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

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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.

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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.

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