a wall inside a building. A second model is similar
to the panel unit, but is built into a protective housing for
operation in remote or outside the plant use. The third and
newest type of sampler utilizes a low flow rate for more efficient
collection of organic substances. Two models of this type of
sampling system have been designed and recently placed In use
following successful field evaluation.
B. DESCRIPTION OF CARBON ADSORPTION COLUMN (CAC)
The CAC consists of a piece of Pyrex glass pipe 3 inches
in diameter and 18 inches long. The ends are fitted with
brass plates and 3/*+-inch galvanized nipples. A stainless
steel screen is fixed in a neoprene gasket at both ends. The
filter unit arrives at the station packed with activated carbon
ready for use. A special shipping container is provided for
returning the filter. The unit, with the shipping container, is
shown in Figure 15. A modified cartridge with a hose tvne con-
nection has been designed for use with the low flow rate sampling
equipment.
-------
-------
PRE SETTLING AND PREFILTERING
Turbid river waters frequently clog the CAC when attempting
to sample 5000 gallons. To permit this amount of water to pass
through the column a presettling tank and prefilter containing
sand and gravel were sometimes required for the manual and
automatic backwash sampling systems designed for use at 0.25 to
0.50 ppm flow rates. This equipment has not been required for
satisfactory operation of the newer low flow rate designs. A
standard hot water tank connected with the inlet at the bottom
and outlet at the top and with a clean-out tap at the bottom,
can serve as a presettling tank. The outlet is connected to
the prefilter containing sand and gravel. The tank must be
flushed at intervals to prevent accumulation of solids.
The sand prefilter consists of a steel pipe 3 feet long
and 3 inches in diameter, threaded at both ends, and equipped
with 3 by 1-inch reducer couplings. Itoo cupped stainless steel
screens are fitted to the reducer couplings. The space between
the screens is packed with gravel and sand as shown in Figure 16.
INSTALLATIONS WITH MANUAL BACKWASH
The presettling tank, the sand prefilter and the CAC are
installed at the most convenient source of raw water. If less
than 15 psi pressure is available, it may be necessary to pump
the water through the system. A drawing of a workable system
is shown in Figure 17,
A water meter located at the end of the system is used to
measure the volume of water sampled. This is usually a disc-type
meter, or oscillating piston-type meter, registering in gallons
and capable of measuring flows as law as \ gallon per minute.
If necessary, a ^-gpm flow regulator or a valve following the
meter can be used to control the flow rate.
Fine carbon dust washes out of the CAC when it is first
started. A few gallons of water are passed through the top
connection and through the CAC drain before the meter is cut
in, to keep the meter free of the carbon.
INSTALLATIONS WITH AUTOMATIC BACKWASH
Preassembled panel units with automatic backwash of the sand
prefilter were developed to ease installation and operation of
the organics sampling apparatus. Figure 18 shows the Model
^O-MIC panel unit designed for installation in water treatment
-------
16-18 MESH
STAINLESS STEEL
SCREEN
STANDARD 3 PIPE
THREADED EACH
END
16-18 MESH
STAINLESS STEEL
SCREEN,SHAPED
TO FIT REDUCER
COUPLING ^ ^
3"x I" REDUCER COUPLING
PACKED WITH GRAVEL
l" l"
8-4 GRAVEL
0.6 TO 0.8 mm SAND
1" 1"
GRAVEL
3" x I" REDUCER COUPLING
FIGURE 16. Details of Sand Prefilter
-------
HOSE CONNECTION
VALVE
VALVE
'—UNION
WATER
METER
— UNION
CARBON
ADSORPTION
COLUMN
SAND PREFILTER
PRESSURE
GAUGE
UNION
UNION
PUMP
REQUIRED)
VALVE
VALV
DRAI
FLOW
REGULATOR
(1/2 GPM)
RAW_
WATER
VALVE
PRE-SETTLING
(IF REQUIRED)
DRAIN
HOSE CONNECTION
VALVE
j- VALVE
figure 17. Schematic Diagram of an Installation With Manual Backwash
-------
smssg
FIGURE 18. Carbon Adsorption Unit Model H^O-MIC With Sand
Prefilter and Automatic Backwash
-------
plants and other buildings. This equipment has an electric
timer and solenoid valves to backwash automatically the sand
prefilter. The panel includes an electric disconnect switch
of fuse-plug-type and grounding-type duplex outlet for pump.
