A Color Reaction for Carotene

Carotene in chloroform solution reacts with formaldehyde in sulphuric acid to form a characteristic deep violet zone between the chloroform and acid layer. The violet zone on shaking disseminates in the acid giving rise to a violet colored lower layer, while the chloroform layer above remains colorless. The addition of 2 to 3 drops of acetic anhydride causes a transitory blue to appear in the acid layer, provided a high concentration of carotene is present in the chloroform solution.

The test, which is considered positive on the appearance of a characteristic deep violet zone, is very sensitive, serving to detect 0.01 mg. of carotene in 2.5 cc. of chloroform. To carry out the test equal volumes of chloroform and formaldehyde-sulphuric acid mixture should be employed. For convenience we have used 2.5 cc. of the chloroform solution with 2.5 cc. of the aldehyde-acid mixture. This mixture is made up in the proportion of 1 volume of 37 to 40% formaldehyde to 50 volumes of concentrated sulphuric acid. Both the formaldehyde and the sulphuric acid should be chemically pure, and the mixture freshly made before using.

Cholesterol, according to Whitby, 1 gives a characteristic reaction with the formaldehyde-sulphuric acid mixture described above. With 1 to 2 mg. of the sterol dissolved in 2 cc. of chloroform he reported a cherry red color in the upper chloroform layer and a brownish red color in the lower sulphuric acid layer, which also showed an intense green fluorescence. When 2 to 3 drops of acetic anhydride were added to the chloroform layer previously transferred to a dry test tube, a blue color formed. With 0.1 mg. of cholesterol in 2 cc. of chloroform he obtained a deep reddish brown color and a very marked fluorescence in the acid layer, but no color in the chloroform layer. With 0.01 mg. of the sterol he could develop only a faint fluorescence in the acid layer and no coloration in the chloroform laver.