Determination of Phenoxy 

Acid Herbicides in Water 

By Electron-Capture and 

Microcoulometric Gas 

Chromatography

GEOLOGICAL SURVEY WATER-SUPPLY PAPER 1817-C
Determination of Phenoxy 

Acid Herbicides in Water 

By Electron-Capture and 

Microcoulometric Gas 

Chromatography

By DONALD F. GOERLITZ and WILLIAM L. LAMAR

ORGANIC SUBSTANCES IN WATER

GEOLOGICAL SURVEY WATER-SUPPLY PAPER 1817-C

Identification, measurement, and 

stability ofphenoxy acid herbicides 

in water samples

UNITED STATES GOVERNMENT PRINTING OFFICE, WASHINGTON : 1967
UNITED STATES DEPARTMENT OF THE INTEPIOR 

STEWART L. UDALL, Secretary

GEOLOGICAL SURVEY 

William T. Pecora, Director

For sale by the Superintendent of Documents, U.S. Government Printing Office 

Washington, D.C. 20402 - Price 15 cents (paper cover)
CONTENTS

Abstract._____________________________________

Introduction. __________________________________

Apparatus.____________________________________

Reagents._____________________________________

Gas-chromatographic procedure._________________

Extraction and hydrolysis___________________

Esterification with diazomethane_____________

Esterification with boron trifluoride-methanol 

Results and discussion__________________________

Summary_ __________________________________

References cited.______________________________

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7

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9

10

18

21

ILLUSTRATIONS

FIGURE 1. Chromatogram of herbicide methyl esters on DC-200 column. 

2. Chromatogram of herbicide methyl esters on QF-1 column. _

Cll

12

TABLES

Page 

TABLE 1. A comparison of two esterification procedures..____________ C14

2. Saponification of selected herbicide esters.______-_____---__ 15

3. Recovery and degradation studies of phenoxy acid herbicides

in surface-water samples__-____---__----_--_--__-_--___ 16

262-370 67-
ORGANIC SUBSTANCES IN WATER.

DETERMINATION OF PHENOXY ACID HERBICIDES IN 

WATER BY ELECTRON-CAPTURE AND MICROCOULCMET 

RIG GAS CHROMATOGRAPHY

By DONALD F. GOEELITZ and WILLIAM L. LAMAR

ABSTRACT

A sensitive gas chromatographic method using microcoulometric titration and 

electron-capture detection for the analysis of 2,4-D, silvex, 2,4,5-T, and other 

phenoxy acid herbicides in water is described. The herbicides are extracted from 

unfiltered water samples (800-1,000 ml) by use of ethyl ether; then the herbicides 

are concentrated and esterified. To allow the analyst a choice, two esterification 

procedures using either boron trifluoride-methanol or diazomethane are 

evaluated.

Microcoulometric gas chromatography is specific for the detection of halogenated compounds such as the phenoxy acid herbicides whereas it does not 

respond to nonhalogenated components. Microcoulometric gas chromatography 

requires care and patience. It is not convenient for rapid screening of 1-liter 

samples that contain less than 1 microgram of the herbicide. Although electroncapture gas chromatography is less selective and more critically affected by interfering substances, it is, nevertheless, convenient and more sensitive then microcoulometric gas chromatography.

Two different liquid phases are used in the gas chromatographic columns 

DC-200 silicone in one column and QF-1 silicone in the other. The performance 

of both columns is improved by the addition of Carbowax 20M. The Gss Chrom 

Q support is coated with the liquid phases by the "frontal-analysis" technique.

The practical lower limits for measurement of the phenoxy acid herl 'cides in 

water primarily depend upon the sample size, interferences present, an-t instrumentation used. With 1-liter samples of water, the practical lower limits of 

measurement are 10 ppt (parts per trillion) for 2,4-D and 2 ppt for silvex and 

2,4,5-T when electron-capture detection is used, and approximately 20 ppt for 

each herbicide when analyzed by microcoulometric-titration gas chromatography.

Recoveries of the herbicides immediately after addition to unftlter^d water 

samples averaged 92 percent for 2,4-D, 90 percent for silvex, and 98 percent for 

2,4,5-T. Studies on the stability of herbicides added to water samples showed that 

2,4-D may be rapidly degraded, especially if the samples are obtained from areas 

which have been repeatedly sprayed with 2,4-D. When degradation was observed, 

added 2,4-D rapidly decomposed within 10 days. At concentrations of ebout 200 

ppt, however, the degradation rate was diminished. In 20 days the concentration 

of 2,4-D was reduced to 160-180 ppt.

Cl
C2 ORGANIC SUBSTANCES IN WATER

INTRODUCTION

Phenoxy acid herbicides are used extensively for weed control. Esters 

and salts of 2,4-dichlorophenoxyacetic acid, 2-(2,4,5-trichlorophenoxy) propionic acid, and 2,4,5-trichlorophenoxacetic acid (commonly 

called 2,4-D, silvex, and 2,4,5-T, respectively) make up most of the 

organic herbicide production in the United States (Air. Chem. Soc., 

1963). Silvex and 2,4-D in the form of water-soluble salts and watersoluble and insoluble esters are effective as aquatic herbicides. The 

general use of phenoxy acid herbicides and especially the increasing use 

of 2,4-D and silvex as aquatic herbicides in lakes, streair s, and irrigation canals makes apparent the need for rapid and convenient methods 

for the identification and measurement of these substances in water.

Early methods of analysis for 2,4-D in agricultural products were 

colorimetric procedures using chromotropic acid (Freed, 1948; Marquardt and Luce, 1951). Yip (1962) reported a gas chromatographic 

method for the analysis of 2,4-D in spinach and wheat which involved 

extracting this herbicide by use of solvents, and then several treatments 

to isolate the acid. The relatively nonvolatile acid was converted to 

the methyl ester by use of diazomethane, and the herbicide was determined by microcoulometric gas chromatography. Since Yip's (1962) 

contribution, many workers have developed gas chromatographic 

methods for the analysis of herbicides in plant and food products 

using different gas chromatigraphic columns, detectors, and other 

esterification techniques.

