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Determination of acid herbicides in water by GC-MS: A modified method

using single extraction and methanol esterification

Article  in  American laboratory · March 2005

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Pradyot Patnaik Jacques N Khoury

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A P P L I C A T I O N
Determination of Acid Herbicides in Water by
GC-MS: A Modified Method Using Single Extraction
and Methanol Esterification
Chlorophenoxy acids are an important
class of herbicides that are widely used Table 1
for weed control in agriculture and
forestry. Most of these compounds
exhibit a low to moderate order of toxic-
ity in humans at varying degrees and a
few are also known to be teratogenic.
The two most common acid herbicides of
this class, 2,4-D and silvex, are usually
found at trace levels in many U.S.
groundwaters. Several chlorophenoxy acid
herbicides are currently under regulatory
control in drinking water by the U.S.
Environmental Protection Agency (EPA).
Analytical methods to identify and quantita-
tively measure these substances in water
involve their extractions into a suitable sol-
vent followed by chromatographic analysis.
The extraction methods mostly include
liquid–liquid extractions (LLE) or solid-
phase extractions (SPE). The compounds in
the extract are determined by GC using an
electron capture detector (ECD), HPLC,
GC-MS, or capillary electrophoresis (CE).
Derivatization of these acids, usually to their
ester derivatives, is often required to improve
the chromatographic separation of such her-
bicides and obtain better sensitivity.
Although the GC-ECD and the HPLC
methods provide high sensitivity and lower
limits of detection, the GC-MS technique is
the most authentic confirmatory tool for
identification of such compounds. Several
derivatization reagents are well-known, and
in most cases the acid herbicides are converted into their
methyl esters. Such common derivatizing reagents used
in environmental trace analysis of acid herbicides are
diazomethane1–5 and methanol-containing boron triflu-
oride.6–8 Other esterifying reagents that are known to
form methyl esters include methanol-H2SO4,9,10
dimethyl sulfate,11 methylchloroformate,12 methyl
iodide,11 and benzyltrimethylammonium chloride.13
Esterifying reagents that have been successfully applied
to produce other alkyl or aryl esters include 2-
chloroethanol-H2SO4,9 pentafluorobenzyl bromide
(PFBBr),2,14–16 benzyl bromide,11 and many tetraalkyl
ammonium (TAA) salts.17 Esterification of herbicide
acids in situ, especially with TAA salts under large-
volume on-line/on-column injection conditions, have
been reported. The method, however, has been applied
selectively to measure a few herbicides only and often
requires the presence of sodium hydroxide. It may be
noted that diazomethane, the most widely employed
derivatizing substance, is toxic, carcinogenic, and explo-
12 / MARCH 2005 • AMERICAN LABORATORY
N O T E
by Pradyot Patnaik and Jacques N. Khoury
Name and chemical structure of acid herbicides studied
sive. Many other derivatizing agents are toxic as well, and
practically all derivatization methods known to measure
acid herbicides in environmental waters are laborious,
time-consuming, and cumbersome.
In the present investigation, the authors have focused
on a simple approach to modify a known method to rou-
tinely analyze acid herbicides in environmental waters
by GC-MS. Seven acid herbicides were investigated in
the study. In this method, the aqueous samples were first
acidified and then shaken with methylene chloride in a
separatory funnel. The acid herbicides were partitioned
from water into the methylene chloride phase and
thereby were extracted into this solvent by LLE in one
single extraction. Methanol was then added to the
methylene chloride extract of acid herbicides along with
one drop of 1:1 HCl. The acids were converted into
their methyl esters during the evaporation of the solvent
extract to a small volume prior to their analysis by GC-
MS. This method avoids the use of any costly or haz-
ardous derivatizing agent and seems to be
simple and straightforward in approach and
practical application.
Experimental
All acid herbicide standards were pre-
pared in acetone. Certified standard
solutions at concentrations of 1000
µg/mL were procured from Supelco Inc.
(Bellefonte, PA). The standard solu-
tions were further diluted in acetone to
give secondary standards. The latter
were spiked into 1 L of reagent-grade
water to produce appropriate concen-
trations of herbicides for this investiga-
tion. One-liter aliquots of prepared her-
bicide samples were acidified and then
extracted with methylene chloride. The
samples were acidified with 5 mL 1:1
HCl prior to their extractions. A single
extraction was carried out in a 2-L sep-
aratory funnel with 50 mL methylene
chloride. In a few experiments, the vol-
ume of the solvent was increased to 75
mL. Methylene chloride extracts con-
taining the herbicides in their acid form
were passed through a bed of anhydrous
sodium sulfate and evaporated to
approx. one-half their initial volume on
a hotplate. After cooling down to ambi-
ent temperature, 5 mL methanol and
one drop of 1:1 HCl were added to the
extracts to convert the acid herbicides
into their methyl esters. The solutions
were then swirled for 1 min and further evaporated
to a small volume between 1 and 2 mL. The sol-
vent extracts were then passed through a thin bed
of anhydrous Na2SO4 to remove any water from
the solutions. The final volumes of the solvent
extracts were precisely measured.
A 5-µL aliquot of sample extract was injected onto a
GC column for analysis. The methyl esters were
identified from their mass spectra and retention
times. Also, other products formed from any com-
peting side reactions were identified from their mass
spectra. The compounds were quantified by analyz-
ing their standard solutions. The GC and MS condi-
tions are outlined below:
• GC column: PTE-5 (Supelco Inc.), 30 m length,
0.25 mm i.d.
• Temperature: oven, 50 °C for 6 min, 8 °C/min to
210 °C, final time, 15 min; injector, 250 °C;
detector, 280 °C
continued
AN_14_A.pg12,1.ps 03.03.05 01:23 AM Page 14

