cIEF for rapid pKa determination of small molecules: a proof of concept.

A capillary isoelectric focusing (cIEF) method was developed for the determination of the ionization constants (pKa) of small molecules. Two approaches used to decrease the electroosmotic flow (EOF) were compared: (i) a hydroxypropylcellulose (HPC) coated capillary in aqueous medium and (ii) the addition of glycerol to act as a viscosifying agent. The cIEF method with the glycerol medium was selected, and the ionization constants of 22 basic and 21 acidic compounds, including 15 pharmaceutical drugs, were determined, resulting in pKa values from 3.5 to 7.4 and 6.4 to 9.3, respectively. cIEF offers an interesting alternative to other techniques for pKa determination with low sample consumption, high throughput and low cost.