Three spectrophotometric
procedures have been described (1, 2) that permit the determination of three hexoses-galactose, mannose, and glucose-in mixtures with each other and with methylpentose. This procedure in its original form had &me drawbacks. One of the reactions, the so-called primary cysteine reaction, is strongly affected by even small amounts of chloride ions which at higher concentrations also depress the color of the so-called secondary cysteine-sulfuric acid reaction. This made it often impossible to carry out the determinations when hydrolysis had to be performed by hydrochloric acid. The present report deals with modifications of the primary and secondary cysteine-sulfuric acid reactions in which ,the sensitivity of the reaction is increased by 100 to 150% and greater stability of the color and independence of the presence of chloride ions are achieved. EXPERIMENTAL (a) Principle. Chloride ions increase the extinction coefficient of hexoses but not of other sugars in the primary cysteine H,SO, reaction, with a maximum effect at 0.75 N in the sugar solution, and depress the secondary cysteine reaction. This depression can be overcome by increasing the concentration of cysteine in the reaction mixture to about 0.7%. The reaction under these conditions proceeds much faster and the extinction coefficient is about 1000/o higher than with the original procedure.
(b) Procedure. To 0.5 <ml of a solution of hexose (40 to 100 pg/ml) is added 0.5 ml 1.5 N HCI. The test tube is placed in an ice water bath ; 5 ml of-a mixture of 1 part H,O and 6 parts concentrated H,SO, (C.P.) is added and the sample is shaken after about 1 min.