Detection of pesticides

Qualitative estimation of pesticide residues.
Steps in TLC Analysis
1. Preparation of TLC plates
2. Extraction of the sample (meat, milk, eggs, stomach contents etc.,)
1. Spotting of the substance
2. Development
6. Visualisation
7. Identification
8. Quantification
1. Preparation of Plates
Wash 3 glass plates of 10 cm x 20 cm x 3 mm. Arrange on a mounting board. Clean glass plates with water, acetone and finally with cotton. Clean the applicator of 250µ thickness. Prepare slurry of silica gel G 7 g in 14 ml water. Shake for one minute. Pour the slurry into the applicator. Draw the applicator uniformly in one motion. Dry at room temperature followed by drying at 110C/10 minutes. Cool in a dessicator.
Preparation of AgNO3 TLC plate (for detection of OC and synthetic pyrethroids)
Arrange 3 plates on mounting board. Clean with water, acetone and cotton. Weigh 7 g Silicagel G. Wash with 200 ml distilled water to remove chlorides in water. (If not AgNO3 forms a precipitate with chloride).
Filter under suction using buchner funnel. Transfer to a stoppered conical flask. Add 6 ml distilled water and 1 ml 0.1 N AgNO3. Shake for 1 minute and pour into applicator. Draw the applicator uniformly quickly. Dry at 1100C/10 minutes (activation). Cool in a dessicator.
2. Extraction of the sample (Eggs/tissues – meat)
The sample is homogenised in a tissue homogeniser. A 10 g of homogenate is taken, to which 60-75 g of sodium sulphate (anhydrous) is added .Mix it thoroughly in pestle & mortar till a clear powder is obtained. Take the powder in a conical flask and add 50ml of acetonitrile. Shake for few min allow it to stand for few more min and collect the supernatant. Repeat this procedure for 3 times. Take this 150ml of acetonitrile in a separatory funnel and add salt solution and distilled hexane 100-150ml, repeat for 3 times. The collected hexane is subjected to florisil “column clean up”. Elute with 6% diethyl ether in petroleum ether 200ml.Ether is removed by evaporating the eluted liquid on a water bath at 50 ºC. the residue is taken in acetone and spotted.

3. Spotting of Sample on TLC plates
Dress the plate by removing silica gel layer (2mm) from the edges. Place the plate on a spotting guide. Clean a microlitre pipette (5 µl) using acetone solvent. Spot the test solution (if serum, apply directly, if other materials like rumen liquor, unlabelled bottle etc., it has to be first extracted – refer above). 2 cm above the bottom edge of the plate and 2 cm inside from the left side edge.
Spot 5 µl each of the standard solution. The standard solution of the pesticide or other substance in question is prepared as follows: Take pure sample, weigh 10g and add 10 ml acetone (1ml=1g). Make it to1ug/ul (1ml=1000µl; 1mg= 1000µg). Leave 1 cm gap between the spots. Clean the pipette after each spotting. Mark at 10 cm from the origin of spotting.
4. Development of TLC plate: Pour a suitable solvent (50-60 ml) into an aluminium tray. Place it in an airtight developing chamber. Allow saturating with solvent vapours for 30 min. Keep the plate vertically in the tray and close with a lid. Allow the solvent to come up to 10cm mark. Remove the plate and allow the vapour to dry for a few seconds.
Developing Solvents
Organo Chlorine - Cyclohexane
Organophosphates - Toluene
Carbamates -Chloroform
Synthetic Pyrethroids - Hexane + Acetone (95+5)
For unknown pesticides - Hexane + Chloroform+Acetone(6.5 + 3.5 + drops)
5. Visualisation of Chromatogram
Different chromatogenic reagents are used for different classes of pesticides.
Organo Chlorine : AgNO3 - Silica gel plate. Expose to UV radiation (254 nm) for 10 min. Steam the plate and re expose to UV for 5 min for better visibility. Brown spots on white background are observed in positive cases. Or
Plain silica gel plate is developed and is sprayed with 2- phenoxyethanol. The advantage is increased sensitivity; 0.01- 0.1µg of the compound can be detected. AgNO30.1g is dissolved in 1ml of water. To this add 10ml of 2- phenoxy ethanol. Dilute this solution with 200ml acetone. Spray on the TLC plate. Dry for 5min at room temperature and for 15 min at 75°C and expose TLC plate to UV light. Development of dark spots indicates the incidence of OC compounds.
Organo Phosphate: Plain silica gel plate is developed and exposed to bromine vapours for 30 sec (Apply a facemask and take utmost precaution while exposing the developed plate to bromine vapours). Spray with Bromocresol green solution (0.5% in methanol). Yellow spots on greenish blue background are observed in positive cases.
Carbamates: Plain Silica gel plate is sprayed with 1 N methanolic NaOH solution followed with a solution of p-Nitrobenzenediazoniumtetrafluroborate (NDFB). (Mix 50 mg of NDFB, 50 ml of Diethyleneglycol, and 50 ml of Acetone).
For carbaryl, blue spots on white background are detected.
In case of carbofuran, purple / violet spots are observed.
Synthetic pyrethroids: Silica gel AgNO3 plate is exposed to UV radiation (254 nm) for 10 min. Steam the plate and re- expose to UV radiation for 5 min for better visibility.
Brown spots on a white background are conclusive for pyrethroids.
6. Identification of Pesticides
Measure the distance of the spot from the origin.
Calculate the Rf.
Rf = Distance Travelled by pesticide / Distance travelled by solvent (10cm)

Spots with same Rf as that of technical are said to be certain.
7. Quantification
By measuring the area of standard and unknown
Wt of std./technical x area of sample x purity of technical
Wt of sample x area of technical/std
Or
Scrap the spot and put it in solvent and elute in GLC or HPLC -especially for quinalphos and fenvalerate.

Qualitative Detection of Thiram in grains
Grains meant for germinating purpose are treated with fungicides like thiram.
Procedure: 100 g grain sample (suspected) and 100 ml chloroform are taken in a conical flask. Shake for 5 min. Filter through whatman No.1 filter paper. Add about 5g charcoal to filtrate and shake for 1-2 min. Filter again through whatman No.1 filter paper. Add few crystals of cuprous iodide to the filtrate (10 ml). Development of amber to brown color indicates the presence of thiram. (Ramasubba Reddy, V. and Rajashekar Reddy, A. 1995).

Fujiwara's test for Halogenated hydrocarbons (DDT)
A small aliquot of the extract (test sample) is heated in a boiling water bath with 1 ml of 20 % aqueous NaOH and 1 ml pyridine. Red colour in the pyridine layer indicates a positive reaction. (NB: do not dissolve the extract in chloroform).
Or
The test sample is dissolved in n-hexane and ethanolic NaOH is added. Mixture is evaporated to dryness in a water bath. After cooling, 4 drops of carbon tetrachloride are added to redissolve the compound. On shaking vigorously with a mixture of sulphuric acid and nitric acid, a green colour develops which lasts for 1 min.
(Toxicity, Mechanism and Methods - Stewart and Stolman Vol.II)