Rapid Determination of Ionization Constants (pK a )b y UV Spectroscopy Using 96-Well Microtiter Plates

We have developed a methodology that enables for the rapid measurement of ionization constants (pKa )o f series of compounds by UV spectrophotometry. This protocol, which is straightforward to set up, takes advantage of the sensitivity of UV spectroscopy and the throughput enabled by the 96-well microplate (as opposed to the use of 1 cm quartz cuvette). The compounds, in stock solutions in DMSO, are dissolved in several aqueous buffer solutions directly in the microtiter plate, allowing the simultaneous determination of the UV spectra as a function of pH. Further treatment of the data provides the pKa values in a medium-throughput manner. The pKa values of 11 new antitrypanosomal dibasic compounds were determined using this methodology.