Aly and Faust (1963) used chromotropic acid for the extermination 

of 2,4-D in surface waters by colorimetric and ultraviolet absorption 

techniques. They stated that the major analytical interferences are the 

naturally occurring color materials in surface water and the impurity, 

2,4-dichlorophenol, in the commercial formulation. Silicic acid column 

chromatography was used to remove these interfering substances. The 

colorimetric method is sensitive to 7 /*g per 1 (micrograms per liter) 

whereas the ultraviolet procedure is sensitive to 30 /*g (micrograms).

Abbott, Hammond, and Thomson (1964) published a thin-layer and 

paper-chromatographic procedure for the determination of six organochlorine herbicides in soil and water. These compounds iroluded 2,4-D 

and 2,4,5-T. The phenoxy acid herbicides were quantitatively extracted 

from acidified water samples by ethyl ether and concentrated before 

chromatographic spotting. The sensitivity stated is about 0.5 /*g on 

the paper chromatogram.

A brief procedure was developed by Gutenmann and Lisk (1964) for 

the electron-capture gas chromatographic analysis of silvex in water. 

The method is sensitive to 0.05 mg per 1 (milligram per liter) based on
PHENOXY ACID HERBICIDES C3

a 25-g (gram) water sample, and the authors suggest using tH technique to trace the disappearance of silvex in water.

Goerlitz and Lamar (1965) published a preliminary report on the 

application of microcoulometric gas chromatography for identification 

and measurement of 2,4-D, silvex, and 2,4,5-T in water. The practical 

limit of measurement for these herbicides in 4-liter water samples was 

reported to be 10 ppt (parts per trillion).

Storrs and Burchfield (1962) reported that 2,4,-D and related compounds were not quantitatively esterified by hydrochloric acid-methanol or boron trifluoride-methanol procedures commonly used to 

esterify fatty acids. Diazomethane was avoided because of its extreme 

toxicity. The use of a suitable internal standard was proposed to bypass 

the many difficulties in residue analysis.

Bevenue, Zweig, and Nash (1962) modified Yip's (1962) procedure 

for the analysis of 2,4-D in dry crops and walnuts. They reported 

quantitative esterification of 2,4-D using diazomethane even with 

traces of water present and also that gas chromatographic column 

efficiency (recovery) was 100 ±5 percent. The method is sensitive to 

0.05 ppm (part per million) of 2,4-D in 25-50 g of crop sample.

Information on the persistence of the phenoxy acid herbicides in 

water supplies is sparse. Newman and Thomas (1950) gave information on decomposition of 2,4-D in soil and liquid media exposed to 

soil that had previously been treated with the herbicide. Aly and 

Faust (1964) studied biological decomposition of 2,4-D and several 

of its esters in lake waters and bottom muds. The 2,4-D persisted for 

120 days in aerated natural lake waters. Esters of 2,4-D were oxidized 

in 7-9 days using a domestic sewage seed, but the evidence indicated 

that only the alcohol part of the esters was involved and th<5 2,4-D 

acid remained. Aly and Faust (1964) found, by adapting th°s microorganisms in lake-bottom mud to increasing amounts of 2,4-D, that 

subsequent doses of 2,4-D were degraded within 24 hours.

The purpose of the research work described in this paper was to 

develop a convenient and sensitive method for the analysis in water of 

2,4-D, silvex, 2,4,5-T, and related compounds. Both electron-capture 

gas chromatography and microcoulometric gas chromatography were 

investigated. Because Storrs and Burchfield (1962) had indicated that 

the boron trifluoride-methanol esterification is not quantitative and 

because some analysts are reluctant to use diazomethane for esterification owing to its extreme toxicity, the evaluation of both procedures 

is pertinent. Further, the results of this research study will provide the 

analyst with substantive grounds for a choice of procedures.

The boron trifluoride-methanol esterification reported by Metcalfe 

and Schmitz (1961) and the diazomethane esterification given by 

Schlenk and Gellerman (1960) were evaluated and modified.
C4 ORGANIC SUBSTANCES IN WATER

This paper also serves to provide information on another significant 

problem, that is, the stability of the herbicides in surface-water samples. Several studies (Alexander, 1965; Aly and Faust, 1964; and Newman and Thomas, 1950) have indicated that 2,4-D is particularly susceptible to rapid degradation, whereas silvex and 2,4,5-T are resistant 

to biological attack. Thus, inf omation concerning the stability of the 

herbicides is important for the reliable analysis of 2,4-D, silvex, 2,4,5-T, 

and related compounds in water.

APPARATUS

Microcoulometric-titrating gas chromatograph. An Aerograph 

Hy-FI, model 550-B gas chromatographic oven specially modified for 

coupling the gas chromatographic column to the combustion unit. A 

quartz liner is used in the injection port, and the carrier gas is regulated 

with a differential-flow controller. The microcoulometric titrating 

system is a product of the Dohrmann Instrument Co. and consists of 

model S-100 sample inlet-combustion unit, model C-200 microcoulometer, and model T-300-S titration cell.

Electron-capture gas chromatograph. An Aerograph Hy-FI, 

model 610-D, equipped with electron-capture detector, quartz-injection port-liner insert, isothermal temperature controller model 328, and 

carrier-gas-differential flow controller.

Concentrating apparatus. A Kuderna-Danish concentrator, 250 ml 

capacity with a 1-ball Snyder column, is used for the initial concentration step. Final concentration is performed in the receiver using a 1-ball 

Snyder microcolumn. A 4.00-ml graduated receiver tube is used for the 

diazomethane esterification, and a 5.00-ml volumetric flask receiver is 

utilized for the boron trifluoride-methanol esterification.

Gas chromatographic columns. The gas chromatographic columns 

are fabricated in a coiled configuration from 5-foot lengths of borosilicate glass tubing. Four analytical columns were prepared and used 

 two each for electron capture and for microcoulometric analysis. 

