In this assignment file i will demonstrate the process involved in Thin layer chromatography for Chlorinated Pesticide determination. if u like this work feel free to share this file. thank you.

1. 1
Bangabandhu Sheikh Mujibur Rahman Science and
Technology University, Gopalganj-8100
An assignment on
“Thin layer chromatography for Chlorinated Pesticide
determination.”
Course Code: ACCE-514
Course Title: Food Processing Engineering.
Submitted by Submitted To
Name: Bapi Mondal
ID No: 20151207052
Year: M.Sc. (Engg.)
Semester: 1st
Dept. of Applied Chemistry &
Chemical Engineering
BSMRSTU
Rifat Ara Masud
Lecturer
Dept. of Applied Chemistry &
Chemical Engineering
BSMRSTU
Date of submission: 25-11-2020

2. 2
“Thin layer Chromatography for Chlorinated Pesticide determination”
1. Pesticide
Pesticides are chemical compounds that are used to kill pests, including insects, rodents,
fungi and unwanted plants (weeds). Pesticides are used in agriculture, to kill pests that
damage crops. Pesticides are a group of chemicals used for the destruction of insects, weeds,
fungi, bacteria, etc. They are generally called insecticides, fungicides, bactericides, herbicides
or rodenticides. Most of the pesticides have the ability to destroy a wide variety of pests or
weeds, but some are developed against specific pests or pathogens.
Pesticides can be grouped according to the types of pests which they kill:
Insecticides –Kill insects, Herbicides – kill plants, Rodenticides -kill rodents (rats and mice),
Bactericides –kill bacteria, Fungicides –kill fungi, Larvicides - larvae.
A pesticide is any substance used to kill, repel, or control certain forms of plant or animal life
that are considered to be pests. Pesticides include herbicides for destroying weeds and other
unwanted vegetation, insecticides for controlling a wide variety of insects, fungicides used to
prevent the growth of molds and mildew, disinfectants for preventing the spread of bacteria,
and compounds used to control mice and rats. Because of the widespread use of agricultural
chemicals in food production, people are exposed to low levels of pesticide residues through
their diets.
Effects of pesticides ion human health: The general population is exposed to low levels of
pesticides through food and water, and these are not typically cause for concern. People at
higher risk of adverse health effects are those who work directly with pesticides, such as
agricultural workers, and those who are in the immediate area when pesticides are applied.
People not involved with applying the pesticide should avoid the area during and
immediately after its use.
After countless studies, pesticides have been linked to cancer, Alzheimer's Disease, ADHD,
and even birth defects. Pesticides also have the potential to harm the nervous system, the
reproductive system, and the endocrine system.
2. Chlorinated pesticides
Chlorinated pesticides are a small but diverse group of artificially produced chemicals
characterized by a cyclic structure and a variable number of chlorine atoms. Most members
of the group are resistant to environmental degradation and relatively inert toward acids,
bases, oxidation, reduction, and heat.

3. 3
Chlorinated pesticides list or Example:
Chlorinated pesticides are chlorinated hydrocarbons used extensively from the 1940s through
the 1960s in agriculture and mosquito control. Representative compounds in this group
include DDT, methoxychlor, dieldrin, chlordane, toxaphene, mirex, kepone, lindane, and
benzene hexachloride.
Chlorinated pesticides list
 Dicofol,
 Eldrin,
 Dieldrin,
 Chlorobenziate,
 Lindane, BHC
 Methoxychloro
 Aldrin, Chlordane,
 Heptaclor, Endosufan,
 Toxaphene,
 Chloro propylate
Most commonly used pesticides: Chlorpyrifos (chlorinated pesticides) one of the most
widely used chlorinated pesticides. Introduced by Dow Chemical in 1965, chlorpyrifos is the
most widely-used pesticide on crops, including corn, soybeans, broccoli, and apples, and is
also widely used in non-agricultural settings like golf courses. hlorpyrifos works by attacking
insects’ nervous systems. At high doses, such as what farmers get exposed to when they
spray pesticides, it can cause people to experience nausea, dizziness, and confusion.
Health effects of Chlorinated pesticides
Exposure to organo chlorine chemicals (Chlorinated pesticides) has been associated with
many deleterious effects on human health, such as, hormone related conditions
(endometrisios, infertility), cancer of male and female reproductive system, immune toxicity,
neurotoxicity and spontaneous abortions.
3. Analytical method for Pesticide determination
The analysis of pesticide in biological samples continues to presents challenges to analysts.
The complexity and the diversity of matrices in biological materials, the low concentrations
of pesticides in samples of fruit and vegetables. Target analytes must, therefore, be isolated
from matrices and then be enriched before the final determination can be undertaken. there
are various method determination of chlorinated pesticides which is listed below:
I. Thin layer chromatography (TLC)
II. Gas Chroamtograpy Mass spectrometry (GC-MS)
III. High performance liquid chromatography(HPLC)
IV. Multi residue method (MRM)
V. Aceton extraction method
VI. QueChers methods
VII. Liquid Chromatography-Mass Spectrometry (LC-MS)

