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Chapter 6

6.1 Introduction

Pesticides are used worldwide in agriculture to protect plants and animals and to

crop damages due to insects. Additionally, they are used against storage and domestic

pests, and to control insect-borne diseases. In India, 20–30% of the total harvest is

destroyed by pests resulting in uncontrolled use of pesticides by the Indian farmers.

Although a wide choice of chromatographic techniques (GC, HPLC) is available

for pesticides analysis, thin layer chromatography remains an important analytical

method for the analysis of pesticides with well-developed standard procedures [1-7].

The acceptance and importance of TLC is mainly due to its simplicity, speed, low cast

and the possibility of analyzing a large number of samples (30 or more samples on

one plate) simultaneously. TLC analysis of pesticides is especially suitable at sites

where the concentrations of pesticides might be high (e.g. in the chemical industry,

and the transport, storage, and distribution of pesticides, and liquidation of dumping

grounds of toxic substances) [8].

Impregnation is one of the most commonly used modification methods for

stationary phases. There are a variety of methods for obtaining impregnated layers,

including the preparation of the TLC plate with slurries containing the stationary

phase and the impregnating reagent, pre- or post-chromatographic dipping, pre- or

post chromatographic development of the plate in a solvent containing the

impregnating agent or spraying. Depending on the kind of interactions between the

reagents and the stationary phases, six basic groups of impregnating agents used in

planar chromatography, and namely:

Non-polar liquids such as saturated and unsaturated hydrocarbons, silicon oils,

and plant oils.

Impregnating agents that are able to form complexes such as

ethylenediaminetetraacetic acid–EDTA, metal ions.

Impregnating agents that are able to form charge transfer complexes such as

caffeine.

171

Substances that lead to the adjustment of pH values such as perchloric acid

and sodium hydroxide.

Impregnating agents that lead to a defined change in the solubility of the

analytes in the liquid stationary phase such as formamide and ammonium

sulphate.

Cationic, anionic and non-ionic surfactants such as dodecylbenzenesulphonic

acid-HDBS, sodium dioctylsulphosuccinate-Na-DSS, TX-100.

The work performed on TLC analysis of pesticides has been well described in

literature [4–17]. According to the literature, the majority of TLC methods for the

analysis of pesticides have been performed on silica gel layers as compared to other

sorbent phases. Literature survey revealed that no work has been reported on TLC of

pesticides on cationic surfactants impregnated silica layers with n-hexane-acetone as

mobile phase. The aim of this work was to develop new stationary phases by

impregnating TLC plates with aqueous solutions of cationic surfactants in order to

utilize the advantages of cationic micelles in stationary phase. For this purpose,

several approaches have been adopted to develop new sorbent phases to realize a

desired separation with a particular mobile phase. n-hexane was selected as reference

organic solvent because its solvent capability is comparable to that of supercritical

CO2 in mild operating conditions. Acetone was chosen in combination with hexane

due to its low viscosity, and miscibility with several organic solvents. Our results

clearly demonstrate that compared to kieselguhr, alumina, and cellulose impregnated

layers, silica gel impregnated layer were exceptionally useful in separation of

coexisting pesticides. As a result five-component mixture of pesticides has been

successfully resolved on silica gel layer impregnated with 0.01% and developed with

n-hexane-acetone, 1:1 as mobile phase.

6.2 Experimental All experiments were performed at 30 ± 2 °C.

6.2.1 Apparatus

A thin layer chromatographic applicator (Toshniwal, India), 20.0 cm×3.0 cm

glass plates and 24.0 cm×6.0 cm glass jars were used for the development of

172

chromatographic plates.

6.2.2 Chemicals and reagents

Silica gel ‘G’ was purchased from Merck, India. Kieselguhr, alumina,

cellulose, cetrimide, and sodium dodecyl sulphate (SDS) from CDH, India; N-cetyl

N,N,N-trimethyl ammonium bromide (CTAB), cetylpyridinium bromide (CPB) from

BDH, India; hexadecyltrimethylammonium chloride (HDTAC) from Merck

(Hohenbrunn, Germany) and dodecyltrimethylammonium bromide (DTAB) were

obtained from Sigma-Aldrich (Steinheim, Germany). All other chemicals used were

of Analytical Reagent (AR) grade.