The two different columns for electron-capture gas chromatography are 

3 mm (millimeters) OD (outside diameter) and 1.5 mn: ID (inside 

diameter), packed with 60-80 mesh Gas Chrom Q support, previously 

coated as follows: (1) with 5 percent by weight DC-200 (viscosity 

12,500 centistokes) and 0.5 percent by weight Carbowax 20M; (2) with 

5 percent by weight QF-1 fluorinated silicone oil (alsc designated 

FS-1265) and 0.5 percent by weight Carbowax 20M. The two different 

columns for microcoulometry are 6 mm OD and 4 mm ID, packed with 

60-80 mesh Gas Chrom Q, support previously coated as follows: (1) 

with 10 percent by weight DC-200 silicone oil and 0.5 percent by weight 

Carbowax 20M; (2) with 10 percent by weight QF-1 anc1 0.5 percent
PHENOXY ACID HERBICIDES C5

by weight Carbowax 20M. The Gas Chrom Q support is coated in each 

instance by the "frontal-analysis" technique (Smith, 1960). Chloroform, is used as the solvent for DC-200 silicone oil and Carbowax 20M 

mixture. A solution of 1:1 v/v (volume per volume) acetone-chloroform is used as solvent for the QF-1 silicone oil and Carbowax 20M.

Recorder. Honeywell Brown Electronik, class 15,0.9166 mv (millivolt) nominal span, 1-second pen speed, %-inch-per-minute chart 

speed. The recorder has a Disc chart integrator.

Sandb\ath. Tecam fluidized sandbath.

Saponification apparatus. Erlenmeyer flasks, 250 ml capacity with 

24/40 ground-glass joint to accept 1-ball Snyder column.

REAGENTS

All reagents should be checked for purity as reagent blanks by the 

gas chromatographic procedure. Much time and effort is saved by 

selecting high-quality reagents that do not require further preparation. However, some purification of reagents may be necessary as 

outlined below. If more rigorous treatment is indicated, the procurement of the reagent from an alternate source may eliminate this 

necessity.

Boron trifluoride-methanol. Esterification reagent, 14 percent 

boron trifluoride by weight.

Benzene. Suitable for pesticide-residue analysis, such as Nanograde (distilled in glass and meeting A.C.S. specifications).

%-(2-Ethoxyethoxy)ethanoL High purity, No 26°C 1.4068.

Water. Distilled water obtained from a high-purity tin-line^ still. 

The feed water is passed through an activated carbon filter. The distillate is collected in a tin-silver-lined storage tank and the water is 

constantly irradiated with ultraviolet light during storage. A gravity 

delivery system is used, and no plastic material other than Teflon 

is allowed to contact the distilled water.

Ethyl ether. Reagent grade, redistilled from an all-glass packed 

column still, after refluxing over granulated sodium-lead alloy for 

8 hours. Purity is checked by gas chromatography after a part is 

evaporated to one-tenth of the original volume.

Florisil adsorbent. A synthetic magnesium silicate 60-100 mesh, 

factory activated at 650°C. The Florisil is stored in an oven at 130°C 

and is checked from time to time for contamination. To determine 

the presence of electron-capturing components, about 1 g of the material is washed with 1 ml of benzene, and the washings are analyzed 

by gas chromatography.

Herbicides. Chlorinated phenoxy acids, analytical grade; 2,4-D 

mp (melting point) 138-139°C, silvex, mp 181-182°C, and 2,4,5-T,

2,62-370 67 2
C6 ORGANIC SUBSTANCES IN WATER

mp 154-155°C. To ensure selection of high-quality standards for this 

study, each phenoxy acid herbicide was obtained from at least two 

different sources and recrystallized from an acetone-water solution. 

The crystals were dried in a vacuum oven at 35°C for 8 hours. Melting 

points were determined. The chlorine was measured vsing microcoulometric gas chromatography, and the methyl esters for this purpose were prepared using diazomethane in 10 percent v/v methanolether solution. The recrystallized acids which exceeded 98 percent 

purity and which appeared as a single component on the chromatogram were selected as standards for this study. Methyl esters of the 

herbicides used to obtain gas-chromatographic calibration data were 

prepared from the recrystallized acids. About 5 g of methyl ester for 

each acid was prepared using boron trifluoride-methanc1 . After reaction, the esters were taken into ether solution, washed once with 

distilled water, once with 5 percent sodium carbonate, and then twice 

with distilled water. The ether solutions were dried over anhydrous 

sodium sulfate, filtered, and the ether removed on a rotary vacuum 

evaporator. The crystallized esters were finally dried r,t 30 °C for 

8 hours in a vacuum oven.

Methanol. Reagent grade, redistilled from an all-glass packed column still after reacting with 5 g of magnesium turnings per liter of 

solvent.

N-methyl-N-nitroso-p-toluenesu2fonamide. Melting rar.^e 60-62 °C.

Potassium hydroxide reagent. A 37 percent aqueous solution, prepared from reagent-grade pellets and distilled water, if treated by 

refluxing for 8 hours to reduce interfering substances. An Ascaritefilled calcium chloride tube is used at the top of the condenser to exclude carbon dioxide.

Silicic acid. Chromatographic grade, 100 mesh, stored at 130°C. 

This material is checked for contamination by washing 1 g with 1 ml 

of ether and by analyzing the washings by gas chromatoprraphy.

Sodium sulfate. Reagent grade, anhydrous, granular; heat treated 

at 300°C for 24 hours. The heat-treated material is divided, and one 

part is labeled "neutral sodium sulfate" and stored at 130°C. The other 

part is slurried with enough ether to cover the crystals and acidified to 

pH 4 by adding a few drops of purified sulfuric acid. To determine the 

pH, a small quantity of the slurry is removed, the ether evaporated, 

water is added to cover the crystals, and the pH is measured on a pH 

meter. The ether is removed by vacuum from the acidified sodium 

sulfate. This fraction is labeled "acidified sodium sulfate" and stored 

at 130°C.

Sulfuric acid. Reagent grade, purified by distilling off water until 

a constant boiling solution remains. The acid is refluxed for about 4 

hours.
PHENOXY ACID HERBICIDES C7 

GAS-CHROMATOGRAPHIC PROCEDURE

All glassware, except volumetric flasks and pipettes, is heat treated 

at 300°C overnight to remove organic contamination. Glassware and 

glass wool which come in contact with the phenoxy acid herbicides are 

rinsed with dilute hydrochloric acid prior to the heat treatment. The 

volumetric glassware is cleaned in the conventional manner with 

sodium dichromate in concentrated sulfuric acid. This cleaning is 

followed by a rinsing in dilute hydrochloric acid and steam cleaning 

for 20 minutes. The steam is generated from an all-glass apparatus 

using a solution of basic potassium permanganate in "organic-free" 

distilled water.

