Determination of Two-Steps Acid Dissociation Constants of L-Ascorbic Acid by Capillary Zone Electrophoresis

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 (pKa1) 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 (pKa2) 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 pKa1 = 4.15±0.01 and pKa2 = 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.