The Colorimetric Determination of Hexosey with Carbazole

The difficulty of applying classical procedures to the qualitative and quantitative determination of carbohydrate in certain biological materials has led to the development of calorimetric methods in which the carbohydrate-containing material is treated with strong mineral acids, causing t,he formation of substances which will react with compounds such as diphenylamine, resorcinol, orcinol, indole, carbazole, et,c., to give distinctively colored products. The rate at which these colored products are formed (1) and the nature of their absorption spectra are frequently sufficiently distinct’ive to allow their use in differentiating the various sugars. One of the most widely used of the above calorimetric methods is the carbazole-sulfuric acid method first described by Dische (2-4) and further developed by Gurin and Hood (5, 6) for the identification and estimation of hexoses and pentoses. The latter procedure was used by Seibert and Atno (7) for the analysis of the polysaccharides present in serum and by Knight (8) for the identification of the sugars present in influenzavirus. Dische (9) has recently described a modification of the carbazole method for the analysis of uranic acids. Although the carbazole-sulfuric acid method has been used extensively, no systematic study of the variables influencing this method appears to have been reported. Difficulties encountered in the quantitative application of the carbazole method have been commented upon (7) and indeed the significance of the carbazole reaction, or other color tests, for the qualitative identification of sugars has been questioned (10). Recently in the course of a study of the polysaccharide fractions from hog gastric mucin, we have had occasion to investigate the more important variables associated with the carbazole reaction as applied to the determination of hexoses. In the course of this study the optimum conditions for the quantitative determination of hexose were determined and certain aspects of the qualitative identification of hexoses were examined.