EXTRACTION AND HYDROLYSIS

The extraction of the phenoxy acids from unfiltered water samples 

and the subsequent hydrolysis are conducted in the following manner:

1. Acidify (pH 2.0) the water sample (approximately 800 ml) with 

concentrated sulfuric acid.

2. Pour the sample into a 1-liter separately funnel. Add 150 ml of 

ether to the sample bottle, rinse the sides, and pour the solvent 

into the separatory funnel. Shake the mixture vigorously for 1 

minute.

3. Allow the contents to separate for at least 10 minutes. Occasionally, 

emulsions may prevent adequate separation. In this event, draw 

off the separated aqueous layer, invert the separatory funnel and 

shake rapidly. Caution : Vent the funnel frequently to prevent 

excessive pressure buildup. Addition of small measured volumes 

of distilled water often aids removal of sediment from tlx °s ether 

layer. Collect the extract in a 250 ml ground-glass Erlenmeyer 

flask containing 2 ml of 37 percent aqueous potassium hydroxide.

4. Extract the sample two more times, using 50 ml of ether each 

time, and combine the extracts in the 250 ml Erlenmeye^ flask.

5. Add 15 ml of distilled water and a small boiling chip to the flask 

containing the ether extract and fit the flask with a 1-ball Snyder 

column. Remove the ether on a steam bath and continue heating 

for a total of 90 minutes.

6. Transfer the concentrate to a 60-ml separatory funnel. Extract the 

basic solution once with 20 ml and two more times each with 

10 ml of ether and discard the ether layers. The herbicides remain 

in the aqueous phase.

7. Add 2 ml of cold 25 percent sulfuric acid to the content? of the 

separatory funnel to bring the pH below 2.0 and extract the 

herbicides once with 20 ml and two times each with 10 ml. of 

ether. Collect the extracts in a 125-ml Erlenmeyer flask contain-
C8 ORGANIC SUBSTANCES IN WATER

ing about 0.5 g of acidified anhydrous sodium sulfr.te. Allow the 

extract to remain in contact with the sodium sulfate preferably in 

an explosion-proof refrigerator for at least 2 hours.

8. Transfer the ether solution into the Kuderna-Danish apparatus 

through a funnel plugged with glass wool; use liberal washings 

of ether. It is necessary to crush any hardened sodium sulfate 

to obtain a quantitative transfer.

9. Concentrate the extract to about 0.5 ml using a fluidized sandbath 

heated to 60°-70°C. Do not allow the extract to evaporate completely.

ESTEBIFICATION WITH DIAZOMETHANE

1. A 4.00-ml graduated receiver tube is used with the Kuderna-Danish 

apparatus when the sample is to be esterified with diazomethane. 

Add a quantity of anhydrous methanol equal to 10 percent of 

the volume of the concentrated extract.

2. Connect two 20 by 150-mm test tubes in series with glass tubing 

through neoprene stoppers so that incoming nitrogen gas bubbles 

through liquid in the tubes. At the outlet, position a piece of glass 

tubing with a right-angle bend and a drawn-out tip so that the 

gas can be bubbled through the sample.

3. Add about 5 ml of ether to the first test tube. To the second test 

tube add 0.7 ml of ether, 0.7 ml of 2-(2-ethoxyethoxy)ethanol, 

1.0 ml of 37-percent aqueous potassium hydroxide rolufcion, and 

0.1-0.2 g of N-methyl-N-nitroso-p-toluenesulfonamide.

4. Immediately position the second test tube and adjust the nitrogen 

flow through the apparatus to about 10 ml per minute. Caution : 

Diazomethane is a toxic and explosive gas. The ure of a good 

fume hood is absolutely necessary.

5. Place the Kuderna-Danish receiver so that the gas bubbles through 

the sample. Allow the reaction to proceed for about 10 minutes, 

or less if the yellow color of diazomethane can be observed to 

persist in the sample tube.

6. Eemove the receiver containing the sample; stopper the receiver 

and allow to stand in the hood for about 30 minute?. Carefully 

discard all waste from the reaction.

7. Add about 0.1-0.2 g of silicic acid to the sample solution to destroy 

excess diazomethane. After the evolution of nitrogen has subsided, pass the solution through a disposable pipette plugged with 

glass wool and packed with 1.5 cm (centimeters) of neutral 

anhydrous sodium sulfate over 1.5 cm of Florisil adsorbent. The 

eluate is collected in a graduated receiver tube. The transfer is 

completed by washing the receiver tube several times" with small 

quantities of ether to a final volume of 2.00 ml.
PHENOXY ACID HERBICIDES 09

8. The tube is stoppered and the contents thorougly mixed ?,nd analyzed by gas chromatography.

9. To approach the practical limit of detectability, further concentration of the esterified extract is necessary. Add a minute boiling 

chip and fit the graduated receiver tube with a small 1-ball 

Snyder column. Concentrate the extract on the sandbath. Accelerate the solvent removal by gently aspirating the vapors from 

the column just above the ball. Do not allow the receiver to go dry. 

Remove the apparatus from the heat and allow the vapors to 

condense and wash down the sides of the tube. The final volume 

of the concentrate, usually 0.05-0.20 ml, depends on the volume 

of the apparatus. The volume may be further reduced by directing a gentle stream of nitrogen on to the surface of the liquid. 

Mix the contents by pumping with a syringe and analyze the 

solution by gas chromatography.

ESTERIFICATION WITH BORON TRIFLUORIDE-METHJ NOL

1. A 5.00-ml volumetric receiver flask is used with the FudernaDanish apparatus when the sample is to be esterified with boron 

trifluoride-methanol reagent. Prior to the concentration step, 

0.5 ml of benzene is added to the extract in the Kuderna-Danish 

apparatus. The extract is concentrated to less than 1 ml, and the 

walls of the flask are washed down with a small amount of ether. 

