Full text of DOI:10.1093/chromsci/38.12.551

Journal of Chromatographic Science, Vol. 38, December 2000
Clean-Up of a Pesticide–Lanolin Mixture by
Gel Permeation Chromatography
1
,
1
2
2
M. López–Mesas *, M. Crespi , J. Brach , and J.P. Mullender
1
Laboratorio de Control de la Contaminación Ambiental, Instituto de Investigación Textil y Cooperación Industrial (INTEXTER), Colón 15,
8222 Terrassa, Spain and Centre de Recherche et de Service CELABOR, Avenue du Parc 38, 4650 Herve, Belgium
2
0
grease is known as lanolin, which is widely used as a moisturizer
in cosmetics (2) and media for some pharmaceutical preparations
3,4) because of its high compatibility with human skin oils.
Unfortunately, pesticides have been found in samples of lanolin,
as revealed by other studies (2,5).
Abstract
(
In this study, the efficiency of a clean-up method by gel permeation
chromatography (GPC) for the separation of pesticides from lanolin
is analyzed. The pesticides analyzed belong to two different families,
organophosphorous and synthetic pyrethroids. Lanolin, a standard
mixture of the pesticides, and a lanolin–pesticides mixture are
injected in a GPC column. The recoveries and elution times from
the GPC column of lanolin (by a gravimetric method) and pesticides
by gas chromatography–electron capture detector) are determined.
From this column, a good separation of the lanolin–pesticides
mixture is observed.
Pesticides that are allowed to be used for sheep are synthetic
pyrethroid and organophosphorous (6). The presence of some
organochlorine pesticides can be because of the ingestion of pas-
tures treated with them, contaminated soil, and illegal use.
When a sample with a high fatty matter content is analyzed, a
three-step procedure is carried out: an extraction stage allowing
the separation of analytes from the sample bulk; a clean-up stage
eliminating the interfering components; and an instrumental
analysis for the separation, identification, and quantitation of ana-
lytes (7). The first two stages are considered to be the most critical
of the analysis (8), because the achievement of the appropriate
fractions needed in the further analysis depends on them.
Lipids from animals or vegetables consist of a primary mixture
complex of long chains of acid and ester alcohols in which pesti-
cides remain strongly retained because of their lipophilic char-
acter. Characteristics of these lipids include polar groups (H
bonds), high content in hydrocarbon, high molecular weight
(
Introduction
The antiparasitic plague control for pasture and wool storage
involves the use of pesticides remaining on wool fibers that
together with the grease secreted by the sebaceous glands of sheep
(
andotherimpurities)mustbeeliminated. Otherwise, theycausean
allergic reaction when wool is used by people. Therefore, it is nec-
essary to ensure a thorough washing of wool, which means the use
of surfactants and abundant water. This process generates a highly
contaminant liquid with biochemical oxygen demand values
between 20,000 and 40,000 mg/L and chemical oxygen demand up
to100,000mg/L(1). Thishigh-contaminantorganiccharge(20–60
times higher than effluents from the dyeing and finishing industry)
causes serious problems in the depuration of these effluents, thus
pesticide determination becomes necessary for their reuse.
(
between 600 and 1500), and low volatility—in other words, char-
acteristics that can be used for their separation from pesticides.
Experimental
Reagents and material
Lanolin solutions
Pesticide-free lanolin from Westbrook Lanolin (Verviers,
Belgium) was weighed directly and dissolved in dichloromethane
to form a stock solution of 25% (w/v).
Some wool scouring industries remove the grease from the
liquid phase, because the product resulting from purifying the
*
Author to whom correspondence should be addressed: e-mail mlopez@intexter.upc.es.
Table I. Chromatographic Method Parameters
Parameters
Mode
Initial temperature (°C)
Pressure (psi)
Flow rate (mL/min)
Average velocity (cm/s)
Injector
Column
Detector
pulsed splitless
constant flow
constant flow in column and make-up
250
100
340
initial 6.25, pulsed 30
nominal initial 6.25
–
purge 25 (0.8 min)
initial 0.9
purge anode 6.0, make-up 40.0
–
20
–
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551

Journal of Chromatographic Science, Vol. 38, December 2000
In order to prepare the spiked lanolin, pure lanolin was weighed
and spiked with a concentrated mixture of the nine pesticides (cal-
culated to have 5 ppm of each pesticide). Dichloromethane was
added and placed into a rotatory system on a waterbath at 40°C.