4. 4
4. Thin-layer chromatography (TLC)
Thin-layer chromatography (TLC) 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. On completion of the separation, each component
appears as spots separated vertically. Each spot has a retention factor (Rf) expressed as:
Rf =
Thin-layer chromatography (TLC) depends on the separation principle. The separation relies
on the relative affinity of compounds towards both the phases. The compounds in the mobile
phase move over the surface of the stationary phase. The movement occurs in such a way that
the compounds which have a higher affinity to the stationary phase move slowly while the
other compounds travel fast. Therefore, the separation of the mixture is attained. On
completion of the separation process, the individual components from the mixture appear as
spots at respective levels on the plates. Their character and nature are identified by suitable
detection techniques.
5. Thin layer chromatography for Chlorinated pesticide determination
A rapid sensitive method for the application of thin-layer chromatography to the detection
and estimation of chlorinated organic pesticide residues has been developed. Compared to
paper chromatography the method is faster, more sensitive, and in most cases more specific.
Chlorinated pesticide residues were identified in extracts of various food products at
concentrations as low as 1 part per billion, these results were verified by gas-liquid
chromatography. Because of its simplicity, speed, and sensitivity, thin-layer chromatography
is suitable as a rapid screening method or as a confirmatory method in conjunction with gas-
liquid chromatography or other analytical methods for chlorinated pesticide residue analysis.
 Apparatus: Volumetric flask (10-200ml), Separating funnel (250-500ml),
Chromatographic column 2 cm diameter, Evaporator, Rotator for evaporation control
under desired pressure.
 Thin layer chromatograph apparatus: Desage applicator, Sandwich chamber, detector,
ultraviolet light source.
 Reagents: Dimethyl formamide, Ethanol. Ethyl ether (anhydrous) Hexane, redistilled
Light petroleum, redistilled. Heptachlor solution Heptachlor epoxide solution. n-Heptane,
n-Hep-tane with 1% or 2% aceton.
a) Developing solvent for TLC: Redistilled hexane and anhydrous ethyl ether.
b) Adsorbents: Aluminum oxide and Silica gel.

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c) Chromogenic agent: Dissolve 0.100 g AgN03 in 1 ml water, add 10 ml 2-
phenoxy-ethanol dilute to 200 ml with acetone, reagent grade, add a very
small drop of 30% H202, and mix.
d) Standard solutions: Stock Solution mixture of aldrin, hepta-chlor, Perthane,
lindane, heptachlor epoxide, methoxychlor. Into one 10 ml glass-stoppered
flask, weigh 0.1 g of each pesticide; into a second flask, 0.05 g of each.
Dissolve in ethyl acetate, dilute to 10 ml, and mix.
e) Stock solutions of individual pesticides: Prepare 10 and 5 mg/ml separate stock
solu-tions of toxaphene, chlordane, and BHC.
f) Diluted stock solution: Dilute each stock solution to contain 2,1,0.5,0.2,
0.1,0.05, 0.02, and 0.01 mg/ml for each pesticide or mix-ture of pesticides
present.
1. Spraying agent Preparation for Chlorinated pesticide determination by TLC.
Fig: Spraying agent preparation for TLC
2. Thin layer plate preparation
Fig: Preparation process of TLC plate.
6. Chlorinated pesticide determination by TLC method
Chlorinated pesticide determination by TLC methods needs some steps and these steps are
very crucial for determining pesticides. The main steps of this experimental procedure are
represented below:
Apply a 0.25
mm thick
layer of
aluminium
oxide and pre
wash the
adsorbent
layer
Activate the
pate at150 0c
for 2 hours
before use
Develop the
plates with 11
cm and sprary
with silver
nitrate
Expsoe thye
short wave
UV light for
30 min to 2
hours with
respect to
concentration.
1Ml of
H2O
200 ml
Aceton
0.01g silver
nitrate
2-phenoxoethanol
30% H2O2