The receiver is fitted with a small 1-ball Snyder column, and the 

extract is concentrated to 0.5 ml in the sandbath.

2. After the benzene solution in the receiver has cooled, 0.5 ml of boron 

trifluoride-methanol reagent is added. The small 1-ball Snyder 

column is used as an air-cooled condenser, and the conterts of the 

receiver are held at 50°C for 30 minutes in a sandbath. Afterwards the reaction mixture is allowed to cool to room temperature.

3. About 4.5 ml of 5-percent aqueous sodium sulfate solution is added 

to the reaction mixture so that the benzene-water interface is observed in the neck of the Kuderna-Danish receiver flask. Close 

the flask with a ground-glass stopper and shake vigorously for 

about 1 minute. Allow to stand for about 3 minutes for phase 

separation. Twirling the flask between the palms of the hands 

from time to time aids separation.

4. The benzene layer is pipetted from the receiver and passed through 

a small column prepared by plugging a disposable pipette with 

glass wool and packing with 2.0 cm of neutral anhydrou^ sodium 

sulfate over 1.5 cm of Florisil adsorbent. The eluate is collected in 

a 2.50-ml graduated centrifuge tube. The transfer is completed by 

repeating the extraction step with small quantities of benzene 

until a final volume of 2.00 ml is attained.
CIO ORGANIC SUBSTANCES IN WATER

5. Add a few crystals of neutral anhydrous sodium sulf ate to the benzene solution and thoroughly mix for gas chromatographic 

analysis.

RESULTS AND DISCUSSION

The gas chromatographic columns used in this procedure are described in the apparatus section. Initially, Chromosorb P, Chromosorb W, and Chromosorb G were tried as solid supports. These were 

found to be unsatisfactory when coated with silicone oils alone. Excessive tailing and poor resolution of the herbicide methyl esters suggested interaction of these compounds with the solid supports. The 

elution characteristics are improved when silanized Chromosorb W 

is used. Carbowax treatment (Lamar and others, 1966), originally used 

to improve the elution characteristics of insecticides, ir also effective 

for the herbicide methyl esters. The addition of 0.5 percent Carbowax 

20M to the silanized Chromosorb W during the coating operation not 

only improves the elution characteristics of the esters from the column 

but also improves the quantitative aspects of the technique. The recovery of the herbicide methyl esters as determined by microcoulometric gas chromatography closely approaches 100 percent of the 

theoretical value when Carbowax 20M is used in the column. The 

columns as described require only about 24 hours of preconditioning 

before use for either electron-capture or microcoulometric gas 

chromatography.

The Gas Chrom Q support was later chosen because it appears to 

allow a more symmetrical elution of the herbicide methyl esters. Figures 1 and 2 are reproductions of chromatograms obtained by using 

the columns described and electron-capture detection. Tl Q uncorrected 

retention times relative to 2,4-D methyl ester on the DC-200 column are 

1.00 for 2,4-D methyl ester, 1.42 for silvex methyl ester, and 1.91 for 

2,4,5-T methyl ester. These values were obtained at a column temperature of 156°C, an injection-port temperature of 190°C, a detector temperature of 196° C, and a nitrogen carrier-gas flow rate of 45 ml per 

minute. The chromatogram of the herbicides separated on the DC200 column is shown in figure 1.

The uncorrected relative retention times on the QF-1 column are 

1.00 for 2,4-D methyl ester, 1.22 for silvex methyl ester, and 1.80 for 

2,4,5-T methyl ester. These data were obtained at, a column temperature of 145°C, an injector-port temperature of 190°C, a detector 

temperature of 177°C, and a nitrogen carrier-gas flow of 45 ml per 

minute. The separation of the herbicides as shown in figure 2 on the 

QF-1 column is not complete but it is adequate. The heavier loaded 

QF-1 column used in the microcoulometric gas chromatograph separates 2,4-D and silvex methyl esters completely. The difference in rel-
PHENOXT ACID HERBICIDES Cll

7 -

6 -

UJ 

CO

z 

o

CL 5 CO ° 

UJ

ce 

cr

UJ

Q

g"

o

UJ

c:

2 -

I _

0 2468 10 12

TIME, IN MINUTES

FIGVRE 1. Chromatograin of herbicide methyl esters on DC-200 coUimn.
C12 ORGANIC SUBSTANCES IN WATER

9 -

7 -

Ul to

z 

O Q. 5

UJ

CC 

a:

UJ

§4

O

O

UJ

ce

I

 sf

0 2 4 .6 8 10 12

TIME, IN MINUTES 

FIGURE 2. Chroniatograin of herbicide methyl esters on QF-1 column.
PHENOXY ACID HERBICIDES C13

a/tive retention times aids identification when the two-column s nalysis 

is used.

Detector-response curves for both chromatographs were prepared 

from purified methyl esters of each acid. Additional standards were 

prepared from stock solutions so that the response curves could be 

reestablished to correct instrument-response fluctuations. During the 

recovery studies, standards were prepared daily, and their con centrations were adjusted so that the quantity injected was equivalent to 

that in the sample. The response was recorded in square inches as a 

function of the amount injected into the chromatograph.

Injections of 5/*l (microliters) of standards and esterified sample 

extracts were used for electron-capture gas chromatography and as 

much as 40/il injections were used for microcoulometric gas chromatography. The practice of reading the syringe before and after 

injection was observed. Septums were replaced after about 15 

penetrations.

When esterification procedures were initially investigated, poor recoveries of the herbicides resulted, especially at low concentrations. 

The reason for these low recoveries was found to be caused by the 

basicity of the glassware and of the anhydrous sodium sulfate. When 

this was corrected by acid treatment, the results were quantitative 

for both esterification procedures. Each step of the analysis was 

checked during the developmental stages. The data in table 1 are the 

results of electron-capture analysis of six prepared samples of herbicides in ether solution carried through the saponification and concentration steps of the procedure and treated in the following manner : Three of the samples were esterified with boron trifluoride-methanol; the other three were esterified with diazomethane. The average 

recovery of the three herbicides using boron trifluoride-methanol was 

98 percent and the average recovery using diazomethane was 94 

percent.

