INTERFERENCE OF PHENOXYACETIC ACID

DERIVATIVES IN THE ESTIMATION OF

MONOCHLOROACETIC ACID BY THE THIOINDIGO
METHOD
G. BALUJA, J. M. FRANCO, and M. A. MURADO
Institute o f Organic" Chemistry
,h~att de la Cierva 3,
~Iadrid -6. Spai~t

The thioindigo test for the detection of monohalogenoacclic acids and/or

derivatives thereof has been adopted for spectrophotometric es|imation of residues
in wines and other substrates. However, this method has been found to lack
specificity because other compounds occurring as residual contaminants, such
as those of phenoxy acetic acid herbicides, are able Io form the thioindigo, presum-
ably through a reaction in which the phenoxy ether bond is split and lhe liberated
acetic moiety is able to coupte w~th the SH group of ~hiosalicylic acid by a
substitution mechanism.

Introduction

Adaptation of the thioindigo test (Horwitz et al. 1970) to the analysis of monohalo-
genoacetic acids in wines (Tercero 1967, and Cabezudo et al. 1971) or preserved food
basically consists of the interaction of thiosalicylic and monohalogenoacetic acids in an
alkaline medium at 100°C, followed by heating at 200°C and oxidation of the residue
with potassium ferricyanide to convert the thioindoxil intermediate into thioindigo for
the spectrophotometric measurement at 550 mtt. When this method was applied by us to
estuarine sediments and mussels, the typical pink thioindigo color was obtained. However,
it was considered unlikely that the values found could be attributable to the presence of
monochloroacetic acid residues, and it was thought that the response could be due to
other compounds. Since this method has been adopted by some laboratories, it appeared
to be worthwhile to investigate the mechanism of the reaction to further the knowledge
of its specificity.

It appears obvious that the halogen of the acetic acid is the vehicle for coupling the
acetate to the thiol group of the thiosalicylic acid, but it seemed clear that any other
compound with a - C H 2 COOtt group linked to a molecule through an ether bond suscept-
ible to being cleaved in an analogous reaction medium would also react with the SH

375

Archives of Environmenta~C~mtamhmtio~ and Toxicology

VaL 1, No. 4, ~973, © ~,c373by
Sprir~ger Verk~gNew York ~nc.
376 G. Baluja et al.

group by a bimolecular nucleophilic substitution. A large number of phenoxyacetic acid

herbicide derivatives (2, 4-D, 2, 4, 5-T and MCPA) that probably occur as contaminants
of many natural substances are compounds of this nature. The residue half life of these
herbicides in the environment is considered to be about 4-6 weeks but in dry soil the
time of disappearance can be longer. Analyses of mud samples have shown significant
residues of 2, 4-D ten months after treatment. Estimation of residues in mussels indicated
that they concentrate 2, 4-D from the surrounding water (Smith and Ison 1967).

Materials and m e t h o d s

Colorless crystals of 2, 4, 5-trichlorophenoxyacetic acid were obtained by repeated

recrystallization from alcohol/water. Pure commercial monochloroacetic acid and its
ethyl ester derivative as well as technical grade aryloxyacetic acids and derivatives were
also used, namely 2, 4, 5-trichlorophenoxyacetic acid and its isooctyl ester, 2, 4-dichlo-
rophenoxyacetic acid and its isobutyl ester, two herbicide formulations containing
different percentages of either ethyl esters of 2, 4-dichloro- and 2, 4, 5-trichlorophen-
oxyacetic acids or isopropyl esters of 2, 4-dichlorophenoxyacetic acid, related to solvents
and other components.

Esterification. One to two mg of either 2, 4, 5-trichlorophenoxyacetic or monochlo-

roacetic acids dissolved in a large excess of absolute methanol containing 10% (w/w) of
boron trifluoride were worked up according to the method of Metcalfe and Schmitz (1961).
Esterification with ethanol was carried out with sulphuric acid by the method of Rogozin-
ski (1964). Conversion of higher esters of 2, 4-dichloro- and 2, 4, 5-trichlorophenoxy-
acetic acids (isolated from formulations) into methyl esters was accomplished by trans-
esterification with boron trifluoride in methanol (Metcalfe and Schmitz 1961).

Fractionation. Aliquots of liquid herbicide formulations representing a suitable

fraction of active ester ingredients were applied to an activated Florisil column
(650°C, 2 hr) and eluted with n-hexane and a gradient mixture of 2.5, 5, and 10% (v/v)
of ethyl ether in n-hexane (Baluja 1969a, b). Separate fractions were collected for the
thioindigo test and EC-GLC.