It also has three 3-way cocks, one to protect the water meter
from fine carbon at the beginning of sampling, one to facili-
tate checking of the flow control valve and water meter, and
one to check backwash performance.
For remote locations the sampling apparatus is installed
in an insulated equipment shelter. An organic sampling field
unit, Model H2O-M2C, containing preassembled panel apparatus,
a 30-gallon presettling tank, electric space heater, and
auxiliary equipment is shown in Figure 19. The pumping system
will vary depending on the needs of the individual sampling
station. A submersible pump was used for the field unit shown
in Figure 19, The equipment shelter has space for a jet
centrifugal-type pump, or other acceptable motor pump unit.
A prefabricated metal building may be provided, where
required, to provide a permanent shelter for equipment and
operating personnel. This type of building is usually installed
on a reinforced concrete base. An organics-sampling panel unit
(Model H2O-MIC), a pumping system, and other sampling equipment
can be installed in this type of facility.
Figure 20 shows the schematic flow diagram with sampling
procedure for the organics sampler. (Models I-^O-MIC and H20-M2C.)
F. LOW FLOW RATE ORGANICS SAMPLING
Studies of optimum sampling rate and samole volume for
maximum recovery of organics by the standard carbon adsorption method
(9) showed that sampling efficiency can be increased by the use
of smaller volumes and lower flow rates. Castelli and Booth (15)
designed a practical system to control flow of raw water through the
carbon column at low rates and measure the throughput. This system
was developed further by Reid and Stierli (16) for field evaluation.
1. Comparative Field Tests
A preliminary field evaluation of two low flow rate
samplers in comparison with conventional sampling apparatus
was conducted at the PHS Water Pollution Surveillance System
Field Test Station on the Little Miami River, Cincinnati,
Ohio, during February and March 1964. Four sampling panel
units were operated in parallel, two conventional units at
the rate of 1/2-gpm flow rate and two prototype samplers at
-------
FIGURE 19
-------
Organics Sampler
Models H20 - MIC and H20 - M2C
A) Sample Type - Organics
B) Sampling Volume - 5000 Ga 11 ons
C) Sampli ng Frequency - HonthIv
D) Information -
Department of Health, Education and Welfare
Water Quality Section
lOU Broadway
Cincinnati 2, Oh i o
Phone: 381-2200, Ext. 322
SCHEMATIC FLOW DIAGRAM — ORGANICS SAMPLING APPARATUS
NORMAL
FLOW BACKWASH
HM ttfTEN
FROM
U rTUM
1 -i.TiKK
CARBON ADSORPTION
COLUMN
to-»U>TC v
MTM.r^gH.
wvOrVrT ipwAt r
TO WASTE
LEQENO
A - SOLENOIO VALVE - CLOSED
ft - SOLENOID VALVE - OPEN
C -SOLENOK> *LVE - OPEN
0 - SOLENOIO VALVE - CLOSED
E ~ J flpm FLOW CONTROL VALVE
F - 2 gpm FLOW CONTROL VALVE
6 -PRESSURE RELIEF VALVE
M - MANUAL CONTROL VALVE
K - GRAB SAMPLE VALVE
P-PRESSURE GAUGE
>• 5-WAV COCK
—»NORMAL FLOW
BACKWASH
SAMPLING PROCEDURE
1) Install carbon filter
2) Flush carbon filter, in place through cock
"X", with raw river water to remove
carbon fines.
3) Begin sampling run and record date and initial
water meter reading on carbon filter log sheet
1) Record daily, If possible, the water meter
readings on the carbon filter log sheet until
the required volume of raw water has been
sampled. (Approximately 7 days)
6) After filtering required volume record removal
date, water meter reading and return carbon
filter and log sheet to Cincinnati, Ohio
within the provided shipping container
6) Upon receipt of used carbon filter in
Cincinnati, a new filter will be returned for
next sampling run
Note: In highly turbid waters, the filter may
tend to clog, turning filter end to end
and/or backwash Ing for 2 to 3 minutes may
be used once to obtain at least a 2000
gal Ion sample
FIGURE 20. Schematic Flow Diagram With Sampling Procedure for
Organics Sampler Models H^O-MIC and H 0-M2C
-------
the reduced rate of 100 ml/min, or less. Sample volumes
for the higher flow rate were approximately 5000 and 1000
gallons and about 250 gallons (1000 liters) for the lower
rates. Figure 21 shows the apparatus as it was installed
at the Field Test Station. The panels in the foreground
and left background operated at 1/2 gpm, while the other two
panels (one behind the center panel and the other in the
right background) operated at the reduced rates of flow.