Initially, a 1:1 v/v benzene-ether solution was used to extract quantitatively the herbicide acids and esters from 4-liter water ramples 

(Goerlitz and Lamar, 1965). The purpose of the benzene was tc retard 

the ether from dissolving in the aqueous phase. It was found, however, 

that benzene was not effective for this purpose. Furthermore, many 

analysts seek to avoid the continued use of benzene because this solvent 

is a chronic systemic poison. Subsequently, 1-liter water samples were 

analyzed and ether alone was used as the extractant. It is necessary 

to use a large quantity of ether in the first extraction because 60-80 

ml dissolve in the aqueous phase. The extraction set forth in the procedure is quantitative, provided the pH of the aqueous phase is 2 

or below. The distribution coefficient for 2.4-D in an ether and water
C14 ORGANIC SUBSTANCES IN WATER

TABLE 1. A comparison of two ester ificat ion procedures

Test

!____ _

2 __ __ __

3__1_____

4_.____ _

5_

6 _-__.

Esteriflcation 

procedure

BF3 -CH3OH 

BF3 -CH3OH 

BF3 -CH3OH 

CHaNj 

CH2N2 

CH2N2

Herbicide

2,4-D _________

Silvex__ ___

9 4 x, T

2,4-D__________

2,4,5-T __ ._. _

2,4-D__________

Silvex___ _ _ _

9 4 PUT

2,4-D__________

Silvex_.____ _ _

2,4,5-T_____-_-_

2,4-D. _________

Silvex_.__ ______

9 4. ^-T

2,4-D _________

2,4,5-T_________

Amount added 

(nanograms) 1

669 

34 

64 

669 

34 

64 

669 

34 

64 

669 

34 

64 

669 

34 

64 

669 

34 

64

Recovered

Nano-jrams

678 

31 

67 

693 

32 

69 

635 

30 

63 

622 

33 

57 

657 

36 

65 

564 

31 

53

Percent

101 

91 

105 

104 

94 

108 

95 

88 

98 

93 

97 

89 

98 

106 

102 

84 

91 

83

1 A nanogram is Ith9 gram.

mixture was found to be greater than 95. This value was determined 

by electron-capture gas chromatographic analysis of a fraction of the 

ether layer after thorough mixing of equal volumes of eth^r and ethersaturated distilled water at pH 2.0. Three independent trials at three 

different concentrations of 2,4-D were made at a temperature of 

25°O±1°C.

To calculate the quantity of herbicide acid removed vrith each extraction, assume a distribution coefficient of 95 for eth«r-ether saturated water at pH 2.0. Extracting 1 liter of water with 50 ml of ether 

removes 82.6 percent of the herbicide. Three such extractions recover 

99.5 percent of the acids. In actual tests, the recovery of the three herbicides from distilled water after three independent trials averaged 96 

percent. The extracts were concentrated and esterified by the diazomethane procedure.

Six esters of the herbicides were obtained for evaluating the saponification step of the procedure. The results of this experiment are shown 

in table 2. Except for the purified methyl ester of 2,4,5-T, the other 

esters were technical-grade compounds. The recovery values from this 

experiment, averaging 97 percent, indicate that the sr.ponification 

is adequate for the analysis of herbicide esters in water.
PHENOXY ACID HERBICIDES

TABLE 2. Saponification of selected herbicide esters

C15

Ester

2,4-D propylene glycol butylether ester. _ _

2,4-D isopropyl ester____________. ________

Silvex butoxyethanol ester __ __________ ___

Silvex 2-ethylhexyl ester ___ ._ _________

2,4,5-T methyl ester_ __ _ _ _ _ _______ ___

2, 4, 5-T butyl ester ________________ .._ _

Amount 

added 

(nanograms)

659 

420 

219 

212 

190 

164

Recovere'1

Nanograms

518 

425 

201 

175 

205 

197

Percent

79 

101 

92 

83 

108 

120

As a final procedure check, recovery studies were made on actual 

samples obtained from surface water in California and Florida. The 

samples were collected in 4-liter bottles and sealed by aluminum-foilcovered rubber stoppers. Each sample was composited in the laboratory in a 5-gallon carboy having a stopcock at the bottom. The 

water-sediment composite was mixed with a Lightnin stirrer and the 

sample was divided, during the constant stirring, into 12 subsamples of 

about 800 ml each.

Purified herbicide acids were weighed into volumetric flasks 

100-ml flasks were used for 2,4-D and 500-ml flasks were used for 

silvex and 2,4,5-T. An excess of potassium hydroxide was added and 

the flasks were brought to volume with distilled water. Appropriate 

amounts of the herbicide solutions were added to six of the subsamples. 

The remaining six subsamples were retained as controls. Duplicate 

subsamples and controls were analyzed immediately, at 10 days, and 

again at 20 days after "spiking" (adding the herbicides). The data 

from these experiments obtained by electron-capture gas chromatography, using boron trifluoride-methanol esterification, are shewn in 

table 3. The samples were stored at 72°-74°F in the dark for the duration of the experiment. The pH of the water was measured before spiking and at the beginning of each operation. No significant pH change 

was noted. The recovery data immediately after spiking show tl °, reliability of the analytical method. These values range from 81 to 110 

percent. The average recovery is 92 percent for 2,4-D, 90 percent for 

silvex, and 98 percent for 2,4,5,-T.

The recoveries for the samples analyzed immediately after r.dding 

the herbicides are acceptable for the levels used, although a few are 

somewhat low. Apparently, a considerable amount of extraneous material eluted with the herbicide methyl esters, because it took at out 20 

minutes for the recorder pen to return to the baseline. Initially, the 

elevated baseline was most apparent for the sample from Eagle Bay 

and the recovery values were lower, averaging less than 85 percent.
C16 ORGANIC SUBSTANCES IN WATER

TABLE 3. Recovery and degradation studies of phenoxy acid herbicides in surfacewater samples

Designation

San Joaquin River near 

Tracy, Calif.

American River at Sacramento, Calif.

Sacramento River at Sacramento, Calif.

