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.

CHROMATOGRAPHY AND ITS APPLICATION 27 Using the "chromatostrip" technique, the 2,4-dinitrophenylhydrazone and semicarbazone derivatives of aldehydes and ketones, and the 3,5-dini- trobenzoate and carbamate derivatives of alcohols were separated using an appropriate solvent in each case. Many investigators have separated the 2,4-dinitrophenylhydrazones of aldehydes and ketones. Using a silica gel-bentonite column, Braddock, et aL (20), were able to separate the cis and trans forms of furfuraL2,4- dinitrophenylhydrazone. White (21), using a similar column and a sol- vent mixture of 75% hexane in ethyl ether, separated 18 pairs of mixtures, representing 12 aldehydes and ketones. He applied this technique to the chromic acid oxidation of crude 2-ethyl butanol. When the crude mixture of the 2,4-dinitrophenylhydrazones was chromatographed, six bands were observed of which the upper two were predominant. The first band yielded the 2,4-dinitrophenylhydrazone of diethyl ketone (identified by its melting point) the lower band, after readsorption, yielded the 2,4-dinitrophenyl- hydrazone of 2-ethyl butyraldehyde. Sorm (22) separated the 2,4- dinitrophenylhydrazones of some volatile aldehydes on a freshly precipi- tated calcium sulfate column, using petroleum ether as the eluting solvent. Rosen (23) used a silicic acid-celite column and benzene as the solvent, in the attempt to resolve 91 pairs of 14 aldehydes and ketones. He was successful in separating 77 pairs. Meigh (24) separated the 2,4-dinitrophenylhydrazones of volatile alde- hydes on paper, using methyl alcohol-heptane as the solvent, and devel- oped the spots by spraying with 10% NaOH. Paper chromatography was used by Uno (25) to separate the benzene-sulfohydroxamic acid deriv- atives of C-1 to C-5 aldehydes using iso-amyl alcohol:acetic acid:water (5:1: 1) as the solvent. A great deal of work has been done on the resolution of the derivatives of alcohols. Siegel, et aL (26), separated the reaction product of mono- hydric alcohols with 3-nitrophthalic anhydride on paper using iso-amyl alcohol-concd. NH4OH-water (30:15:5), as the solvent, and identified them with umbelliferone under ultraviolet light. Momose, et aL (27), used 3,5- dinitrophthalic anhydride to form the acid esters with both lower and higher alcohols and separated them on paper, using butanol saturated with acetic acid and butanol-acetic acid-water (8:1:2), respectively, as the solvents. The spots were detected by first spraying with NaOH and then with aceto- acetic ester, to yield orange-red spots. The resolution of the 3,5-dinitro- benzoates of alcohols on paper has been effected by Rice, et aL (28), and Meigh (29), and on columns by Holley and Holley (30), and White, et aL (31). Kariyone, et aL (32), separated the potassium xanthogenates on paper. Davenport and Sutherland (33), prepared the phenylazophenyl- urethans of terpene alcohols and separated them on an alumina column, identifying them by their melting points.