292 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS oil constituents, their separation from isomeric or azeotropic mixtures, and preparation in very pure form has been accomplished by the skillful use of chromatography. Taking advantage of different versions and techniques it is now possible to isolate trace compounds which, although present in an essential oil only in minute quantities, nevertheless exert great influence upon its odor and flavor (60, 133, 183). (Kirchner and Miller (84) have shown that chromatography can be applied also for the preparation of terpeneless oils.) The classical method of chromatography is that of selective adsorption on a column of active solid, development of the column, and elution with a series of solvents. Automatic fraction collectors add to the efficiency of the method. Another technique is partition chromatography (either paper or column partition), which also allows the examination of minute quanti- ties of material (173). Variations are the chromatostrip (100) and the chromatoplate (137) methods which permit the use of very small quanti- ties for simple and rapid characterization of an essential oil. New possibilities are being opened by the recent introduction of gas chromatography which gives very sharp separation (82). Once the nec- essary spectra have been accumulated and properly interpreted, gas chromatography will be a relatively simple and highly efficient tool in the hands of the essential oil and terpene chemist. Striking results have been obtained by a combination of chromato- graphic and spectroscopic methods: isolation of very small quantities of a substance in pure form, and rapid identification by means of its infrared spectrum. Entirely new vistas appear on the horizon with the emergence of Raman, mass, microwave, nuclear and magnetic resonance spectra (173), but in this far-reaching new field much more experience must be gathered, and better apparatus deyeloped before the techniques can be applied effec- tively in the essential oil laboratory. ELUCIDATION OF STRUCTURAL FORMULAS OF ESSENTIAL OIL CONSTITUENTS Reviewing the work of the early researchers (Wallach, Semmler, etc.) in the field of terpene chemistry toward the close of the last century one cannot help being amazed at how much was accomplished with the rela- tively primitive tools and techniques available at a time which has justly been called the "Elizabethan Age of the essential oil industry." Within less than thirty years the structures of most of the sesquiterpenes and their oxygen derivatives occurring in essential oils were established only the configurations of the more complex sesquiterpenes and azulenes defied at- tempts at elucidation. Representing formidable problems, these com- pounds could be attacked only after fundamentally new techniques of

RECENT DEVELOPMENTS IN ESSENTIAL OILS 293 research (high-efficiency fractional distillation, chromatography and spectrophotometry) had been introduced. With these new tools, terpene chemistry has entered a second rapid era of progress, and within the last five or six years the structures of a number of the most puzzling sesquiter- penes and azulenes have been solved. For example, •orm, Herout and their collaborators--a very progressive and successful research group in Czechoslovakia--have developed efficient methods of isolating and identi- fying sesquiterpene compounds. One of their methods is based upon careful fractional distillation, in combination with repeated adsorption chromatography, the latter controlled by infrared spectroscopy (146). Employing this technique, it is relatively easy to separate sesquiterpenes, in very pure form and without loss, from complex mixtures. In the labora- tory, the particular fraction is removed from the essential oil by fractional distillation, dissolved in petroleum ether and percolated through ten times its quantity of strongly active aluminum oxide. The eluate con- tains the hydrocarbons, free from all oxygenated admixtures. The mix- ture of hydrocarbons is then fractionated in high-efficiency columns, the distillation being followed by determination of the physical properties of the various fractions. In most cases the portions thus obtained have to be further fractionated by repeated adsorption chromatography on active aluminum oxide. Fractions containing the compound in question are dissolved in petroleum ether and chromatographed over one hundred times the quantity of adsorption medium. Again the course of chromatography is followed by determination of the physical properties, including infrared spectra. If these properties show accumulation of several chemical com- pounds the mixture is further chromatographed until the substances are obtained in absolutely pure form. In some instances distillation even at low temperatures may be harmful to certain components of the essential oil under investigation--for example, oils of chamomile or wormwood. In such cases the oil is not distilled, but is diluted with petroleum ether and submitted directly to repeated chromat- ographic separation. Mere fractionation by distillation is not suited to the separation of oxy- gen-containing sesquiterpenes. The experience of gorm, et al., has shown that in such cases repeated adsorption chromatography with a great excess of only slightly active aluminum oxide gives much better results. It has also been found that for the actual identification of the separated sesquiterpene compounds infrared spectrophotometry offers an almost ideal tool, particularly in view of the fact that only very small quantities of the material (20 to 30 milligrams) are required. The method can also be used most successfully for the elucidation of the structural formulas of the isolated compounds. Taking advantage of these new techniques gorm and his associates suc-