Colour Reactions of Sterols

(a) Liebermann­Burchard reaction: A chloroform solution of a sterol, when treated

with acetic anhydride and conc. H2SO4 gives a grass­green colour. The usefulness
of this reaction is limited by the fact that various sterols give the same or similar
colour. This reaction forms the basis for a colorimetric estimation of cholesterol
by Sackett’s method (Basis of the reaction—see above).

(b) Salkowski test: When a chloroform solution of the sterol is treated with an

equal volume of conc. H2SO4 develops a red to purple colour. The heavier acid,
which forms a layer below assumes a yellowish colour with a green fluorescence,
whereas the upper chloroform layer becomes bluish red first, and gradually
turns violet-red.

(c) Zak’s reaction: When glacial acetic acid, (aldehyde free), solution of cholesterol

is treated with ferric chloride and conc. H2SO4, produces a red colour. This reaction
forms a basis for the colorimetric estimation of choles- terol (Zak’s method).