]. Soc. Cosmetic Chemists, 21, 119-128 (Feb. 4, 1970) Characterization of Natural and Synthetic Waxes Using Combined Chromatographic Techniques VINCENT ABATE, B.S., VIOLETTE BADOUX, B.S., S. Z. HICKS, B.S., and MONROE MESSINGER, B.S. *• Presented May 8, 1969, New York City Synopsis--A detailed thin-layer, gas-liquid, and column CHROMATO( RAPHIC PROCEr•URE for the evaluation of single WAxEs and various mixtures normally found in cosmetic preparations is presented. The thin-layer chromatographic systems developed have been found to give the best possible class separations of the wax components, which has facilitated the analysis of mixtures. The gas-liquid chromatographic evaluation utilized short columns (16-18 in. long) on the hydrocarbon, alcohol, and acid components of the various waxes. Thus the alcohols with chain lengths to Ca, were easily resolved without resorting to derivatives. This combination of the various techniques described resulted in the identification of individual waxes. INTRODUCTION The analysis and characterization of waxes have been receiving in- creased attention recently. Several workers (1-7) have attempted to characterize various waxes using the well-known chromatographic tech- niques. Recently, Holloway and Challen (8) have presented a systematic approach to the analysis of natural waxes using thin-layer chromatog- raphy (TLC) combined with various detection methods. However, a systematic approach to the characterization of natural and synthetic waxes, using combined chromatographic techniques, has not been re- ported. Plant and animal waxes are, generally speaking, compositions made up largely of nonglyceryl esters formed in nature by the union ot• higher alcohols with the higher fatty acids and with which are associated one or more of the following components: free fat and wax acids, free * Chesebrough-Pond's Inc., Clinton, Conn. 119

120 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS monohydric alcohols, and sterols, hydrocarbons, lactones, and other con- densation compounds. This paper describes a detailed systematic ap- proach to wax characterization utilizing TLC, liquid chromatography, and gas-liquid chromatography (GLC). The analysis proceeds in three steps: 1. The use of analytical or diagnostic TLC which separates the waxes into "characteristic" classes. 2. Column chromatography to separate quantitatively on a prepara- rive scale the five main classes of constituents observed on the analytical TLC plate. 3. GLC to further characterize some of the fractions obtained via column chromatography. In the TLC step, the waxes are looked at in their "natural" state, using three mobile phases, two developed by Carlier et al. (2) and one which was developed in this laboratory. The column chromatography, which was developed as a preparative step to reflect the class separation achieved via TLC, results in five main fractions: hydrocarbons, simple esters, a mixture of esters (containing possible diesters, triesters, acid esters, etc.), alcohols, and acids. GLC is used on some of the fractions obtained from the column to separate these complex mixtures into as many individual components as possible. Though the hydrocarbons can be easily fractionated via a GLC procedure, the less volatile and more polar compounds such as the alcohols and acids present greater difficulties. Downing et al. (9) circumvented these obstacles by converting oxygenated components of beeswax into hydrocarbons containing the same number of carbon atoms by a process of chemical reduction. In this laboratory, the GLC "short column"* (an 18-in. long X 1/4-in. diameter copper tube filled with acid-washed Chromosorb G coated with 1.5% Apiezon grease) was developed. This simple column allows an efficient resolution of the hydrocarbons and alcohol fractions directly and the acid fractions as their methyl ester derivatives. An added advantage of the use of short columns is the decreased retention time. For example, the separation of the C24-Ca2 alcohols requires less than 10 minutes. The combined chromatographic techniques evolved have been ap- plied to the study of many of the natural and synthetic waxes used by the cosmetic chemist, some of which will be presented here. Also, these *Subsequent work has resulted in a more efficient column, a 24-in. long X ¬-in. diameter stainless steel tube filled with AW/DMCS Chromosorb W coated with 5% Sili- cone Gum Rubber SE30 (methyl).