80 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS EXPERIMENTAL Qualitative determination of solubility of cholesterol in isopropyl myristate: Cholesterol* and isopropyl myristate-I- were heated to 95-100øC. The mixtures were stirred until solu- tion was complete. The solutions were then cooled to room temperature (2 iøC) and observed for the presence or absence of crystals. Quantitative determination of solubility of cholesterol in isopropyl myristate.' Cholesterol and isopropyl myristate were mixed together and stored in covered beakers for at least 2 weeks. An aliquot of each solution (or an aliquot of the supernatant liquid) was with- drawn and diluted with an equivalent volume of anhydrous chloroform. Optical rota- tion measurements for the resulting solutions were made using the Perkin Elmer Model 141 Polarimeter½ equipped with a 5 cc capacity, 1 decimeter cell. RESULTS Qualitative measurement.' A series of solutions of cholesterol in isopropyl myristate were made in the concentrations as is shown in the chart that follows. •Solution (per cent by weight)-- A B C D E F G H I Cholesterol 9.8 8 6 5.5 5.25 5 4.5 4 2 Isopropyl myristate 90.2 92 94 94.5 94.75 95 95.5 96 98 At t = 0, all solutions, except A and B, which were a mass of crystals, were placed at 5øC. Two and one-half hours later, solutions C, D, E, and F formed crystals and solu- tions G, H, and I were devoid of crystals. The mixtures were then placed at room temperature (21øC). Eighteen hours later, while at room temperature (21øC), there were no crystals in .E, F, G, H, and I C and D remained as a slurry of crystals and A and B were almost a solid mass of crystals. Quantitative measurement.' A series of mixtures of cholesterol in isopropyl myristate was made. The system compositions ranged from 1.0 to 8.0 per cent by weight cholesterol. Table I indicates the observed rotations for all the samples made. A plot of the per cent composition versus observed rotation, at both 589 and 365 nm (as is shown in the chart that follows), over the concentration range of 1 through 5 per cent (by weight) choles- terol in isopropyl myristate was found to be linear. The rotations of the supernatants from solutions 6, 7, and 8 were used to calculate the cholesterol concentration in solu- tion by use of the calibration curve obtained from the rotation data obtained for solu-. tions 1 through 5 of Table I. *U.S.P. grade supplied by Amerchol, Edison, N.J. 08817. q-Armak, Chicago, I11. 60690. $Norwalk, Conn.

SOLUBILITY OF CHOLESTEROL 81 Per cent by weight of cholesterol in isopropyl myristate (starting mixture) Per cent by weight of cholesterol soluble in isopropyl myristate (supernatant solution) 589 nm 365 nm 5.5 5.14 5.28 6.0 5.37 5.39 8.0 5.26 5.15 Average 5.26 5.27 This procedure would enable one to determine unknown cholesterol concentrations in isopropyl myristate accurately using the Perkin Elmer Model 141 Polarimeter (or equivalent) at least one order of magnitude below that reported for the lowest concentration shown in Table I. An 0.05 per cent solution of cholesterol in isopropyl myristate chloroform (1:1) has a rotation of -0.017 ø at 589 nm and -0.057 ø at 365 nm. Incidentally, at 365 nm, there is still sufficient readability of the rotation to go down to a 1 to 5 dilution. Table I Observed Rotation a Percent by Weight of Cholesterol in Solution Number Isopropyl Myristate • 589 • 365 1 1.0 -0.163 ø -0.562 ø 2 2.0 -0.366 ø -1.217 ø 3 3.0 -0.540 ø -1.760 ø 4 4.0 -0.695 ø -2.382 ø 5 5.0 -0.868 ø -2.920 ø 6 5.5 b -0.897 ø• - 3.103 ø• 7 6.0 b -0.938 øe -3.168 • 8 8.0 b -0.918 ø½ -3.028 • aRotation of cholesterol/isopropyl myristate solution diluted by an equivalent volume of chloroform. Read- ings were taken at 24øC. bCrystals of cholesterol present. CSupernatant solution assayed. CONCLUSION The estimation of cholesterol solubility in isopropyl myristate by the classical approach .::::of visually ascertaining when the system composition can no longer support a homo- geneous condition requires a large number of experiments in order to put upper and lower limits on solubility. The use of optical rotation yielded a precise solubility figure with a minimum of effort. In principal, one need only examine the supernatant of a single isopropyl myristate/cholesterol system for cholesterol content which contains solid in equilibrium with solution. The simple optical rotation procedure indicates cholesterol is soluble in isopropyl myristate to the extent of 5.26 per cent (w/w). This technique conceivably can be