TLC STUDY OF AN ANTIMICROBIAL PRESERVATIVE 539 but as the preservative is up to 50•o aqueous soluble, the relatively small proportion of water in the mixture, methanol-water (70: 30) was found to be satisfactory and was used for all the products examined. It was shown to be essential to dry the spots well, after application, other- wise distortion and lack of sensitivity resulted. No decomposition of the imidazolidinyl urea zones was detected on heating the applied spots with a warm-air dryer, even when heated to a considerably greater extent than that required for a good chromatogram. As imidazolidinyl urea was almost insoluble in any solvent other than water, in which it was very soluble, the correct flow solvent required a certain water content but this needed to be modified by a non-polar solvent, chloroform was chosen as a convenient solvent after considering its position in the eluotropic series. These two solvents were rendered homogeneous by the mutually-miscible solvent methanol. A mixture of chloroform-methanol-water (45:45: 10) was found to give satisfactory Rf values but rather diffuse spots. As a change in pH will often produce a 'sharpening up' of diffuse spots, the following solvents were investigated. Chloroform-methanol-acetic acid-water (20: 30: 40: 10), although compacting the spots, produced an increase in Rf's and failed to separate the preservative from other components in the formulations. Chloroform- methanol-ammonia (50:40: 10) gave very low Rf's and even after the carefully-attempted removal of the ammonia, a poor background colour reduced the limits of detection considerably. Chloroform-methanol-acetic acid-water (50: 30: 10: 10) having a pH of 2.5 was found to yield compact zones separated from all other visible components. This solvent also gave a good separation of the imidazolidinyl urea components in Germall itself. A number of spray reagents were investigated, several being general reagents, whilst the remainder were intended to be more specific for the imidazolidinyl urea grouping. Of the twelve reagents tried, few successfully detected less than 25 gg of imidazolidinyl urea (Table I). Only Erlich's reagent and ninhydrin are sufficiently sensitive to detect 0.5•o imidazoli- dinyl urea in a product, i.e. 2.5 [tg for the loading used. The sensitivity of the ninhydrin spray is increased by a factor of ten when viewed by trans- mitted uv light of 366 nm. There seems to be little record of compounds which react with ninhydrin being more sensitively detected using uv light rather than colours visible in daylight. It seems likely that the difference in sensitivity of detection found when viewed by reflected and transmitted uv .light may be mainly due to the difference in intensity of light on the plate,

540 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table I. The limits of detection of imidazolidinyl urea type (Germall 115) using various reagents Colour of Limit of Reagent reaction detection Ninhydrin (daylight) Red 2.5 lag Ninhydrin (reflected UV) Yellow 1 lag Ninhydrin (transmitted UV) Yellow 0.25 lag Dragendorff's reagent No reaction -- Blue Salt Irga B No reaction -- Cobalt thiocyanate Blue 25 lag Erlich's reagent Yellow 2.5 lag Silver nitrate No reaction -- Iodine No reaction -- Fluorescein No reaction -- Rhodamine 6G Yellow in UV 10 lag Prochazka reagent No reaction -- Chromic acid No reaction -- Pinacryptol yellow Blue in UV 10 lag i.e. 450 [tW cm 2 at 18 in for reflectance and 1900 gW cm 2 at the surface for transmission. The extent to which plates were sprayed was found to be critical, thus in order to establish optimum conditions, plates were sprayed until varying states of wetness were obtained. Plates which were observed to be not wetted, i.e. not darkened, plates which were just wetted and even plates which were fairly well wetted, gave poor results. Well-wetted plates and those which were soaked, i.e. of a shiny appearance were satisfactory. The correct spray was therefore judged to be well wetted until the first shiny appearance was observed, which normally required 20 ml of reagent for a 20 x 20 cm plate, although due to variance in spraying techniques the appearance of the plate rather than the volume used is the better criterion. Chromatograms heated at 105øC, the normally recommended tempera- ture for ninhydrin failed to give good sensitivity even when heated for up to 90 min. At 150øC little reaction was noted for up to 10 mins heating but the maximum sensitivity was achieved after 15 min and no change was detected after further heating for 30 min. Thus heating at 150øC for 20 min is preferred. The spots once developed are stable for several days if pro- tected from daylight. As when examined at a higher loading, the preservative revealed six components, i.e. two major and four minor, the possibility of decomposition