Abstract
The reagent consists of 0.2 per cent. aqueous solution of orcein (Kahlbaum) and 85 per cent. phosphoric acid (Eimer and Amend) in separate containers.
The qualitative test is carried out as follows: to 1 c.c. of the solution under investigation in a test tube, add from 6 to 8 drops of the orcein solution and 1 c.c. of the phosphoric acid. The test tube is heated to boiling over a free flame and then placed in a boiling water-bath for ten minutes. If levulose is present, a yellow color appears which is deeper for greater concentrations of the sugar. A yellow precipitate settles out on cooling. Upon the addition of alkali (KOH or NaOH) sufficient to neutralize the phosphoric acid, the yellow color changes to a distinct orange. The characteristic orange color developed was made the basis for the quantitative determination of levulose.
The procedure for the quantitative test is essentially the same. The standard for colorimeter comparison is a definite known solution of levulose in distilled water. The strength of the standard is arbitrary but should be as near the concentration of the unknown solution as possible to favor a more accurate comparison. Hence the advisability of performing a preliminary test to determine the optimum standard. The standard and unknown solutions are similarly treated, to 1 c.c. of each in separate test tubes add 8 drops of the orcein solution and 1 c.c. of phosphoric acid. The solutions are, as before, boiled over a free flame and heated in a boiling water-bath for ten minutes. The test tubes are then removed and the contents of each are transferred quantitatively to separate ten c.c. volumetric flasks and made up to the mark with 5 N NaOH. The solutions are at once placed in the colorimeter chambers and the orange colors compared.
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