Analysis of Pesticides using HPLC Muhammet Mammetkuliyev November 27, 2013 Abstract A high performance liquid chromatography (HPLC) method was developed to quantify the p,p´ -dichlorodiphenyltrichloroethane (DDT) dissolved in acetonitrile. Colorimetric detection was used with 236.4 nm incident light. The absorbance showed a linear increase in the concentration range of 10-25 ppm. A test solution with known con- centration was analyzed with the developed method. The measured and actual concentrations of test solution differed by 4.91 ppm and the percent deviation is 37.3 %. The reason for this deviation is proposed. Introduction Dichlorodiphenyltrichloroethane (DDT) was first synthesized in 1874 by a German chemistry student Othmar Zeidler. He was unaware of the insec- ticidal property of DDT. The available insecticides at the time were either expensive natural products or ineffective insecticides. The only available in- secticide was arsenic which is toxic to both animals and humans [1]. The 1
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Analysis of Pesticides using HPLC
Muhammet Mammetkuliyev
November 27, 2013
Abstract
A high performance liquid chromatography (HPLC) method was
developed to quantify the p,p-dichlorodiphenyltrichloroethane (DDT)
dissolved in acetonitrile. Colorimetric detection was used with 236.4
nm incident light. The absorbance showed a linear increase in the
concentration range of 10-25 ppm. A test solution with known con-
centration was analyzed with the developed method. The measured
and actual concentrations of test solution differed by 4.91 ppm and the
percent deviation is 37.3 %. The reason for this deviation is proposed.
Introduction
Dichlorodiphenyltrichloroethane (DDT) was first synthesized in 1874 by a
German chemistry student Othmar Zeidler. He was unaware of the insec-
ticidal property of DDT. The available insecticides at the time were either
expensive natural products or ineffective insecticides. The only available in-
secticide was arsenic which is toxic to both animals and humans [1]. The
1
discovery of the insecticidal property of DDT was done in 1939 by Paul Her-
mann Muller. His first experiment was on a fly in a cage. Later, larger scale
tests of the DDT against Colorado potato beetle, mosquito, louse and flea
proved the effectiveness the insecticide [1]. DDT was patented in Switzerland,
US, Australia and UK within 4 years of its discovery and mass production
had begun. It’s benefits came with a cost, however. It was banned in Sweden
in 1970 due to ecological reasons [2]. Its production slowed down such that
only three dedicated factories are operational in the world and they are in
US, France and India [2].
The main route for DDT intake in human is through ingestion. It has
been proposed that the main parts in the body targeted by DDT is the
nervous system and the liver [2]. In the liver, DDT is linked to the carcino-
genic activity [2]. Another study elucidated the effect of DDT on the sperm
health in men [3]. The key findings from the study are that the decrease in
sperm concentration and the decrease in the motility of the sperm in men
are associated with the DDT presence in the body [3].
Gas chromatography and colorimetric methods are widely used for quan-
tification of DDT. With gas chromatographic method, the electron capture
detectors are good for quantification of DDT as they have electronegative
chlorine atoms. Colorimetric methods, on the other hand, relies on the broad
absorption of DDT in the range of 200-285 nm. In this study, high perfor-
mance liquid chromatography (HPLC) used to identify and quantify the DDT
dissolved in acetonitrile.
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Experimental
The ideal wavelength of 236.4 nm to be used in HPLC was found using
UV/Vis spectrometer. DDT was dissolved in acetonitrile and the absorption
spectrum in 220-450 nm range was recorded. The absorbance due to acetoni-
trile was subtracted from the DDT solution. The HPLC method also used
acetonitrile a mobile phase. Four standard solutions and the test solution
were prepared using pure p,p- dichlorodiphenyltrichloroethane (Chem Ser-
vice, Lot 63-45A, Purity 97.0%) with the concentration in the range of 10-25
ppm and 13.16 ppm, respectively. The flow rate for the HPLC runs were
tested to get good peak separation in relatively short scan length. With the
resultant solvent programming, the DDT peaks were eluted at 2.13 min.
Results and Discussion
The DDT peak areas for each standard solution concentrations are shown in
Table 1. The linear fit and the experimental data agree very well as shown
in Figure 1. Our unknown solution peak area was 8385 and the measured
concentration is thus 8.25 ppm compared with the actual value of 13.16 ppm.
The difference between the measured and actual concentrations is 4.91 ppm
and the percent deviation is 37.3 %.
Concentration (ppm) 9.40 16.45 18.80 22.56
Peak area 9361 15778 17893 21089
Table 1: DDT concentrations and peak areas at 236.4 nm.
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There are two possible explanation for the observed disparity: either the
test solution concentration is indeed 13.16 ppm and that the instrumental
factors affected the measurements or that the test solution concentration is
8.25 ppm and a random error was done during the dilution. The former
reason is not convincing since the standard curve shown in Figure 1 fits very
well to the experimental data thus suggesting that the reproducibility of
the tests with our instrument is very good. Moreover, analyzing the peak
shapes of the standard and test solutions support this initial conclusion.
All peaks have a Gaussian shape and are free from irregular features such
as shoulders as shown in Figure 2. Therefore, a random error during the
unknown preparation is thought to be the real cause of the disparity.
10 15 20DDT concentration (ppm)
10000
15000
20000
Pea
k a
rea
(AU
)
Experimental data
Linear fit.y=894.0x+1008.3 (R
2 = 0.9998)
Figure 1: Absorbance of Cr(VI) standard solutions at 540 nm.
4
Conclusion
In this study, a method was developed to quantitatively analyze the pure
DDT concentration. For the separation of DDT and its analysis, HPLC
was used with acetonitrile as the mobile phase. The detector was set to
236.4 nm since DDT and acetonitrile absorptions are strong and negligible,
respectively, at this wavelength. The linear fit to the experimental DDT
absorbance values vs. concentration were in very good agreement. Therefore,
the 236.4 nm absorption of DDT molecules in the concentration range of 10-
25 ppm with acetonitrile as a solvent shows a linear behavior.
References
[1] World of Anatomy and Physiology. Gale Cengage, 2002.
[2] DDT and its Derivatives. Technical report, World Health Organization,
1979.
[3] S.E. Martenies and M.J. Perry. Environmental and occupational pesticide
exposure and human sperm parameters: A systematic review. Toxicology,
307:66, 2013.
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9.4 ppm std
16.45 ppm std
18.80 ppm std
22.56 ppm std
13.16 ppm test
Figure 2: DDT absorbance peaks for standard and test solutions at 236.4 nm.