Abstract
Colorimetric methods for the determination of inulin in blood serum or plasma and urine based on the reaction of inulin with diphenylamine have been described in recent years. The most widely used procedure is that of Alving, Rubin and Miller, 1 in which the inulin-containing solutions are heated in closed tubes with diphenylamine and hydrochloric acid dissolved in ethyl alcohol. Tightly capped tubes are necessary because of the volatile solvent, and because the solution must be heated for 60 minutes at 100°C to obtain the maximum color. In this laboratory as well as in others, this method has been troublesome and the results not consistently accurate.
For this reason a modification has been devised in which a less volatile solvent is used, thus permitting the use of open tubes. With this reagent a shorter period of heating is necessary for maximum color development and accurate results have been obtained in the determination of inulin in blood serum or heparinized plasma in concentrations as low as 4 mg per 100 cc. The determination can be carried out on 0.2 cc.
Method. Reagent. Three grams of diphenylamine, reagent grade, are dissolved in 100 cc of glacial acetic acid and to this solution are added 60 cc of concentrated hydrochloric acid.
This reagent is stable if kept in an amber bottle in a refrigerator at 6°C, and the color production with inulin is found to remain constant for at least one month. A few crystals of diphenylamine may precipitate out of solution but this does not affect the reagent. If kept at room temperature the color in the blank determination becomes progressively darker, but if correction be made for the blank as is automatically done in a photoelectric colorimeter of the Evelyn type the calibration curve is not changed.
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