Capillary Electrophoresis Measurements

CE determination of pKa is new, compared to the other techniques [144-147]. It has the advantage of being a rather universal method since different detection systems can be coupled to CE. Because it is a separation technique, sample impurities seldom are a problem. A fused-silica capillary, with an inner diameter of 50-75 mm and 27-70 cm in length is filled with a dilute aqueous buffer solution (ionic strength

0.01-0.05 M) [144]. About 10 nL of a sample solution, whose concentration is ^50 mM, is gathered at one end of the capillary, and a 20-30-kV potential is applied between the ends of the capillary dipped into each of two beakers. Sample consumption is roughly 0.2 ng per injection. Sample species migrate according to their charge and fluid drag. Apparent electrophoretic mobility is determined, which is related to the migration time, the length of the capillary, and the applied voltage. The mobility of ionizable compounds is dependent on the fraction of the compound in the charged form. This, in turn, depends on the pKa. The plot of the apparent mobility versus pH has a sigmoidal shape, with the midpoint pH equal to the pKa. The practical range for buffer pH in CE is 2-3 at the low end and 11-12 at the high end. When UV detection is used, the limit of detection for a molecule having the molar absorptivity of benzoic acid at 220 nm is ^2 p.M [144]. Ishihama et al. [145] were able to determine the pKa of multiprotic molecules by CE, one molecule having seven ionization groups. They reported a 10 p.M limit of detection for verapamil. Its reported pKa, 8.89, compares well to that determined by poten-tiometry, 9.07 [pION].

Ishihama et al. [147] have describe a rapid screening method for determining pKa values of pharmaceutical samples by pressure-assisted CE, coupled with a photodiode array detector. Each CE run was completed in less than 1 min, so a 96-well microtiter plate could be measured in one day. Determinations of the pKa values of 82 drugs illustrated this interesting new method.

Since most drug discovery projects deal with very sparingly soluble compounds, the usual CE sample concentration would lead to precipitation. The handling of ''real'' drug candidate molecules is poorly developed in CE applications, in comparison to the most robust potentiometric method.

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