A P P L I C A T I O N N O T E

Among the seven acid herbicides studied, six

Table 2 Primary and molecular mass ions and retention times of methyl esters of herbicide acids chlorophenoxy alkanoic acids exhibited a
Ester derivatives Primary ion (m/z) Molecular ion* (m/z) Retention time (min) lower (minimum) detection level at concentra-
Dicamba methyl ester 203 234** 22.530 tions varying from 0.5 to 1.5 µg/L than the
methoxychlorobenzoic acid, dicamba, which
MCPA methyl ester 141 214 23.412 could only be detected at a much higher con-
centration of 40–50 µg/L, assuming 5 µL of
Dichloroprop methyl ester 162 248 24.124
sample extracts, respectively, were injected
2,4-D methyl ester 199 234 24.598 onto the GC. The primary and the molecular
ions of the methyl esters of acid herbicides and
Silvex methyl ester 196 282 26.280 their retention time are presented in Table 2.
2,4,5-T methyl ester 233 268 26.776 The minimum detection levels of the seven
herbicides tested under the conditions of these
2,4-DB methyl ester 101 262** 27.849 experiments are shown in Table 3.
*The most abundant molecular mass ion based on Cl-35 isotopic mass.
**Detected only in trace amounts.
Discussion
Among all the esterifying agents that were used
This study indicates that the herbicides in the sol- in this study to convert the acid herbicides in
Table 3 Lowest detection limits of acid
vent extract may also be determined in their acid the solvent extract to their ester derivatives,
herbicides* form by GC-MS without any esterification. methanol showed a distinct advantage over n-
Lowest detection Although esterification did not lower the detec- butanol and the dialkylacetals of N,N-
Herbicides limits (µg/L) tion limit of the compounds to any appreciable dimethylformamide. Under the conditions of
Dicamba 40 extent, the ester derivatives enhanced the resolu- the experiments, the yield of butyl esters from
tion of compounds on the GC column. In a few n-butanol were much smaller, especially for the
MCPA 1.2 experiments, another class of esterifying reagents larger carbon chain chlorophenoxy acids, than
that includes N,N-dimethylformamide diethylac- the yield of methyl esters obtained from
Dichloroprop 0.7 etal, N,N-dimethylformamide propylacetal, and methanol. Thus, 2,4-DB acid with n-butanol
2,4-D 1.5 N,N-dimethylformamide dibutylacetal were produced 1,1-dibutoxybutane and butanoic acid
employed to esterify the acid herbicides. Although butyl ester as the two major products, while
Silvex 0.5 such reagents were able to convert the acid herbi- giving a much smaller yield of 2,4-DB butyl
2,4,5-T 0.8 cides into their alkyl esters, separation of degraded ester, as shown in Figure 1.
amide residues from the solvent extracts required
2,4-DB acid 0.7 additional clean-up steps; therefore, any further Also, similar peaks were detected on esterifica-
*The values are approximate. Experiment conditions: investigation on potential applications of these tion of silvex with n-butanol, the major products
1-L sample, acidified; single extraction with 75 mL esterifying reagents was not pursued further. In a being 1,1-diisopropoxybutane and isopropanoic