6. 6
Fig: Chlorinated pesticide determination by TLC methods process flow diagram.
For best results, prewashing the prepared coatings with distilled water to remove chlo-
rides prior to analytical use is recommended. This is particularly important for silica gel
coatings. On unwashed plates of A1203 G, the maximum sensitivity that can be
attained is 0.05 µg for all compounds examined except chlordane. The spotting technique
for thin layer chromatography, in general, is similar to that for paper
chromatography. Exposure times for prewashed plates of A1203 are not critical
because backgrounds do not darken after a prolonged period of exposure to
ultraviolet light (3-4 hours) nor on subsequent standing. Thus plates should be
exposed as long as necessary to fully de-lineate spots of the lowest concentration
sought. Exposure times for silica gel are very critical. Spots appear rapidly during
expo-sure to ultraviolet light, but the background also darkens very rapidly and
continues to darken on standing. Therefore these plates should be exposed the
minimum time necessary to fully delineate spots of lowest con-centration sought,
and evaluated without delay.
 Chlorinated pesticide determination in Butter, Vegetables oils and Similar Foods
Melt 100 g of butter and margarine and filter through a folded paper. Transfer the 40.0 g of
vegetable oil or filtered fat to a 200 ml volumetric flask and make up to the mark with
hexane. Transfer 50 ml of the sample solution to a a 250 ml separating funnel and add 1 ml of
standard solution. Shake with 50 ml of dimethyl formamide solution for 2 min. after
separation transfer the dimethylformamide layer to a separating funnel containing 400 ml of
2% Na2SO4 solution. Repeat the process for 3 times and transfer the extracts to the funnel.
Add 50 ml of hexane to the combined extracts and shake vigorously for 2 min. Discard the
aqueous layer and wash the hexane layer with 200ml of Na2SO4 solution. Transfer the
hexane layer to the evaporator and rinse the separating funnel with small portions of hexane
and evaporate the combined extracts and washing at reduced pressure at 350
c to 2 ml.
Pack a chromatographic column with 10g of activated Al2O3.and put a 3.5 cm layer of
anhydrous Na2SO4 on the top. Pre wet the adsorbents with 25 ml of hexane.
Preparation of adsorbent layer
Prewashing of adsorbent layer
Sample spotting.
Development of plates.
Spraying of plates
Exposure

7. 7
Transfer the evaporated extracts quantitatively to the column and elute with 100 ml of
hexane. Evaporate the elute at reduced pressure at 350
c to 5ml.transfer quantitatively to a
volumetric flask and make up to the mark with hexane. Examine the solution by gas
chromatography and after further concentration by thin layer chromatography. Using this
clean up an amount of extracts corresponding to 10 g of fat can be analysis. And finally the
presence of chlorinated pesticide can be checked by using detector of TLC.
 Chlorinated pesticide determination in Milk
Chlorinated pesticide determination in Milk is a quite simple method and that is understood
easily by a process flow diagram. And this process flow diagram for chlorinated pesticide
determination by TLC method is represented below:
 Chlorinated pesticide determination in Meat
1. Mince or slice 100 g of meat in mincing machine and mix thoroughly .Homogenize
the meat with 50 ml of ethanol.
2. Transfer the mixture to a 500 ml centrifuge flask with 90 ml of water. Rinse the
homogenize with 50 ml of ethanol and transfer to the flask.
3. Add 1 ml of heptachloro epoxide soluion and shake for 10 min. add 50 ml of ethyl
ether and shake it.
4. Finally add 50 ml of light petroleum and check the TLC detector machine and
determine the chlorinated pesticide in meat of fish food.
• Add 1 ml of heptachloro epoxide solution , 1g of potasium oxalate and 100 ml of
ethanol to 100 g of milk in each of two 500 ml centrifuge bottles and shake for 10
min .
• add 50ml of ethyl ether and shake vigorosuly and also add 50 ml of light
petrolium and shake for 50 min.
• Until the phase separation is complete centrifuge the solution for 45 min.Then
transfer the solution into light petrolium ,ethyl ether layers from both bottles with
a pipette to the separating funnel.
• Shake the residue in centrifuge flask with 50ml of light petrolium, ethyl ether for
5 min.also centrifuge the rest extraction and repeat it.
• collect the light petrolium ethyl ether in 1 L of separating funnel and shake with
100 ml of water.
• Evaporates the organic solvents at reduced pressure at 35 0c .transfer the resiude
ith 50ml of hexane to a 250 ml separating funnel.
• finally shaking the solution with 50 ml of dimethyl formamide solution and check
via the TLC detector and determine the chlorinated pesticide in milk .

