A micromethod for the determination of serum calcium.

The standard method for the determination of serum calcium based upon the precipitation of calcium oxalate and titration with permanganate does not lend itself to a micromethod because of the difficulty in handling very small precipitates. This difficulty is not encountered in the direct titration of calcium with the chelating agent, disodium dihydrogen ethylenediaminetetraacetate (EDTA). Schwarzenbach et al. (1) studied the formation of complexes of the alkali earth metals with EDTA, and the reagent was adopted for use in water analysis largely owing to the work of Diehl et al. (2) and Beta and No11 (3). The latter authors demonstrated that ammonium purpurate at pH 12 is a specific indicator for calcium and can be used in the titration of calcium with EDTA. Recently Elliott (4) presented a macromethod for the determination of serum calcium by this direct titration method. Unfortunately, the end-point of the titration is not sharp, but there is a gradual change from a red to a purple color. Also, when serum with a high icteric index is encountered, the end-point is further obscured. For these reasons, the end-point cannot be determined visually with the necessary degree of accuracy for the microdetermination of serum calcium. However, it has been found that the end-point can be determined with considerable precision with the aid of a spectrophotometer, and accurate determinations of calcium may be obtained with only 0.1 ml. of serum. While this work was in progress, Kibrick e2 al. (5) independently developed a micromethod for serum calcium, using these same principles. However, it is felt that the method to be presented has definite advantages over that of Kibrick et al. Apparently the calcium equivalence of the EDTA changes with varying calcium concentrations when the titration is carried out by their method. This would seem to indicate that the true end-point is not determined by their method. This difficulty is not encountered in the graphical method of determining the end-point to be presented, since the calcium equivalence of the EDTA remains constant with changing calcium concentrations. Also, in the method of Kibrick et al., errors may result due to dilution, fading of the indicator, and drift of the spectrophotometer. In the method to be presented, all these factors