94 ABBOTT, BUNTING AND THOMSON: THE DETERMINATION OF RESIDUES [AfldySt, VOl. 91 The Determination of Residues of Dimethoate with Multi-band Chromatoplates BY D. C. ABBOTT, MRS. J. A. BUNTING AND J. THOMSON (Ministry of Technology, Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, S.E.1) A method is proposed for the determination of dimethoate residues in vegetable material. Tripartite multi-band plates are prepared and used to separate the dimethoate from co-extracted materials ; spot-area measurement is used t o give a quantitative determination of the amount of pesticide. CONSIDERABLE interest has been shown1 l2 l3 3* in the thin-layer chromatographic separation of standard organophosphorus pesticides. However, little use of these procedures has been reported for the determination of residues on crop samples.A molybdenum-blue colorimetric method has been described5 for the determination of dimethoate and its oxygen analogue after they have been separated on a silica-gel chromatoplate developed with acetone + chloro- form, (3 -t 1). This chromatographic technique gives a degree of identification that is lacking in most other colorimetric meth0ds,~9~7* but the operations required are cumbersome and time-consuming. Recent work by Kovacsg has illustrated the uses of thin-layer chromato- graphy as a semi-quantitative process applied to extracts of kale, lettuce, strawberries, apples and carrots, co-extractives causing little interference with those organophosphorus pesticides studied. The use of similar techniques as clean-up and identification stages before gas- chromatographic determination and infrared examination has proved invaluable,1° but the apparatus required is expensive and considerable time is required for the satisfactory completion of the exercise.EXPERIMENTAL The use of spot-area measurement as a means of determining pesticide residues on thin- layer chromatograms offers an inexpensive and rapid method of combining identification with accuracy. Graphs of the square root of the area of the spot against the logarithm of the weight of material contained have been shown to be linearll; such a system has already been used successfully for the determination of some triazine herbicides in soil and water.12 Where clean extracts are readily obtained, similar procedures may be used for many organophosphorus-pesticide residues on vegetable tissue,l0 silica-gel chromatoplates being developed with hexane + acetone, (9 + 1). Dimethoate [dimethyl S-(N-methylcarbamoyl- methyl)phosphorothiolothionate] is not amenable to this treatment since, under these con- ditions, it remains at the origin together with a proportion of co-extracted interfering materials; its presence is thereby masked and determination is not possible.If the polarity of the mobile solvent is increased, higher RF values are obtained, as shown in Table I, but only at the cost of similarly increasing the mobility of the residual co-extractives with resultant streaking and malformation of the dimethoate spot. TABLE I RF VALUES OF DIMETHOATE I N VARIOUS SYSTEMS Mobile solvent Nitromethane + carbon tetrachloride, 1 + 1 .. .. Nitromethane + dichloromethane, 1 + 1 .. .. Light petroleum 40" to 60" + 2-butoxy-ethanol, 1 + 1 . . Light petroleum 40" to 60" + ethyl methyl ketone, 1 + 1 Butanol + benzene, 1 + 1.. . . * . .. ., Butanol + toluene, 1 + 1 . . . . . . . . . . Chloroform + acetone, 9 + 1 . . .. . . .. Chloroform + acetone, 9+ 1 . . . . . . . . Chloroform + acetone, H+ 1 . . . . . . . . Chloroform + acetone, 9 + 1 . . .. . . . . Chromatoplate RF value Silica gel 0.17 Silica gel 0.72 Silica gel 0.36 Silica gel 0.48 Silica gel 0.55 Silica gel 0.39 Alumina 0.90 0.52 0.35 Silica gel 0.21 Kieselguhr + silica gel, 1 + 1 Multi-band plate as in methodFebruary, 19661 OF DIMETHOATE WITH MULTI-BAND CHROMATOPLATES 95 The use of a tripartite multi-band ~hromatoplatel~ has been found effective in over- coming most of the difficulties experienced with this compound