As described in the Methods section, final pH was measured for each aqueous sample and was plotted against the LogC (concentration). This data was fitted to the Henderson-Hasselbalch equation to determine the pKa. An example is shown in Figure . The experimental pKa for the example of SAMPL8-7 was calculated to be 6.63 (R 2 = 0.997). pKa determination, and concluded that HPLC analysis is a strongly favorable approach with good precision, but is limited at the far ends of the lower and upper pH scale . The use of HPLC instrumentation is reported, also by Reijenga, as a time-consuming experimental approach that is only effective if the analyte has a chromophore. The novelty of this work is in the use of automation to streamline the data collection process. However, due to a limited amount of drug substance available for these studies, repeated replicate samples could not be prepared, which we acknowledge could be a risk for uncertainty in the reported pKa values. Settimo et al. reported that pKa predictors may provide a degree of inaccuracy due to a significant molecular weight difference between the generic organic molecules used in modeling software and the larger and more complex molecules typically found in drug research . In some cases of the molecules investigated, the experimentally determined pKa did not match the predicted values, and this was likely due to pKa values that were at the extreme ends of the upper or lower pH scale and therefore could not be experimentally measured. An example of this is SAMPL8-13, which had a predicted pKa of 13.86, which could not be confirmed experimentally. This same approach was used for all twenty-three compounds, with the results provided in Table .