Photoelectric Study of Liebermann-Burchard Reaction and Its Significance in Determination of Cholesterol

Abstract

The common colorimetric method for the determination of cholesterol is based upon the Liebermann-Burchard 1 reaction, in which acetic anhydride and concentrated H₂SO₄ are added to a dilute solution of cholesterol in chloroform. The color produced is at first blue, then becomes green and finally, on long standing, a yellow-brown. This reaction, in spite of its apparent simplicity, has proved difficult to control, for the intensity as well as the shade of the color is markedly influenced by small differences in the concentrations of the reagents, the presence of traces of water or other impurities, time, and temperature. The many previous efforts to control these factors by modification of the technique of Autenrieth and Funk 2 and of Bloor 3 have been for the most part empirical. A study of the reaction, however, with the aid of the photoelectric colorimeter and varying filters gives a clue to the nature of the reaction and the principles that must be followed for exact, duplicable, colorimetric analysis of cholesterol.

A mixture of 15 cc of acetic anhydride, 1 cc of concentrated H₂SO₄ and 24 cc of chloroform, C.P. was used as the reagent. This mixture was prepared immediately before using, since it decomposes on standing for more than an hour. Five cc were added with thorough mixing to 5 cc of a chloroform solution containing 0.48 mg of cholesterol in a glass-stoppered cylinder. The vessel was immediately placed in a water bath maintained at the proper temperature. This procedure, which was also followed in the routine analysis of cholesterol, eliminated errors due to inaccuracy of measurements of small quantities of acetic anhydride and H₂SO₄ and to irregular initial temperatures produced by direct addition of H₂SO₄, and allowed accurate measurement of the effect of time and temperature.