Cosumnes River at Michigan 

Bar, Calif.

Mokelumne River near 

Clements, Calif.

Eagle Bay at Okeechobee, 

Fla.

Herbicide

2,4-D _______

Silvex__ _--_

2,4,5-T__.____

2,4-D. _______

Silvex _ __

2,4,5-T_______

2,4-D.

Silvex._______

9 4. rrT"

*> 4-D

Silvex_______.

2,4,5-T..__.__

2,4-D. _______

*> 4- 'i-T

2,4-D. _______

Silvex_

2,4,5-T_._____

Amount 

added (nanograms)

308 

70 

92 

308 

70 

92 

308 

70 

92 

470 

126 

140 

470 

126 

140 

515 

222 

221

Percent recovered after 

aldition

Immediately

94 

87 

86 

82 

99 

110 

90 

81 

104 

91 

91 

86 

97 

86 

96 

99 

98 

104

10 days 

later

66 

84 

68 

98 

97 

90 

95 

111 

104 

92 

92 

85 

75 

76 

73 

3 

100 

80

20 days 

later

52 

84 

79 

110 

123 

109 

99 

100 

108 

82 

81 

87 

74 

85 

81 

0 

101 

80

This water was highly colored (170 units on the platinum-cobalt scale) 

by "humic acids" (Lainar and Goerlitz, 1966). Whenever the baseline 

is noticeable raised on the electron-capture chromatogram, the response of the detector is somewhat reduced. A similar situation may be 

observed when column material from a newly prepared column elutes 

through the detector.

Recoveries averaging 100 percent were obtained for the Eagle Bay 

sample after a large amount of the extraneous material was removed by 

the Florisil treatment. The baseline was only slightly elevated, about 

the same as for a benzene solution of pure methyl esters. These observations indicate that the Florisil treatment effectively reduces the 

interferences from the natural organic matter.

Table 3 also shows the results of analyses following periods of sample 

storage after spiking. Disappearance of added 2,4-D in the sample 

from Eagle Bay clearly shows that degradation in the srmple bottle 

does occur. This sample is the only one that contained 2,4-D (180 ppt) 

prior to spiking. It was obtained from an area which had been sprayed 

with 2,4-D amine about bimonthly for more than a year. Apparently, 

acclimated micro-organisms capable of decomposing 2,4-D were present in the sample. Losses of added 2,4-D were also observed in the
PHENOXY ACID HERBICIDES C17

sample from the San Joaquin Eiver, Calif. Because the San Joaquin 

Kiver passes through an area of intensive agricultural activity, it is 

conceivable that acclimated micro-organisms were present. The- other 

California rivers selected for study have less exposure to agricultural 

activity, and this fact may explain why no significant degradation was 

observed for these streams.

When degradation was observed, the added 2,4-D rapidly decomposed to about 200 ppt (parts per trillion) within 10 days. At this concentration, however, the rate of degradation was diminished. In 20 

days the concentration of 2,4-D was reduced to the 160-180 ppt level. 

The reduction of 2,4-D to this level occurred (1) when 2,4-D was present in the sample as collected, (2) when 2,4-D was added to a sample 

which contained this herbicide, and (3) when 2,4-D was added to another sample which did not contain 2,4-D. It is significant that this 

degradation of 2,4-D occurred only in samples suspected of having 

acclimated micro-organisms from areas subject to repeated spraying 

with 2,4-D.

In an earlier work (Goerlitz and Lamar, 1965), surface-water 

samples from the Loxahatchee National Wildlife Eefuge, Palm Beach 

County, Fla. were analyzed. Herbicide spraying, principally 2,4-D 

and silvex, had taken place in the area, and some of the sampler were 

collected at the time of, and in the vicinity of, spraying operations. 

Very little 2,4,5-T (90 ppt or less) was detected because it was sprayed 

on few occasions. However, silvex was found at concentrations as 

high as 51,500 ppt. Although spraying records obtained by the authors 

show that most of the spraying was done with 2,4-D, the analytical 

results indicated that the 2,4-D in the samples at the time of analysis 

was not greater than 250 ppt. A subsequent field study designed to 

determine the distribution of applied 2,4-D amine in the canal cross 

section resulted in finding levels below the anticipated concentrations. 

Even though the water samples were analyzed promptly after receipt 

in the laboratory, it is apparent that 2,4-D degradation had already 

occurred. Decomposition of 2,4-D in the water was considered, but 

the rapidity of the degradation was not anticipated.

The indications that degradation of 2,4-D had occurred in the Loxahatchee studies prompted investigation of phenoxy acid herbicide 

stability in various surface-water samples. In retrospect, it is noted 

that whenever 2,4-D was found in the water, it was at concentrations 

less than 250 ppt. The observations from previous investigations and 

the stability studies described in this report support a conclusion 

that 2,4-D may be rapidly decomposed by acclimated micro-organisms 

but that at about 200 ppt the rate of degradation is significantly 

reduced.
CIS ORGANIC SUBSTANCES IN WATER

SUMMARY

A sensitive gas chromatographic method using microcoulometric 

titration and electron-capture detection for the analysis of 2,4-D, 

silvex, 2,4,5-T, and other phenoxy acid herbicides in water is described. The herbicides are extracted from unfiltered water samples 

(800-1000 ml) by use of ethyl ether, and are then corcentrated and 

esterified. Two esterification procedures boron trifluc^ide-methanol 

and diazomethane are evaluated. Two types of analytical columns 

are used DC-200 and QF-1, which are improved by the addition 

of Carbowax 20M. Recovery studies and stability of the herbicides 

with time are presented. Degradation of 2,4-D by acclimated microorganisms is related to water from areas which have b^en repeatedly 

sprayed with this herbicide.

The analysis of a variety of water samples is difficult if different 

interfering substances are present. The microcoulometric gas chromatograph was selected because it provides specific detection of halogenated compounds. This detection system is least affected by interfering substances. Microcoulometric gas chromatography, however, 

requires care and patience by the analyst, and it is not suitable for 

rapid screening of 1-liter water samples that contain less than 1 /*g 

of the herbicide.

