When an ionizable compound in a sample, the pH of the mobile phase or buffer is the most significant variable in manage of separation and retention in a reversed-phase chromatography (RP-HPLC). But, if it is uncontrolled, it can be the source of many problems in separation. As many molecules analyzed by RP-HPLC have one or more acidic or basic functional groups, mainly mobile phases need pH control. This is a reason that buffers are widely used in HPLC. Because the retention time of ionizable analytes is extremely sensitive to the pH of the mobile phase, it is required to control the pH by the addition of buffer. When a small amount of acid or base is added a buffer maintains the pH.It is compulsory to know the pKa of the component before the selection of buffer pH.
It is suggested for symmetrical peak shape the buffer 02 pH units above or below pKa of the component. If the molecule pKa is not known, then, try different pH of the mobile phase could provide the appropriate results. For all chromatographic separation, determine and adjust the pH on the aqueous phase, prior to mixing with the organic phase, to give the most precise and reproducible results.