Electrophotometric Determination of Phosphorus by the Method of Fiske and Subbarow

The substitution of the filter photometer for the visual matching of solutions directs attention forcibly to the fact that many of the reactions employed in quantitative colorimetric microanalysis yield colors that are highly variable with respect to their rates of intensification and fading. An attempt to carry out Bodansky's directions 1 for the determination of serum phosphatase activity with a filter photometer∗ demonstrated the procedure of Kuttner and Lichtenstein 3 for the determination of phosphorus to be particularly troublesome in this respect. The essay was made during an excessively hot and humid period of the summer when the daily fluctuations of temperature sometimes amounted to 10°C. Under these circumstances it was found impossible to reproduce with satisfactory accuracy from day to day the standard curves expressing the relation of the phosphorus concentration of the solutions to their absorption of light. The difficulty manifested itself in the irregularity of the values that were obtained for K₁, the proportionality constant in the familiar equation, C = 2—log G/K₁, where C is the concentration of phosphorus; 2, the logarithm of 100, representing complete transmission of light; and G, the reading of the galvanometer. Efforts to modify the influence of temperature by icing the solutions before mixing were defeated by the condensation of a film of moisture on the absorption tubes.

The replacement of stannous chloride, the reducing agent in the Kuttner and Lichtenstein method, by 1-amino-2-naphthol-4-sulfonic acid, as recommended by Fiske and Subbarow, led to more reproducible results: the effect of fluctuations of temperature was comparatively inconspicuous. However, the blue color did not attain maximum intensity within a short time, as we had hoped, but gained in intensity at a diminishing rate for as long as 72 hours, the limit to which we continued our observations.