IP International Journal of Forensic Medicine and Toxicological Sciences 2021;6(1):17–19

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IP International Journal of Forensic Medicine and

Toxicological Sciences
Journal homepage: http://www.ijfmts.com/

Original Research Article

Detection of Chlorpyrifos in human blood using thin layer chromatography

Sneha Yadav1,2, *
1 National Institute of Criminology and Forensic Science (LNJN), Rohini, New Delhi, India
2 Dept. of Forensic Science, Galgotias University, Greater Noida, Uttar Pradesh, India

ARTICLE INFO ABSTRACT

Article history: Chlorpyrifos is considered to be a broad- spectrum pesticide. It is a most commonly used kind of pesticides
Received 19-01-2021 for killing of variety of insects, pest’s worms in agricultural sector. It is easily available in market and
Accepted 20-03-2021 is highly toxic in nature. The suicidal cases, accidental cases due to poisoning of chlorpyrifos have been
Available online 28-04-2021 reported in large number due to easy to access for this pesticide. Even though many instrumental techniques
like UV, HPLC are available for its identification but the analysis cost is high. In this paper we have detected
the chlorpyrifos in human blood by using TLC (Thin Layer Chromatography) using different types of
Keywords: solvent system. This technique is fast, reliable, cost effective and less expensive than other sophisticated
Pesticides
techniques.
Toxicity
Chromatography © This is an open access article distributed under the terms of the Creative Commons Attribution
Chlorpyrifos License (https://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited.

1. Introduction
Chlorpyrifos is an organophosphate pesticide which is used
to killing variety of insects, pests, worms in agricultural
sector. The IUPAC name is O, O-Diethyl O-3,5,6-
trichloropyridin-2-yl phosphorothioate its chemical formula
isC9 H11 CI3 N03 PS with molar mass 350.57g/mol9(Eaton et
al;2008). 1 It was introduced by dowed chemical company
in 1965 specially targeting or controlling mosquitoes, flies
many foliage crops pests, household pests and aquatic larvae Fig. 1: Chemical structure
(Rathod et al;2017). 2 The high use of chlorpyrifos results
into poisoning to non-target species like human beings. It
has been observed that in recent years the suicidal cases, Chlorpyrifos falls under the category of organophosphate
accidental cases of poisoning have been increased but pesticides and is derived through thiophosphoric acid and is
still due to being a broad-spectrum pesticide it cannot be a type of neutral non-volatile poison (Racke et al; 1993). 5
eliminated from market (Ambali et al ;2007). 3 It has been It is available in various formulation like liquids form
reported worldwide that chlorpyrifos causes 1000 deaths per commonly as sprays, wet table powders, in dry and flow
year (Pawar et al;2015). 4 able solutions and many more. The chlorpyrifos powder
The common names for chlorphyrifos are Brodon, can be formed in many different colours from colourless
Bolton Insecticide, chlorpyrifos –ethyl, Detmol UA, Dowco crystals, gray crystals, yellow crystals, to various granular
179, Empire, Eradex, Nufos, Tricel, Lorsban, Hatchet etc. form. Mainly it smells like sulphur compounds and is
practically insoluble in water and completely soluble in
* Corresponding author. different organic solvents like acetone, ether. Due to the
E-mail address: snehay2108@gmail.com (S. Yadav). chlorine containing group in its structure the lipid solubility

https://doi.org/10.18231/j.ijfmts.2021.005
2581-9844/© 2021 Innovative Publication, All rights reserved. 17
18 Yadav / IP International Journal of Forensic Medicine and Toxicological Sciences 2021;6(1):17–19

