Retention of ionizable compounds on HPLC. 12. The properties of liquid chromatography buffers in acetonitrile-water mobile phases that influence HPLC retention.

The addition of acetonitrile to aqueous buffers to prepare RP HPLC mobile phases changes the buffer properties (pH and buffer capacity). This variation is studied for ace tate, phosphate, phthalate, citrate, and ammonia buffers in acetonitrile-water mixtures up to 60% in acetonitrile (v/v). Equations are proposed to relate pH and buffer capacity change of these buffers to the initial aqueous pH value and to the volume fraction of acetonitrile added. It is demonstrated that the pH change of the buffer depends not only on the initial aqueous pH of the buffer and on the percentage of acetonitrile added but also on the particular buffer used. The proposed equations allow an accurate prediction of this ionization for the studied buffers. Since the retention of acid/base compounds shows a strong dependence of their degree of ionization, the equations are used to predict the change in this ionization with addition of acetonitrile when the RP HPLC mobile phase is prepared. This prediction allows estimation of the retention of an acid/base compound in a particular acetonitrile-water buffered mobile phase.