The metal ion solutions (1.0%) studied, were prepared from the nitrates of

Th3+, Zn2+, Tl3+, and Pb2+, the chlorides of Ni2+, and Hg2+, the sulphates of Cu2+.

6.2.3 Pesticides studied

The following pesticides (Table 6.1) were used as analyte.

6.2.4 Test solution

Test solutions (2%, each) of tabulated pesticides were prepared in methanol. 6.2.5 Detection

All pesticides were detected as dark brown/yellow spots by exposure to iodine vapour

about 1 or 2 min.

6.2.6 Stationary phase

The stationary phases used are listed in Table 6.2.

6.2.7 Mobile phase

The mobile phases used were the mixtures of hexane and acetone in 4:1 (M1), 1:1

(M2) and 1:4 (M3) ratios by volume.

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6.2.8 Preparation of TLC plates

6.2.8.1 Plain silica gel thin layer plates

TLC plates were prepared by mixing silica gel ‘G’ with double distilled water

in 1:3 w/v ratios with constant shaking for 5 min until homogeneous slurry was

obtained. The resultant slurry was coated on the glass plates with the help of a

Toshniwal applicator to give a 0.25 mm thick layer. The plates were first air dried at

room temperature and then activated by heating at 100 ± 5◦C for 1 h. After activation,

the plates were kept in air tight chamber until used. Similar procedure was applied for

the preparation of kieselguhr, alumina, and cellulose TLC plates.

6.2.8.2 Impregnated TLC plates

The activated silica gel plates were impregnated with desired concentrations of

CTAB (0.001, 0.01, 0.1 and 1%), HDTAC, CPB, DTAB, cetrimide and SDS (0.01%)

by developing silica gel plates in aqueous solution of impregnant, followed by drying

of the plates at 100 ◦C in an electrically controlled oven for 1 h. Activated kieselguhr,

alumina or cellulose plates were impregnated with 0.01% CTAB similarly.

6.2.9 Scanning electron microscopy (SEM)

Electron micrographs were recorded for silica and silica-CTAB by using

scanning electron microscope at 20.0 KV. The magnification was kept constant at

20,000 X.

6.2.10 Fourier transforms infra red spectroscopy (FTIR)

For FTIR analysis, 10 mg (dry mass) of the adsorbent (silica-CTAB) was

thoroughly mixed and powered with100 mg (dry mass) of KBr. A transparent disc

was formed by applying a pressure of 80 psi (1 psi = 6894.76pa) in a moisture free

atmosphere. The FTIR absorption spectrum was recorded in between 500-4000 cm-1.

6.3 Procedure

Thin layer chromatography was performed on unimpregnated (or plain) and

174

impregnated (with CTAB, HDTAC, CPB, DTAB, cetrimide, or SDS) silica gel layers

in glass jars. Test solutions (10 µL) were applied by means of micropipette about 2

cm above the lower edge of the plates. The spot was allowed to dry and then the

plates were developed in the chromatographic chamber (glass jar) presaturated for 5

min with desired solvent system using ascending technique. The solvent ascent was

kept upto 10 cm from the point of application. After development, the plates were

dried at room temperature. All pesticides were detected as dark brown/yellow spots

by exposure to iodine vapour. The RF values were calculated from the values of RL (RF

of the leading front) and RT (RF of the trailing front): RF = 0.5 (RL + RT)

6.3.1 Separation

For separation, equal volumes (1.0 mL each) of pesticides to be separated

were mixed and 10 µL of the resulting mixture was loaded on CTAB (0.01 %)

impregnated silica gel TLC (S2) plates, which was then dried in air. The plates were

developed with mobile phase hexane─acetone (1:1) and detection performed as

described above. The separated pesticides were identified on the basis of their RF

values.

