MICROCHEMICAL JOURNAL 58, 31–38 (1998)

ARTICLE NO. MJ971517

Liquid–Liquid Extraction for Sample Preparation prior to Gas

Chromatography and Gas Chromatography–Mass Spectrometry
Determination of Herbicide and Pesticide Compounds

Bashir M. Mahara,1 J. Borossay, and K. Torkos

Department of General and Inorganic Chemistry, Eötvös Loránd University, H-1518 Budapest 112
PO Box 32, Hungary
Received January 6, 1997; accepted May 21, 1997

Liquid–liquid extraction (LLE), compared with solid-phase extraction (SPE), has been found
to be effective for the isolation of herbicide and pesticide organic compounds from industrial
wastewater samples and also from complex matrices. LLE was followed by concentration in a
Kuderna–Danish evaporator, and the final analytical determination was carried out on a capillary
gas chromatograph coupled to a mass spectrometer. The method was optimized to obtain maxi-
mum analyte recoveries, and performance characteristics were assessed and compared with those
of SPE. To assess the accuracy of the method duplicate water samples were spiked or fortified
with known amounts of each herbicide or pesticide and analyzed with every set of industrial
wastewater samples. The recoveries were better by about 10% for LLE, whereas the detection
limits for both the LLE and SPE methods were found to be between 5 and 10 mg/liter and
between 8 and 15 mg/liter for herbicides and pesticides, respectively. q 1998 Academic Press

INTRODUCTION
In environmental analysis, hazardous compounds are usually present at low concen-
trations and are often masked by complex patterns of interfering components. There-
fore, preconcentration and separation procedures are mandatory for the determination
of most contamination (1). Many trace organic compounds, especially those present
in industrial effluents, have toxic and carcinogenic properties. The determination of
herbicides and pesticides in food and water has received much attention (2–4), and
analytical protocols are beginning to focus on health-based limits. Pesticides are intro-
duced into the environment through a variety of compounds such as insecticides,
herbicides, and fungicides. These compounds have been monitored by several authors
(5–7). Atrazine is an interesting example of herbicides, and the liquid–liquid extrac-
tion technique has been elaborated for its determination (8–10) with the use of methyl-
ene chloride or similar organic solvents (11–13).
EXPERIMENTAL
Liquid–Liquid Extraction
A 10-ml volume of saturated sodium chloride and 10 ml internal standard (3-
nitrophenol) was added to 250 ml of an industrial wastewater sample followed by the

1
To whom correspondence should be addressed.

31
0026-265X/98 $25.00
Copyright q 1998 by Academic Press
All rights of reproduction in any form reserved.

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32 MAHARA, BOROSSAY, AND TORKOS

