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A simple method for the accurate determination of free calcium concentration in calcium ethylene glycol bis beta aminoethyl ether n n n' n' tetra acetic acid solutions

, : A simple method for the accurate determination of free calcium concentration in calcium ethylene glycol bis beta aminoethyl ether n n n' n' tetra acetic acid solutions. American Journal of Physiology 242(5): C404-C408

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 (KCa) 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 2.45 .+-. 0.04 .times. 106 M-1 in 100 mM KCl, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 1 mM EGTA at pH 7.00 and C. Total [EGTA] varied with supplier but was always less than quoted.


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