A Test for Cholesterol Based Upon the Use of Sulphuric Acid Containing Selenious Acid.

Cholesterol dissolved in chloroform gives a characteristic color test with concentrated sulphuric acid containing in solution 125 mg. of sodium selenite for every 25 cc. of acid. The test is made by adding to a chloroform solution of cholesterol an equal volume of the selenious-sulphuric acid reagent. The reaction is positive when the top layer assumes a deep beautiful purple, while the lower layer assumes a red brown to a very dark brown color, but does not show any green fluorescence.

The test is more sensitive than the Salkowski test. The chloroform solution containing 0.0025% of cholesterol does not yield the typical color layers observed with the concentrated sulfuric acid in the Salkowski test, which is concentrated sulphuric acid. Such solution does not give the cherry red color in the top layer, and does not give in the bottom layer a red color by direct light and a green fluorescence in transmitted light. Instead it yields only a faint trace of pink in the top layer, and in the bottom layer a slight yellow color in direct light and a slight green fluorescence by transmitted light. On the other hand, the same strength solution of cholesterol with an equal volume of the selenious-sulphuric acid reagent still gives a deep purple in the top layer, and in the bottom layer a reddish brown without fluorescence.

With a 0.001% cholesterol solution the reaction is still positive with the new reagent, while with the sulphuric acid in the Salkowski test the reaction is atypical, no color being present in the top layer and but a very faint yellow color in the bottom layer, which by transmitted light gives a very slight green fluorescence.