Analysis of ionic compounds by reversed-phase HPLC is usually performed with the pH of eluent controlled using an acid or buffering agent. However, a separation using a pH range which is not optimum for the compound of interest can cause problems such as split peaks or peak broadening. Even if the peak shape is satisfactory, retention times can in some cases be non-reproducible.

The relation between retention of benzonic acid and pH is shown in the figure below. Although the k' falls within relatively narrow limits in the pH range from 2 to 3.5, it varies widely in the pH range from 3.5 to 4.5. The pKa of benzonic acid is 4.2 and it is noticeable that the region where the k' varies most widely is near the pKa. If the eluent pH is adjusted to the region near the pKa, the result will not be reproducible because a slight change in the pH will result in a large change in the k' and have a significant effect on the separation. In fact, an eluent pH variation of just 0.1 significantly affects separation. Therefore, it is desirable that the eluent pH should be more than 1 unit away from the pKa. If the pKa is unknown, the eluent pH should be adjusted to within the region where the impact on separation seems minimal, after having carefully considered the relation between the eluent pH and the retention time.

When considering the pH value, it is also important to confirm the influence on the separation using several different eluents with their pH values slightly different from each other.