100 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS From Fig. 1, the soluble beeswax alcohols which will be associated with the 0.239-gm. insoluble beeswax alcohols = 0.044 gin. ' Total beeswax alcohols in 0.5-gin. sample = 0.239 gm. + 0.044 gm. = 0.283 gm. But total alcohols = 0.906 gin. (Table 8). ' Beeswax alcohols in whole sample 0.906 - 0.5' X 0.283 = 0.513 gm. From Tables 4 and 5 beeswax alco- hols = 53.8% (total unsaponi- fiable material) - 13.5% (hy- drocarbons) = 40.3% of the bees- wax. 0.513 .'.Beeswax - 0.40• - 1.273 gm. (103.6% recovery). Total spermaceti alcohols = total alcohols - beeswax alcohols = 0.906 gm. - 0.513 gm. = 0.393 gm. and spermaceti alcohols = 49.7% of spermaceti (Table 2) 0.393 .: Spermaceti - 0.497 - 0.791 gin. (96.5 % recovery). Calculation of Non-Beeswax 'Hydro- carbons Total hydrocarbons = 5.693 gm. (Table 8). Beeswax hydrocarbons = 1.273 X 0.135 = 0.172 gm. ß Non-beeswax hydrocarbons = 5.693 gm. - 0.172 gm. = 5.521 gin. (99.5% recovery). EXPERIMENTAL PROCEDURE Saponification of Sample and the Chromatography of the Unsaponi- offable Fraction. S•/ponify sample by retluxing for two hours with 25 ml. of absolute alcohol, 1 gm. KOH, and 50 ml. of benzene. Transfer ß the saponified mixyure to a separa- tory funnel, add 75 ml. of hot water, shake well and draw off the aqueous layer.* Continue the ex- TABLE 8--ANALYTICAL DATA OF MIXTURE OF HYDROCARBONS• BEESWAX• AND SPERMA- CETI Gm. Sample: U.S.P. Mineral Oil ............... 5. 549 U.S.P. Beeswax ................. 1. 229 U.S.P. Spermaceti..: ............ 0. 820 Fatty acids ....................... 1. 034 Unsaponifiable matter ............. 6. 606 Unsaponifiable matter insoluble in heptane and petroleum benzin (insoluble beeswax alcohols) ...... .0.314 Unsaponifiable matter soluble in hep-. • tane and petroleum benzin (by difference) ...................... 6. 292 First petroleum benzin fraction* .... ' 5.690 Second petroleum benzin fraction... 0.003 First hot alcohol fraction ........... 0. 586 Second hot alcohol fraction ......... 0.006 Solubility of 0.5-gm. sample of com- bined alcohols in cold methyl alco- hol ............................ 0. 261 * Rate 4 ml. p.b./minute. traction with two additional 50-ml. portions of hot benzene. (Reserve the extracted aqueous solution.) Combine the benzene extracts wash with three 30-ml. portions. of 30% alcohol, and add the washings to the reserved extracted aqueous solu- tion. Filter the washed benzene •xtract through a' cotton plug into a 250-ml. tared beaker, evaporate the benzene on the steam bath, dry the residue in an oven at 100øC for 15 minutes, cool in a vacuum desiccator, and weigh as unsaponi- fiable m•.tter. Repeat the heating of the unsaponifiable matter in the oven until the weight is constant to 1-2 mg. • * Troublesome emulsions can be broken by fastening the stopcock of the separatory funnel with a rubber band and partially im- mersing the separatory funnel in a steam bath until the contents begin to boil.

MIXTURES OF HYDROCARBONS, Acidify the reservecf aqueous solu- tion with HC1, extract kith three 30-ml. portions of chloroform, and wash the combined chloroform ex- tracts with w:ater. Filter the washed chloroform extract through a cotton plug into a tared beaker, evaporate the chloroform on the steam bath, dry the residue in an oven at 100øC. for 15 minutes, cool and weigh as fatty acids. Repeat the heatting of the fatty acids until the weight is constant to 1-2 mg. Dissolve the unsaponifiable frac- tion in 50 ml. of boiling heptane and cool the solution to room tem- perature with stirring. Filter the resulting mixture through filter paper, wash the residue well with petroleum benzin, and reserve thee flitrate. Dissolve the residue on the filter paper by pouring hot chloroform through the filter, evap- orate the chloroform solution on the steam bath, dry the residt•e in an oven at 100øC. for 15 minutes, cool in a vacuum desic'cator, and weigh as unsaponifiable material insoluble in heptane and petroleum benzin. Re- peat the drying in the oven until the weight ik,constant to 1-2 mg. Evaporate the reserved petroleum benzin-heptane flitrate on the steam bath, take up the residue in 50 ml. of warm petroleum benzin (b.p. 30-75øC.), cool the solution to room temperature, transfer solution with the aid of 25 ml. petroleum benzin tO oa chromatograph tube contain- ing a 9 X a/4-in. activated A1203 column ("Alorco" grade F-20 mesh 80-200), and allow the solution to flow through the column at a rate BEESWAX, AND SPERMACETI 101 of 3.3 to 5 ml. per minute. Follow this solution with 175 ml. of petro- leum benzin, combine the flitrates, and label as first petroleum benzin fraction. Pass another 50 ml. of petroleum benzin through the •col- umn and label flitrate as second petroleum benzin fraction. Re- move the petroleum benzin by pass- ing 50 ml. of 95% alcohol, under air pressure, through the column. Follow this with 125 ml. of boiling alcohol which is also forced through the column at a rapid rate (4-7 minutes) with air pressure. Mark the combined two tiltrates as the first hot alcohol fraction. Pass an- other 50 ml. of boiling alcohol through the column and label as second hot alcohol fraction.* Evaporate the various fractions on the steam bath. Dry the tWO petroleum benzin fractions in an oven at 100øC. for 15 minutes, cool in a vacuum desiccator, and weigh. Repeat drying in oven until weight is constant to 1-2 rag. Redissolve each of the hot alcohol fractions in 10 ml. of chloroform, evaporate chloroform on steam bath, and dry and weigh residues in the manner described for the petroleum benzin fractions. Solubility Procedure. Weigh sam- ple into a 150-ml. beaker, add 80 ml. methyl alcohol, cover beaker with watch glass, and heat on hot plate until solution' is complete. Transfer beaker to ice bath and stir solution vigorously wi{h thermom- * The second fraction obtained in each case above should contain negligible amounts of non-/'olatile material.