When the mixture was homogeneous, an amount of dichloro-
methane was added until a 2% final solution (w/v) was obtained.
mesh (Bio-Rad, Eke, Belgium, ref. 152-2750). All connections
1
were made in a Teflon pipe with a length of / inch (0.8-mm i.d.)
16
1
and / inch (1.5-mm i.d.).
8
Procedure
All injections were always made at least by duplicate.
Clean-up method
Before initiating the clean-up study, the filling state of the gas
Pesticides
Nine of the pesticides that are used for sheep or wool treatment
were selected for this study—five of them belonging to the syn-
thetic pyrethroids (cyhalothrin, cypermethrin, deltamethrin, fen-
valerate, and tetramethrin) and four organophosphorous
Table II. Statistical Analysis of the Capacity Factor for the
Nine Pesticides
(
carbophenothion, chlorpyriphos-methyl, diazinon, and pro-
Relative
standard
deviation
95%
Confidence
intervals
petamphos). A stock solution of 1000 ppm of each pesticide was
prepared in ethyl acetate or cyclohexane. A mixture of 50 ppm of
each pesticide was prepared from the stock solutions and used as
a stock solution for the calibration standards. Purities were
higher than 99%.
All solvents used were of Pestiscan-grade from Lab-Scan
Analytical Sciences (Madrid, Spain).
Standard
deviation
Pesticide
k' average
Propetamphos
Diazinon
Chlorpyrifos-met
Carbophenothion
Tetramethrin
Cyhalothrin
Cypermethrin
Fenvalerate
Deltamethrin
1.87281
1.92759
2.37789
5.0705
0.00043
0.00048
0.00043
0.00085
0.00142
0.00106
0.00101
0.00097
0.00113
0.02278
0.02464
0.0179
0.01677
0.02342
0.01546
0.01185
0.01055
0.01147
0.00031
0.00034
0.0003
0.00061
0.00101
0.00076
0.00072
0.00069
0.00081
6.0507
6.88809
8.49533
9.20656
9.89721
Gas chromatograph
A Hewlett-Packard (Geneva, Switzerland) HP6890 gas chro-
matograph (GC) fitted with an automatic injector (Series
Injector) was equipped with an HP-5 column (30 m × 320 µm ×
0
.25 µm) using helium as a carrier gas. Detection was made by a
Model HP6890 ECD. Data were collected and statistically treated
by Chemstation HP Software.
Table III. Statistical Analysis of the Selectivity for the
Nine Pesticides
Gel permeation chromatography
Relative
standard
deviation
95%
Confidence
intervals
Equipment fitted with a high-performance liquid chromato-
graph Varian (Middelburg, The Netherlands) Vista 5500 liquid
chromatograph pump, an ultraviolet (UV)–visible (vis) Varian 634
detector, and Varian Star chromatography software integrator
was used. The injector used was a Model 7000 stream switching
valve with a 2-mL sample loop. Two chromatographic columns
were used (Figure 1) that were made of transparent glass (450- ×
Standard
deviation
Pesticide
S average
Propetamphos
Diazinon
Chlorpyrifos-met
Carbophenothion
Tetramethrin
Cyhalothrin
Cypermethrin
Fenvalerate
Deltamethrin
1.06084
1.02925
1.05919
1.01385
1.02371
1.02372
1.00484
1.04422
1.01817
0.05247
0.00011
0.00014
0.00009
0.00006
0.00003
0.00004
0.00005
0.00004
4.94645
0.0107
0.03754
0.00008
0.0001
0.00007
0.00004
0.00002
0.00003
0.00003
0.00003
0.01293
0.00929
0.00576
0.00325
0.00367
0.00465
0.00431
15-mm i.d.) and slurry packed with BIO-BEADS S-X3 200–400
Figure 1. Configuration of the GPC columns used.
