Determination of An Acid Dissociation Constant of Hydrolyzable <i>p</i>-Nitrophenyl Phosphate by Capillary Zone Electrophoresis

An acid dissociation constant of hydrolyzable p-nitrophenyl phosphate (NPP), pKa2, was determined in this study through its effective electrophoretic mobility in capillary zone electrophoresis (CZE). NPP could undergo hydrolysis in an aqueous solution to produce p-nitrophenol (NP), and a reliable value for its pKa2 has not been reported previously. When NPP solutions were freshly prepared each day and analyzed by CZE on the same day, only a peak assigned to NPP was detected in the electropherograms, with no detectable peak corresponding to NP. The effective electrophoretic mobility of anionic NPP increased with pH, and analysis of the mobility change yielded a second acid dissociation constant of pKa2 = 5.46±0.02. The pKa2 value was also determined by spectrophotometric titration without interference from hydrolyzed NP, giving pKa2 = 5.53±0.08. These two pKa2 values were in good agreement, demonstrating that the pKa2 of NPP was successfully determined in this study.