Thioindigo reaction. The procedure was carried out according to the methods
quoted (Baluja 1969, Cabezudo et al. 1971, and Tercero 1967) and the optical density
or the percentage of transmission of the thioindigo color was measured at 550 m/~ in a
spectrophotometer (Hitachi Perkin-Elmer, Mod. 139). Aliquots of technical 2, 4, 5-T and
herbicide formulations (20 mg) containing various percentages of ethyl and isopropyl
esters of 2, 4-D and 2, 4, 5-T were washed with ice water (10 ml) and the thioindigo
reaction was performed on both the aqueous solution and the remaining insoluble residue.
A blank was also made with the reagents only. Chloroform extracts from aqueous
solution, insoluble residue and blank showed a light pink color, strong red color and a
 Interference of Phenoxyacetic Acid in Estimating Monochloroacetic Acid 377

colorless solution, respectively. Characteristic thioindigo color was also developed in an

aliquot of a dry residue of technical 2, 4, 5-T (200 rag) after washing with 200 ml of
water at room temperature, Chloroacetic acid is very soluble in water and the solubility
of 2, 4, 5-T is 19 mg per 100 ml o f water at 20°C.

Operating conditions for EC-GLC. A gas chromatograph apparatus fitted with an EC

detector, concentric type, tritium or Ni-63 source was used. Two 1.5 m glass columns
3 mm i,d. were used: A) 3,025~, Carbowax 1500 on 80/100 mesh Gas-Chrom Q; Temp.:
colunm 80°C, detector 150~C. B ) m i x t u r e of 15'2~ QF-1 and 10,c2 DC-200 on 60/80 mesh
Gas-Chrom Q; Temp.: eolulnn 200°C~ detector 205°C. Cohunns A and B were operated
with tritium and Ni-63 source detectors, respectively.

In the column A the retention time of ethyl mouochloroacetate (in n-hexane)was 1.8
rain. and the recorder response was 507; f.s. {at 5 x 10 1 l amp.) for an injection of 10 ng.
This column was used for detection and estimation of methyl and ethyl esters o f mo-
nochloroacetic acid. Esters of 2, 4-D and 2, 4, 5-T showed very high retention times.
Colunm B was inadequate for chloroacetic esters because of their short retention tilne
at the temperature used but it worked well with esters of 2~ 4-D and 2, 4, 5-T. Identifica-
tion was attained by the retention times relative to aldrin (tR 9.2 rain.}: 2, 4-D methyl
ester, 0.40: 2, 4, 5-T methyl esler, 0.63: 2, 4-D isobutyl ester, 0.76: and 2, 4, 5-T isooctyt
ester, 3.21.

Herbicide formulations assayed were previously washed with water to extract any
monochloroacetic acid and/or their inorganic salts, the alkaline aqueous solution (pH 10)
evaporated to dryness and the residue esterified with methanol in presence of boron
trifluoride or ethanol with sulphuric acid. Accordingly, one and five mg of pure monochlo-
roacetic acid were also esterified under the same operating conditions. After appropriate
injections (t-2 gl) of the ether extracts into the chromatographic colmnns A and B well
defined peaks from direct monochloroacetic acid esterification were obtained but none
from the aqueous extracts of the formulations. Furthermore, a mixture of equal volmnes
of two herbicide formulations containing ethyl and propyl esters of 2, 4-D and 2, 4, 5-T,
which also develop the thioindigo test, did not yield any peak of monochloroacetic acid
esters. This mixture was previously fractionated on an activated Florisil column and
aliquots of fractions were injected into the chromatographic column B. Peaks of ethyl
and propyl esters o f 2, 4-D and 2, 4, 5-T were easily recognized by their retention times
relative to aldrin. Confirmation was attained t h r o u # conversion to methyl esters by
transesterification with methanol. Analogous results were obtained from technical grade
isobutyl and isooctyl esters of 2, 4-D and 2, 4, 5-T.

Results and discussion

Since 2, 4, 5-T and/or its esters developed the pink color after applying the thioindigo
test it appeared unlikely to be due to the presence o f monohalogenoacetic residues.
378 G. Baluja et al.

However, it is well known that the phenoxyacetic acid derivatives 2, 4-D and 2, 4, 5-T are
generally made by reaction of monochloroacetic acid with suitable chlorophenols. There-
fore technical products may contain some impurities of chloroacetic acid which will give
a positive thioindigo test, although as the synthesis is carried out with an excess of
chlorophenol the presence of chloroacetic residues is improbable. Nevertheless to further
this knowledge the thioindigo reaction was performed on technical 2, 4, 5-T and herbicide
formulations containing ethyl and isopropyl esters of 2, 4-D and 2, 4, 5-T as described in
the experimental section.

