Acid dissociation constants (pKa) of nine kinds of flavin analogues as molecular catalyst candidates were determined by CZE. Although some of the analogues are instable and degradable under the light exposure or in alkaline aqueous solutions, the effective electrophoretic mobility of the flavin analogue of interest has been measured with the residual substance. The pKa values of the flavin analogues were analyzed through the changes in the effective electrophoretic mobility with varying pH of the separation buffer. One or two steps pKa values were determined by the analysis. One of the degraded species from the flavin analogues, lumichrome, was also detected in the CZE analysis, and its pKa values were also determined. While coexisting impurities generated over the storage conditions were found in some analogues, the pKa values of the target analogues were successfully determined with the help of the CZE separations. A pressure‐assisted CZE was utilized for the determination or the estimation of the pKa values of such analogues as possessing carboxylic acid moiety.
Two-steps acid dissociation constants of L-ascorbic acid (AA) were determined through the changes in the effective electrophoretic mobility in capillary zone electrophoresis. Although ascorbic acid is oxidatively degradable in an aqueous solution, especially in alkaline conditions, the effective electrophoretic mobility of AA was successfully measured in the pH range between 1.87 and 11.97. In the analysis of the first-step acid dissociation constant (pK a1 ) at weakly acidic pH conditions, a coated capillary with 1,3-propanesultone and a pressure-assist were utilized to detect anionic AA. In the analysis of the second-step acid dissociation constant (pK a2 ) at weakly alkaline pH conditions, AA was successfully detected at the pH range up to 11.97 with the help of Cu catalyst to remove the dissolved oxygen in the separation buffer. Acid dissociation constants were independently determined as pK a1 = 4.15±0.01 and pK a2 = 12.07±0.04 by non-linear least-squares analyses. AA did not fully dissociate at the weakly alkaline pH range, and the effective electrophoretic mobility of the dianion form of AA was extrapolated for the analysis.
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