抄録
Some factors affecting the skatole-hydro-chloric acid reaction for fructose were examin-ed. The chromogen extracted into chloroform was rather unstable and faded under the diffuse light or during a lengthy measurement in a spectrophotometer. One half volume of chloroform to one volume of the aqueous reaction mixture was found to be sufficient for complete extraction of the chromogen from the aqueous phase. By this procedure, the sensitivity of the method can be doubled so that a more dilute solution of fructose can be analyzed by this procedure. By modifying the incubation conditions to 60°C for 50min and omitting the chloroform extraction procedure, a stable color and a higher absorbance value per unit amount of fructose was obtained than the original method. Under these conditions, a straight calibration curve was obtained between fructose concentrations from 0.5 to 8.0 μg per 0.1ml of the sample solution (final volume, 1.0 ml; ε×10-3=22.5; color intensity ratio of fructose to glucose, ca. 86). This modified method has the advantage of fairly good sensitivity to fructose, but has the disadvantage of rather poor specificity to fructose. Moreover, the reagent blank has a rather high absorption and varies according to batch of the skatole reagent. Chloroform extraction procedure was not much effective in reducing the absorption of the reagent blank.
The time course of color development in the β-indolylacetic acid-hydrochloric acid reaction was studied at 40°C and 50°C. At a higher temperature, the rate of color development was much faster and the color yield was much higher, but the color intensity ratio of fructose to glucose was much lower, than at a lower temperature. It is suggested that fructose can be determined under any one of the conditions according to the need of sensitivity and/or specificity in the determination. When the amount of glucose in the sample solution does not exceed 10-fold over that of fructose to be determined, the modified procedure at a lower temperature (i.e., incubation for 80min at 40±0.1°C) is recommended for routine use. This method is simple in operation and gives reproducible results. The reagent is stable when kept in a refrigerator and the reagent blank absorption is very low.