Included in the study were tests with and without
presettling and sand prefiltering. The performance of
the low flow rate equipment was satisfactory without
presettling and prefiltering even though turbidities of
up to 1750 Jcu were measured for the raw water.
The study indicated that approximately double the
amount of total organic materials is recovered per gallon
of water passing through the "regular"30 carbon column by
reducing the throughput from 5000 to 1000 gallons. Approxi-
mately five times the amount of total organic substances
was recovered per gallon of water by decreasing the rate of
flow from 1/2 gpm to 100 ml/min and reducing the throughput
from 5000 gallons to approximately 1000 liters. The use of
"all fines"31 carbon columns further increased the recovery
of organic materials when sampling at conventional and low
flow rates.
The comparative tests with and without presettling and
prefiltering indicate significantly greater recovery of
organic materials from raw water samples receiving no pro-
cessing prior to flow through the carbon column. Six to
eight times the amount of total organic substances per
gallon of water were recovered from "all fines" carbon
columns operated with low flow rates and no preprocessing
of turbid water as compared with parallel "regular" carbon
columns receiving presettled and prefiltered water at a
flow rate of 1/2 gpm and a 5000-gallon throughput. The filter-
ing action of either "all fines" or "regular" carbon columns
can be utilized in the low flow rate samplers to obtain an
organics sample which includes much of the silt and other
particulate matter transported by a river. This is of
special importance for measurement of pesticides in water
as the transported material may carrv specific substances
of concern.
OQ
The "regular" carbon column is packed with two types of carbon
(see page 8.)
31
The "all fines" carbon column is packed only with 30 mesh Nuchar C-190
carbon. (See page 8.)
-------
FIGURE 21. Equipment Installed in Field Test Station for Tield
Evaluation of Low Flow Rate Samplers in Comoarison
With Conventional Sampling Apparatus
-------
Additional field tests of low flow rate samplers were
conducted on the Missouri River at Omaha, Nebraska, and the
Arkansas River at Little Rock, Arkansas. The low flow rate
samplers installed at these two sites are now in routine
use.
Low Flow Rate Organics Sampler, Model LF-1
Figure 22 shows a prototype Model LF-1 organics
sampler for water. The carbon column and accessory equip-
ment is assembled on a plywood panel 2'6" wide by 3' high.
Raw water enters the sampling system at the left and passes
through a 1-gpm flow control valve located behind the pressure
gauge. An adjustable pressure relief valve to the left of
the pressure gauge is used to control the pressure for
operation within 3 to 15 psi. The sample water passes
through a Teflon tube to the carbon column. After passing
up through this column, the water flows through a rubber
hose to a peristaltic action type pump for control of flow
at approximately 100 ml/min.
The water goes from the pump to the volumetric measur-
ing tank which contains probes for control of the solenoid
valve below it. When water in this tank reaches the top
probe it activates the liquid level control and solenoid
valve to drain one liter of water and close the valve.
This volume is automatically recorded in liters on the digi-
tal counter at the top of the panel. Normally, a one-week
sampling period is used to collect a sample with approximate-
ly 1000 liters throughput.
Low Flow Rate Organics Sampler, Model LF-2
A prototype Model LF-2 organics sampler is shown on
Figure 23. This sampler is similar to the Model LF-1
sampler except it includes a constant head tank between
the carbon column and peristaltic action type pump. A
float in the constant head tank controls the flow of water
from the carbon column. The flow through the constant head
tank is regulated by the pump during operation.
The volumetric tank, liquid level control, solenoid
valve, digital counter and fused electric disconnect switch
operate similarly on Models LF-1 and LF-2.
Satisfactory operation can be obtained with the
Model LF-2 sampler with pressures ranging from 5 to 50 psi.
The constant head tank enables the peristaltic action type
pump to operate with a minimal variation in flow rate.
However, flow rates and throughputs for the Model LF-2
are from 10 to 25% lower than Model LF-1 for parallel sampling.