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Chlorinated pesticide in Meat determine by TLC Process represented by a figure:
So finally we can say that the Thin layer chromatography technique for determine chlorinated
pesticide is better with comparing among all the analytical technique for chlorinated pesticide
determination. And this technique is advantageous.
7. References
1. https://www.who.int/news-room/q-a-detail/chemical-safety-pesticides
2. https://www.niehs.nih.gov/health/topics/agents/pesticides/index.cfm
3. https://www1.health.gov.au/internet/publications/publishing.nsf/Content/ohp-enhealth-
manual-atsi-cnt-l~ohp-enhealth-manual-atsi-cnt-l-ch5~ohp-enhealth-manual-atsi-cnt-l-
ch5.7
4. https://www.nature.com/scitable/blog/green-science/the_dangers_of_pesticides/
5. http://sitn.hms.harvard.edu/flash/2018/widely-used-pesticide-one-year-later/
6. https://link.springer.com/chapter/10.1007/978-1-4613-9538-6_6
7. https://dhss.delaware.gov/dhss/dph/files/organochlorpestfaq.pdf
8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464684/
9. https://byjus.com/chemistry/thin-layer-chromatography/
10. https://academic.oup.com/jaoac/article-abstract/46/5/884/5732029
11. https://www.hsj.gr/medicine/mechanisms-of-actions-and-health-effects-of-
organochlorine-substances-a-review.php?aid=3667
12. https://www.researchgate.net/publication/226241207_Improved_analytical_procedure_fo
r_determination_of_chlorinated_pesticide_residues_in_human_serum_using_solid_phase
_disc_extraction_SPDE_single-step_clean-up_and_gas_chromatography
13. https://www.tandfonline.com/doi/abs/10.1080/04345546609415378?journalCode=ttpma2
0
Shake
for 10
min
50 ml
Ethanol
Slice
100 g of
meat
500 ml
centrifuge
flask with 90
ml of water
Flask
50 ml of
ethyl ether,
heptachloro
epoxide
Flask
50 ml of light
petroleum
Detector to
determine
Pesticide

9. 9
14. https://pubmed.ncbi.nlm.nih.gov/4179951/
15. Determination of Some Chlorinated Pesticides in Vegetable Oils, Margarine, Butter,
Milk, Eggs, Meat, and Fish by Gas Chromatography and Thin-Layer Chromatography by
Norén, Koidu; Westöö, Gunnel. DOI number: 10.3891/acta.chem.scand.22-2289. Pages:
2289-2293.
16. Kovacs, B. M. F. (1963). Thin-Layer Chromatography for Chlorinated Pesticide Residue
Analysis. 46, 884–893.
17. https://www.tandfonline.com/doi/abs/10.1080/10826076.2017.1298024?scroll=top&need
Access=true&journalCode=ljlc20
18. https://academic.oup.com/jaoac/article-abstract/46/5/884/5732029
19. https://www.longdom.org/open-access/chromatographic-determination-of-pesticides-in-
foods-and-food-products-2155-9600.1000126.pdf
20. https://www.sciencedirect.com/science/article/abs/pii/002196739285688P