Abstract
Dissociation constants (pKa) of calcein which had been purified by recrystallization and reprecipitation were determined by a potentiometric method.
The procedures of purification of the reagent were as follows:
(i) Recrystallization: Commercially available calcein was dissolved in 50% aqueous methanol at 60°C, and residue was filtered off by using low ash filter paper. The filtrate was allowed to stand in an ice bath for 12 days. Crystals formed on the glass wall were collected on a glass filter, washed with methanol and dried in a sulfuric acid vacuum dessicator. (ii) Reprecipitation: The commercially available calcein was dissolved in a small amount of 0.1 NNaOH. The solution was diluted with 510% aqueous methanol, acidified to pH 2.5 with HCl, and allowed to stand in an ice-bath overnight. Crystals formed was treated in the same way as mentioned above.
The paper electrophoresis at pH 7.0 of the purified calcein was compared with that of the raw calcein. The purified samples gave three spots which moved towards the positive side while the raw calcein gave six separate spots; one of them moved towards the negative side, four spots towards the positive side and the other one was immovable. The four, which moved towards the positive side showed fluorescence and the intensity of the spot located in the most positive side was strong but that of the other three was weak. No difference was found in the electrophoresis of the samples prepared by the two purification methods.
Elemental analysis of the samples agreed with the composition, to be C30H26O13N2. H2O, and the results of potentiometric titration indicated the purity to be 98%. From these results, the purification of calcein by the above methods was concluded to be enough effective for the determination of pKa.
Two pH jumps appeared in the titration curve: one in the place of the 2 equivalent point, and the other in the 4 equivalent point. This indicates that each of the protons of the carboxylic and phenolic groups dissociated almost simultaneously. For the determination of pKa of the carboxylic groups, titrations with 0.1 N KOH were carried out in dioxane solution at various concentrations and the pKa values obtained were extrapolated to the zero concentration of dioxane. For the determination of pKa of the phenolic proton and those of amino groups, the sample was dissolved in 0.1 N KOH and back titration was carried out with 0.1 N HCl. The pKa values which are close to each other were estimated using an equation similar to that presented by Speakman. The values of pKa1-pKa6 obtained were 2.1; 2.9, 4.1; 5.4, and 10.1; 12.0 for the carboxylic and phenolic protons and those of amino groups, respectively.
These pKa values of calcein were compared with those of phthaleine complexon which was synthesized from ο-cresol phthalein, and it was concluded that the two methyliminodiacetic acid groups in calcein substitute the 4 and 5 position, 2 and 5 position or 2 and 7 position of fluorescein.