methylene chloride; 5 mL methanol and 1 drop 1:1 few experiments, n-butanol was employed for ester- acid butyl ester. Silvex butyl ester, on the other
HCl added to extract; final volume of extract evapo- ification under the same experimental conditions. hand, formed in much smaller amounts. Such
rated 1 mL; 5-µL injection; MS scan mode. The objective was to generate butyl derivatives smaller yields of butyl esters of longer carbon
that had larger molecular mass ions than their cor- chain chlorophenoxy acids, such as 2,4-DB acid,
responding methyl derivatives, and thus lower the silvex, or dichloroprop, thus offset any advan-
• MS conditions: electron impact ionization; elec- detection level of such herbicides. However, the tage of low-level detection of larger molecular
tron energy, 70 V; scan mode, 1.57 scans/sec; mass authors have found that such esterification reac- mass ions of such butyl esters that one would
range, 35–550. tions with n-butanol did not exclu-
sively produce butyl esters since many
side reactions occurred, forming prod-
Results ucts such as 1,1-dibutoxybutane and
Extraction of seven acid herbicides in aqueous 1,1-diisopropoxybutane.
matrix by LLE followed by GC-MS determination
under scan mode was applied to develop a simple, The results of this study indicate that
rapid, and confirmatory method for their analysis. esterification of acid herbicides with
While one of these herbicides, dicamba, was a methanol was complete by the time
methoxychlorobenzoic acid, the other six herbi- the solvent extract concentrated
cides were the chlorophenoxy derivatives of acetic, down to a small volume, and there was
propanoic, and butanoic acids. The common no competing side reaction that pro-
names, chemical names, CAS registry numbers, and duced any undesired product(s) to
chemical structures of these compounds are listed interfere with chromatographic sepa-
in Table 1. ration of methyl esters. Such esterifi-
cation readily occurred in the methyl-
Acidification of samples to a pH below 2 parti- ene chloride phase in the presence of
tioned all acid herbicides into the extraction one drop of 1:1 HCl under heating
solvent, methylene chloride. One single extrac- during concentration of sample
tion from 1 L of aqueous sample taken in a 2-L extracts. It may be noted that only a
separatory funnel using a 50-mL aliquot of meth- minute quantity of HCl, just one drop,
ylene chloride sufficed for this purpose. was needed to catalyze the esterifica-
However, increasing the volume of the solvent tion of acid herbicides in the solvent
from 50 to 75 mL enhanced the efficiency of extract with methanol. While no
extraction by approx. 10%. No further attempt reaction occurred in the absence of
was made in this study to increase the quantity HCl, increasing the amount of the lat-
of solvent above 75 mL. Keeping the solvent ter tended to revert the reaction,
volume low makes the process cost-effective and reducing the yield of the herbicides as
shortens the time needed for sample concentra- methyl esters and reverting back into
tion in routine analysis. their acids. Figure 1 Reactions of 2,4-DB acid with n-butanol.