These reactions appear to confirm the absence of monochloroacetic acid and/or

derivatives as contaminants of either technical 2, 4, 5-T or the formulations assayed.
Furthermore, the thioindigo test for monochloroacetic acid is highly sensitive. Then
thioindigo formation must be likely developed from a -CH2COOH group originated
through cleavage of 2, 4, 5-T and/or ester derivatives of 2, 4-D and 2, 4, 5-T of the
formulations under the energetic conditions of the reaction. It was also found that the
thloindigo color increased when the alkaline media or the time of heating were increased.
Cleavage of the phenoxy ether bond to originate the - CH2COOH moiety may be a
common reaction, as phenyl esters cleave when heated with pryidine hydrochloride
(Prey 1941).

Kinetics of the development of the thioindigo coloring could be followed on increasing

the concentration of 2, 4, 5-T in the reaction media. Optical density or the transmittance
at 550 m# of the chloroform extracts plotted v e r s u s mg of 2, 4, 5-T added are shown in
Figure 1, where the absorbance yields a curve of exponential type.

Phenol liberated by the cleavage of 2, 4, 5-T does not develop the colored reaction
under the same operating conditions used for the thioindigo formation as would be
expected, since the method of Marquardt and Luce (1961) uses the phenol moiety from
2, 4-D for spectrophotometric estimation. On the contrary, when thiosalicylic acid is not
present in the reaction media a colorless solution is produced. However, the 2, 4, 5-
trichlorophenol liberated is isolated with chloroform from the reaction medium, the
chloroform removed and the phenol detected with ferric chloride in an alcoholic solution
of the residue.

Therefore it appears to be plausible that the cleavage of the phenoxy ether bond is
enhanced by the electrophylic behavior of the chlorinated phenol moiety. Then the
SN2 process for the thioindigo formation may take place according to the following
scheme:
0
C~OCH2COOR -- COOH cI OH ii
OH
J" -'F r ~ / CH-COOR.--...~Thioindigo
%Y_/L.s/
 Interference of Phenox-y-acetic Acid in F.stimating Monochloroacetic Acid 370

Tr O.D.
(%)

"N i
I
\ /
\ !

50 \ / 1.o
\, /
\/

10 \ .,. o.1

1 5 m9

Fig. 1. Plot of absorbance I - - - ) and percent of transmittance 1 - - - - - ) 1550 mg) of

thioindigo ps. mg of 2, 4, 5-T.

Absence of monochloroacetic acid and its methyl or ethyl esters in the commercial
2, 4, 5-T and herbicide t\)rmulations assayed was also confirmed by EC-GLC. Then it
would appear that monohalogenoacetic acid and/or ester derivatives are absent from the
technical grade products 2, 4-D and 2, 4, 5-T and their herbicide formulations. Therefore
these phenoxyacetic derivatives can also develop tile thioindigo reaction under the same
operating conditions established for monohalogenoacetic acid. Since these compounds are
used very frequently as herbicides in agriculture they can be found as residues in soil,
water, vegetables, fruits, and animal tissues, and consequently the thioindigo test can not
be applied as a specific method for monohalogenoacetic detection on complex substrates.

References

Baluja, G.: Extraction and purification of residues of pesticides and their metabolites for
simultaneous determination by gas chromatography. I. Nonionic pesticides. Rev.
Agroquim. Technol. Aliment. 9, 377 (1969a).
--.:Extraction and purification methods for pesticide residues and metabolites for
multiple gas chromatographic determinations. B. Ionizable pesticides. C. Inter-
feting substances. D. Solvent purification. Rev. Agroquim. Technol. Aliment. 9,
536 (1969b).
380 G. Baluja et al.

Cabezudo, M. D., E. F. Gorostiza, and J. M. Garrido: Determination of halogen derivatives

of acetic acid. Rev. Agroquim. Technol. Aliment. 11,145 (1971).
Horwitz, W., P. Chichilo, and H. Reynolds: Offic. Methods of Analysis Assoc. Offic.
Anal. Chemists, 1 lth ed., p. 399. Benjamin Franklin Station, Washington, D.C.
(1970).
Marquardt, R. P. and E. N. Luce: A new basic procedure for determining phenoxy acid
herbicides in agricultural products. J. Agr. Food Chem. 9,266 (1961).
Metcalfe, L. D. and A. A. Schmitz: The rapid preparation of fatty acid esters for gas
chromatographic analysis. Anal. Chem. 33,363 (196t).
Prey, V.: Cleavage of phenol ethers with pyridine hydrochloride. Ber. 74 B, 1219 (1941).
Rogozinski, M.: A rapid quantitative esterification technique for carboxylic acids. J. Gas
Chromatog. 2, 136 (1964).
Smith, G. E. and B. G. Isom: Investigation of effects of large-scale applications of 2, 4-D
on aquatic fauna and water quality. Pesticide Monitor. J. 1, 16 (1967).
Tercero, C.: Investigation and estimation of monochloro- and monobromoacetic acids.
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national Office of the Vineyard and the Wine. F. V. No. 224. Bull. O. I. V. 40, 963
(1967).

Manuscript received Nov. 11, 1972; accepted Aug. 15, 1973