and a method is now proposed for the determination of dimethoate in vegetable tissue.The layers of adsorbent comprising the chromatoplate have been so chosen and arranged that the pesticide is caught quantitatively on a narrow band of silica gel, while migrating interfering materials move away rapidly across a band of inactive kieselguhr; chloroform + acetone, (9 + l), has been chosen as mobile solvent for this purpose. Other commonly used organophosphorus pesticides such as parathion, malathion, chlorthion, fenchlorphos, phorate, diazinon, azinphos-methyl and carbophenothion do not interfere as they migrate close to the solvent front; elution of the adsorbent in this area with dichloromethane yields a clean extract suitable for examination for the presence of other compounds on a silica-gel chromatoplate. Schradan, dichlorvos and dimethoate oxygen analogue remain on the lower kieselguhr + silica-gel band.Spots are rendered visible by means of a Brilliant green + bromine treatment which is sensitive to 0.1 pg of dimethoate. Although the spots are slightly distorted in shape, a linear relationship is observed between the square root of the area and the logarithm of the weight, as is illustrated in Fig. 1, over the range 0.2 to 1Opg. The extraction procedure used is a modification of that previously advocated for gas - liquid chromatographic determinations.14 'I- 1 5 1 7 1 9 I 0 0 3 0 5 0 7 0 9 Log (weight, pg) Fig. 1 .Quantitative determination of dimethoate METHOD APPARATUS- Multi-band spreader-Insert two close-fitting spacers into the body of a thin-layer spreader as described by Abbott and Thomson.12 The Desaga (Camlab Ltd, Cambridge) and Shandon (Shandon Scientific Co. Ltd, London) spreaders are suitable for this purpose Cork spacers covered with aluminium foil are suitable. Carrier $lates-20 x 20-cm glass plates. Chronzatographic tank--22 x 21 x 9.5 cm internal measurements. Chronzatographic spray-The laboratory Spray Gun (Shandon Scientific Co. Ltd, London) Top-drive macerator. Air-oven-Set at 120" C, suitable for drying and activating thin-layer chromatoplates. Use analytical-reagent grade materials whenever possible. Ethyl methyl ketone.Hexane-Kedistilled, boiling-point 68" to 70" C. Acetone. Dichloromethane. Chloroform. Sodium suZphate-Granular anhydrous material. Sodium sulphate solution, 5 per cent. w/v, aqueous. is suitable. REAGENTS-96 ABBOTT, BUNTING AND THOMSON : THE DETERMINATIOK OF RESIDUES it 5 per cent. v/w of water and mix the solid thoroughly in a closed vessel. [Analyst, Vol. 91 Aluminium oxide-Heat aluminium oxide at 800” C for 4 hours, cool, carefully add to Silica-gel G-For thin-layer chromatography. Kieselguhr G-For thin-layer chromatography. Brilliant green soldon-A 0.5 per cent. solution of Brilliant green (C.I. 42040) in acetone. Bromine. Standard dimethoate solutions-Prepare a stock solution containing 2 pg per ml of dimethoate in acetone. PROCEDURE- Preparation of multi-band chromato$lates-Position the two spacers within the body of the spreader at distances of 5 cm and 8 cm from one end.Simultaneously prepare the layer-mixes described in Table 11, shake them for l$ minutes and pour each mix into the appropriate compartment before layering 5 carrier plates, each 20 x 20 cm, in the usual way. Activate the 250-p thick layers by heating at 120” C for at least 1 hour. Dilute with acetone as required. TABLE I1 COMPONENTS OF MULTI-BAND CHROMATOPLATE Weight of Volume of Compartment Width, Material material, water, cm g m! 1 5 Kieselguhr + silica gel, 1 + 1 11.0 22 2 3 Silica gel 6.5 13 3 12 Kieselguhr 25 30 Extraction and clean-Ufi-Macerate 50 g of sample with three successive portions each of 75 ml of a mixture of ethyl methyl ketone + hexane, (3 $- a), and separate the solvent extracts by filtration.Reduce the combined extracts to a volume of about 5 ml and transfer the solution with 20 ml of hexane into a separating funnel containing 500ml of sodium sulphate solution. Shake the funnel