Ca/EGTA solutions: comparison between measured and calculated free calcium ion concentrations in the micromolar range

We prepared a series of Ca/EGTA solutions and compared the pCa2+free values obtained by calculation and measurement with an ion sensitive electrode. Two possible reasons for the discrepancies between these values were studied: (1) the effect of various association constants used for the calculation of pCa2+free, and (2) the influence of deviations of the calcium total concentration in the Ca/EGTA solutions from the values used for the calculation. The results obtained showed that the main reason for differences between calculated and measured pCa2+free values was the nonequimolarity of both stock solutions CaCl2 and EGTA. A linear dependence of measured electrode potentials with Nernstian behaviour on the calculated pCa2+free in the Ca/EGTA solutions has been obtained with carefully equalized stock solutions.