6.3.2 Interference

For investigating the interference of metal cations on chromatographic

behaviour and separation of pesticides, an aliquot (10 µL) of 1% aqueous solution of

cations was spotted along with the mixture (10 µL) of pesticides. The chromatography

was performed with hexane–acetone, 1:1 (mobile phase) and CTAB (0.01 %)

impregnated silica gel (stationary phase) system. The spots were detected and the RF

values of pesticides were determined.

6.3.3 Detection of pesticides in cereals, vegetables and fruit grains

The extraction of pesticides from contaminated cereals, vegetables and fruit

grain samples (1.0 g each) were carried out by immersing in methanol (10 mL) for 18

h. The resulting extract was concentrated to approximately 2.5 mL in a rotary

evaporater. Aliquot (10 µL) of the resultant extract sample was loaded on CTAB

impregnated silica layer (S3) and developed with mobile phase (hexane─acetone, 1:1),

175

followed by drying at room temperature. Spots were detected by exposure to iodine

vapour.

For identification with preliminary separation of mixture of pesticides (1, 2,

3, 4/5 and 8) in a sample of maize grains, the grains were immersed in the mixture of

pesticides solution (equal volume of each). The contaminated sample grains (one

gram) were treated with methanol (10 mL) for 18 h. The resulting extract of pesticides

in methanol was concentrated to ≈2.5 mL, and then used for further analysis.

6.3.4 Validation

For validation and reproducibility of developed method the chromatographic

parameters such as standard deviation (SD), ΔRF, and α (separation factor) were

determined from resolution of mixture of pesticides in six replicate RF value

measurements at a concentration of 200 µg zone-1 of the mixture.

6.3.5 Limit of detection

Limits of detection for Pesticides (1, 2, 3, 4, 5, and 8) were determined by

spotting different amounts of the pesticides on the TLC plates, developing the plates,

and detecting the spots. The method was repeated with successive lowering of the

amounts of pesticides applied until no spot was detected. The lowest amount of

pesticide detectable on the TLC plate was taken as the limit of detection.

6.4 Results and Discussion

The results of the present study have been summarized in Tables 6.3–6.9 and

Figures 6.1-6.2

The results presented in Table 6.3 indicate that the chromatographic

behaviour of eight pesticides on plain silica gel layer is influenced by the volume

composition of the n-hexane─acetone mobile phase. The RF values of pesticides 1, 2,

3 or 8 increased with increasing the concentration of acetone in mobile phase.

Peculiar behaviour was observed for pesticides 2, 4, 5, 6 or 7. These pesticides

furnished single, double or multiple spots irrespective of the composition of mobile

phase (M1-M3). This formation of multiple spots indicates the possible occurrence of

different species of pesticide 2, 4, 5, 6 or 7. In view of better chromatographic

176

performance, mobile phase M2 was selected for further studies.

Results listed in Table 6.4 deals with the use of silica stationary phases

impregnated with N-cetyl N,N,N-trimethyl ammonium bromide (CTAB) at four

different concentration levels [i.e. 0.001% (S2)─1% (S5)] for the chromatography of

pesticides using selected mobile phase M2 (n-hexane─acetone, 1:1). Our results of

Table 6.4 demonstrate that the RF values were affected by the variation of CTAB

concentration from 0.001 to 1% in the stationary phase. As compared to plain silica

gel layers, spots were more compact on impregnated silica gel layers. Slightly lower

mobility of chlorpyrifos (3) was observed for higher concentrations (1%) of CTAB in

stationary phase (S5). Malathion (4) on S3 and methyl parathion (5) on S3-S5 layers

produced single spot. Dimethoate (6) furnished triple spots over the concentration

range (0.01%-1.0%) of CTAB. Dichlorvos (7) could not be detected on layers S3-S5.

The mobility of isoproturon (8) decreases with increasing the concentration of CTAB

in stationary phases (S2-S4), but at higher concentration on S5 layer, it was not

detected. The best results with greater separation possibilities (i.e. great difference in

RF values of pesticides 1, 2, 3, 4 or 5, and 8) were realized on layer impregnated with

0.01% CTAB (S3). It was therefore, selected for detailed study.

The morphology of plain silica gel and silica gel impregnated with CTAB (S3)

were examined by scanning electron microscopy (SEM). Figures 6.1 (a) & (b) show

the micrographs of untreated and impregnated silica gel respectively. In Figure 6.1

(b) the silica gel particles are coated with CTAB as a result of impregnation.