addition of 30 ml methylene chloride solvent. The mixture was poured into a 500-
ml-volume separator funnel and then shaken several times over 10 min. The organic
layer was allowed to separate from the residual water phase for at least 15 min. In
the second and third steps of the extraction, only 10 ml of solvent was applied, and
the three fractions were collected in a 100-ml flask. The extract was dried over
anhydrous sodium sulfate and concentrated to a volume of 1 ml by using a Kuderna–
Danish evaporator (14). For chromatographic measurements the injected volume of
the concentrated extract was 1 ml.
Solid-Phase Extraction
A 250-ml volume of water sample to which was added 20 ml of internal standard,
as above, was extracted using cartridges with silica-based, monofunctionally bonded
octadecyl derivative (C18) packing. The tube volume was 3 cm3, the weight of packing
500 mg, the pore size 125 Å, particle size 55–105 mm, and surface pH (for 10%
slurry) 7.0. The cartridge were first conditioned by the addition of 5 ml methanol,
then flushed with 5 ml of double-distilled water. Water samples were sucked through
the cartridge by vacuum at 20–30 ml/min flow rate. Finally, the cartridge was washed
once more with purified water and left under water-jet vacuum for 2 min to dry.
Analytes were eluted from the cartridge manually with 2 ml methanol, and the extract
was dried over anhydrous sodium sulfate and stored in Teflon-sealed vials in the
refrigerator until analysis (15, 16).
Instrumentation and Operation
The determination was performed on a gas chromatograph (Hewlett–Packard
5790A) coupled to a mass spectrometer (VG 12-250) with a capillary column [25 m
1 0.32 mm i.d., 0.52 mm 5% phenyl methyl silicone (HP Ultra 2)]. The oven tempera-
ture was between 70 and 2807C, and the heating rate, 87C/min. The injector temperature
was 2507C, and the carrier gas was helium of 99.9999% purity flowing at 50 cm/s.
Splitless mode (valve time 1 min) was applied. The mass spectrometer was equipped
with an electric ionization source at an electron energy of 70 eV. The MS ion source
was maintained at 2007C, the GC–MS interface temperature was 2507C. The MS was
scanned using a scan rate of 0.9 s per mass range (29–500 amu). Data processing
was performed with the LAB-BASE 2.1 software system.
Wastewater samples were injected into the gas chromatograph also and the parame-
ters are listed below. A Hewlett–Packard 5730a gas chromatograph with Hewlett
Packard 3392 integrator was used. Resolution was accomplished on a wide-bore
column [30 m 1 0.53 mm i.d., 2.65 mm 5% methyl silicone (HP 1)]. The oven
temperature was between 70 and 2807C with a heating rate of 87C/min. The injector
temperature was 2507C, and the flame ionization detector was set at 3007C. Hydrogen
carrier gas (99.9995% purity) was chosen with a flow rate of 4 cm3/min and split vent
of 80 cm3/min. Nitrogen (99.99% purity) at a flow rate of 307C/min was used as
makeup gas.
RESULTS AND DISCUSSION
Identification
GC–MS measurements were made for the mixture of standard materials containing
3-nitrophenol as internal standard (ISTD). These standard materials are N,N-di-n-

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 LIQUID–LIQUID EXTRACTION 33

FIG. 1. Compounds studied: (1) N,N-di-n-propyl-S-ethylthiocarbamate (EPTC), (2) N-isopropyl-2-chloro-

acetanilide (Propachlor), (3) N-dichloroacetyl-1-oxo-4-aspiro-4,5-decane (AD-67), (4) 2-chloro-4-ethylam-
ino-6-isopropylamino-s-triazine (Aktinit), (5) N-ethoxymethyl-2-ethyl-6-methylchloroacetanilide (Acet-
ochlor).

propyl-S-ethylthio carbamate (EPTC), N-isopropyl-2-chloroacetanilide (Propachlor),

N-dichloroacetyl-1-oxo-4-azaspiro-4,5-decane (AD-67), 2-chloro-4-ethylamino-6-iso-
propylamino-s-triazine (Aktinit), and N-ethoxymethyl-2-ethyl-6-methylchloroacetani-
lide (Acetochlor). Their chemical structures are shown in Fig. 1.
In Fig. 2 is shown the chromatogram of the mixture of these compounds except
for Acetochlor and ISTD, where the peaks represent ion intensities of the individual
compounds that are not resolved according to mass/charge scale. In Fig. 3 is shown
the mass spectrum of the chromatographic peak of 837 s (Fig 2, top) in comparison
with the reference LAB-BASE spectrum of EPTC (lower). As can be seen, there is
good agreement between the peaks of the two mass spectra; i.e., the chromatographic
peak of 837 s belongs to EPTC. By similar mass spectroscopic identification it was
proved that the chromatographic peaks at 1068 s, 1208 s, and 1255 s belong to
Propachlor, AD-67, and Aktinit, respectively.

Calibration
Standard stock solutions were prepared by dissolving 0.01 g of each compound in
10 ml methanol. Dilutions were made by pipetting 0.05-, 0.1-, 0.2-, and 0.5-ml volumes

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34 MAHARA, BOROSSAY, AND TORKOS

FIG. 2. Total ion chromatogram for standard mixture (retention time in seconds).

FIG. 3. Mass spectrum of EPTC (top) in comparison with library spectrum of EPTC (bottom).

