Figure 6.  Quantitative measurement of concen­tration gradients by flow dialysis.  As mentioned above, the filtration assay for accumulation is relatively quantitative for charged and neutral substrates, but accu­mulation of weak acids, in particular, is drastically un­derestimated because of high passive permeability in the protonated state.  This causes accumulated weak acids to leak rapidly from the vesicles during dilution and filtration.  Therefore, flow dialysis is used.  By this means, the concentration of any small molecular weight solute in the medium surrounding the vesicles can be measured continuously without manipulation.  A flow dialysis unit consists of a Lucite block with 2 chambers separated by a dialysis membrane and inlet and outlet ports in the lower chamber through which oxygenated buffer is pumped to either a fraction collector or a continuous flow scintillation counter.  The basic idea is that vesicles in the upper chamber are much too large to pass through the dialysis membrane.  In contrast, a radiolabeled weak acid or base, a lipophilic ion or a sugar, amino acid or another transported substrate in the external medium can pass through the dialysis membrane.  Therefore, the concentration of the radiolabeled compound in the solution bathing the vesicles in the upper chamber is measured continuously by monitoring the solution flowing out of the lower chamber.  If the vesicles accumulate the radiolabeled material in the upper chamber, the dialyzable concentration drops which is reflected by a decrease in radioactivity in the flow-through from the lower chamber.  Conversely, if the radiolabeled compound accumulated by the vesicles is released, the level of radioactivity in the flow-through increases.  The technique is extremely powerful, as it allows quantification of concentration gradients of the probes used to measure DY or DpH, as well as transported substrates under identical conditions.