and half -life in body increases as a result more slow and 2.3. Extraction of Pesticide from blood
persistent lowering of enzyme cholinesterase level in body
The 10 ml of blood sample solution was mixed with 10 ml of
if compared to other thiophosphoric derived pesticides. It
10 % sodium tungstate solution and 15 ml of sulphuric acid
has been observed that there are cases of poisoning of
in a beaker and it is shaken for two minutes and then filtered.
chlorpyrifos reported mainly accidental or intentional or
The filtrate was kept reserved, the residue was washed
suicidal among them homicidal is the rarest form. And
with 15 ml portions of 0.1 N sulphuric acid, the washings
there are various factors responsible for effect of this
are collected, mixed with filtrate and then transferred into
pesticide on body which includes health of individual,
a separating funnel and then extracted thrice with 20ml
various environmental conditions, dose, frequency. This
portion of n hexane, later on the hexane layers are collected
chlorpyrifos works by inhibiting the activity of acetyl
& combined and followed by passing it through anhydrous
cholinesterase as a result it get converted into chlorpyrifos
sodium sulphate and solvent is removed by passing a stream
–oxon which is toxic than chlorpyrifos itself and act as
of air.
respiratory poison and causes more damages to neurons
and ganglia (Jung et al; 2009 and Srivastava et al;2011). 6,7
After poisoning due to ingestion of chlorpyrifos it has been 2.4. Thin layer chromatography
seen that its analysis in of very important for that purpose TLC Plates were activated at 110◦ C for about 25-30 min and
urine, blood sample is most commonly sent for toxicological then let it be cooled down at room temperature.
analysis. In our study we have extracted the residues of
chlorpyrifos from spiked blood sample and identified it the
2.4.1. Spotting
help of thin layer chromatography by using various mobile
The extract obtained through extraction is evaporated to half
phases (Jaiswal et al; 2008). 8
and then this extracted sample was spotted on TLC plates
with the help of capillary tube, the spotting was done at 1
2. Materials and Methods cm away from bottom of TLC plate, and the diameter of
spot was kept small, one more spot of standard solution
2.1. Material and reagent of chlorpyrifos was spotted on TLC plate at some distance
from sample spot. The spots were allowed to dry for few
minutes, at the sample spot, 10-15 times spotting was done
Acetic acid, Acetone, hexane, Amyl alcohol, carbon
by giving time for earlier spot to get dry.
tetrachloride, chloroform, formaldehyde, methanol, Ethanol
absolute, Platinic chloride, Potassium Iodide, Methanol,
Hydrochloric acid, Sodium sulphate anhydrous, Sodium 2.4.2. Development
Tungstate, sulphuric acid, sulphanilic acid all chemicals After spotting is completed the TLC plate is kept in
use are of Analytical grade /HPLC grade. Stoppered Saturated Mobile solvent system in TLC developing
Borosil test tubes(10ml), Borosil test tubes (10ml), chamber, here 9 different types of solvent system were used
Borosil Beakers (250ml, 500ml), Glass rod, China the plates were run in these solvent systems the plates were
dish, Glassgropper, Conical Flask (250ml), Glass Plate removed from chamber when the solvent run almost to the
(10cmx10cm), Solvent Chamber, Silica crucible, Borosil top of plate.
Measuring Cylinder (10ml, 100ml), spray bottle(Atomiser).
Chlorpyrifos purchased from local market, Blood sample 2.4.3. Visualization
collected from (Jaipur golden Hospital, Delhi) The TLC plates were air dried for 5-10 min and then
to make the spots visible the spraying reagent palladium
chloride was sprayed. Yellow color spot were observed in
2.2. Method both sample & standard. This reaction was instantaneous
and colour formation was clearly distinct and was not faded
The standard stock solution ofchlorpyrifos10ppm was out with time.
prepared by dissolving 0.01mg of chlorpyrifos in 10 ml of n- The R f value is used in quantifying the movement of the
hexane. The blood sample solution was prepared by spiking material throughout the plate. R f is equal to the distance
2mg of Chlorpyrifos in 10ml of human blood and keeping travelled by substance divided by the distance travelled
is aside for 24 hrs at 35 degree Celsius. by the solvent, the value lies between 0-1.R f value of
chlorpyrifos extracted from blood was found neatly equal to
that of standard used ll the 9 different solvent systems were
2.2.1. Preparation of Palladium chloride spraying reagent
analyzed and found good to run the sample. The R f values
Palladium chloride (prepared by 0.5g of Palladium chloride lies between (0.55-0.966). Ethyl Acetate: Cyclohexane (9:1)
in 100ml of Distilled water containing few drops of (25% resulted in highest R f value i.e. 0.96. Therefore according to
HCL). highest R f value it can be considered as good solvent system
 Yadav / IP International Journal of Forensic Medicine and Toxicological Sciences 2021;6(1):17–19 19

Table 1: TLC results obtained with different solvent systems

S. No Mobile solvent system Ratio Rf value Extracted Rf value Standard Colour of
sample sample spot
1 Hexane: Acetic acid 9:1 0.57 0.599 Yellow
2 Ethyl acetate: Cyclo hexane 9:1 0.966 0.965 Yellow
3 Benzene: Hexane 8:2 0.866 0.855 Yellow
4 Benzene: Carbon tetrachloride 6:4 0.77 0.78 Yellow
5 Formaldehyde: Acetone 6:4 0.85 0.833 Yellow
6 Benzene: hexane 5:5 0.699 0.71 Yellow
7 Amyl alcohol: Cyclo hexane 9:1 0.955 0.977 Yellow
8 Amyl alcohol: chloroform 7:4 0.56 0.55 Yellow
9 Chloroform: Methanol 9:1 0.65 0.67 Yellow

in comparison to other 8 otherwise all 9 solvent system 7. Source of Funding

can be used for detection of chlorpyrifos. The calculated
None.
R f value in 9 different solvent systems are summarize in
Table 1.
References
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The TLC of sample and standard solution performed and neurodevelopment. Crit Rev Toxicol. 2008;1(2):1–25.
R f values calculated which suggest that the Rf values 2. Rathod AL, Garg RK. Chlorpyrifos poisoning and its implications
in human fatal cases: A forensic perspective with reference
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chlorpyrifos was extracted out successfully and was able to 3. Ambali S, Akanbi D, Igbokwe N, Shittu M, Kawu M, Ayo J, et al.
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detect by TLC with the help of 9 different solvents systems.
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Mobile phases can be used for detection of chlorpyrifos in 1993.
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district. Hum Exp Toxicol. 2011;30:1458–63.
The present work describes that the detection of chlorpyrifos 8. Jaiswal AK. Extraction and detection of morphine from viscera by new
extracted from blood can be done using TLC which is solvent systems using High Performance thin layer Chromatography
a less expensive, fast and reliable technique by using 9 Plate. Indian Police J. 2008;p. 72.
different types of solvent system. The main advantage of
using TLC is minimum sample preparation is needed and Author biography
simultaneously many samples can be analyzed.
Sneha Yadav, Assistant Professor

5. Acknowledgment
I sincerely would like to appreciate and thanks Dr. J.R Sethi Cite this article: Yadav S. Detection of Chlorpyrifos in human blood
for his guidance and support during the work. using thin layer chromatography. IP Int J Forensic Med Toxicol Sci
2021;6(1):17-19.

6. Conflicts of Interest
All contributing authors declare no conflicts of interest.