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 LIQUID–LIQUID EXTRACTION 35
TABLE 1
Detection Limits and Average Recoveries in the Concentration Range 5–100 ppm

SPE LLE

Detection Detection
limit Recovery limit Recovery
Component (mg/liter) (%) (mg/liter) (%)

EPTC 6.5 80 { 7.2 5 89 { 7.5

Propachlor 6.5 81 { 6.9 5 90 { 7.2
AD-67 13 75 { 9.2 10 85 { 6.6
Aktinit 13 75 { 9 8 85 { 6.7
Acetochlor 6.6 80 { 7.1 5.5 90 { 7.4

of stock solution into 1000 cm3 double-distilled water. These diluted standard solutions
were extracted using the sample preparation technique as described above. Calibration
curves were also set up with pure standard components, i.e., without applying sample
preparation techniques, to determine the recoveries in the range between 5 and 100
ppm. The calibration curves of all the components were linear up to 90% and the
deviation was 10% at 100 ppm.
Liquid–liquid extraction (LLE) gave good recoveries for all the components be-
tween 85 and 90%. Solid-phase extraction (SPE) gave recoveries between 75 and
80%, as shown in Table 1. The extraction efficiency was calculated by relating the area
of the analyte peak in the chromatogram of the extract with that in the chromatogram of
a standard solution prepared in the same solvent:

recovery (%) Å [peak area (standard)/peak area (sample)] (1) 100.

The detection limits and recoveries for LLE and SPE are summarized in Table 1.

Analysis of Wastewater Samples by GC and GC–MS

Wastewater samples were injected into the gas chromatograph and the resulting
chromatogram is shown in Fig. 4. It can be seen that the chromatogram of industrial
wastewater sample K14 is much more complex than the chromatogram of the standard
mixture (Fig. 2). From separate identification studies it turned out that the peak of
EPTC was interfered with by the peak of trichlorophenol (PhCl3). To achieve separa-
tion of these two compounds, wastewater samples were subjected to GC–MS with
modification of the elution parameters as follows. A split injection mode was applied
with a ratio between 1 and 20; the initial temperature was 807C for 2 min, the final
temperature was 2407C maintained for 5 min, the heating rate was 47C/min, and the
injector temperature was 2007C.
Use of the modified elution parameters demonstrated that dichloro- and trichlorophe-
nols could easily be identified from total ion chromatograms of industrial wastewater
samples by using reference library spectra. In addition, the peak of trichlorophenol

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36 MAHARA, BOROSSAY, AND TORKOS

FIG. 4. Gas chromatogram of industrial wastewater sample K14 .

could be separated from the peak of EPTC. The concentrations of seven contaminants
found in wastewater samples using LLE are listed in Table 2.

CONCLUSIONS
This paper deals with the determination of EPTC, Propachlor, AD-67, Aktinit,
Acetochlor, dichlorophenol, and trichlorophenol compounds that are widely used and
manufactured in Hungary. It is shown that LLE can be applied successfully to the

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 LIQUID–LIQUID EXTRACTION 37

FIG. 5. Total ion chromatogram of industrial wastewater sample K14 .

extraction of these pollutants from industrial wastewater with recoveries between 85

and 90%. SPE yields recoveries between 75 and 80%, which are less by about 10%
than those obtained by LLE. This lower recovery is due to partitioning of the analytes
into the head space of the flasks, to evaporative losses during sample storage, or to
passage of too large a sample through the cartridge. If the cartridge is dried for more
than 2 min after loading and if there is a significant delay between drying and elution
of the cartridge, low recoveries will result. If the cartridge is eluted under vacuum
treatment, irreproducible losses of the solvent and analytes may occur.

TABLE 2
Concentrations of Contaminants in Wastewater Samples (mg/liter)

Sample PhCl2 PhCl3 EPTC Propachlor AD-67 Aktinit Acetochlor

Wastewater sample K14 177 22 20 õ4 46 õ5 177

Wastewater sample K15 75 19 23 õ6 37 õ6 57

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38 MAHARA, BOROSSAY, AND TORKOS

We found that LLE yielded slightly better detection limits and recoveries for these
compounds than did SPE.
ACKNOWLEDGMENTS
The authors thank Professor T. Kántor for his advice and helpful discussion in preparing the manuscript.

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