Liebermann-Burchard Reaction with Carotene

Abstract

Conclusions

Carotene can readily be detected in as low a quantity as 0.01 mg. dissolved in chloroform by means of acetic anhydride and concentrated sulphuric acid. The reaction is similar in method of performance and in final color production to the one employed in the Liebermann-Burchard test for cholesterol. The difference in the duration of the green color developed may serve, however, to differentiate one compound from the other. The green color obtained with carotene develops its maximum intensity at once and fades within 2 minutes, leaving a dirty yellow brown color. The green color obtained with cholesterol develops its maximum intensity at the end of 15 minutes.

Carotene does not interfere with the quantitative estimation of cholesterol in the blood. The extraction with chloroform of a mixture of blood and plaster of Paris removes the cholesterol, but does not release the carotene present.

We have postulated some common chemical configuration, actually present or easily produced as a result of chemical interaction, in sterols, in carotene, in the fat-soluble vitamins A, D, and E, in terpenes, and in 5-membered monoheterocyclic compounds. 1 We have recently demonstrated that a mixture of formaldehyde and concentrated sulphuric acid constituted a very sensitive reagent for the detection of carotene in chloroform solution. 2 Whitby, 3 employing the same reagent, reported for cholesterol a color reaction which, however, is not identical with the one obtained for carotene. In this communication we report our findings with reference to the Liebermann-Burchard reaction with carotene. The reaction is the familiar one used in the qualitative detection and in the quantitative estimation of cholesterol. We have already shown that this reagent reacts with heterocyclic compounds like thiophene, furfurane and derivatives. 4

Carotene gives a positive response with the Liebermann-Burchard reagent, using 10 cc. of chloroform solution, 2 cc. of acetic anhydride and 0.2 cc. concentrated sulphuric acid. With low concentrations of carotene a green color is obtained, which matches in color the one developed when cholesterol is treated with the same reagent. In higher concentrations the green color is tinged faintly blue, but can still be matched with the color obtained with cholesterol. Copper-colored crystals of carotene exposed to sunlight and air turn yellow brown. Carotene, thus oxidized, does not give the characteristic color when dissolved in chloroform and treated with acetic anhydride and sulphuric acid. A brown color instead of the green develops. Old solutions of carotene in chloroform also react in the negative. Baumann and Steenbock 5 have recently shown that carotene in organic solvents deteriorates on standing. We therefore used freshly prepared chloroform solutions of carotene for our tests.