A Colorimetric Method for the Determination of Levulose in Blood and Urine. ∗

Abstract

One volume of the solution to be analyzed, a half volume of concentrated HCl, and a tenth volume of a 20% alcoholic solution of diphenyl amine, in a large test tube are heated in a boiling water bath for 15 minutes and then cooled. It has been found convenient to close the tube with a one-hole rubber stopper with the hole stuffed with glass wool. Shaking the solution with a third volume of liquid (melted) phenol causes the immediate absorption of the diphenyl amine together with the color. The addition of a half volume of 95% ethanol renders the mixture homogenous and suitable for colorimetric comparison, which may be made immediately, although this is not necessary. The color tends to darken slightly on standing. Standards are prepared similarly and simultaneously from solutions of levulose. It is felt preferable to make these latter at fairly frequent intervals from a 1% stock solution, using of course a preservative as toluene. Since 1 mg. of levulose per cc. of solution gives a fairly intense color, the standards have been made to range downwards from this concentration. Similarly if necessary the solutions to be analyzed have been diluted to within this range.

This method has been found satisfactory in the analysis of aqueous solutions, considerably diluted urines and tungstic acid blood filtrates. In more concentrated urines interference has been encountered, the color obtained being darker than it should be and frequently of a somewhat different shade. Tungstic acid filtrates contain substances that cause the development of a faint greenish blue coloration, that increases slightly the apparent color given by levulose. While theoretically dilutions and standards should probably be made from tungstic acid filtrates, as the influence is rather constant, it has been felt possible to use water, making a small frequently determined correction, without introducing significant error, except possibly when the levulose concentration is very low.