Using Capillary Electrophoresis to Measure pKa

The acid-base dissociation constant (pKa) is a critical measurement to obtain in early drug discovery. pKa values can strongly influence the solubility, permeability, and ADME properties of a compound. However, high-throughput pKa measurement, whether using capillary electrophoresis, potentiometry, or spectrophotometry, is notoriously cumbersome.  Experimental measurements can be influenced by a number of factors such as the method used, the ionic strength of the test article, and temperature, making in-silico predictions insufficient to use as a standalone approach to guide decision making. 

Analiza uses a multiplexed capillary electrophoresis method to measure pKa. Advantages of this method include:

1. 30μL sample requirement
2. Increased accuracy of measurements of low solubility compounds by use of a cosolvent method
3. Increased accuracy of measurements of multiprotic compounds with closely spaced pKa values through use of a 24-point buffer system
4. The ability to obtain measurements of compounds in the presence of degradants or impurities

The example below shows how the pKa is determined for norfloxacin, an analytical standard used in every pKa analysis. The effective mobility (μ_eff) of the test compound is calculated using the migration time between the compound and a neutral marker (DMSO) in buffer across 24 equidistant pH’s between 1.8 and 11.2.  

Using proprietary software, a titration curve is generated by plotting compound μ_eff as a function of pH. The inflection point(s) correspond to pKa(s).