Spectroscopic data for steroids subjected to Zak color reaction.

HE COLOR TEST OF Zak et al. (1, 2, 3), in which a mixture of concentrated sulfuric acid, ferric chloride, and acetic acid is added to a solution of a steroid in glacial acetic acid, gives a purple color for steriods with the 5-ene,3$-ol grouping, and has proved useful for the clinical determination of cholesterol. These authors have qualitatively described the colors given by 22 other steroids in the visual spectral range. We have investigated the feasibility of qualitative and quantitative determinations in the range of 320 to 700 m of a number of steriods not previously studied by this reaction. In the clinical procedure (2) the ferric chloride-sulfuric acid solution is added to the solution of steroid in acetic acid. This step involves the liberation of considerable heat; to reduce the variability of heat liberation in mixing, we have modified the test as follows: To 0.01-1.0 mg. steroid obtained from an ethanol solution evaporated to dryness in a test tube is added 1.0 ml. glacial acetic acid. After solution of the steroid is effected, 4.0 ml. of a previously prepared and cooled mixture, consisting of 0.5 ml. of a solution of 2.5 Gm. FeCls. 61120 in 25 ml. glacial acetic acid, 50 ml. 96% sulfuric acid and 50 ml. of glacial acetic acid, is added by underlaying and then mixing. Two hours after mixing, the solution is read in a spectrophotometer against a reagent blank. The steroids investigated were of a high degree of purity, unless otherwise indicated; those with hormonal properties were obtained