FTIR spectroscopy is useful tool to ascertain the bonding modes of any

compounds. The IR spectrum of silica-CTAB composite material exhibited various

peaks characteristics of the functional groups present in CTAB and silica gel. The

negative shift in the bands compared to the free non-bonded silica and CTAB

confirms that the silica moieties are bonded to the CTAB fragments. A broad band at

≈3400 cm-1 is assignable to the presence of water molecules as well as OH groups in

the complex [18]. A medium intensity peak at 1647 cm-1 is characteristics of H-O-H

bonding mode of vibration. Si-H bond is ascertained by the presence of a peak at 2363

cm-1 [18]. The presence of silicate group is confirmed by a medium intensity peak at

1094. The FTIR spectrum is clearly indicated that the silica gel and the CTAB are

embedded with each other in a well organized manner.

To examine the effect of nature of surfactant as impregnant, cationic

(HDTAC,CPB, DTAB, and cetrimide) and anionic (SDS) surfactants were

177

introduced in silica stationary phase by impregnating silica gel with aqueous solutions

(0.01%) of these surfactants and the chromatography of pesticides on these stationary

phases (S6–S10) was performed using M2 as mobile phase. The results presented in

Table 6.5 further, demonstrate that the separation of 1, 2, 3, 4 or 5, and 8 is possible

only on S3 layer. Thus, cationic surfactant (CTAB) impregnated silica layer is most

favourable for selective separation of coexisting five pesticides from their mixture.

To establish the effectiveness of S3, the retention behaviour of pesticides was also

examined on different sorbent layers (S11─S13), and the obtained results have been

listed in Table 6.6. From the results presented in Table 6.6 following trends are

apparent:

o As compared to S3 layer, pesticides 1 and 2 (on S11 and 12) show high mobility

range from 0.95─0.98.

o Malathion (3) had almost an identical RF value irrespective of the nature of

adsorbent.

o Methyl parathion (4) produced tailed spot on S11.

o Except acephate (2, where RF could not be measured), on S13 all the pesticides

show high mobility (RF in the range 0.95–0.98).

Although increase in RF value of certain pesticides were realized on kieselguhr

(S11), alumina (S12), and cellulose (S13) layers, but separation of coexisting five

pesticides is possible only on S3, because of differential mobility of certain pesticides.

The position of spots appeared on S3 plate is depicted in Figure 6.2. From Figure 6.2

it is clear that, with mobile phase M2 (hexane─acetone, 1:1, v/v) five-component

mixture of pesticides is clearly resolved.

To widen the applicability of developed TLC system (S3–M2), the separation

of 1, 2, 3, 4 or 5, and 8 was examined in the presence of metal cations (Th, Zn, Tl, Ni,

Hg, Cu, and Pb) as impurities. From the results listed in Table 6.7, it is clear that in

case of all metal cations isoproturon (8) was not detected. However, 3 (in the presence

of Tl or Pb) and 1, 2, or 3 (in the presence of Hg) were also not detected. A marginal

changes in RF of 1, and 2 were seen in the presence of Th, Zn, Tl, Cu, and Pb, the RF

values of 1 vary between 0.02 to 0.07 and 2 vary between 0.31 to 0.37. In case of Zn,

Ni, Cu and Pb, there is increasing trend in the RF values of 4 and 5 as compared to

178

their standard RF value (0.85 and 0.84 respectively). Thus, separation of pesticides (1,

2, 3, 4 or 5, and 8) is effected by the presence of metal cations as impurities.

Effect of metal cations on chromatographic parameters (∆RF and α) have also

been calculated for representative separations and the results were presented in Table

6.8. The marginal increase or decrease in values of ∆RF and α was due to the

increase/decrease in spot size of the analyte as a result of various interactions with

metal cations.The standard deviations in RF values for the resolved spots of pesticides

(1, 2, 3, 4, 5, and 8) were calculated. The low values of standard deviation (S.D.) 1 (±

0.001), 2 (± 0.02), 3 (± 0.05), 4 (± 0.04), 5 (± 0.04), 8 (± 0.03), obtained after

resolving the mixture 1, 2, 3, 4 or 5, and 8 in six replicate RF value measurements

indicated the robustness of the developed TLC method.

