478 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS An often-overlooked factor in thin-layer chromatography is that of the volume of the development chamber. Tanks supplied with the various types of commercial spreading apparatus are usually only slightly larger than the standard 20 x 20 cm carrier plate and about 5 to 10 cm wide. Paper chromatographic development chambers are usually rather larger than this and since they are already available in most pesticide residue analytical laboratories, their conversion to thin-layer use has frequently occurred. Whichever size of tank is chosen it must be closely adhered to for all accurate comparative work. Provided that known standard pesticides are spotted on to every plate and that confirmation of identity is all that is required, small variations in tank-volume will be relatively unimportant. The humidity of the atmosphere within the development chamber has been found to have a profound influence on the separation of some 2, 4- dinitrophenyl-hydrazones (20). In the absence of water vapour excellent resolution of six compounds was obtained, while all substances migrated with the solvent front when atmospheric moisture was not excluded. In order to obtain such satisfactory separations it was necessary to devise a tank through which dry nitrogen could be circulated prior to allowing the mobile solvent to contact the chromatoplate. This apparatus was similarly found useful for preventing oxidation effects during the chromato- graphy of methyl esters of some unsaturated fatty acids. Geiss and Schlitt (56) have also shown the variation of R F value of polyphenyl hydrocarbons which could be induced by large changes in the relative humidity of the development chamber atmosphere. Differing views have been expressed in the literature as to the extent of the effect of variations in the temperature at which development proceeds upon the R F values observed on thin-layer chromatoplates. Stahl (10), studying essential oils on silica gel chromatoplates developed in a hexane- acetic acid mixture, found no alteration in running time on changing the temperatures from 20 ø to either 4 ø or 28øC. The effects of insecure closure of the tank and variation in the depth of immersion of the layer in the mobile solvent were found to be more important. Brenner et al (57) similarly found that raising the temperature of development from 18 ø to 38øC had virtually no effect on the RF values of a nmuber of amino-acids developed with a phenol-water mixture, although the reproducibility of these values became poorer. Muller and Honerlagen (58), on the other hand, in their study of the thin-layer chromatography of cinchona bark alkaloids developed with a

THIN-LAYER CHROMATOGRAPHIC TECHNIQUES IN RESIDUE ANALYSIS 479 mixture of kerosene-diethylamine-acetone as mobile solvent, found that the observed RF values were strongly temperature dependent they advised the use of a controlled 25øC as being most convenient and suitable for their purpose. Similar effects were described by Harthon (5.9) who advised the inclusion of a reference compound on each chromatoplate to act as a check against variations in R•., value due to temperature fluctuation. Use of the effect of temperature on R F value has been made by Malins and Mangold (t30) in their separation of palmitic and oleic acids on silica gel chromatoplates developed at 4-6øC, these compounds being unresolved at normal ambient temperatures. Stahl (138) has recently devised a chamber for isothermal operation at temperatures ranging from --50 ø to + 50øC. Development at low temperatures allows the use of low boiling solvents as mobile phase and also renders p6ssible the chromatographic separation of compounds that are appreciably volatile at room temperatures. His results indicate that the effect of variation of temperature on R F value is more dependent on the nature of the mobile solvent than upon the material under investigation. The procedures described above relate in general to a single linear development in one direction. Thin-layer chromatography is also suitable for two-dimensional separations, either by means of different mobile sol- vents in directions perpendicular to one another (131), or by a combination of normal solvent development with an electrophoretic separation (132). Multiple or step development techniques may also be applied, either by repeated application of the same solvent (1313) 9r by sequential use of different solvents (134,135). Gradient elution techniques, whereby the polarity of the mobile solvent is continually changed while the develop- ment proceeds, have also been used, suitable apparatus having been described by Rybicka (1313). Visualization of residues The application of a visualization reagent is usually required before the developed chromatogram may be properly observed. Most reagents suitable for paper chromatographic indication purposes may similarly be applied to thin-layer chromatograms. Additionally, corrosive reagents and elevated temperatures may be used where necessary. Visualization agents fall readily into two classes, general or universal reagents and specific or semi-specific indicators. The reagent is usually applied in the form of a spray for the best results the droplet size must be very small and also uniform, the reagent must be evenly applied and only the minimum