TLC is a useful technique for qualitatively and semi-quantitatively analyzing chlorinated pesticide residues in food products. The procedure involves preparing standard and sample solutions, spotting them on a TLC plate coated with silica gel, developing the plate in n-hexane, detecting spots using silver nitrate under UV light, and comparing sample Rf values to standards to identify residues. TLC allows for easy, inexpensive, and portable detection of pesticides in foods and has been established as an effective method by the AOAC. However, TLC only provides semi-quantitative results and has lower accuracy than techniques like HPLC.

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THIN LAYER CHROMATOGRAPHY FOR
IDENTIFICATION OF CHLORINATED
PESTICIDES IN FOOD PRODUCTS
Introduction
Pesticides, in general, are chemicals used worldwide in
agricultural production to destroy or control weeds, insects,
fungi and other pests. Some of these remain on food as
residues or contaminants, thus posing significant health risk
to consumers.
These are usually present in trace amounts and so it is very
difficult to detect and analyse them. But many analytical
methods are now available for analysis of pesticides in food
products. The important chromatographic methods include
TLC (Thin Layer Chromatography), GLC (Gas Liquid
Chromatography) and HPLC (High Pressure Liquid
Chromatography).
TLC is useful for both qualitative as well as semi-
quantitative analysis of chlorinated pesticides in food
products.
What are chlorinated pesticides?
Chlorinated pesticides or chlorinated hydrocarbon pesticides
are nerve agents used in agriculture as pesticides, around
homes as termicides and in grains as fungicides. These
chemicals were designed to attack the nervous system of
pests which leads to overstimulation of nerves and

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eventually death. Some properties of chlorinated pesticides
include:
 They have low polarity and so water insoluble.
 They degrade in the environment slowly and their half
lives generally vary from 2 to about 16 years in soil.
 They are lipophilic and thus show bioaccumulates in
fatty tissues of animals.
 They show biomagnification in food chain.
Examples include aldrin, methoxychlor, DDT, BHC,
heptachlor, dieldrin.

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The food stuffs which may contain chlorinated pesticide
residue include:-
1. Leafy and root vegetables like spinach, cucumber
(absorbed alongwith nutrients from contaminated soil).
2. Fruits (via dust particles).
3. Dairy products (since chlorinated pesticides get stored in
fatty tissues of animas, dairy products may contain
these).
4. Meat (since they have property of fat solubility and
bioaccumulation, these may be present in meat of
animals exposed to them by their natural environment or
ground water contamination).
5. Fish (from contaminated water).
TLC for Analysis of Chlorinated
Pesticides
Thin Layer Chromatography (TLC) is a well established and
widely used separation technique having innumerable
applications in the areas of food analysis. Chlorinated
pesticides are best detected and analysed by TLC.
Standard AOAC (Association of Analytical Chemists)
method for detection and identification of chlorinated
pesticides recommends n-hexane as the developer and Silica
gel – G as the stationary phase. The stationary phase is
either mixed with AgNO3 or dipped in 0.1% aqueous
AgNO3 after separation. When the plate is radiated with
short wavelength UV radiations in the presence of chlorine
substance, AgCl will be formed, which reacts with light to
elemental Ag; thus giving black or dark spots on the plate.
Identification is done by comparing the Rf values so

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calculated with the Rf value of the standard pesticide
solution.
PROCEDURE
Preparation of Standard Pesticide Solution: First of all a
stock solution is prepared which is then diluted to required
working standards.
1. Preparation of stock solution (1mg/mL) – Weigh 10 mg
of pesticide standard reference and transfer in 10mL
volumetric flask. Dissolve and make up in n-hexane or n-
heptane.
2. Preparation of intermediate solution (10 μg/mL) – Pipette
out 1 mL of stock solution in a 100 mL volumetric flask
and make up the volume with n-hexane or n-heptane.
3. Preparation of working standard (for example, 0.1
μg/mL) – Pipette out 1 mL of 10 μg/mL solution in 100
mL volumetric flask and make up the volume with n-
hexane or n-heptane.
The analysis of chlorinated pesticide residues in food
products includes the following steps:-
1. Sample extract preparation: The food sample to be
analysed is chopped, blended and homogenized. This
thoroughly mixed test portion is extracted with
acetonitrile (in case of high water containing foods) or
aqueous acetonitrile (in case the food has low water
content).
Fat is extracted from fatty food and partitioned between
petroleum ether and acetonitrile. The petroleum ether
layer containing extracted fat is discarded.