Figure 2. Oven temperature program.
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Journal of Chromatographic Science, Vol. 38, December 2000
permeation chromatograph (GPC) columns was verified by
injecting a phthalate sample. The retention time and peak area
were compared with the standard injected when the columns
were just packed.
Detection method
The optimized parameters for the detection method by
GC–ECD are shown in Table I. The temperature gradient used for
the separation of the nine pesticides is summarized in Figure 2.
Two milliliters of a sample was injected for successive analyses.
The solvent used for the elution of the grease and pesticides was
dichloromethane at a flow rate of 4 mL/min. Detection was set at
Results and Discussion
254 nm.
Detection method
Figure 3 shows the chromatogram obtained
when a mixture of the nine pesticides was ana-
lyzed in the method described previously. The pes-
ticides were eluted in a total time of 28 min.
Verification of the method
Verifications were necessary in order to estab-
lish if the parameters of the process developed
were adaptable to further applications. The
parameters studied in this work were: the capacity
factor (k'), selectivity, linearity, and repeatability.
In order to carry out this analysis, a 0.5-ppm
standard mixture was injected 10 times, and the
chromatograms were analyzed in terms of the
parameter studied.
Capacity factor
The k' value was defined as:
Figure 3. Chromatogram of the injection of a nine-pesticides standard sample containing
ppm of each.
t – t
r
o
k' = ———
Eq. 1
5
t
o
where t is the retention time for a compound and
r
to is the dead time (both in minutes). Results for
the nine pesticides and the deviation of the
average of the 10 injections are shown in Table II.
Selectivity
Selectivity (S) is the capacity of analyzing a
compound in the presence of certain interfer-
ences. It is measured by the equation:
k'
(
b)
S = ———
k'
Eq. 2
(
a)
given that t (a) < t (b). Table III shows the selec-
r
r
tivity values obtained for each pesticide.
Linearity
As it is known, the response of an analytical
method is proportional to a given value. This is a
linear proportionality determined from a series of
injections of standard mixtures at different con-
centrations (in this case 0.01, 0.05, 0.1, 0.5, 1, 2,
and 5 ppm). They were injected by triplicate in the
GC–ECD. The average of the pesticide areas were
plotted, and the calibration curve was obtained for
each component.
Figure 4. Calibration curves for the nine pesticides.
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Journal of Chromatographic Science, Vol. 38, December 2000
For each pesticide, the response was linear until 2 ppm—
except for diazinon, which slightly deviated at this concentration
laboratory. Results are indicated in Table IV.
Clean-up method
Lanolin elution
(Figure 4).
Repeatability
A 25% lanolin solution in dichloromethane was injected. This
solution showed saturation in the UV–vis detector, thus a 2%
solution was prepared and injected (Figure 5).
The repeatability of the system was measured in order to deter-
mine the reliability of the method. In order to do this, a series of
the same standard sample was injected and the results were sta-
tistically analyzed. Repeatability was obtained when the analysis
was made in a short time by the same equipment, operator, and
Table VI. Recovery Percentages of the Nine Pesticides
Analyzed
Pesticide
%Recovery
Table IV. Statistical Analysis of the Retention Time
Repeatability for the Nine Pesticides
Propetamphos
Diazinon
Chlorpyrifos-met
Carbophenothion
Tetramethrin
Cyhalothrin
Cypermethrin
Fenvalerate
Deltamethrin
97.6
98.8
92.5
Relative
standard
deviation
95%
Standard
deviation
Confidence
intervals
86.3
Pesticide
Average
107.5
122.5
97.6
101.2
97.1
Propetamphos
Diazinon
Chlorpyrifos-met
7.1864
7.3235
8.4499
0.0011
0.0012
0.0011
0.0021
0.0035
0.0027
0.0025
0.0024
0.0028
0.0148
0.0162
0.0126
0.0140
0.0201
0.0135
0.0106
0.0095
0.0104
0.00076
0.00085
0.00076
0.00152
0.00254
0.00191
0.0018
Carbophenothion 15.1856
Tetramethrin
Cyhalothrin
Cypermethrin
Fenvalerate
Deltamethrin
17.6375
19.7323
23.7529
25.5320
27.2597
A
0.00174
0.00203
B
Figure 5. Elution spectrum of lanolin at 254 nm.
