A blue color develops when ketohexoses or sugars which liberate ketohexoses upon hydrolysis are heated in the presence of a strong acid and concentrated urea. Among the acids tested, perchloric acid produced the most intense and stable color. The absorption maximum (605 nm) was specific for ketohexose. Although aldohexose and aldopentose are less reactive than ketohexose, they exhibited characteristic absorption spectra. This reaction provides a simple calorimetric determination of ketohexoses.
Various methods for the calorimetric determination of sugars have been devised using combinations of strong acids and organic reagents (mostly aromatic compounds). Urea was used as an organic reagent in some reports; the use of sulfuric acid, stannous chloride, and urea for qualitative determination of ketohexoses (1,2) and sulfuric acid and urea for quantitative determination of fructose (3) has also been reported.
In this study, the conditions for the coloration reaction between fructose and urea in the presence of various acids were investigated. The reaction specificities of different sugars were also tested. The specificities demonstrated in this method are comparable to those of other methods in which sulfuric acid-cysteine-carbazole ( 4)) hydrochloric acid-resorcinol (5)) and hydrochloric acid-Fe3+-resorcinol (6) were used.
Methods
Materials. D-Deoxyribose was purchased from Sigma Chemical Co., St. Louis, MO.; D-(-)ribulose from Calbiochem, San Diego, Calif.; and D-tagatose from Nakarai Chemicals, Kyoto, Japan. Other sugars and sugar metabolites, 60% perchloric acid, urea, biuret, and sodium cyanate were obtained from Katayama Chemical Industries Co., Ltd., Osaka, Japan. The materials used were of reagent or analytical grade. Standardprocedure.
To sugar solution (0.5 ml) in a test tube, 1.2 ml of 60% perchloric acid and 1.3 ml of 8 M aqueous urea were added. After 349