By the use of chromatographically purified cholesterol, the coloration developed by SbCl3-CHCl3 reagent was studied. As shown in Table 1, the larger the amount of cholesterol present, the deeper becomes the pink color, the more adds an orange shade and the quicker in its development. As is shown in Fig. 1, the absorption maxima of the reaction product are found at 423 and 500mμ. The E values calculated from each of the maximum differ depending on the reaction time of the observation, and on the amount of cholesterol used. As is shown in Fig. 2, when cholesterol is 70 micrograms per ml. of the reagent, the increase of absorption at both 423 and 500mμ shows even after about 5 hours. In the case of 200 micrograms, however, the absorption at 500mμ increases much faster than that at 423mμ, and in contrast with the apsorption at 423mμ that reaches an approximate constant after changing its position into 408mμ via 415mμ. When a color solution, the development of which is still increasing, is diluted with another aliquote of reagent, the apsorption at 423mμ decreases in proportion to the quantity of newly added reagent, while the 500mμ maximum does not (Fig. 3). On the contrary, a color solution, when the development of which has ceased to increase, the color is diluted in proportion to the quantity of newly added reagent.
The reaction products of cholesterol with the reagent were recovered by treating the colored solution with KOH and ether, and chromatographically separated into several fractions (Table 2 and Fig. 5).
On the basis of the data, the process of the color development of cholesterol can be traced as follows: Cholesterol→ a substance with a maximum at 235mμ→ a substance with maxima at 265, 275 and 287mμ→ a substance with maxima at 292, 305 and 320mμ.