NMR spectra of Liebermann-Burchard color reagent

The intensities of colors of ergosterol dissolved in various mixtures of acetic anhydride, glacial acetic acid and sulfuric acid (Liebermann-Burchard reagent) have been investigated by inspecting NMR spectra of the reagent. A positive relationship has been found between color intensity and chemical shift of methyl proton of the reagent mixture. The chemical shift of methyl group in acetic acid in the reagent moves to low field, its amount depending on the concentration of sulfuric acid which will enhance the formation of CH3CO+. The movement has been enhanced with increasing concentration of acetic anhydride in 025 mole % sulfuric acid, and with increasing concentration of glacial acetic acid in 25 mole% or more of sulfuric acid concentration. It seems that the magnitude of intensity of color developed in Liebermann-Burchard test is dependent largely on the concentration of CH3CO+ in the reagent, and is also propotionate to the movement of chemical shift of methyl proton to low field. A reaction mechanism of Liebermann-Burchard color test has been given from these results.