-------
FIGURE 22. Prototype Low Flow Rate Qrganics Sampler, Model LF-1
-------
FIGURE 23. Prototype Low Flow Rate Organics Sampler, Model LF-2
-------
II. PUMPING SYSTEM
Pumps, piping, and accessories are selected to suit the
specific conditions of each station. Shallow and deep well-type
jet centrifugal pump systems are in use at many stations to bring
raw water from a representative sampling point to the sampling
apparatus. Submersible pumps with helical screw rotors and synthe-
tic rubber stators, rotary pumps with flexible impellers, and other
pumping mechanisms may be installed to meet individual needs.
It is important that the pump does not contaminate the
sample through grease-type packing or other sources. The pump
must have a greaseless-type rotary shaft seal or special packing
material to avoid contamination. Laboratory control procedures
are employed to assure that the pump does not contaminate the
sample. New pumps are sometimes grease-coated and must be thorough-
ly cleaned before being put into service. Piping strainers, check
valves, and all other accessories that come in contact with the raw
water pumped to the CAC filter are also cleaned (See Precautions,
paragraph below.)
III. PRECAUTIONS
The purpose of the CAC is to adsorb small amounts of organic
impurities from the water in as great quantity as possible. It is
important to avoid contamination of the carbon from other organic
sources. Hence the following precautions are observed:
A. New strainers, pipe fittings, and other accessories are usually
coated with oil or grease. The oil is removed by washing in
kerosene or chloroform followed by a detergent wash before
fittings are used for making connection to the CAC.
B. Ordinary organic pipe jointing compounds are not used. Red
lead (lead oxide) mixed to a paste with water can be used for
this purpose.
C. Except as noted below, plastic hose is avoided, and if rubber
hose is used in any connections it is flushed thoroughly before
being connected to the CAC. Copper tubing is ideal for
connections. NOTE: Polyethylene pipe and PVC (polyvinyl chloride)
pipe meeting National Sanitation Foundation (NSF) standards for
drinking water use are acceptable. Teflon hose also is satisfactory
for use.
IV. USE OF CARBON COLUMN DATA SHEET
Accurate flow measurements are important. Figure 24 shows a
sample data sheet used to record flow and other pertinent inform-
ation ,
-------
DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
WATER QUALITY SECTION
1014 Broadway, Cincinnati 2, Ohio
CARBON FILTER DATA
STATE
STATION LOCATION (River Mileage)
TYPE OF SAMPLE (Raw, Finijhed, Other)
COLLECTED BY: (Company or Agency)
DATE CARBON FILTER STARTED _ STOPPED
TOTAL GALLON5 FILTERED
DATE
METER READING
IN GALLONS
REMARKS
~ ATE
METER READING
IN GALLONS
REMARKS
DISTRIBUTION: White Copy to Addrai* Above; Pink Copy lor Retention by Collecting Laboratory or Agency.
PMS-JMS-7 WAJH QUMJIYMSICMUKK»T (CAUONmmt FORM APPROVED:
(9-SS) BUDGET BUREAU NO. 48-R& 34
FIGURE 24. Carbon Filter Data Sheet
-------
APPENDIX TWO
CHROMATOGRAMS, SAMPLE CALIBRATION CURVES,
INFRARED SPECTRA,
AND STRUCTURAL FORMULAE
-------
BLBCTRCN CAPTURE GAS CHRGMATOGRAM OF A STANDARD
MIXTURE
TLC SECTION II
Dieldrin
Endrin
i-i
FIGURE 25. EC Gas Chromatogram of Standard Pesticides in TLC
Section II (Dieldrin, Endrin)
-------
ELECTRON CAPTURE GAS CHROMATOGRAM OF STANDARD
MIXTURE
TLC SECTION III
1. Lindane
2. Heptachlor epoxide
