Deviation from the “Beer Law” in the Kuttner-Cohen Method for Determination of Phosphorus.

Recently, when determinations of inorganic phosphate in mixtures containing sodium glycerophosphate (used as a substrate in plasma phosphatase determinations) were required, the use of heat involved in the Benedict-Theis method rendered it obviously unsuitable. The many advantages of the Kuttner-Cohen method, 1 with the modifications later suggested by Kuttner and Lichtenstein 2 and Raymond and Levene, 3 recommended themselves to us. We found this method capable of yielding very accurate results (within ±2% of the known quantities), after correction for the considerable deviations from the “Beer law”. These deviations were determined by a large number of analyses, charts were constructed and tables compiled for more convenient reading.

We use 1 cc. of plasma when it is expected to contain 5 mg. of phosphorus per 100 cc. or more, and 2 cc. of plasma when it is expected to contain less. Ten cc. of water are added and 5 cc. of 10% trichloracetic acid. About 14 cc. of filtrate are available. Four cc. of the acid-molybdate mixture is added to an aliquot diluted to 5 cc, the tube is mixed by tapping, and 1 cc. of stannous chloride solution∗ is added. Several standards of different concentrations may be employed in a series of tests.

We found it convenient to employ a single standard containing 0.02 mg. per 5 cc. We usually include in one series about 18 tests, triplicate 0.02 mg. standards, and, as a check upon all foreseen and unforeseen sources of error, several solutions of known concentration covering approximately the same range as the unknowns. After calculation of the results the correction is applied.