Table V. Recovery Percentages of Lanolin in Each Fraction
Collected from the Discharge of the GPC Column
Elution time
%Lanolin recovered
10~12
12~14
14~16
16~18
18~20
20~22
1.34
12.04
17.16
24.88
32.14
5.6
Figure 6. Recovery percentages of the different pesticides in each of
the different fractions collected at the output of the GPC column: (A)
organophosphorous pesticides and (B) synthetic pyrethroid pesti-
cides.
554

Journal of Chromatographic Science, Vol. 38, December 2000
Figure 7. Chromatogram of an eluted sample from the GPC column after injection of a lanolin–pesticides mixture showing the interference by the
remaining lanolin in the base line.
In order to determine exactly when lanolin is eluted from the
GPC column, 2 mL of a 25% solution was injected, and the frac-
tions eluted were collected in calibrated vials every 2 min from 10
to 26 min. The solvent was evaporated to dryness under a
nitrogen atmosphere, and by difference, percentage recoveries
were calculated for each eluted fraction (Table V). The maximum
percentage of lanolin eluted was found between 18 and 20 min.
which makes further analysis easier. The separation of the bulk of
the lanolin from the pesticides is also possible. The presence of
small residues of lanolin in the fraction of the pesticides eluted
does not make analysis by GC–ECD difficult when obtaining
recovery percentages greater than 85%.
Elution of the pesticides mixture
Acknowledgments
A 5-ppm standard mixture was injected in the GPC with the
same conditions as the lanolin injection, and a single fraction was
collected between 16 and 38 min. The eluted fraction was evapo-
rated to dryness and reconstituted in 5 mL of cyclohexane and
injected in the GC system. After analysis, recovery percentages
higher than 85% for each pesticide were obtained (Table VI).
In order to determine when the pesticides were exactly eluted
from the GPC column, eluted samples after the injection were
collected from 16 to 38 min every 2 min. The recovery percentage
of each fraction versus the elution time was represented (Figure
The Ministry of Education and Culture of Spain is kindly
acknowledged for their financial support (AP96 36187769).
References
1
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6
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phorous was eluted between 22 and 28 min.
215–220 (1991).
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Elution of a lanolin–pesticide sample
3
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Two milliliters of a 2% lanolin dissolution spiked with a 5-ppm
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lected every 5 min. A GC analysis showed a chromatogram
4. E.W. Clark. New concepts of lanolin manufacturing. Chemist 7:
18–23(1990).
(
Figure 7) with a non-flat baseline because of the interference of
5. D.L. Heikes and J.C. Craun. Rapid multiresidue procedure for the
determination of pesticides in anhydrous lanolin and lanolin-con-
taining pharmaceutical preparations utilizing gel permeation chro-
matography cleanup with gas chromatographic and mass
spectrometric techniques. J. Agric. Food Chem. 40: 1586–1590
lanolin. Nevertheless, all peaks were able to be integrated.
Recovery percentages higher than 85% (with the exception of
diazinon) were found.
(
1992).
6
. T. Shaw. Agricultural chemical in raw wool and the textile industry.
J. Inst. Waters Eng. 8: 287–290 (1994).
Conclusion
7
. R. Lázaro, S. Bayarri, P. Conchello, A. Ariño, and A. Herrera.
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determinación de residuos organoclorados en alimentos. Grasas y
Aceites 46(1): 35–38 (1995).
. R.V. Geene and J.M. Wimbush. The 9th International Wool Textile
Research Conference, II. 1995, pp 321–330.
The GC–ECD method for the detection of the nine pesticides
developed is valid and surpasses the 95% reliability test.
By means of the clean-up system, the separation of the pesti-
cides into two groups constituted by each of the two families
8
(
organophosphorous and synthetic pyrethroids) is possible,
Manuscript accepted July 13, 2000.
555