Irving T. Glover' Oak Ridge Associated Universities Oak Ridge, Tennessee 37830 and Ann P. Minter Roane State Community Col ege Rockwood, Tennessee 37854
Analysis of Chlorinated Hydrocarbon Pesticides
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Experiments for nonscience majors
The nossible adverse ecological effects of persistent pesticide iesidues in the environment with their associated controversial issues and sometimes heated arguments are familiar and meaningful topics among most college students and the general public alike.2 Even with the nearly total ban against the use of 1,l-his(p-chloropheny1)-2,2,2trichloroethane (DDT) by the Environmental Protection Agency in 1972, there is continued disagreement with regard to the use of this and other chlorinated hydrocarhon .~ and its pesticides, particularly aldrin and d i e l d ~ i n DDT metabolite^,^ 2,2-his@-chloropbeny1)-l,l-dichloroethylene (DDE) and 4,4'-dichlorobenzophenone (DCBP), may be readily separated by thin-layer chromatography (tlc) and quantitatively determined by ultraviolet (uv) spectrophotometry. Alternatively, the compounds may be separated and quantitated by gas-liquid chromatography (glc). This article describes experiments designed to take advantage of the students' identification with the "environment crisis" and to demonstrate the analytical techniques of tlc, uv, and glc. TLC Analysis
TLC d a t e s coated with Silica Gel mav . be nrenared . . bv student; or p u r c h a ~ e dEastman .~ tlc sheets0 are also sati;. factorv. Senaration of DDT. DDE. and DCBP is achieved with hexaie as the chromatography solvent. The components of the chromatogram may he visualized in iodine vapor or by uv illumination. A standard solution of DDT is prepared by dissolving a few mg of DDT in about 5 ml of CHC13. Standard solutions of DDE and DCBP' are prepared similarly. TLC plates are lightly lined with pencil about 1.5 cm from the bottom and spotted on the line with the standards about 1.5 cm apart using a caoillarv tube. Unknowns containing 1, 2, or ail 3 components in C H C are ~ ~spotted in the same manner on tlc plates. The plates are placed in chromatography tanks (wide-mouth jars 17.5-cm high are convenient tanks) containing about 0.5 cm of hexane and the tanks are covered. After the solvent front has moved 12-15 cm (about 30 min), the plates are removed, the position of the solvent front is marked, and the plates are dried and placed in a second tank containing a few crystals of iodine. Spots begin to appear on the plates within 1 min (spots may also be visualized under uv illumination). The plates are removed and the spots circled with a pencil tip or stvlus. The comnonent(s) of the unknowns are identified by comparison of the spot positions of thp unknowns and the standards. K , values are calculated for the three compounds distance traveled hy component distance traveled by solvent front UV Determinations
Quantitative analysis of DDT and DDE can be accom.. plished by scraping the resolved spots of these pesticides and an internal standard from the tlc plate, leaching each component into 95% ethanol, and measuring the uv ah-
sorbance relative to the internal standard. Benzophenone (BP) is a convenient intemal standard because it has a large uv extinction coefficient and separates readily from DDT and DDE bv tlc. Use of an internal standard makes i t unnecessary to" deliver known or reproducible volumes of solutions when spotting the chromatogram ~ l a t e s . ~ A calibration plot i' prepared b y chromatographing mixtures containing known concentrations of DDTQ and BP. The separated components are visualized under uv illumination, scraped off the tlc plates into 25-ml beakers, and leached into 6-8 ml of 95% ethanol. The solutions are filtered into 10-ml volumetric flasks, diluted to the mark, and the absorbancies are readlo (DDT a t 237 mfi and BP at 253 mc). A linear calibration plot is obtained when the ratio of the absorbance of the pesticide to that of the standard is plotted against the corresponding concentration ratios. Unknowns containing DDT in CHCI. are sniked hv the students with a knownuconcentration 07 B P i; C H C and ~~ diluted to 10 ml in a volumetric flask. The samples are chromatographed as before, the components leached into 95% ethanol, diluted to 10 ml in volumetric flasks and the absorbancies read. The ratio of the absorhancies of DDT to BP is calculated and the corresponding concentration ratio is read from the calibration olot. Finallv. the concentration of DDT in the unknown'is calculat~dfrom the concentration ratio and the known concentration of BP in the spiked sample. GLC Determination
The main analytical tool for the determination of pesticide residues today is glc. Chlorinated hydrocarhon pesticides have been separated by means of a variety of glc stationary phases.ll This experiment uses an SE-30 column at 225°C with He as the carrier gas at a flow rate of 62.5 ml/min and a flame ionization detector.12 Retention times observed for DDT, DDE, and DCBP are 6.2, 3.8, and 2.1 min, respectively. Presented in part at the Southeastern Regional Meeting of the American Chemical Society, Charleston, South Carolina, Novemher 7, 1973. To whom correspondence should he addressed. 2Ehrlieh, P. R., and Ehrlich. A. H., "Population, Resources, Environment," W. H. Freeman and Company, San Francisco, 1970, pp. 129-34. Carter, L. J., Science, 181, 143 (1973). Focht. D. D.. and Alexander. M.. Science. 170.19 (1970) rinkm man cbrooration. westbur". New ~ o r k ~~~~
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Ea.tman Kodak Cornpsny. ~ u c h r & r , N e w \'ark.
