24 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS different rates, thereby separating into a series of zones generally containing a single component. It has been likened to a fractionation procedure where it resembles a fractional distillation through a packed fractionating column. One can consider that the vapors are being fractionated by selective dis- tribution between the mobile gas phase and the refluxed liquid phase (1, 2). In spite of the obvious advantages of the adsorptive process, certain disadvantages are also present: the adsorption may be slowly reversible, resulting in a poor separation and the subsequent incomplete recovery of the solutes the components may oxidize, polymerize, or rearrange on the column, but this is a rather infrequent occurrence, and the adsorption process is dependent on the physical state of the adsorbent material. This last disadvantage leads to an unfortunate situation because the lack of an adsorbent with uniform properties makes it necessary to do some pre- liminary work on an established procedure in order to effect the resolution in question. In paper chromatography, the immobile phase has been replaced by an untreated paper strip, a paper strip having some adsorbent, such as alumina, magnesia, etc., on its surface, or a paper strip that has been treated with various reagents, such as acetic anhydride while the mobile phase is still a solvent or a solvent mixture. Any of the Whatman grade papers may be used, depending upon the rapidity of the solvent flow on the paper strip. As for the mobile phase, any solvent or solvent mixture may be used, but, again, no set rule can be followed. For the development of a paper chromatogram, a drop of the solution is placed near one end of the strip, which is then dipped into the solvent. The paper strip may be held vertically or be bent across a horizontal glass rod, so that gravity hastens the flow of the solvent and, thereby, aids in the development of the chromatogram. The development of the paper chro- matogram must be performed in a closed vessel in order to prevent evapora- tion of the solvent and the disturbance of the solvent equilibrium. After the paper is dried, either at room temperature or in an oven, depending upon the material on the paper strip, the spots can be located and identified by various methods: by comparison with known substances on pilot strips, by determining the Rf value (Rf = distance zone moved/distance solvent moved), by ultraviolet fluorescence, by spraying with specific functional group reagents--such as 2,4-dinitrophenylhydrazine for aldehydes and ketones, Ag(NH3)2 + for reducing sugars, bromphenol blue for acids and ninhydrin for amino acids--by biological printing (bioautograph) (3), by radioactivity, or by elution of the zones from the paper and the perform- ance of chemical or physical tests. In paper chromatography, extremely small quantities of complex mix- tures may be completely resolved once the solvent system has been de- termined. In this respect, it is a more adaptable technique than columnar

CHROMATOGRAPHY AND ITS APPLICATION 25 chromatography, although it takes more time (18-24 hours for a paper chromatogram, compared to a few hours for a columnar chromatogram to complete). For instance, two-dimensional chromatography has been used by Dent (4) to separate 61 amino acids from each other. In this variation, a drop of the solution is placed near one corner of a square sheet of paper, an adjacent edge of which is then dipped into a developing solvent contained in a long narrow dish or trough. After the chromatogram has been formed, the paper is dried and the edge adjacent to the chromatogram is developed farther in the direction at right angles to that of the first development. Under these conditions, the solutes appear as a series of spots distributed in a specific pattern. on the paper. Attempts have been made to explain chromatography mathematically, but, to date, the problem has not yet been completely resolved. One can only suggest that the early papers of Martin and Synge (5), Consden, Gordon, and Martin (6), M•ller and Clegg (7), and Dent (4) be studied, in order to get an insight into the mathematical complexity of this compara- tively simple analytical tool. In general, however, the objectives of both columnar and paper chroma- tography are identical: the resolution of mixtures and the identification of the components qualitatively, and quantitatively where possible, and the comparison of substances, especially those suspected of being identical or adulterated. To date, there has been very little published work on the application of chromatography in cosmetic chemistry, and only within the past few years have we seen information on its application to the essential oil industry. I, now, would like to show how this technique has been used. D•shusses (8) separated the dyes in lip rouge, face paints, etc., by drop- ping a solution of the product on an alumina disk and eluting the spot with various solvents, such as pentane, benzene, carbon tetrachloride, ethyl ether, acetone, and water. Weiss (9), using columnar chroma- tography with various adsorbents and solvent systems, and Tilden (10), using paper chromatography, were able to resolve a large number of coal tar dyes and identify them by their color reactions with concd. H2SO4, concd. HC1, 10% NaOH, and concd. NH4OH. Ramsey and Patterson (11) separated the C-11 to C-19 straight chain fatty acids from each other on a silicic acid column, using a solvent mix- ture containing furfuryl alcohol, 2-aminopyridine and n-hexane. They were equally successful in the separation of C-12 to C-18 straight chain fatty acids. However, the authors were net able to separate the odd- numbered fatty acids from the even-numbered homologues. Vanden Heuvel and Hayes (12), using a silicic acid column and the solvent mix- tures of chloroform containing 5% butanol and chloroform containing 10% butanol, were successful in separating sebacic acid, azelaic acid, suberic