Richard Jarosch
Universitv of Wisconsin Center-Sheboygon Sheboygon, Wisconsin 53081
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The Determination of Pestitide Residues A laboratory experiment
The recent worldwide interest in chlorinated pesticides and their long-range effects on the environment, plus some personal research on the subject, has resulted in an experiment performed in our sophomore organic laboratory involving their qualitative determination in aquatic samples. By varying the basic procedure, one can analyze water samples (from rivers, ponds, etc.), soil samples (from fields, river bottoms), and aquatic life. The determinative step can also be varied depending on the instrumentation available. Thin-layer chromatography is used for qualitative work hut a suitable gas chromatograph allows one to make qualitative and quantitative determinations. The basic procedure involves: (1) extraction of the sample with a solvent such as hexane or mixed solvents such as hexane with ethyl ether, benzene, or acetone; ( 2 ) partial concentration of the extract; (3) "clean-up" to remove interferences (waxes, oils, etc.) using column chromatography, followed by further concentration of the column eluate; and (4) determinative steps-thin-layer and gas chromatography. Water samples for the experiment can either he actual samples taken from area waters (collected in glass bottles, not plastic) or laboratory water that has had small amounts of pesticides added to it by the instructor. Because of the extremely small quantities of pesticide which may be present in natural waters, it is wise to use as large an aqueous sample as is possible in your lab situation. Pesticide standards are available from various suppliers for "spiking" purposes and foruse as standards in tlc and gc. [CAUTION-pesticides a r e dangerous chemicals and should be handled with care.] The water sample is extracted twice with a 15% ethyl ether-hexane solvent, saturated sodium sulfate solution is added (about 35 ml/l sample), and a third extraction with hexane only is performed. Use about 60 ml of high purity solvent/l sample for each extraction. The separatory funnel used should preferably have a Teflon stopcock. The extracts are combined, dried with anhydrous NazS04, placed in a beaker and partially evaporated to 40-50 ml in a water bath a t about 70°C using no air or vacuum assist. Soil and fish samnles are extracted with a Soxhlet extractor. Soil samples are dried a t room temperature for 4-5 davs before extraction. Fish samnles are homoeenized with a-portion of the solvent in a blknder. The sample is placed in the extractor and extracted for 8 hr or longer with about 200 ml of hexane or hexane-acetone (9:l). The sample extrad is then dried with anhydrous NazS04 and suhiected to the clean-uo orocedure. waxes, oils and otf;& interfering substances are removed by a clean-up procedure utilizing column chromatography with Florisil (TM, Floridin Co., Pittsburgh) which is stored in an oven a t 130°C prior to use. The column is prepared by adding a half-inch layer of anhydrous NazS04, followed by the Florisil (about 15 g in a 19 mm column), and an inch layer of anhydrous NazS04 on top. About 50-60 ml of hexane is used to pre-elute the column and then is discarded. The sample extract is added to the column and the pesticides eluted with 300-400 ml of 1015% ethyl ether in hexane. The column eluate is then
evaporated down to 1 ml or less by a warm water bath a t 40-50°C with a gentle stream of clean dry air. Silica gel plates are used for tlc of the cleaned-up extract. These can be coated in the lab (0.25 mm thick) or purchased as a pre-coated sheet. The plate is activated a t 110°C for an hour and stored in a desiccator until use. Carbon tetrachloride or other low-polarity solvents can be used for developing the chromatogram. Samples and standards are spotted on the plate, keeping spots as small as possible. The use of a spotting template is recommended. The thin-layer plate is placed in the development chamber and allowed to equilibrate. The plate is then developed until the solvent front has migrated 10 cm (or other convenient distance), removed from the chamber, and air dried. The developed plate is then sprayed with a fairly heavy coat of the visualization reagent Rhodamine B (0.1 mg/ml in ethanol), allowed to dry thoroughly, and exposed to ultraviolet light. (Other visualization reagents are used, see references.) Pesticides show up as dark areas on a lighter background. Some typical Rr values for common pesticides and metabolites are given in the table. These values will vary with different adsorbents, developing solvents, and other conditions. Gas chromatography can also be used provided an iustrument equipped with a very sensitive detector is available. Most thermal conductivity detectors are not sufficiently sensitive, and hydrogen flame ionization detectors may or may not be acceptable. Most researchers use an electron capture detector for pesticide residue analysis. Since these are usually not available or a t least not to undergraduates, I will refer interested readers to the bibliography for further information. Finally some cautions-the small quantities involved make lab technique important so this experiment should be done bv students who have had some prior experience in organic lab techniques and procedures. So as not to further contribute to pollution, sample extracts should not he discarded a t the end of the experiment, but should be placed in a small container specifically meant for this purpose. I also repeat the caution mentioned earlier concerning the dangers involved in handling pesticides. Many variations are possible in analysis procedures of this type and I again refer readers to the publications listed in the bihliwaphv - - .for further information. Acknowledgment
I wish to thank the University of Wisconsin Center System Research Committee for partial support of a research project which led to development of this experiment. Typical R, Values for Common Pesticides and Metabolites
Pesticide dieldrin endrin lindane DDD (TDE) heptaehlor DDT
Rf
0.18
DDE
aldrin Volume 50, Number 7, July 1973
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507
Bibliography
ton, D.C. WZ42, 1966. (21 "FWPCA M e i h d for Chlorinated Hydmearhon Pesticides i n W a b r and Wastowa-
508
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Journal of Chemical Education
ter," Rdersl Water Pollution Control Administration. U S . Department of the lntsrin7Warhindnn
15)
"
id^ Identification
C M?d? Anril
191i9~
at the &idue Level." No. 16, Advancer in Chemistry Series. American Chemical Society, Wsshington, D.C.. 1971.