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For removal of co-extractives, the aliquot (in case of non-
fatty foods) or entire solution (in case of fatty foods) of
the acetonitrile layer is diluted with water and then
extracted in petroleum ether; the aqueous layer is
discarded. Subsequent washings are done with water.
The solvent layer is then transfer in glass stoppered
cylinder and anhydrous Na2SO4 is added to it for removal
of remaining moisture; the extract should not remain with
Na2SO4 for more than one hour as it may adsorb
chlorinated pesticide residues. It is then filtered and
concentrated (by evaporation) to about 5 – 10 mL.
Now, this concentrate is taken in a separatory funnel and
100 mL of petroleum ether saturated with acetonitrile is
added to it. After vigorous shaking, the petroleum ether
layer is discarded and the acetonitrile layer is collected
and concentrated (by evaporation) to about 10 mL.
Finally, the chlorinated pesticide residues are extracted
from this acetonitrile using n-hexane.
2. Application of the sample spot on the TLC plate and
development of the plate: Thin coating of Silica gel G
on a glass plate is used as the stationary phase in the
analysis of chlorinated pesticides. The sample is spotted
almost 2 cm from the bottom of the plate.
Standard syringe with a flattened needle (which uses
solvent flush technique) is used for spotting the sample.
About 1 μL of solvent is drawn into the barrel, followed
by an air space and then required amount of sample. The
exact sample value is read and then it is discharged onto
the plate.
Similarly, a spot of the standard is also discharged on the
plate, at an appropriate distance from the sample spot.

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The spotted plate is then introduced into the developing
tank; containing n-hexane as developer for chlorinated
pesticides. The developing tank is tightly closed and
sheets of heavy filter paper soaked with
solvent/developer are used to line the inside of the tank to
achieve vapour saturation.
3. Detection: After the chromatogram has developed for a
distance of about 10-20 cm, it is taken out of the
developing chamber, dried and then sprayed with
detecting reagent, i.e., 0.1% aqueous AgNO3 solution
followed by exposure to sunlight or UV light for about 10
minutes. Black or brownish spots are visible.
4. Identification: The identification is made from the
migration distance of spots. In the resulting
chromatogram, spots are characterized by Rf values. The
Rf value is defined as:-
𝑅 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑓𝑟𝑜𝑚 𝑠𝑡𝑎𝑟𝑡𝑖𝑛𝑔 𝑙𝑖𝑛𝑒 𝑡𝑜 𝑐𝑒𝑛𝑡𝑟𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑝𝑜𝑡
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑡𝑎𝑟𝑡𝑖𝑛𝑔 𝑙𝑖𝑛𝑒 𝑡𝑜 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
The Rf value determines the velocity of the movement of
spot relative to that of developer front.
An agreement of about 2 mm in migration distances of
the unknown and standard spot is considered adequate for
tentative identification, since the sample spot may be
slightly affected by co-extractives despite clean up.

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The Rf value range of some common chlorinated
pesticides (developer – n-hexane) is:-
PESTICIDE Rf VALUE RANGE
Aldrin 0.78 – 0.81
DDT 0.59 – 0.62
Perthane 0.48 – 0.50
BHC (Lindane) 0.39 – 0.41
Dieldrin 0.17 – 0.19
Methoxychlor 0.10 – 0.12
5. Semi – Quantitative Analysis: For quantitative
estimation, visual comparison between samples and
standard is accurate to about ±20%. The comparison is
done by taking in consideration either the intensity of the
spot or the area of the spot, or in some cases both
intensity and area of the spot. The pesticide content of the
sample is calculated by the formula:-
𝑃𝑒𝑠𝑡𝑖𝑐𝑖𝑑𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡(𝑝𝑝𝑚) =
𝜇𝑔 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑝𝑜𝑡 𝑓𝑟𝑜𝑚 𝑣𝑖𝑠𝑢𝑎𝑙 𝑒𝑠𝑡𝑖𝑚𝑎𝑡𝑖𝑜𝑛
𝑇𝑜𝑡𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛 (𝑔) × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 =
𝜇𝐿 𝑠𝑝𝑜𝑡𝑡𝑒𝑑
𝑇𝑜𝑡𝑎𝑙 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 𝑣𝑜𝑙𝑢𝑚𝑒
Merits and Demerits
MERITS:-
1. TLC analysis can be preformed anywhere with ease.
They can be moved away from laboratory into field
without losing their ability to detect pesticide.

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2. Since sophisticated instrumentation is not required, it is
relatively inexpensive compared to other methods.
3. It is easy to use and less time consuming.
DEMERIT:-
The only demerit of TLC method is that the degree of
accuracy is low.
References:-
1. fssai Manual of Methods of Analysis of Foods
PESTICIDE RESIDUES.
2. U.S. Congress, Office of Technology Assessment,
Pesticide Residues in Food: Technologies for Detection.
3. Thin-Layer and Liquid Chromatography and Pesticides
of international importance, Volume 7;edited by Joseph
Sherma, Gunter Zweig
4. Handbook of Food Science, Technology, and
Engineering; edited by Y. H. Hui, Frank Sherkat
5. Analytical Chemistry; B.K. Sharma
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