NATURAL AND SYNTHETIC WAXES 123 Figure 3. [bin-layer cbromatographic sep- aration of classes of constituents. Mobile phase: chloroform, eth)l ether, acetic acid (90:10:2). See Figs. ! and 2 for code The warm plates were evenly sprayed with the sulfuric acid-potassium dichromate reagent. At this point various constituents such as steroids and triterpenoids appeared as colored spots characteristic of lanolin and candelilla wax. They were recorded before the final charring of the spots was obtained by further heating (25 rain at 120øC). Permanent records of each chromatograph were made by photography. The total time for the TLC and charting was 1.5 to 2 hours. Colurn •'• Chromatography By monitoring the chromatographic column with TI•C, a procedure was developed to reflect the separation of the five main classes of con- stituents observed on analytical TLC (mobile phase A). The use of column chromatography is superior to preparative TLC in collecting large amounts of the various fractions for further study and routine analysis. Alumina* (225 g) was stirred for 10 minutes with 1 1. of 1% HCL aqueous solution. The mixture was allowed to settle and was decanted. The ahunina was then dried at room temperature and activated at 110øC for 16 hours. The dry treated alumina (48 g) was poured into a 12-in. X 1-in. glass column (fitted with a stopcock) and rinsed with 100 ml of * No. 9296, Matheson-Coleman, East Rutherford, N.J.

124 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS n-heptane. One gram of wax dissolved in a small volume of n-heptane was carefully introduced onto the top of the prepared column. The use of gradient dudon resulted in five major wax fractions (Table I). Table I Wax Fractions Obtained using Gradient Elution Column Chromatography Eluates Fractions A. 250 ml ofn-heptane B. 400 ml of 4% ethyl ether in n-heptane C. 250 ml of 25% ethyl ether in n-heptane D. 250 ml of 3.5% n-propanol in n-heptane E. 250 ml of 25% acetic acid in n-heptane Hydrocarbons Esters I Esters II Alcohols Acids The eluates were taken to dryness without further treatment, except for eluate E, which was thoroughly washed with distilled water to elimi- nate the acetic acid prior to evaporation. The dried residues were weighed and then checked by TLC to control the efficiency of the separa- tion and to make sure that no major hydrolysis had occurred. The resi- dues were also checked by infrared spectrophotometry (IR) as a thin film between sodium chloride plates. Gas-Liquid Chromatography The separation of the class constituents into their individual com- ponents is best achieved via GLC. The objective was to analyze the fractions from the cohmm without further chemical modification. This was partially realized by the development of a short column containing an inert support with a nonpolar coating. This one column effectively separates the "as is" hydrocarbon and alcohol fractions and the methyl ester derivatives of the acid fraction. Total time for all three separa- tions is less than 45 minutes. Table II gives the instrument parameters and sample concentration used. RESULTS AND Discussion With TLC as a diagnostic tool, several natural waxes were studied as were synthetic waxes, wax substitutes, and mixtures of waxes. Most natural and synthetic waxes were separated by column chromatography (Table III). Depending upon the waxes analyzed, the total recovery of waxy •naterial varied from 82--99%. Those waxes which showed low recoveries contained very polar substances that were retained on the col u ran.