14 / MARCH 2005 • AMERICAN LABORATORY

AN_16_A.pg12,1.ps 03.04.05 12:53 PM Page 16
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A P P L I C A T I O N
expect over the methyl esters of the same herbi-
cide acids. It may be noted that the acid-
catalyzed esterification reactions of the six
chlorophenoxy acids with methanol did not pro-
duce any accompanying side reactions, unlike
the side reactions that occurred with n-butanol.
The products with methanol were exclusively
the methyl esters.
The three dialkylacetals of N,N-dimethylfor-
mamide that were probed in this investigation as
alternative derivatizing reagents for the acid herbi-
cides also formed their alkyl derivatives. The sepa-
ration of degraded amides and other extraneous
products from the sample extracts required tedious
cleanup steps and, therefore, the process cannot
serve as a viable alternative to the relatively sim-
pler and more straightforward acid-catalyzed esteri-
fication with methanol.
Repeated solvent extractions of acid herbicides
from aqueous matrices were expected to improve
extraction efficiency; the authors, however, uti-
lized only one single extraction. The compounds
were extracted from 1-L aqueous samples using
either 50- or 75-mL methylene chloride. Such
single extractions reduced the time and cost of
analysis and the degree of exposure to toxic sol-
vent vapor.
The methoxychlorobenzoic acid, dicamba, unlike
the chlorophenoxyalkanoic acids, could not be
detected in the sample chromatogram at the con-
centration levels at which the latter compounds
were identified and quantified. The minimum
detection limit of dicamba was found to be
between one and two orders of magnitude greater
than those of the chlorophenoxyalkanoic acids.
This was attributed to the fact that the rate and
extent of esterification of the benzoic acid are
severely inhibited by steric hindrance arising par-
ticularly from chlorine and the methoxy group at
the proximity of the ortho positions on the ben-
zene ring. On the other hand, in chlorophe-
noxyalkalnoic acids, no such steric repulsion
would arise toward esterification of the terminal
carboxylic acid groups that are on the aromatic
side chains and away from the chlorine atoms on
the aromatic ring.
Conclusion
This study indicated that the chlorophenoxy acid
herbicides in aqueous samples can be determined by
GC-MS following a single LLE in a separatory fun-
nel. The method involves partitioning these herbi-
cides in acidified waters into methylene chloride,
followed by esterification with methanol in the
presence of one drop of 1:1 HCl during the sample
concentration step, and then analyzing the methyl
esters in the solvent extract by GC-MS. No
cleanup of sample extracts is required in such analy-
ses. For identifying the ester derivatives, one must
take into account both the primary characteristic
mass ions and the molecular mass ions of such
esters. This method, however, is less sensitive for
the measurement of dicamba, the esterification of
which apparently is affected by steric factors.
Although the GC-ECD determination gives detec-
tion limits that are well below those obtained by
MS, the major advantage of the latter is the
authentic identification of compounds and confir-
N O T E
mation of their presence in aqueous samples in rou-
tine environmental analyses.
The single LLE-methanol esterification-MS method
presented in this paper offers certain advantages over
most other known methods in terms of simplicity,
speed, cost of analysis, and safety.
References
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Works Association and Water Environmental Federation.
Standard Methods for the Examination of Water and
Wastewater, 20th ed. Method 6640. American Public
Health Association, Washington, DC, 1998.
2. U.S. EPA. Solid Waste 846, Method 8151A, National
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Dr. Patnaik is with the Laboratory of Environmental Chemistry,
Interstate Environmental Commission, 6S-106, 2800 Victory
Blvd., Staten Island, NY 10314, U.S.A.; tel.: 718-982-3792;
fax: 718-698-8472; e-mail: prpatnaik@yahoo.com, and the
Center for Environmental Science, College of Staten Island of the
City University of New York, NY, U.S.A. Mr. Khoury is in the
Earth and Environmental Science Dept., Graduate Center of the
City University of New York. The authors wish to thank the
Interstate Environmental Commission, New York, for supporting
this investigation. They would also like to thank Dr. William
Wallace, Center for Environmental Science, City University of
New York, College of Staten Island, for providing laboratory space
for sample preparative and extraction work.