The lowest possible detectable microgram amounts of all four pesticides

obtained on S3 TLC plates developed with M2 was ≈20 µg spot-1. It shows that the

developed method is reasonably suitable for identifying these pesticides at trace level.

6.5 Application The applicability of the proposed method (S3–M2 system) for the identification

of pesticides (1, 2, 3, 4, 5, and 8) in cereals, vegetables and fruit grains was also

tested. The results listed in Table 6.9 clearly demonstrate that pesticides (1, 2, 3, 4, 5,

and 8) contamination of a variety of samples can be detected on S3 TLC plates

developed with M2 mobile phase. Pesticides 1, 2, 3, 4 or 5, and 8 if present as

contaminant in a sample of grains can be easily separated by using S3-M2 TLC system

(Figures 6.2 a & b).

6.6 Concluding Remarks

The proposed thin layer chromatographic system comprising of CTAB (0.01

%) impregnated silica layer as stationary phase and hexane─acetone (1:1) as mobile

phase was the most favorable for the separation of pesticides from their multi

component mixture (Figure 6.2). Being selective, the proposed method could be

implemented as a reliable analytical tool for the analysis of pesticides in cereals,

vegetables and fruit grain samples. More interestingly, the method could be

successfully applied for the separation of pesticides (1, 2, 3, 4 or 5, and 8) from

contaminated maize grains.

179

References

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Retrospective View for the Third Millennium, Nyiredy Sz. Ed., 585 (2001) 607.

[2]. T. Tuzimski, Chromatographia. 56 (2002) 379.

[3]. C. F. Poole, J. Chromatogr. A. 1000 (2003) 963.

[4]. R. Akkad, W. Schwack, J. Chromatogr. B 878 (2010) 1337.

[5]. J. Sherma, Acta Chromatographica. 15 (2005) 5.

[6]. A. Ambrus, I. F. Uzesi, M. Sus´an, D. Dobi, J. Lantos, F. Zakar, I. Kors´os, J.

Ol´ah, B. B. Beke, L. Katavics, J. Environ. Sci. Health 40 (2005) 297.

[7]. J. Sherma, J. Chromatogr. A 880 (2000) 129.

[8]. T. Tuzimski, E. Soczewin´ski, J. Chromatogr. A 961 (2002) 277.

[9]. E.G. Sumina, S.N. Shtykov, N.V. Tyurina, J. Anal. Chem. 58 (2003) 720.

[10]. A. Mohammad, A. Moheman, J. Planar Chromatogr. 24 (2011) 113.

[11]. A. Mohammad, I. A. Khan, N. Jabeen, J. Planar Chromatogr. 14 (2001) 283.

[12]. S. A. Nabi, A. Gupta, M. A. Khan, A. Islam, Acta Chromatographica. 12

(2002) 201.

[13]. D. W. Armstrong, R. Q. Terrill, Anal. Chem. 51 (1979) 2160.

[14]. R. P. Singh, R. Kumar, Soil Sediment Contamination. 9 (2000) 407.

[15]. H. S. Rathore, T. Begum, J. Chromatogr. 643 (1993) 271.

[16]. J. Sherma, J. AOAC Int. 84 (2001) 993.

[17]. M. Hamada, R. Wintersteiger, J. Planar Chromatogr. 16 (2003) 4.

[18]. J. Sherma, J. AOAC International. 86 (2003) 602.

[19]. K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination

Compounds, Wiley-Interscience, New York Publication, (1886) 191.

