This document discusses the process of pesticide residue analysis, which involves 4 main steps: 1) sampling the crop, 2) extracting the pesticide, 3) cleaning up the extract, and 4) analyzing the residue. It provides details on various techniques used for each step, including different extraction, clean-up, and analysis methods like gas chromatography, thin layer chromatography, and spectrophotometry. The goal of pesticide residue analysis is to accurately measure extremely small amounts of pesticides remaining in food after harvesting or storage.

1. Sampling, extraction, clean- up
and estimation of insecticide
residues by various methods
Presented By
Aaliya Afroz
Ph.D. Scholar
Department of Entomology, IGKV, Raipur (C.G)

2. Introduction
 When crops are treated with pesticides very small amounts
may remain in or on the crop after harvesting or storage.
 In order to determine any pesticide residue present in
foodstuffs analytical methods capable of accurately measuring
extremely small amounts of pesticides are required.
 Gas chromatography is the most used equipment in pesticide
analysis others include High performance liquid
chromatography (HPLC) .

3. Determination of Pesticide Residue analysis
involves 4 major steps-
1. Sampling
2. Extraction
3. Clean up
4. Analysis

4. 1. Sampling:
 Sampling can be defined as the procedure or step adopted to
obtain a representative quantity from the large consignment,
so that selected representative quantity can be handled
conveniently.
a. Collection of samples
b. Packaging and Transmission of laboratory samples
c. Sub-sampling
d. General Guidelines
e. Compositing
f. Homogenization of sample composite
g. Sample storage
h. Reserve storage

5. a. Collection of samples

6. 2. Extraction:
 Extraction is a fundamental process in pesticide residue
analysis because no in-situ technique has yet been
invented.
 It is the process by which the toxicant is transferred from the
treated bulky biological material into a solvent.
 The selection of solvents depends on the physical and
chemical properties of the toxicant which is to be extracted
and on the type of substrate where it persists.

7. Methods for extraction:
 Surface Rinsing:
This method is employed for the extraction of toxicant
from the surface of the substrate.
 Soxhlet Extraction:
This method is employed for the extraction of dry
samples.
 Macerating & Blending:
This method is used to remove the external and
internal residues of insecticides from the treated biological
material.

8. There are several extraction approaches employed such as:
 Pressurized liquid extraction(PLE)
 Supercritical fluid extraction (SFE)
 Solid-phase extraction (SPE)
 Solid-phase micro extraction (SPME)
 Microwave-assisted extraction(MAE)
 Accelerated solvent extraction (ASE)
 QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe)
method
 Matrix solid phase dispersion (MSPD)

9. 3. Clean Up
 Isolation of toxicant from interfering substances or solvent is
called clean up.
 Clean-up is done to obtain uncontaminated products for
analysis.
Commonly Used Methods in Clean up
(i) Steam Distillation
(ii) Partition
(iii) Sweep Co-Distillation
(iv) Coagulation
(v) Oxidation

10. 4. Analysis
 The estimation of residues is possible by adopting any one
of the analytical techniques.
A. Chemical assay:
The Commonly used chemical methods are
1) Spectrophotometric method
2) Ultraviolet Spectrophotometry
3) Visible spectrophotometry

11. Spectrophotometric methods:
 This technique utilizes the property of selective absorbtion of radient
energy by chemical substances.
 The quantitative analysis of insecticides in given samples is based on the
Beer & Lambert’s law.
Ultraviolet Spectrophotometry:
 This method is used for the determination of residues of BHC, DDT,
Diazinon and Parathion.
 In this method the commonly used range is from 220 to 380 nanometers
Visible spectrophotometry:
This method is most commonly used in residue analysis. In this method the
range used is from 400 to 600 nanometers.

12. Chromatographic Methods:
 4 types of Chromatographic methods are generally used in residue
analysis. They are
Column Chromatography:
 Column chromatography in chemistry is a chromatography method
used to isolate a single chemical compound from a mixture.
 Chromatography is able to separate substances based on
differential adsorption of compounds to the adsorbent; compounds
move through the column at different rates, allowing them to be
separated into fractions.

13. Paper Chromatography:
 It is an analytical method used to separate colored chemicals or
substances[1]. It is primarily used as a teaching tool, having been replaced
by other chromatography methods, such as thin-layer chromatography.
 A paper chromatography variant, two-dimensional
chromatography involves using two solvents and rotating the paper 90°
in between. This is useful for separating complex mixtures of compounds
having similar polarity, for example, amino acids.

14. Thin layer Chromatography:
 This technique is used in residue analysis for cleanup,
Identification & quantification of some of the organochlorines,
OPs and Carbamates. It is a chromatography technique used to
separate non-volatile mixtures. Thin-layer chromatography is
performed on a sheet of glass, plastic, or aluminium foil, which
is coated with a thin layer of adsorbent material, usually silica
gel, aluminium oxide (alumina), or cellulose.

15. IV. Gas Chromatography:

16. Gas-liquid chromatography GC-MS
 GC-MS is usually used in the analysis of mid- polar to non-polar compounds
whereas HPLC technique used for polar compounds. Organophosphate and
carbamate pesticides are mid polar in nature thus gas chromatography is well
suited for their analysis. GC-MS systems are equipped with extensive mass
spectral “libraries” that can be extremely useful for identification and
characterization of unknown compounds.

17. Advances in pesticides residue analysis
 Their has been a trend in advancement of analysis. Chromatography was the 1st
invention back in 1906. Gas chromatography came into use in 1960 until late
1900. High performance liquid chromatography was later used in 1970- 1990 era.
Gas chromatography mass spectrometry with advances came into use from
2000-to date.

18.  In this method the living organisms are used for determining the insecticidal
residues. Among insects, Drosophila, Adult house flies and larvae of mosquitoes
are commonly selected as test insect. The primary disadvantage of this method is
an inherent lack of specificity.
B. BIO ASSAY: