The HPTS assay can be modified to determine the chloride (or other anions) vs proton or hydroxide transport selectivity due to the potential for H + or OH -transport to be the rate-limiting step. The anionic fatty acid headgroup cannot freely diffuse through the lipophilic membrane interior. However, complexation by a transporter allows the carboxylate head group to form a charge-masked complex allowing it to diffuse through the hydrophobic membrane core. This, combined with the protonation of the headgroup, free diffusion of the neutral species, and eventual deprotonation of the headgroup, completes a proton transport cycle (Figure ), allowing an anion transporter to facilitate H + transport synergistically with a fatty acid. Anion transporters can also facilitate H + /OH -transport via fatty acid-independent mechanisms, including transporter deprotonation and OH -transport. Hence, a comparison between the two conditions must be made to determine the influence of the fatty acid flip-flop mechanism. The first condition to be tested is vesicles containing fatty acid impurities (Figure , a), while the second is where the fatty acids have been sequestered from the membrane (Figure ).