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A simple method for the accurate determination of free Ca in ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-buffered Ca solutions is described. This method is useful for calibration of Ca macro- and microelectrodes to low free Ca and should improve the reliability of calculated free Ca in more complex solutions. Briefly, free Ca in Ca-EGTA solutions is measured with a Ca electrode, bound Ca is calculated, and Scatchard and double-reciprocal plots are resolved for the total EGTA and the apparent Ca-EGTA association constant (K'Ca) in the solutions used. The free Ca is then recalculated using the determined parameters, giving a more accurate knowledge of the free Ca in these solutions and providing an accurate calibration curve for the Ca electrode. These solutions can then be used to calibrate other Ca electrodes (e.g., Ca microelectrodes) or the calibrated Ca electrode can be used to measure free Ca in solutions containing multiple metal ligands. This method allows determination of free Ca, K'Ca, and total EGTA in the actual solutions used regardless of pH, temperature, or ionic strength. It does not require accurate knowledge of K'Ca or EGTA purity and circumvents many potential errors due to assumption of binding parameters. K'Ca was found to be 2.45 +/- 0.04 X 10(6) M-1 in 100 mM KCl, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 1 mM EGTA at pH 7.00 and 23 degrees C. Total EGTA varied with supplier but was always less than quoted.
Donald M. Bers (Sat,) studied this question.