The HPTS Anion Gradient Assay Synthetic POPC vesicles (200 nm) containing NaCl (100 mM), HEPES (10 mM), and HPTS (1 mM) were made using the same method described in the HPTS transport selectivity assay. However, the vesicles were suspended in different external solutions containing the anion of interest as NaX salts (X = Cl -, Br -, NO3 -, I -, and ClO4 -). No base pulse was added before transport was initiated by adding the transporters as DMSO solutions (5 μL) at t = 30 s. The transport experiments continued until the rate of transport plateaued or the fluorescence intensity reached an inflexion point. Before pH data could be obtained from the recorded fluorescence data, a calibration curve was constructed by using the same vesicles and conditions as the HPTS assay and monitoring the fluorescence intensity of the HPTS probe as the pH of the system was adjusted incrementally using a NaOH solution. The collected If data was then normalised and graphed as a function of the overall assay pH before the data was fitted with a modified Henderson-Hasselbach equation (Equation ). Where y is the system pH, x is the If value, and a, b, and c are all constants which are derived during the data fitting process. These three constants are then used to calibrate the remaining fluorescence data obtained through the transport experiments.