THIN-LAYER CHROMATOGRAPHIC TECHNIQUES IN RESIDUE ANALYSIS 481 Chiba and Morley (76) in a study of carbaryl, and by Katz (77) for locating DDE and dichlorobenzophenone. Fluorescent compounds have enjoyed wider popularity among pesticide residue analysts, the spots usually being located and marked by observing the quenching effect of the compound on the background fluorescence when viewed under UV light. Kirchner et al (9) used zinc-cadmium sulphide as a built-in phosphor to indicate the presence of biphenyl on their chromatostrips. Fluorescein, either built-in or spray-applied, has been used by Spickett (78) for pyrethroids and by Salo et al (79) for organo- phosphorus pesticides, exposure to bromine vapour showing the yellow spots on a red background, Walker and Beroza (74) added silver nitrate to this fluorescein-bromine combination and give limits of detection ranging from 0.5 to 10 •g for 59 pesticidal compounds for the organochlorine com- pounds 7 min irradiation with UV light was also required. Dichloro- fluorescein has been found useful for organo-phosphorus pesticides (34). Some esters of MCPA and mecoprop have been located by means of Rhodamine B (63). Among the more specific visualization agents two have been widely used. Palladous chloride has been found preferable for organo-phosphorus compounds by B/iumler and Rippstein (30), Blinn (80) and Steller and Curry (81). Silver nitrate, with UV irradiation, has been most popular for chlorine-containing pesticides. The "chromogenic reagent" of Mitchell (82), i.e. silver nitrate with 2-phenoxyethanol, has been universally used for visualizing paper chromatograms (18,74) and has been widely adopted for similar purposes on thin-layers. On silica gel or alumina plates, how- ever, it is not very satisfactory because dark backgrounds are obtained which limit its sensitivity. A simple 0.5% ethanolic solution of silver nitrate has been found to be more satisfactory (83). Other variations on this theme include ammoniacal silver nitrate (39,84), ethanolamine or potassium hydroxide with silver nitrate (72), silver nitrate-formaldehyde- potassium hydroxide (79), and silver nitrate-nitric acid (63). Abbott, Egan and Thomson (85) have described spray reagents con- sisting of a combination of silver nitrate with one of several pH indicator compounds. Bromophenol blue gave yellow spots on a blue background with organo-chlorine pesticides without irradiation with UV light. Bromo- cresol green also showed promise but required irradiation and further heating for the best results. Plates containing built-in silver nitrate are suitable for the detection of organo-chlorine pesticides, although a rather "grainy" background may be obtained. For organo-phosphorus com-

482 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS pounds, however, such plates are quite unsuitable as considerable streak- ing of the pesticide occurs. One benefit given by the use of silver nitrate-UV irradiation as an indicator system is that Since only a very small amount (10%) of the pesticide is affected by the irradiation, the bulk may be extracted for examination by other means (34). These workers have also studied the properties of a number of other indicator compounds, belonging in the main to the azo and triphenylmethane classes of dyestuffs, when applied as visualization agents for pesticidal compounds. Brilliant green (colour index No. 42040) showed very useful reactions and good sensitivity. With the aid of this material it is possible to locate organo-chlorine, organo-phosphorus and triazine compounds and to dis- tinguish between them. On spraying the silica gel chromatoplate with an 0.5% solution of brilliant green in acetone, organo-chlorine pesticides are observed as pale yellow spots on a green background and may be readily marked. On placing the sprayed plate into an atmosphere of bromine vapour (the plate must still be damp with acetone) the green background and the spots of organo-chlorine pesticide disappear but triazine herbicides are located as semi-transient green spots on white and organo-phosphorus pesticides appear as permanent dark green or yellow spots. Among the specific visualization methods mention must be made of the use of esterase inhibition procedures for the detection of organo- phosphorus compounds and their metabolites. The sensitivity of this procedure varies very markedly with the compound studied, for example the anti-cholinesterase activity of phorate oxygen analogue sulphone is one thousand times that of the parent phorate and its limit of detection is correspondingly lower. Methods used on paper chromatograms (86,87) have been similarly applied to thin-layer separations (88). The developed paper or plate is briefly exposed to bromine vapour and placed in contact with a sheet of filter paper which has been impregnated with out-dated human plasma. After incubation for 10 to 20 min at 35 to 40øC the paper is removed and sprayed with acetylcholine bromide and bromo- thymol blue or bromophenol blue. After a short while (5-10 min) the transferred spots appear in blue on the yellow background. Using tech- niques of this nature sensitivities down to about 10ng have been obtained from developed thin-layer chromatograms. The documentation of chromatograms As thin-layer chromatography increased in importance and widened