THIN-LAYER CHROMATOGRAPHIC TECHNIQUES IN RESIDUE ANALYSIS 487 The second class of methods used for the quantitative evaluation of thin-layer chromatograms follows more closely those techniques which have been well established in the field of paper chromatography (107). Spot density measurements have been used by Privett and Blank (108) in the determination of component triglycerides. The areas under the densitometer curves were found to be directly proportional to the amount of sample applied for the saturated triglycerides and glyceryl residues of the unsaturated glycerides, spots being located by charring with 50% sulphuric acid. However, the area given varied with the type of structure of the compound. Squibb (109) made densitometric measurements on amino-acids located with ninhydrin and found a coefficient of variation of q- 6.90/0 . Reflectance densitometry as used for the paper chromato- graphic determination of herbicides (83) has been found to be less success- ful with thin-layer chromatograms of similar compounds, largely owing to the fragile nature of the layer surface and the difficulty in obtaining a sufficiently clean and uniform background w•,•h the silver nitrate reagent used. A review of photometric methods has been given by Klaus (110). Methods based upon the measurement of spot area avoid the difficulties associated with elution of the material from the absorbent and the possi- bilities of further pollution of the purified sample. Seher (111) simul- taneously chromatographed samples of the unknown and a series of standards applied to the chromatoplate in equal volumes of solution. After rendering the developed chromatogram visible, the areas occupied by the standard sample spots were determined and plotted against the corresponding weight of material. Reference of the area of the unknown sample spot to this curve gave a measure of the material present. Purdy and Truter (112) showed that the square root of the area of the spot is a linear function of the logarithm of the weight of the material it contains. Statistical evaluation (113) showed that this relationship was preferable to those of area against logarithm of the weight or logarithm of the area against logarithm of the weight. Planimetric means were used by Aurenge et al (114) to determine spot areas graphs of area squared against weight of material were linear. They also traced the chromatograms, cut out the traced spots and weighed them in order to determine the area of the spot more accurately, the weight per cm • of the tracing paper being known. (Received: 14th July 1965)

488 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table I gives a few examples of the wide variety of chemical substances whose separation, purification and identification have been effected by the use of thin-layer chromatographic techniques. Table I Thin-layer chromatographic separations Class of Compound Adsorbent Mobile phase Visualization agent Reference Alkaloids Silica gel Chloroform, Iodoplatinate 115 diethylamine (90 + •o) Antibiotics Silica gel Acetone, Iodine azide 116 methanol (so + 80) Antioxidants Silica gel Chloroform Phosphomolybolic 117 acid Fatty acids Silica gel Petroleum Sulphuric acid 118 ether, diethyl ether (90 q- 10) Food dyes Cellulose n-Propanol, Self indicating 119 ethyl acetate, water, (60 q- 10 q- SO) Pesticides (A) Organo-chlorine (B) Organo-phosphorus (C) Herbicide Plasticizers SteroIs Sugars Vitamins Silica gel Hexane Silver nitrate 85 Silica gel Hexane, Brilliant green and 34 acetone bromine (90 q- 10) Silica gel, Cyclohexane, Silver nitrate 83 kieselguhr benzene, (40 q- 60) acetic acid, liquid paraffin (20 q- 3 q- 2 q- 1) Silica gel Benzene, ethyl Fluorescence 120 acetate (45 q- 5) Silica gel Benzene, ethyl Phosphoric acid 121 acetate (90 q- 10) Alumina, Butanol, Aniline phthalate 122 silica gel acetic acid, (1 q- 1) methanol (20 + 20 + 60) Alumina Benzene o-To]uidine q- KI 123