Avoid missing pK_as: High-throughput workflow using solution pH-metric in tandem with UV-metric measurements

We describe a new high-throughput automated pK_a workflow using potentiometry starting with 10 mM DMSO stock (solution pH-metric). Two approaches using either neat DMSO stock solution or removal of DMSO were evaluated with different sample amounts and cosolvent schemes. These were validated against traditional potentiometric measurements for optimal conditions. Further, we detail how high throughput solution pH-metric experiments are performed in tandem with established UV-metric measurements to capitalize on the advantages of both approaches. This new workflow maintains the sample and time savings required for measuring a large number of samples in a drug discovery setting, while avoiding "missing pK_as" due to lack of sufficient UV chromophores. The combination of the two assays is key to tackle the challenges of low solubility, overlapping pK_as, and preliminary assignment of pK_as for Structure-Activity Relationship (SAR) understanding.