' O 2 4 6 fi 10 12 14
RBTBNTICN TUB HI MJNUTBS
FIGURE 26. EC Gas Chromatogram of Standard Pesticides in TLC
Section III (Lindane, Heptachlor Epoxide, DDD)
-------
ELECTRON CAPTURE GAS CHRCMATOGRAM OF A STANDARD
MIXTURE
TLC SECTICN IV
Heptachlor
Aldrin
p, p' -DDE
—+--
RETENTION TIME IN MINUTES
FIGURE 27. EC Gas Chromatogram of Standard Pesticides in TLC
Section IV (Heptachlor, Aldrin, DDE, DDT)
-------
MICROOOULCMETRIC TITRATICW GAS CHRCMATOGRAM
OF A STANDARD MIXTURE
TLC SECTION II
Dieldrin
Bndrin
-5 —
-3-
2--
12
R^TBNTip Til
innrrss
FIGURE 28. MCT Gas Chromatogram of Standard Pesticides in TLC
Section II (Dieldrin, Endrin)
-------
MICROCOULCMETRIC TITRATION GAS CHRCMATOGRAM
OF A STANDARD MIXTURE
TLC SECTICN III
Lindane
Heptachlor epoxide
DDD
V
12
10
RETORTION ITIW IN MB UCHS
FIGURE 29. MCT Gas Chromatogram of Standard Pesticides in TLC
Section III (Lindane, Heptachlor Epoxide. DDD)
-------
-9
MICEOCOULOMETRIC TITRATION GAS CHROMATOGRAM
OF A STANDARD MIXTURE
TLC SECTION IV
o,p-DDT
p, p' -DDT
1. Heptachlor
2. Aldrin
3. p,p'-DDE
5
1
12
10
TIME
IN MI JUTES
FIGURE 30. MCT Gas Chromatogram of Standard Pesticides in TLC
Section IV (Heptachlor, Aldrin, DDE, DDT)
-------
9
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SAJCLB OP
STANDARD DIBLDRIN CURVE
-------
FIGURE 32. Sample Calibration Curve for Dieldrin (MC
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.60
.70
1.0
CO
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2000 1500
¦ i i i ¦ i i i i i
1000 900
800
700
J_L
I37-12B0
7 8 ? 10 U 12 13 14 15
WAVELENGTH (MICRONS)
SPECTRUM NO
f^AAAPI F doe
ORIGIN cietgy Acmair.-
i FnFKin
RFMARKS J
TURAL. CHEMICALS DIVISION
NEW YORK
i
i
STANDARD
PURITY 99-99%
0
PHASF MINERAL OIL MJUL
HATF 7/7/64
THICKNESS
OPERATOR uk.
'3
-------
4000 3000
00
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c
£
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01
1000 900 80u
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7 8 9 10 11
WAVELENGTH (MICRONS)
SPECTRUM NO
SAMPLE
QR1G1N1 NUTRITIONAL BIO-
CHEMICAL. CLEVELAND, OHIO
LEGEND
1,
REMARKS
DDT STANDARD
PURITY 77¦ ^ p-p'~^scxaer
?
MINERAL OIL MULL
DATF 7/7/64
THICKNFSS
OPERATOR LH-
m
:Q
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-------
ALDRIN
DIELDRIN
ENDRIN
C-C-CI
CI
HEPTACHLOR
CI
CI
CI
CI
CI
LINDANE
C-C-C
HEPTACHLOR
EPOXIDE
CI
CI
c
CI-C-CI
I
CI
DDT
CI CI
v„y
c
CI-C-CI
I
H
DDD
CI
CI
r^i
C
II
CI-C-CI
DDE
FIOURE 41. Structural Formulae of Nine Chlorinated Hydrocarbon
Pesticides
-------
Table 1
ELECTRON CAPTURE RETENTION DATA.
PESTICIDE
RETENTION DATA
itATTfi (Retention time of pesticide)
(Retention time of aldrin)
Distance
(mm)
Time
(min.)
Lindane
15
1.20
0.48
Heptachlor
24
1.92
0.77
Aldrin
31
2.48
1.00
Heptachlor epoxide
52
4.16
1.68
Dieldrin
71
5.68
2.29
DDE
72
5.76
2.32
_ « , 26
Endrin
78, 107, 160
6.24, 8.56, 12.80
2.52, 3.45, 5.16
o,p DDT
90
7.20
2.90
DDD
96
7.68
3. 10
p,p » DDT
120
9.60
3.87
See footnote 25.
The multiple peaks associated with endrin have been associated with the thermal isomerization of endrin on
-------
Table 2
MICROCOULOMETRIC TITRATION RETENTION DATA
RETENTION DATA
(Retention
time of pesticide)
PESTICIDE
Distance
(mm)
Time
(rain.)
RATIO
(Retention
time of aldrin)
Lindane
21
1.68
0. 50
Heptachlor
33
2.64
0.79
Aldrin
42
3.36
1.00
Heptachlor epoxide
59
4.72
1.41
DDE
76
6.08
1.81
Dieldrin
77
6.16
1.83
Endrin
86
6.88
2.05
DDD
104
8.32
2.48
o,p DDT
109
8.72
2.60
p,p ' DDT
135
10.80
3.21
See footnote 27.
