NATURAL AND SYNTHETIC WAXES 121 techniques have been used to identify individual waxes in possible mix- tures of waxes used in cosmetic products. In this report, beeswax will be used as a reference wax to illustrate the techniques evolved. EXPERIMENTAL Thin-Layer Chromatography Glass plates (20 54 20 cm) were prepared by spreading a slurry of 30 grams of Silica Gel G in 60 ml of distilled water on five plates at a 0.25-mm thickness. The air-dried plates were further activated by heat- ing at 105øC for 30 minutes. The prepared plates were stored in wooden cabinets over a drying agent and used within two days. Shortly before use, the preparod plates were predeveloped in a mixture of chloroform/ methanol 80/20 v/v. The solvent mixture was run to the upper edge of the plate as a washing procedure. The predeveloped plates were then dried again for 10 minutes at 105øC, and cooled at room temperature before use. Sample Preparation Quantities of 0.5 g of waxes were dissolved and diluted to 10 ml with warm chloroform. The waxes which were slightly soluble in chloro- form (e.g., carnauba wax) were maintained in solution in a lukewarm water bath until used. One or 2 v1 of solution representing 50 and 100 vg of wax were applied at 2 cm above the bottom edge of the pre- developed plate, and after complete drying of the applied solutions, the plates were placed into the developing chambers. Developing Chambers The fully saturated atmosphere in the chambers was obtained by lin- ing the walls with Whatman No. 1 filter paper. To obtain a fully equili- brated solvent system, the well-mixed mobile phases were transferred to the lined chambers at least one hour prior to development. Mobile Phase (v/v) The following mobile phases were prepared: A. n-Heptane-ethyl ether anhydrous-acetic acid (90:10:2) B. n-Heptane-ethyl ether anhydrous-acetic acid (95:5:2) C. Chloroform-ethyl ether anhydrous-acetic acid (90:10:2) The mobile phases A and B (2) were adopted to obtain the best reso- lution of the main classes of constituents. The fast moving spot adjacent

122 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS to the solvent front represents the hydrocarbon portion. The next spots, in descending order, are simple esters, mixture of esters, acids, and alco- hols (Figs. 1 and 2). Mobile phase C was developed for the study of the polar portion of waxes: the alcohols, acids, etc., are moved to a higher Rt (Fig. 3). All plates were developed 12 cm above the origin. All solvents were analytical grade reagents used without further treatment. Figure 1. Thin-layer chromatographic sep- aration of classes of constituents. Mobile phase: n-heptane, ethyl ether, acetic acid (90:10:2) 1. Beeswax, 50 2. Beeswax, 100 3. n-Docosane, 10 Figure 2. Thin-layer chromatographic sep- aration of classes of constituents. Mobile phase: n-heptane, ethyl ether, acetic acid (95:5:2) 4. Sperraaceti esters, 10 5. Docosanoyl alcohol, 10 6. Docosanoic acid, 10 Methods o[ Detection Detection of constituents was accomplished by exposure to iodine vapors and by use of a sulfuric acid-potassium dichromate reagent. This reagent was prepared by slowly adding 180 ml of concentrated sulfuric acid, under cooling, to a solution of 2.25 g of potassium dichromate in 120 ml of distilled water. The developed plates were thoroughly dried to eliminate the acetic acid and cooled at room temperature before the exposure to iodine vapors. Any iodine reactive spots were rapidly recorded and the plates were heated again for a short period of time (10 min at 90-100øC).