The determination of reducing sugars by titration of ferricyanide.

THE use of ferricyanide for the quantitative estimation of sugar was introduced by Hagedorn and Jensen [1923]. Their method has been extended to other sugars by Hanes [1929] and Hulme and Narain [1931]. These methods involve a preliminary heating for 15 minutes and a subsequent back titration. The fact that ferricyanide is only slightly coloured would seem to make it valueless for a direct titration method. In spite of this Hawkins and Van Slyke [1929] have introduced a timing method for the estimation of glucose in blood and urine, based on the time required for the decoloration of alkaline ferricyanide when immersed in a boiling water-bath. They state that a little practice is necessary to train the eye in judging the moment at which the yellow colour vanishes. I personally find it extremely difficult to know when to stop the watch 'but am more concerned with the fact that they make no mention of the variation due to alterations in barometric pressure. I discarded a timing method based on the decoloration of an alkaline copper solution when immersed in boiling water owing to this difficulty. Lane and Eynon [1923] introduced methylene blue as an internal indicator for the detection of the end-point when titrating Fehling's solution with reducing sugars. I have used this method with good success in class work for some years. It is admirable for the estimation of pure sugar solutions, but my students find it difficult to detect the end-point when applying it to urine. The colloidal substances in urine inhibit the aggregation of the cuprous oxide to the red form, so that the end-point is seen by a slight decrease in total colour rather than in a disappearance of the blue tint. It is particularly difficult to see this by artificial light. I find that the addition of methylene blue to an alkaline solution of ferricyanide enables. one to estimate reducing sugars by direct titration very rapidly and accurately. The indicator is not reduced until the whole of the ferricyanide has been reduced and the end-point, being a change from a blue or violet solution to one that is colourless, is unmistakeable.