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

26 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS acid, pimelic acid, glutaric acid, adipic acid, and succinic acid from each other. :, ß Leone and Guerritore (13) used paper chromatography in separating anthranilic acid from 3-hydroxyanthranilic acid.' They used the descend- ing technique with iso-amyl alcohol as the solvent. After locating the spots by ultraviolet fluorescence, the zones were cut out, extracted with 1 NHCt, and determined spectrophotometrically. Kurtz (14) separated the C-18 esters, such as methyl stearate, methyl oleate, methyl linoleate, and methyl linolenate, from each other on a silica gel column using a solvent mixture of petroleum ether and benzene. V•lon (15) separated the liquid hydrocarbons from the solid hydrocarbons in cosmetics by columnar chromatography on alumina oxide and by their selective solubility in methyl ethyl ketone. In the essential oil industry, the problem of separating and identifying the components of an oil is both diflScult and very tedious. Now, with the chromatographic techniques at our disposal, the problem is being made com- paratively simple. For instance, Kirchner and Miller (I6-19) have made an extensive chromatographic study on the citrus oils, in order to deter- mine some of their minor constituents. They prepared terpeneless oils on a laboratory scale by passing the crude oil dissolved in hexane through a silicic acid column. The terpenes came through and the terpeneless oil was then eluted with a more polar solvent-like ethyl acetate. The terpene and non- terpene fractions were then chromatographed, using a novel technique that combined both types of chromatography discussed previously. Kirch- her and Miller used a "chromatostrip," which was prepared by coating a piece of glass with a slurry of silicic acid and some binder. The dried "chromatostrip" was spotted with the terpene or terpeneless oil and chro- matographed, using an appropriate solvent. The separations were excellent and the identification on the "chromatostrip" were performed easily, using such reagents as fluorescein, followed by bromine vapor to detect olefin double bonds, concd. H2SO4-HNO3 mixture to detect hydrocarbons, by the reduction of aldehydes and ketones with lithium aluminum hydride to the corresponding alcohol, and by the oxidation of the alcohol with chromic acid to the corresponding aldehyde or ketone. Camphene, limonene, and a-pinene were separated using methyl-cyclo- hexane as the solvent. Citral, lauric aidehyde, cinnamylaldehyde, and furrural were'separated using 15% ethyl acetate in hexane and were de- tected by spraying the "chromatostrip" with o-dianisidine. Carvone, methyl heptenone, pulegone, and camphor were separated using 50% iso- propyl ether in hexane. Methyl anthranilate, ethyl anthranilate, and N-methyl methyl anthranilate were separated using 30% isopropyl formate in hexane. Numerous other complex mixtures were resolved using the Kirchner procedure.