180

Table 6.1: The pesticides investigated Pesticide Assigned

No

Chemical structure

Glyphosate 1

Acephate 2

Chlorpyrifos 3

Malathion 4

Methyl Parathion 5

181

Dimethoate 6

Dichlorvos 7

Isoproturon 8

Table 6.2: The stationary phases used Code Stationary phase

S1 Silica gel ‘G’

S2 S1 impregnated with CTAB (0.001%)

S3 S1 impregnated with CTAB (0.01%)

S4 S1 impregnated with CTAB (0.1%)

S5 S1 impregnated with CTAB (1%)

S6 S1 impregnated with HDTAC (0.01%)

S7 S1 impregnated with CPB (0.01%)

S8 S1 impregnated with DTAB (0.01%)

S9 S1 impregnated with cetrimide (0.01%)

S10 S1 impregnated with SDS (0.01%)

S11 Kieselguhr impregnated with CTAB (0.01%)

S12 Alumina impregnated with CTAB (0.01%)

S13 Cellulose impregnated with CTAB (0.01%)

182

Table 6.3: RF values of pesticides on plain (i.e. non-impregnated) silica gel (S1)

layers using hexane–acetone, 4:1 (M1), 1:1(M2) or 1:4 (M3) volume/volume as mobile

phase

Mobile phase

Pesticide M1 M2 M3

1 0.00 0.02 0.07

2 0.03 0.03, 0.40 0.08

3 0.88 0.92 0.98

4 0.02, 0.52 0.10, 0.80 0.09

5 0.04, 0.26, 0.93 0.07, 0.75 0.04, 0.13, 0.26

6 0.03, 0.07, 0.71 0.11, 0.42, 0.64, 0.77, 92 0.03, 0.10, 0.42

7 0.00 0.14, 0.86 0.00

8 0.19 0.84 0.91

Each value is an average of six replicate determinations

Table 6.4: RF value of pesticides on non-impregnated plain silica layer (S1) and CTAB

(different concentration levels) impregnated silica layer using hexane–acetone, 1:1 (v/v)

as mobile phase

Stationary phase

Pesticide S1 S2 S3 S4 S5

1 0.02 0.02 0.02 0.92 0.78

2 0.03, 0.4 0.03 0.30 0.22 0.03

3 0.92 0.92 0.95 0.97 0.84

4 0.10, 0.80 0.10, 0.84 0.86 0.08, 0.96 0.03, 0.86,

5 0.07, 0.75 0.07, 0.80 0.83 0.88 0.81

6 0.11, 0.42,

0.64, 0.77, 92

0.14, 0.45,

0.64, 0.77,

0.92

0.46, 0.67,

0.91

0.1, 0.40,

0.75, 0.95

0.03, 0.56,

0.93

7 0.14, 0.86 0.27, 0.88 n.d. n.d. n.d.

8 0.84 0.80 0.71 0.66 n.d.

Each value is an average of six replicate determinations; n.d. = not detected.

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Table 6.5: RF values of pesticides obtained on different cationic and anionic micelles

(0.01%) impregnated silica layers using hexane─acetone, 1:1 as mobile phase

Stationary phase

Pesticide S3 S6 S7 S8 S9 S10

1 0.02 0.04 0.09 n.d. 0.05 0.02

2 0.30 0.17 0.37 0.25 0.20 T 0.41

3 0.95 0.98 0.91 0.90 0.95 0.94

4 0.86 0.93 0.87 0.92 0.88 0.05,

0.90

5 0.83 0.81 0.87 0.91 0.82 0.67

6 0.46, 0.67,

0.91

0.19, 0.52,

0.87

0.08, 0.62,

0.91

0.85 0.87 0.04,

0.65

7 n.d. 0.00 0.00 0.93 0.86 n.d.

8 0.71 0.91 0.84 0.90 0.80 n.d.

Each value is an average of six replicate determinations; T = tailed spot (RL + RT ≥

0.30)

Table 6.6: Comparative RF values of pesticides on different stationary phases

using hexane─acetone, 1:1 (v/v) as mobile phase

Stationary phase Pesticide

S3 S11 S12 S13

1 0.02 0.98 0.98 0.98

2 0.30 0.95 0.17, 0.98 −

3 0.95 0.94 0.96 0.97

4 0.86 0.63 T 0.94 0.97

5 0.83 0.98 0.26 T, 0.88 0.96

6 0.46, 0.67, 0.91 0.55 0.46, 0.83 0.96

7 n.d. 0.91 0.22 T, 0.95 0.95

8 0.71 0.97 0.91 0.97

Each value is an average of six replicate determinations; − = not determined

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Table 6.7: Separation of pesticides in the presence of cations impurities on CTAB