-------
Table 3
SOME COLUMN PACKINGS USED FOR GAS CHROMATOGRAPHIC ANALYSIS
OF CHLORINATED HYDROCARBON PESTICIDES
SOLID SUPPORT LIQUID PHASE REFERENCE
Chromosorb W (60-80 mesh) 57, Dow 11 Silicone (11)
Chromosorb W (60-80 mesh) 107., S.E. 30 Silicone Gum Rubber (24)
Chromosorb W (60-80 mesh) 10% S.E. 30 Silicone Gum Rubber (24)
preceded by Na2C02
Chromosorb P (60-80 mesh) 57, Dow Corning D1C 200 Silicone
Oil (25)
Chromosorb P (30-60 mesh) 15 to 30% High Vacuum (29)
Silicone Grease (31)
Chromosorb P (30-60 mesh) 20% S.E. 30 Silicone Oil (32)
Chromosorb P (30-60 mesh) 2.5% Epon Resin 1001 (33)
Fluoropak 80 preceded by 12% Dow 11 Silicone and 67o Epon (26)
CaC^ Resin 1001
Anakrom ABS (90-100 mesh) 10% Dow Corning 200 Silicone Oil (3^)
-------
APPENDIX THREE
EQUIPMENT, SOLVENTS AND REAGENTS
-------
x. EQUIPMENT. SOLVENTS AND REAGENTS USED TO COLLECT AND PROCESS
WATER GRAB SAMPLES. (Per Sample)
1 1-liter glass bottle with Teflon liner in cap.
1 2-liter separatory funnel with Teflon stopcock.
1 1500-ml beaker.
2 300-ml Erlenmeyer flasks
1 chromatographic column (19 mm I.D.).
1 Kuderna-Danish Evaporator with ampoule graduated
in 0.01 ml.
anhydrous sodium sulfate A.R. grade
300 ml of hexane - redistilled A.R. grade
1 each 10-ul, 50-ul, and 100-ul syringes for gas chromato-
graphic injections.
II. EQUIPMENT, SOLVENTS AND REAGENTS USED TO COLLECT AND PROCESS
CCE SAMPLES^ (Per Sample)
A. EQUIPMENT
Collection equipment (see Appendix One.)
1 Drying oven.
1 1-gallon paint can with crimp lid.
1 Extraction assembly, includes:
a. 2 3-liter round bottom flasks
b. 1 large Soxhlet extractor
c. 1 Friedericks condenser
d. 1 3-liter heating mantel
(Glas-Col Series M)
e. 1 Variable transformer
(Powerstat, Type 116)
1 Manifold for filtering and warming air for
drying carbon in Soxhlet.
1 10-ml beaker.
1 50-ml beaker.
1 100-ml beaker,
2 300-ml Erlenmeyer flasks.
12 125-ml Erlenmeyer flasks.
1 125-ml vacuum flask
1 60° sintered-glass funnel
11 5-dram glass vials
1 chromatographic column (19 mm I.D. with medium
porosity fritted disc).
1 100-ml graduated cylinder,
1 each 10-ml, 50-ml and 100-ml syringes for gas chromato-
graphic injections.
-------
B. SOLVENTS (all redistilled A.R, grade)
3 liters chloroform
3 liters ethanol (95%)
100 ml of methanol
100 ml of isooctane
100 ml of benzene
300 ml of ethyl ether
C. REAGENTS
50 ml HC1 (conc.)
50 ml HC1 (5%)
50 ml NaHCCU (5%)
50 ml NaOH (5%)
NaOH (25%) or pellets
20 grams activated silica gel (Davison Code 950-08-08-226,
60-200 mesh)
D. PESTICIDE STANDARDS
III, EQUIPMENT, SOLVENTS AND REAGENTS FOR THIN LAYER CHROMATOGRAPHY
Plates (8"x8M), 200 mm x 200 mm glass
Chamber, glass developing, with lid, 8-1/2" x 4" x 8-1/2"
Spreader, variable thickness
Thickness gauge
Plate holder, plastic
Plate carrier
Spotting template
Chromatography sprayer
Desiccator to accommodate 200-mm plates
UV light box
Micropipets, 1 ul - 10 ul, 100 ul
Eye droppers
Spatulas
Graduated centrifuge test tubes, capacity 15 ml
Glass wool, pre-extracted with chloroform
Pesticide standards
All pesticides 0.1% w/v (1 mg/ml) in hexane
Chromogenic agents
5% bromine in carbon tetrachloride
Fluorescein solution
MC £ B fluorescein, water soluble, U.S.P., 25 mg. in
100 ml dimethylformamide; 10 ml of this concentrated
solution are diluted to 50 ml with ethanol 95% for
spraying.
