Determination of reducing sugars by oxidation in alkaline ferricyanide solution.

Abstract A suggested procedure is described which is applicable to 0.3–13 mg quantities of glucose. Five milliters of sample solution and 5–15 ml 0.04 M potassium ferricyanide in 5.0% Na2CO3 are heated 30 min in a water bath at 80°C. The reasons for choice of 80° are discussed. Ferricyanide is determined spectrophotometrically at 418 mμ wavelength, or iodometrically by the Mohr method. Ferricyanide solutions in Na2CO3. when well protected from light, have been kept at least six months at room temperature without any evidence of decomposition whatsoever. The molecular absorbance of ferricyanide is 1020 under the conditions of alkalinity of the method. The factors which affect the accuracy of the Mohr method were studied; the conditions which must be met for accurate determination of ferricyanide are summarized. A linear relation between glucose and ferricyanide reduction is obtained up to about 75% reduction of ferricyanide, and often the relation is linear to almost 90% reduction. Equations are given for calculation of glucose under prescribed conditions of procedure. In the boiling water bath, about 95°C at Boulder, Colorado, altitude 5300 ft, the reduction of ferricyanide is quite similar to that of the cupric-tartrate complex of the Shaffer-Somogyi reagent when both reagents contain the same concentration of Na2CO3, NaHCO3, and oxidizing agent. The rates are parallel and the reaction is more than 95% complete at about 30 min; approximately 8.1 moles of both oxidizing agents is reduced per mole of glucose, M 1 M 2 . At the altitude of Chicago (600 ft) the same degree of reduction is attained in 15 min. But when both reagents contain Na2CO3, and no NaHCO3, the reactions of both approach completion within 5–10 min in the boiling water bath, 95°C; M 1 M 2 , although of approximately the same magnitude with both reagents, is considerably reduced. At 80°C, only the ferricyanide reagent, with Na2CO3 alone, gives satisfactorily complete reduction within less than 30 min; the reaction is complete within about 20 min, and M 1 M 2 agrees closely with that at 95°C. The following sugars give approximately the same relative reduction per milligram (glucose = 100%) respectively by the authors' and the Shaffer-Somogyi methods: glucose 100, 100; fructose 99, 95; galactose 78, 80; sorbose 86, 86; xylose 97, 96; lyxose 96, 96; and arabinose 88, 87. Greater relative reductions per milligram are given by the authors' the disaccharoses whereas the copper reagent apparently oxidizes only the reducing moiety. The advantages of the greater relative reducing ability of ferricyanide are pointed out. The following variables do not significantly affect the reduction of ferricyanide in Na2CO3 solution: (1) size of test tube, from 18 to 31 mm diameter, and volume of reaction mixture, from 4 to 20 ml, provided that the rate of heating is rapid and uniform; (2) time of standing of the reaction mixture up to as long as 30 min before heating; (3) concentration of Na2CO3 from 0.625 to 4.37% in the reaction mixture, provided the molar ratio of Na2CO3: ferricyanide is greater than about 10: 1; (4) the time of standing of the reaction mixture after heating and cooling. Immediate determination of residual ferricyanide is recommended, although ferrocyanide reoxidation is extremely slow, if at all, less than 2% occurring within 24 hr. Three factors greatly affect the oxidation: (1) presence of NaHCO3, either in the reagent or produced by unneutralized acid in the sample; (2) ratio of Na2CO3: ferricyanide; (3) concentration of ferricyanide. The first two are ruled out when the authors' recommended reagent is used and the sample is properly neutralized. The third factor, therefore, is the most important variable. The following general equation may be used for approximate calculation of milligrams glucose, g, in the sample when the milliequivalents of ferricyanide reduced, T, and the molar concentration of ferricyanide, F, in the reaction mixture are known: g= T 0.02884+0.2127F