(0.001%) impregnated silica phase (S2) using hexane─acetone, 1:1 as mobile phase

Impurities Separation (RF)

Without Impurities 1(0.02 ± 0.001) / 2 (0.31 ± 0.02) / 3 (0.97 ± 0.05) / 4 (0.85 ± 0.04)

or 5 (0.84 ± 0.04) / 8 (0.69 ± 0.03)

Th 1(0.07 ± 0.004) / 2 (0.35 ± 0.02) / 3 (0.98 ± 0.05) / 4 (0.85 ± 0.04)

or 5 (0.82 ± 0.05)

Zn 1(0.06 ± 0.003) / 2 (0.34 ± 0.02) / 3 (0.98 ± 0.05) / 4 (0.90 ± 0.04)

or 5 (0.88 ± 0.04)

Tl 1 (0.09 ± 0.005) / 2 (0.37 ± 0.02) / 4 (0.85 ± 0.05) or 5 (0.83 ±

0.04)

Ni 1 (0.02 ± 0.001) / 2 (0.31 ± 0.02) / 3 (0.97 ± 0.05) / 4 (0.87 ± 0.04)

or 5 (0.86± 0.04)

Hg No separation

Cu 1 (0.07 ± 0.004)/ 2 (0.35 ± 0.02)/ 3 (0.99 ± 0.05) or 4 (0.92 ± 0.05)

or 5 (0.90 ± 0.05)

Pb 1 (0.07 ± 0.004)/ 2 (0.36 ± 0.02)/ 4 (0.88 ± 0.04) or 5 (0.87 ±

0.04)

Each value is an average of six replicate determinations

185

Table 6.8: Effect of cations impurities on ∆RF values and separation factor (α) of

resolved pesticides

Metal cations ∆RF Separation factor (α)

Without

impurities

3-4 (0.12), 3-5 (0.13), 4-8 (0.16),

5-8 (0.15), 8-2 (0.38), 2-1 (0.29)

3-4 (5.86), 3-5 (6.33), 4-8 (2.55), 5-8

(2.36), 8-2 (0.50), 2-1 (22.0)

Th 3-4 (0.13), 3-5 (0.16), 4-2 (0.50),

5-2 (0.47), 2-1 (0.28)

3-4 (8.8), 3-5 (10.55), 4-2 (10.55), 5-2

(8.47), 2-1 (7.15)

Zn 3-4 (0.08), 3-5 (0.10), 4-2 (0.56),

5-2 (0.54), 2-1 (0.28)

3-4 (5.55), 3-5 (6.8), 4-2 (17.48), 5-2

(14.27), 2-1 (8.06)

Tl 4-2 (0.48), 5-2 (0.46), 2-1 (0.28) 4-2 (9.67), 5-2 (8.34), 2-1 (5.94)

Ni 3-4 (0.10), 3-5 (0.11), 4-2 (0.56),

5-2 (0.55), 2-1 (0.29)

3-4 (4.96), 3-5 (5.4), 4-2 (14.93), 5-2

(13.73), 2-1 (22.02)

Hg ─ ─

Cu 3-4 (0.07), 3-5 (0.09), 4-2 (0.57),

5-2 (0.55), 2-1 (0.28)

3-4 (8.6), 3-5 (11.1), 4-2 (21.59), 5-2

(16.72), 2-1 (7.15)

Pb 4-2 (0.56), 5-2 (0.51), 2-1 (0.29) 4-2 (13.06), 5-2 (11.92), 2-1 (7.47)

186

(a)

(b) Figure 6.1: SEM images of (a) plain silica gel and (b) impregnated silica gel

187

Figure 6.2: Chromatograms obtained for pesticides with preliminary separation

from extracts of contaminated maize grain and from mixture of control solution

on S3 plates developed with M2 as mobile phase; (a) mixture of 1+2+3+4 +8, and

(b) mixture of 1+2+3+5+8