Silver nitrate solution
1.7 g AgN0_ in 5 ml distilled water is added to 10 ml
2-phenoxyethanol (MC 6 B, technical), and diluted to
200 ml with acetone.
-------
Adsorbents:
Silica Gel G
Aluminum Oxide G
Solvents:
All solvents are redistilled before use
Hexane
Carbon Tetrachloride
Acetone
Dye Mixture:
Sudan Yellow, Sudan IV, Azobenzene, 0.1% in benzene
-------
APPENDIX FOUR
GENERAL COMPOSITION OF CARBON
CHLOROFORM AND CARBON ALCOHOL
EXTRACTS
-------
I. CHLOROFORM EXTRACTS
The organic residue recovered from the carbon adsorption
column by chloroform is very complex. It is desirable to separate
the crude extract into certain broad chemical classes, and this
can be done on the basis of solubility differences. The various
classes or groups and their general significance are discussed
briefly below.
A. ETHER INSOLUBLES
This group is usually a brown, humus-like powder, appar-
ently composed to a large extent of carboxylic acids, ketones,
and alcohols of complicated structure. Origin of the group,
which is an indicator of "old" pollution, is believed to be
partially oxidized sewage and industrial wastes. For example,
the Ohio River at Cincinnati has been exposed to much industrial
and sewage pollution, and hence large amounts of ether-insoluble
materials are found. Streams with little or no pollution his-
tory have little or no ether insolubles. Chloroform extracts
contain from 0 to 30 percent of ether-insoluble material.
B. WATER SOLUBLES
These substances are largely acidic and undistillable at
moderate temperatures, but their solubility in ether indicates
that the molecules are smaller and probably simpler than the
ether-solubles. On the other hand, their water solubility
practically requires the presence of several functional groups,
such as hydroxy-acid, keto-acid, and keto-alcohol. Such com-
pounds probably originate from partial oxidation of hydrocarbons
or they may be natural substances. They have very little odor.
These materials usually make up 10 to 20 percent of the total
extract.
C. WEAK ACIDS
This group is characterized by being removed from ether
solution with sodium hydroxide but not with sodium bicarbonate.
Phenols are the best known weak acids, and if present in the
water, appear in this group. Other weakly acidic compounds
include certain enols, imides, sulfonamides, and some sulfur
compounds. This group of materials also occurs in nature. The
weak acids are odorous, and commonly constitute 5 to 20 percent
of the chloroform extract,
D. STRONG ACIDS
These acids are usually carboxylic acids such as acetic,
benzoic, salicylic or butyric. Although classified as strong
-------
in reference to carbonic acid, they are actually weak when compared
with a mineral acid, such as sulfuric. Many of the compounds are
used industrially, but may also be produced by natural processes,
such as fermentation. Some of the materials are highly odorous. This
fraction makes up from 5 to 20 percent of the total. The significance
of the strong acids can be interpreted only in the light of
stream pollution conditions.
E. BASES
These compounds are organic amines. Such materials as
aniline and pyridine are amines of commerce. Lower amines
may occur as a result of decomposition. Although odorous,
the low concentrations found are not likely to cause objec-
tionable conditions. However, in the case of snecific amine-
containing wastes the compounds can be of considerable signi-
ficance. Generally, only 1 or 2 percent of the total extract
is made up of the bases.
F. NEUTRALS
This group frequently constitutes the major portion of
the chloroform extract. Neither basic nor acidic, the materials
are less reactive and tend to persist in streams longer than
many other types. Hydrocarbons, aldehydes, ketones, esters,
and ethers are examples of neutral materials. The group lends
itself to further fractionation by chromatographic separation
into aliphatic, aromatic, and oxygenated subgroups:
1. Aliphatics;
Thisportion represents petroleum-type hydrocarbons in
a considerable state of purity, and is usually made up of
mineral oil type of material. The percentage of aliphatics
present yields important information about the possible
source of pollution, since petroleum is the most likely
source.
