CHROMATOGRAPHY AND ITS APPLICATION TO THE ESSENTIAL OIL AND COSMETIC INDUSTRIES* ., By ALEX POST Laboratories of Polak's Frutal l/Forks, Inc., Middletown, N.Y. SINCE THE OBSERVATION by Tswett in 1906 on the separation of plant pigments on a, column containing powdered chalk, chromatography, in its many variations, has grown tremendously culminating, but not ending, with the work of the Nobel prize winners, Martin and Synge, in 1952. Its use both in research and analysis has given the investigator an insight to problems that hitherto were considered unsolvable or an- alytically impracticable. First, let us talk about some of the principles and techniques of both columnar and paper chromatography. In columnar chromatography, the resolution of organic compounds de- pends upon the difference in the adsorption characteristics of these com- ponents toward a given adsorbent. Many adsorbents have been used, such as: alumina, magnesia, charcoal, silicic acid, talc, magnesium silicate, calcium sulfate, etc. Any adsorbent that may seem of interest to an in- vestigator may be used. However, he should consult the literature to see what adsor, bents have been used previously. In ,general, a columnar chromatographic analysis is performed as follows: the sample, in a suitable solvent is adsorbed at the top of the adsorbent column. The adsorption is usually indicated by a coloring at the top of the adsorbent. Then various.:. solvents or solvent mixtures are passed through the column in order to desorb one of the components from the adsorbent. This is continued until this component is completely separated from the other components. It may then be analyzed by chemical or physical means after the zone has been separated from the column by either pushing out the entire column, cutting away the zone and eluting it, or by further washing the column with the eluting solvent until the zone is completely washed free from the column. Because of the components' selective distribution between the mobile solvent phase and the immobile adsorptive phase, the solutes migrate at * Presented at the December 10, 1953, Meeting, New York City. 23

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