INTRODUCTION:

The pesticides were created for control of insect born deceases affecting man and in the protection of crops and live stock. Carbamate group of pesticides are also use for the same manner. Because of the ease of their availability and low cost the insecticides are often misused for homicidal and suicidal and sometime in accidental poisoning. Identification of those pesticides becomes essential to protect the ecology/mankind from the adverse effect of pesticide residues and forensic toxicology and criminology purposes.

Thin layer chromatography and spot tests are found to the best choice for routine work in toxicology purposes for detection of carbamate pesticides. Thus several chromagenic reagents and solvent systems are studied which shows different colour spots after reaction with these insecticides and thus help in their identification ⁽¹⁾.

Pesticides have great value in the control of insect borne deceases affecting man and in the protection of crops and live stock. They are widely used in homes, industries and shops and by farmers, gardeners and foresters. They are classified as insecticides, herbicides, rodenticides, fungicides, nematocides, algaecides and acaricidies on the basis of their field of use.

Insecticides: Insecticides are widely employed chemicals in controlling insects and other pests.

Herbicides: Herbicides are chemicals that kill plants. Both selective and non selective herbicides are available, selective herbicides kill only certain types of plants and non-selective herbicides kill all plants.

Fungicides: Fungicides are used to eradicate fungal decease of plants.

Rodenticides: Rodenticides are used against rodents such as rats and mice.

Commercial formulations of pesticides form sub-classes. The most important sub-classes are organophosphorous (op), Organochloro (oc), carbamates and pyrathraoids.

The carbamate pesticides namely carbaryl, carbofuran, methomyl, propoxur, thiophanate methyl and cartap are widely used for the same purposes. Different manufacturing companies prepare this carbamate group of pesticides due to low manufacturing cost and highly effective nature of them and so these carbamates are available in the market.

Number of analytical and advanced instrumental methods viz. gas chromatography and mass spectroscopy can be used for the identification of these compounds. Though these techniques are rapid, specific and sensitive, but these techniques cannot be used for identification of compound extracted from biological matrix as the purity of sample is in question. Hence for routine toxicological examination TLC and spot test are more practical technique for the identification of sample extracted from biological matrix like soil, food, plant and animal tissues.

A number of solvent system and chromagenic reagents are available for the detection of pesticides. The identification of carbamate group of pesticides namely carbaryl(1-napthyl-N-methyl carbamate), carbofuran(2,3,-dihydro-2,2-dimethyl-benzofuran-7-dimethyl carbamates), methomyl(o-methyl-thio aceto hydroxamate), propoxur(2-isopropoxy-N-methyl carbamate), thiophanate methyl(dimethyl-4,4’-(o-phenylene)-bis-(3-thioalophanate) and cartap[s,s’-(2, 2’-dimethyl-amino trimethylene-bis-thio-carbamate)] by using TLC technique and spot test for routine work and initial identification are reported in this paper ^(2-4).

MATERIAL AND METHOD:

For study of the analytical techniques and their validation the standard chemicals of analytical grades were used.

1) Preparation of sample solution: A standard solution of 1mg/ml strength of commonly used carbamate insecticides namely carbaryl, carbofuran, methomyl, propoxur, thiophanate methyl and cartap were separately prepared in methanol.

2) Preparation of chromatographic plates: Standard glass plates (10cmX20cm) were coated with slurry of silica gel in water to a uniform thickness of 0.25mm. The plates were activated by heating at 110⁰C for one hour before use.

3) Preparation of reagents:

i) 10% aqueous solution of sodium hydroxide: 50gm of sodium hydroxide dissolved in 500ml of distilled water.

ii) 10% aqueous solution of potassium hydroxide: 50gm of potassium hydroxide dissolved in 500ml of distilled water.

EXPERIMANTAL PROCEDURE:

Standard pesticides solution in n-hexane was spotted on the TLC plates by using 2ml micropipette and the following steps were carried out ^(2-5).

STEP-I

The spotted plates were inserted in the TLC chamber.

Solvent system:-

(i) Dichloromethene : Ethyl acetate :: 9:1(v/v) (ii) Toluene : Ethyl alcohol :: 4:1(v/v)

(iii) Benzene : Acetonitrile :: 9:1(v/v)

Spray reagent: 2.5 gm of potassium haxacyanoferrate was dissolved in 50ml of 10% sodium hydroxide solution and sprayed on the TLC plates.

Observation: The reagent shows violet spot for carbaryl and orange spot for propoxur.

STEP-II

The spotted plates were inserted in the TLC chamber.

Solvent system:-

(i) Dichloromethene: Ethyl acetate::9:1(v/v)

(ii) Toluene: Ethyl alcohol::4:1(v/v)

(iii) Benzene: Acetonitrile::9:1(v/v)

Spray reagent: 2.5 gm of sodium nitroproside was dissolved in 50ml of 10% sodium hydroxide solution and sprayed on the TLC plates.