' hldrich Chemical Cornpan,. Inr.. Mhvnukee. Wisconsin RThli ilwlmev that there is no fractionatmn of unknown relative to standard throughout the procedure. DDE may be determined in an identical fashion and ahsarbance read at 237 mp. lo A Beckman DU spectrophotometer was used. "Lynn, T. R., Hoffman, C. L., and Austin, M. M., "Guide to Stationary Phases far Gas Chromatography," Analahs, Inc., North Haven, Connecticut, 1970, pp. 63-9. l2 A Beckman model GC-45gas chromatograph wasused. Volume 51. Number 70. October 1974
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DCBP is used as the internal standard because it is eluted from the SE-30 column with a peak position clearly resolved from the DDT and DDE peaks and gives a comparable response in the flame ionization detector. A linear calibration plot results when known mixtures of DDT and DCBP in hexane are chromatographed and the ratio of the peak area of the DDT peak to that of the DCBP peak is plotted against the corresponding concentration ratio. A similar calihration plot is prepared for DDE and the standard. Under certain conditions, peak height ratios may he used instead of peak area ratios.13 Student unknowns are prepared containing mixtures of DDT and DDE in about 5 rnl of hexane. The unknowns are "spiked" with 1 ml of a standard DCBP solution containing 2.00 mg/ml in hexane and diluted to the mark with hexane in a 10-ml volumetric flask. The students are required to inject varying volumes of their unknown at different instrument attenuations until a satisfactory chromatogram is ohtained. The ratios of the peak areas of DDT and DDE to the DCBP neak are calculated and the corresponding concentration ratios are read from the calibration nlots. The concentration of the pesticides in the unknown are calculated from the concentration ratios and the known concentration of the standard. Results
Compovnd
Mean
Range
DDT DDE DCBP
0.44 0.65 0.17
0.38+.52 0.55-0.71 0.1W.30
Standard deviation' 0.035
0.045 0.053
Table2. Student Results on UVAnalysir DDT concentration (mg/lO ml)
Sam&
percent
Table 3. Student Results on GLC Analysis. DDT Concentration (mgllo ml) -
Sample number
Given
Found
Average
percent ermr
and Discussion
Tahle 1 lists student R, values ohtained with this tlc system. The range and standard deviation of the values indicate good resolution of the components. Excellent separations are usually ohtained with this system whether or not the chromatography plates are activated by heating. The separations are simple to perform and the materials are inexpensive and well within the budget of any educational institution. Quantitative determination of the pesticides by uv spectrophotometry of the separated components is somewhat more tedious and the results of this experiment were often poor. The laboratory instructor ohtained reliable results in this analysis, but in the hands of inexperienced students, the results were usually unreliable. Tahle 2 eives the best student results obtained on uv analysis by a class that was carefully supervised. Since the s"cce& of this exoeriment aDpears to depend in considerable degree on the'experimenial technique of the analyst, the u v ~ d e terminations might he used hetter as a project for an ambitious student or group of students rather than a lahoratory exercise. Student results on glc analysis are presented in Table 3. As might be expected, the samples containing the least amount of DDT and DDE produce the largest error. Students can perform the analysis quite satisfactorily as indi-
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Table 1. Experimental TLC R, Valuer
/ Journalof Chemical Education
DDE Concenlmfion 1.75
2.64
3.80 5.24 5.98 7.64
b
1.65
2.80 3.88 5.26 6.04 7.48
Fmm area ratio versus eoneentration ratio calibration plot. Fmm peak height ratio versus concentration ratio calibration plot.lr
cated by a mean percent error of less than five. Calihration plots for this experiment are more rapidly constructed on the basis of peak height ratios than peak area ratios and the data in Tahle 3 show comparable precision and accuracy for both methods with this instrument. Since glc is an important analytical technique and is frequently used in the analysis of environmental samples, this exoeriment mieht well he incornorated in the lahora" tory for general chemistry, analytical chemistry, or instrumental analysis, as well as courses for nonscience majors. '3If the burning time of a component in the flame ionization detector is approximately constant (independent of the mass of component),then the peak height is directly proportional to peak area hecause the base ofthe peak is constant.
