One Teflon-coated flea stir-bar was added to each vial to facilitate mixing of the constituents. 500 µL of buffer media was then added to each individual vial using a Rainin multichannel pipette, ensuring that the drug substance was in a saturated state before continuation. The vials were capped, and the entire 96-well plate was placed on an Unchained Labs Freeslate CM3 platform and stirred for 24 hours at 500 RPM with temperature controlled at 22 °C. At the conclusion of stirring, the magnetic stir bars were removed from the vials and the supernatant from the samples were filtered using the Hamilton Microlab NIMBUS liquid handler (The 100X with (50:50 v/v) acetonitrile/water mixture as a diluent. An appropriate dilution factor was applied to ensure that the final concentration of the sample was within the linear range of the calibration curves collected, between 0.001 mg/mL to 1 mg/mL. At this point, the samples were ready for chromatography analysis on an Agilent 1290 HPLC. At the conclusion of each HPLC run, the plates were returned to the Freeslate CM3 to measure the final pH of the solutions for confirmation. pH-solubility was plotted using Synergy Software's Kaleidagraph data analysis application (Synergy Software, Reading PA) based on the chromatography results, and a curve was fitted for each compound using the Henderson-Hasselbalch solubility equations . The approach used for modeling the pKa was originally established by Jagannadham and Sanjeev, and later confirmed by Bahr et al, whereby the final pH of the solution is plotted against the log of the concentration, and the resulting plot is modeled against the solubility equations provided in Table 2 . Each curve was unique to the nature of the compound, for example there were unique equations for acids, bases and ampholytes as well as for mono-and di-protic acids and bases. The model curve for the pKa determination, chosen by the software, was based on the compound's acidic or basic functional groups. The curve fitting equations used to solve the optimization and determine the pKa for each drug molecule are shown in Table .