Observation: The reagent shows orange spot for cartap, other carbamates not respond with this reagent.

STEP-III

The spotted plates were inserted in the TLC chamber.

Solvent system:-

(i) Benzene: Hexane: Acetone :: 7 : 2 : 1(v/v)

(ii) Hexane: Benzene : Methanol : Glacial Acetic Acid :: 5:4 : 0.75 : 0.25(v/v)

(iii) Iso-octane : Iso-butanol : Chloroform :: 7 : 2 : 1(v/v)

Spray reagent: 2ml methanol mixed with 50ml of 10% potassium hydroxide and sprayed on the TLC plates and heated for five minutes at 110⁰C, then Folin ciocalteaus reagent.

Observation: A dark blue spot obtained for propoxur and methomyl, other carbamates not respond with this reagent.

STEP-IV

0.5gm of cupric chloride dissolved in 50ml distilled water.

Spot test:- 1% cupric chloride applied on the standard pesticides plotted plates followed by 10% sodium hydroxide.

Observation: A green coloured spot obtained for thio-phanate methyl, other carbamates not respond with this reagent.

STEP-V

Step-I to Step-IV were carried out by using the standard solution of 1mg/ml strength of commonly used Organophosphorous namely malathion, parathion, organochlorine namely D.D.T, B.H.C, carbamates carbosulfane and synthetic pyrathroids namely alphamethrin and cypermethrin separately in methanol for evaluating specificity of the reagent.

Observation: No characteristic colour spot observed.

RESULT AND DISSCUTION:

The result of thin layer chromatography and spot tests are convenient to identification of commonly used carbamates namely carbaryl, carbofuran, methomyl, propoxur, thiophanate methyl and cartap for routine work in Toxicology purposes.

1. The reagent alkaline potassium haxacyanoferrate responded and reacted with carbaryl and propoxur pesticides gave violet and orange spot respectively and other pesticides described were failed to react or respond with any characteristic colour. Thus this reagent is found suitable for their detection and identification.

2. The reagent sodium hydroxide-sodium nitroprosside responded and reacted with cartap pesticide gave orange colour spot and other pesticides described were failed to react or respond with any distinguishing characteristic colour. Thus this reagent is found suitable for their detection and identification.

3. The reagent alcoholic potassium hydroxide-folin-ciocalteau’s reacted with methomyl, carbofuran and propoxur and gave dark blue spot with different hRf values. The other described pesticides are failed to react with this reagent. The propoxur compound responded in step-I, so the carbofuran and could be identified by comparing their hRf values in different mobile phase.

4. The reagent 1% cupric chloride-10% sodium hydroxide gave green colour spot for thiophanate methyl and the colour persists. The other described pesticides were failed to show any reasonable colour. Hence this reagent is found suitable for detection and identification of thiophanate methyl.

Though there are uses of hyphenated techniques like FT-IR, GC-MS etc for the study of different pesticides which can give a more reliable and reproducible result but there is certain problem in use of such techniques. Firstly, if it a pure compound and analysis is meant for qualitative or latch conformity then there is little need for purification. The samples can be diluted to desirable quantity and injected to the instrument. But when it comes from other medium then it is different to predict the nature of impurities present in it. For this it is advisable to be scraped from a preparative TLC and then applied to the instrument. Secondly no many laboratories have access to such sophisticated hyphenated techniques or state of the art equipment as these are very costly, difficult to maintain and running cost is very high. In this regard the TLC is a cost effective time tested technique and the repeatability and also reproducible.

REFERENCES:

1. Jagtap R.K., Kokare R.N., Jawale M.M., Malve M.K., Pandav V.S: Identification of Organo Thiophosphorous insecticide by new chromagenic reagent, XVIII all India Forensic Science Conference-2007, Kolkata, West Bengal. P (107-109)

2. Bhatia Jitesh, Sharma J.D, Thin Layer Chromatographic Detection of Carbaryl and Propoxur by alkaline potassium hexacyanoferate spray reagent, XVIII all India Forensic Science Conference-2007, Kolkata, West Bengal. P (36-38)

3. Bhatia Jitesh, Sharma J.D, Detection of Cartap by TLC and Spot test using Sodium hydroxide and Sodium nitroproside reagent, XVIII all India Forensic Science Conference-2007, Kolkata, West Bengal. P (39-41)

4. Bhatia Jitesh, Sharma J.D, Detection of Thiophanate Methyl by Spot test method, XVIII all India Forensic Science Conference-2007, Kolkata, West Bengal. P (31-35)

5. Laboratory Procedure Manual, Forensic Toxicology (2005), Directorate of Forensic Science, Ministry of Home Affairs, Govt. of India New Delhi. P (135-192)

Received on 06.09.2012 Modified on 27.09.2012

Asian J. Research Chem. 5(11): Nov., 2012; Page 1365-1367