DETERMINATION OF PESTICIDES
(ORGANOPHOSPHATE) IN SOIL SAMPLE USING
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
RYAN MBAKO KUSASA
BSc GEOLOGY
17000476
OBJECTIVE
To determine the concentration of organophosphate n the soil sample using HPLC technique.
INTRODUCTION
Soil is an important part of the ecosystem, and closely related to human survival. As part of
the human environment, contaminated soil may cause a serious risk to human health.
Organophosphorus pesticides (OPPs) enter the soil ecosystem because of direct spraying on
the soil surface during pesticide application in agriculture; the drop from the foliage and
stems by the washing of rain and the rotting of plant bodies containing OPPs residues in the
soil. The majority of OPPs show high toxicity. Therefore, the analysis of OPPs residue in the
soil is vital in environmental protection and human health (Baig, S.A.,et.al (2009).
Sample pre-treatment is one of the most important and key procedures used when doing a
pesticide residue analysis. In the past normally, the determination of trace levels of pesticide
residues depended on the use of these methods; liquid–liquid extraction (LLE) and solidphase extraction (SPE). However, some of the main disadvantages of LLE include its high
expense, prolonged run time, and the large volumes of toxic organic solvents it requires.
Hence with the SPE typically is less time-consuming than LLE, but it requires column
conditioning and elution with organic solvents. In the latest decade, more environmentally
friendly techniques, such as supercritical fluid extraction, microwave-assisted extraction, and
accelerated solvent extraction have been developed to determine pesticides in soil samples.
Seebunrueng, K., et,al(2015)
All these techniques have the advantages of a short extraction time and the require fewer
organic solvents, but the instruments required are expensive. Hence, there is an increasing
demand to develop a rapid, easy, and sensitive sample pre-treatment method for the
determination of such OPPs in soil.
In this experiment, concentration of organophosphate in the soil sample is determined using
the HPLC technique.
MATERIALS/ CHEMICALS







Acetonitrile 1L
Acetone
Hexane
Soil 100g
Volumetric flask
Conical flasks
Glass syringe
EXPERIMENTAL PROCEDURE
25004.5mg of soil was took and extracted with acetone: hexane (1:1 v/v,250 mL), and was
agitated on a shaker (30min), the eluate was then collected, filtered and concentrated to
100mL.A stock solution of the reference standard of pesticide in acetone hexane was
prepared. Serial dilutions form the stock reference standards of pesticides for calibration was
carried out and labelled as standard 1, standard 2 and standard 3. HPLC was run as per SOP,
the initial mobile-phase composition was acetonitrile-water (55-45 v/v), which was held
constant from 0.00 to 11 min. At 11.00min ,the acetonitrile was increased to 65%.From 11.10
min, the gradient was programmed linearly to a final composition of acetonitrile-water (70-30
v/v)at 34.00 min. Separations were performed on a 250mm × 4.6 mm C-18 column with a
particle size of 5µm.A guard column (10mm ×4.6 mm) was used along with the analytical
column. The flow rate was kept at 1.0 mL/min, and injection volume was 20µL.The
absorption spectra of the organophosphate pesticides were obtained by scanning the
wavelengths in the range 190-350 nm in 2-nm increments. The individual wavelengths were
monitored based on sensitivity and/or interference considerations. The individual
wavelengths monitored were 202,207,230,250,274, and 314 nm. HPLC was run again at
11.00min, and the acetonitrile was increased to 65%. From 11.10 min, the gradient was
programmed linearly to a final composition of acetonitrile-water (70-30 v/v) at 34.00 min.
The aliquot of clear solution from prepared sample was taken and the sample was injected.
RESULTS
standards
Peak Area( mAu)
Concentration (ppm)
1
2550.4
2
2
4132.4
4
3
5623.5
6
4
6797.0
8
5
6961.8
10
sample
4855.0
2.5
Table 1: results of HPLC showing peak area with corresponding concentration
Fig 1: graph showing concentration of standards to corresponding peak areas
Calculations
Concentration of sample = 𝐴 − 𝐼 ÷ 𝑚
Where A: peak area
= 4855- 4638.55÷ 0.97437
= 94.1
I: Y- Intercept
m: slope
DISCUSSION
High performance liquid has the ability to separate, identify and quantitate the compounds that
are present in any sample that can be dissolved in a liquid.
The output of the HPLC was recorded as a series of peaks - each one representing a compound
in the mixture passing through the detector and absorbing UV light. One Can also use the peaks
as a way of measuring the quantities of the compounds present. The area under the peak is
proportional to the amount of sample which has passed the detector, and this area can be
calculated automatically by the computer linked to the display. If the solution of sample was
less concentrated, the area under the peak would be less (Engelhardt, H. (1985), although the
retention time will still be the same. The area under the peak was used to calculate
concentration of the sample as shown on the calculations above. Some problems encountered
during this experiment are a lot of chemical spillage throughout the process which could end
up giving wrong results because of insufficient chemical or inaccurate measurement proportion
used which could have been avoided by carefully pouring solution into beakers and flasks and
onto the filter.
CONCLUSION
The concentration of polyaromatic hydrocarbon anthracene in pesticides found in soil sample
is 94 ppm.
REFERENCE
Baig, S.A., Akhtera, N.A., Ashfaq, M. and Asi, M.R., 2009. Determination of the
organophosphorus pesticide in vegetables by high-performance liquid
chromatography. American-Eurasian Journal of Agriculture and Environmental
Science, 6(5), pp.513-519.
Betowski, L.D. and Jones, T.L., 1988. The analysis of organophosphorus pesticide samples
by high-performance liquid chromatography/mass spectrometry and high-performance liquid
chromatography/mass spectrometry/mass spectrometry. Environmental science &
technology, 22(12), pp.1430-1434.
Engelhardt, H. (1985). HPLC [1] by HPLC [2]. Chromatographia, 20(6), pp.333-334
Seebunrueng, K., Santaladchaiyakit, Y. and Srijaranai, S., 2015. Vortex-assisted low density
solvent liquid–liquid microextraction and salt-induced demulsification coupled to high
performance liquid chromatography for the determination of five organophosphorus pesticide
residues in fruits. Talanta, 132, pp.769-774.