well, discard the hexane layer and wash the aqueous solution with 25ml of hexane. Extract with three 25-nil portions of dichloromethane an6 dry by passage through a short column of anhydrous sodium sulphate, then reduce the volume of the combined extracts to 1 ml. Transfer the solution to a column of aluminium oxide (8 cm x 1.8 cm i.d.) and elute with 200 ml of dichloromethane. (With samples con- taining little wax or oil this step may be omitted.) Reduce the solution to dryness, take up the residue in 40 p1 of acetone and spot 20 pl on to the mixed kieselguhr + silica-gel band about 2 cm from the lower edge of the chromatoplate. Apply to the same plate a series of spots of standard dimethoate solutions, each in 2Opl of acetone, covering the range 0.2 to 10pg.DeveZopment and visualisation of the chrornato9Zate-Develop by ascending chromato- graphy with chloroform + acetone, (9 + 1) for 45 minutes; in this time the solvent front will travel about 12 cm. Spray the developed plate with Brilliant green solution and then place it immediately in an atmosphere o f bromine. After 30 seconds, remove the plate and allow the bromine to evaporate; dimethoate spots are shown as yellow areas with blue “haloes” at about the centre of the silica-gel band. Fig. 2 shows a typical range of standards so obtained.Quantitative rneasurement-Circumscribe the well defined spots carefully and measure their area by any suitable method. It may be more convenient to measure the area of the spot on a direct copy15 of the developed plate than on the layer itself. Plot a graph relating the square root of the area of the standard spots to the corresponding logarithm of the weight of dimethoate. By reference to this linear graph determine the amount of dimethoate in the sample spot. RESULTS Dimethoate was added to a variety of samples to give concentrations varying from 0.025 to 1.0 p.p.m. Recoveries obtained are listed in Table I11 and these are close to 100 per cent., only celery giving low yields of 80 per cent. All blanks carried out on untreated samples were negative.February, 19661 OF DIMETHOATE WITH MULTI-BAND CHROMATOPLATES 97 t I Kieseiguhr +Silica gel ( 1 + 1 ) I * * * * * * * * 0 2 5 0 5 I 2 4 6 8 1018 Fig.2. Multi-band chromatoplate veloped in chloroform + acetone, (9 + de- 1) TABLE I11 RECOVERY OF DIMETHOATE ADDED TO VARIOUS SAMPLES Sample Weight of sample, Dimethoate added, Dimethoate recovered, g p.p.m. p.p.m. 50 0.5 0.52 50 0.2 0-18 50 0.1 0.09 Apple . . .. .. J I Brussels sprouts . . Celery . . .. .. 50 50 I 50 50 i 50 0.5 0.2 0.1 0.2 0.1 0.54 0.22 0.10 0-16 0.08 Sugar beet . . .. 50 0.2 0.18 Strawberries . . .. Water . . .. .. 50 50 200 200 0.2 0.1 0.05 0.025 0.20 0.09 0.045 0-024 Permission to publish this paper has been given by the Government Chemist, Ministry of Technology. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. REFERENCES IValker, K. C., and Beroza, M., .J. Ass. Off. -4grir. Chent., 1963, 46, 250. Baumler, J., and Kippstein, S., Helv. Chiwi. Acta, 1061, 44, 1162. Bunyan, P. J., Analyst, 1964, 89, 615. Stanley, C. W., J . Chramat., 1964, 16, 467. Steller, \V. A., and Curry, A. N., J . Ass. Off. Agric. Chem., 1964, 47, 645. Heath, D. F., Cleugh, J . , Other, 1. K. H., and Park, P. O., J . Agric. Fd Chem., 1956, 4, 230. Chilwell, E. D., and Beecham, P. T., J . Sci. Fd Agric., 1960, 11, 400. Laws, E. Q., and Webley, D. J., Analyst, 1961, 86, 249. Kovacs, M. F., J . Ass. On. Agric. Chem., 1964, 47, 1097. Abbott, D. C., Crosby, N. T., and Thomson, J., in Shallis, P. W., Editor, “Proceedings of the Purdy, S. J., and Truter, E. V., Analyst, 1962, 87, 802. Abbott, D. C., Bunting, J . A., and Thomson, J., Ibid., 1965, 90, 356. Abbott, D. C., and Thomson, J., Chem. G. Ind., 1965, 310. Egan, H., Hammond, E. W., and Thomson, J., Awalyst, 1964, 89, 175. Abbott, D. C., Egan, H., and Thomson, J., J. Chronzat., 1964, 16, 481. SAC Conference, Nottingham, 1965,” W. Heffer & Sons Ltd., Cambridge, 1965, 121. Received June loth, 1966