2. Aromatics:
These are principally the coal tar hydrocarbons such
as benzene, toluene, and a host of others, and their pre-
sence in any significant amount is a reliable indication
of industrial pollution. Further, the materials can fre-
quently be identified by infrared spectrophotometry.
Some aromatic compounds which have been found in our
rivers—and in our drinking water—include DDT, aldrin,
dieldrin, endrin, phenyl ether, orthonitrochlorobenzene,
pyridine, phenol, and others. Some of these materials
are highly odorous; others may also be toxic. Their
appearance in any quantity as pollutants should receive
careful evaluation.
-------
3. Oxygenated Compounds (Oxys):
These are the neutral compounds containing oxygen
in aldehyde, ketone, or esters groups. They may have
originated by direct discharge or may represent oxidation
products from both natural and industrial materials. Thev
help to indicate the "age" of the pollution, since pollution
exposed to oxidation forces for a long time would be ex-
pected to contain large amounts of oxvs. The oxy materials
are odorous.
G. LOSSES
Manipulative losses inherent in this type of separation
may amount to 10 to 15 percent. Losses greater than this may
indicate that volatile components were lost from the sample.
Such volatiles may have significance as pollutants.
II. ALCOHOL EXTRACTS
The alcohol extractables generally consist of materials more polar
than the chloroform extractables. They often contain synthetic
detergents, carboxylic acids and humic materials which may
originate naturally or from oxidized products of domestic and in-
dustrial wastes. These classes of substances are not quantita-
tively recovered by the alcohol extraction. For example, this
extraction recovers only 20 to 30 percent of the synthetic detergents
present. On waters of mixed industrial and domestic pollution, the
chloroform and alcohol extractables may be about equal. On some
streams where the industrial pollution is rather low and much natural
pollution or sewage is present, the alcohol extractables may exceed
the chloroform extractables by a factor of 4 to 6.
The alcohol extract is usually only partially soluble in water
and most ordinary solvents. Very little further chemical separation
of this material is currently practical. However, tests have re-
vealed that synthetic detergents may make up 1 to 12 percent of the
alcohol extract.
-------
APPENDIX FIVE
GLOSSARY
-------
GLOSSARY
-3
mg milligram (10 gram)
• 6
ug microgram (10~ gram)
—9
ng nanogram (10 gram)
12
pg picogram (10 gram)
-3
ml milliliter (10 liter)
C
ul microliter (10 liter)
CAM carbon adsorption method
CAC carbon adsorption column
CCE carbon chloroform extract
CAE carbon alcohol extract
EI ether insolubles
WS water solubles
B bases
SA strong acids
WA weak acids
N neutrals
AL aliphatics
AR aromatics
0XY oxygenated substances
IR infrared
TLC thin layer chromatography
GC gas chromatography
ECGC electron capture gas chromatography
MCTGC microcoulometric titration gas chromatography
Rf distance travelled by a given substance
distance travelled by solvent front
-------
REFERENCES
1. H. Braus, F.M. Middleton, and G. Walton, Anal. Chem., 23,
1160 (1951). —
2. F. M. Middleton, W. Grant, and A. A. Rosen, Ind. Eng. Chem.,
48, 268 (1956)
3. F. M. Middleton and A. A. Rosen, Public Health Reports, 71,
1125 (1956).
4. F. J. Ludzack, F, M, Middleton and M. B. Ettinger, Sewage 6
Ind. Wastes, 30^, 662 ( 1958).
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Recovery and Identification of Organic Chemicals in Water.
Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio.
-------
13. F. M. Middleton, A. E. fireenberg, and fi. F. Lee, Tentative Method
for Carbon Chloroform Extract (CCE) in Water. J.A.W.W.A., 5_4,
223 (1962).
14. R. L. Booth, Reproducibility of Carbon Adsorption Method (CAM),
Memo to Sub-committee on Applicability of Carbon Adsorption
Technique to the Mission at the National Water Quality Network,
Jan. 31, 1963.
15. J. A. Castelli and R. L. Booth, Metering and Measuring Liquids
at Low Flow Rates, PHS Internal Report (in press).
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-------
25. J. I. Teasley and W. S. Cox, J.A.W.W.A., 55, 1093 (1963).
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with Electron Capture Ionization Detection for Rapid Identification
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992 (1963).
-------
Mention of products and manufacturers is
for identification only and does not imply
endorsement by the Public Health Service
and the U.S. Department of Health, Edu-
cation, and Welfare.
------- |