Figure 5.  Accumulation of weak acids or weak bases is used to measure DpH.  If DpH is interior alkaline, accumulation of non-metabolized permeant weak acids [e.g. acetate, salicylate, or dimethyloxazolidinedione (DMO)] is measured.  If DpH is interior acid, accumulation of non-metabolized weak bases (e.g. methylamine) is measured.  The basic principle is that there is an equilibrium between protonated and unprotonated weak acids and weak bases which is determined by the pH of the immediate environment, and the uncharged species of the acid (i.e. protonated) or base (unprotonated) is membrane permeant.  Therefore, if the internal space is alkaline relative to the external space, a higher percentage of the weak acid will be unprotonated inside than outside, and the unprotonated, charged species will accumulate. 

Conversely, if the internal space is acid relative to the external space, a higher percentage of the weak base will be protonated inside than outside, and the protonated, charged species will accumulate.  As with lipophilic ions, measurement of DpH is facilitated by the use of radioactive weak acids or bases.  Alternatively, DpH (interior acid) can be measured fluorimetrically with 9-aminoacridine or acridine orange, but in many systems, DpH is overestimated by this means.  [³¹P_i]NMR has also been used for quantitative DpH measurements in E. coli, since the valence state of P_i can be observed directly using this technique.  Values similar to those obtained by measuring accumulation of weak acids have been obtained.