For convenience, electron-capture gas chromatography was used 

in most of the developmental stages of the procedure. Although the 

electron-capture detector is less selective and requires more attention 

to cleanliness of apparatus and reagents, it has several distinct advantages over the microcoulometric-titrating detector. The electroncapture detector is more sensitive by several orders of magnitude. 

It is easier to operate and consequently a greater number of analyses 

may be performed per unit time.

Water-soluble amine salts and insoluble esters of 2,4- D, silvex, and 

2,4,5-T are often used in and around water courses. It is possible 

for a given chlorinated phenoxy acid herbicide to exist in the water in 

various forms salts, esters, and ester-hydrolysis products. Because 

of this possibility, it was decided to simplify the analysis by employing 

a saponification step to isolate the important acid part of the herbicide.

Many investigators prefer to avoid using diazomethane for esterification because of its dangerous nature. Boron trifluoride-methanol 

reagent was investigated as an alternative. Both boron trifluoridemethanol and diazomethane provided quantitative results. Boron trifluoride-methanol can also be used as an interesterification agent for 

preparing methyl esters from herbicide esters of the higher alcohols, 

and may be done without going through the saponification step. However, to perform interesterification or esterification at this point the 

concentrated extract must be relatively free from extraneous halogen-
PHENOXY ACID HERBICIDES C19

ated or electron-capturing components otherwise a meticulous "cleanup" procedure to remove interfering compounds is necessary.

The saponification step is in itself a "cleanup." Ether-soluble compounds are removed by extraction of the basic aqueous solution. Water 

samples with little organic matter usually need no more "cleanup" than 

the saponification. However, removal of extraneous matter if desirable, especially for analysis of highly colored water samples. The extractable organic material from the water reduces the separating 

ability of the gas-chromatographic columns. Even though this does 

not appear to affect the quantitative aspects of the microcoulometrictitrating detector, a sharper, more distinctive chromatogram is obtained if the esterification reaction mixture is passed through a small 

amount of Florisil adsorbent. If this procedure is used in the analysis 

of water samples having moderate amounts of agricultural or similar 

pollution, a gas-chromatographic column will function normally for 

more than a month of almost continuous use. This experience shows 

that it is more convenient to prepare gas-chromatographic columns 

than to process each sample through a laborious "cleanup" procedure. 

This finding also applies to electron-capture gas chromatography 

where, in addition to column degradation, detector foulirg is a 

problem.

In regard to compounds that may cause interference with the 

herbicide analysis, boron trifluoride-methanol reagent is used to advantage for electron-capture gas chromatography whereas diazomethane reagent is the choice for microcoulometric gas chromato^raphy. 

Boron trifluoride-methanol reagent reacts specifically with carboxylic 

acids to forms the methyl esters. Diazomethane reacts not only with 

carboxylic acids but it may also react with phenols and other compounds with relatively active hydrogens. The action of diazonethane 

may increase the volatility of the extraneous substances by allowing 

them to "bleed" through the chromatograpliic column and thus interfere with the electron-capture detector. This "bleeding" does not appear to hamper the operation of the microcoulometric detector, and 

it may extend the usef ulness of the gas-chromatographic column over 

a longer period of time. Of the two esterification procedures, diazomethane is more convenient to use than boron trifluoride-methanol.

The practical lower limits for measurement of the phenoxy acid 

herbicides in water primarily depend upon the size of the sample and 

also the instrumentation used. If the extract from a 1-liter water 

sample is concentrated to 2.00 ml and a 5.0-/J part of the concentrate 

is injected into the electron-capture gas chromatograph, reliable measurement of 50 ppt of 2,4-D and 10 ppt of silvex and 2,4,5-T is feasible. 

Concentrating the extract to 0.50 ml permits detection of approximately 10 ppt of 2,4-D and 2 ppt of silvex and 2,4,5-T when 5.0 pi
C20 ORGANIC SUBSTANCES IN WATER

aliquots are analyzed. The sensitivity of the electron-capture detector 

is often adversely affected by extraneous material in the sample or 

reagents. Concentrating the extract may progressively amplify this 

complication. Owing to this problem, the practical lower limits of 

measurement for the electron-capture gas chromatographic method are 

difficult to define.

The minimum reliable value or practical lower limit of measurement is approximately the same for each herbicide when analyzed 

by microcoulometric gas chromatography. Compared to the electroncapture detector, the relative insensitivity of the microcoulometric 

detector is somewhat offset by its mode of response. Because microcoulometric detection is specific, sensing only halogenated compounds, 

larger injections from more concentrated extracts are possible, and 

the lower limits of measurement are more easily defined. The injection 

of 40 )«,! from 2.00 ml of concentrate extract permits quantitative determination down to about 1,000 ppt for each of the herbicides. Concentrating the extract to 0.05 ml and injecting 40 /al (80 percent of the 

total sample) allows a minimum reliable measurement of approximately 20 ppt.

The practical limits of quantitative analysis were calculated 

from gas-chromatographic peaks having measureable areas and were 

not obtained from a statistical treatment. Because the absolute lower 

limit of detection (2:1 signal to noise ratio) is variable, depending on 

the sample and analytical conditions, no attempts were made to estabblish the specific minimum limits of detection for th^ compounds 

studied.

The surface-water samples obtained for this investigation were 

analyzed by use of both DC-200 and QF-1 chromatographic columns. 

The herbicides, detected by electron-capture gas chromatography, were 

also analyzed by microcoulometric gas chromatography to provide an 

additional parameter of identification.

Studies on the fate of the herbicides in water samples showed that 

2,4-D may be rapidly degraded by biological action. Thir degradation 

was related to acclimated micro-organisms from areas subject to repeated spraying with 2,4-D, because samples from other areas did not 

show significant degradation in 20 days. The degradation was pronounced when the water supply had been repeatedly sprayed with 

2,4-D.

When degradation was observed, 2,4-D added to samples rapidly 

decomposed to about 200 ppt within 10 days. At this concentration, 

however, the rate of degradation was diminished. The concentration of 

2,4-D, found in the controls or found after the addition of this herbicide to the samples, was not reduced below the 160-180 ppt level in 20 

days.
PHENOXY ACID HERBICIDES C21

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