liebermann–burchard test liebermann–burchard test the liebermann–burchard or acetic anhydride test is used for the detection of cholesterol. the formation of a green or green-blue colour after a few minutes is positive. liebermann–burchard testpurposedetection of cholesterol lieberman–burchard is a reagent used in a colourimetric test to detect cholesterol, which gives a deep green colour. this colour begins as a purplish, pink colour and progresses through to a light green then very dark green colour. the colour is due to the hydroxyl group (-oh) of cholesterol reacting with the reagents and increasing the conjugation of the un-saturation in the adjacent fused ring. since this test uses acetic anhydride and sulfuric acid as reagents, caution must be exercised so as not to receive severe burns. method: dissolve one or two crystals of cholesterol in dry chloroform in a dry test tube. add several drops of acetic anhydride and then 2 drops of concentrated h2so4 and mix carefully. after the reaction is finished, the concentration of cholesterol can be measured using spectrophotometry.