Why is pH so important in buffers and additives in reversed-phase HPLC or LC-MS?

When samples contain ionisable compounds, mobile phase pH can be one of the most important variables in the control of retention in a reversed‑phase HPLC (RP-HPLC) separation. However, if it is not controlled properly, pH can be a source of many problems. Since most compounds analysed by RP-HPLC contain one or more acidic or basic functional groups, most mobile phases require pH control. For this reason, buffers are widely used.

contributed by VWR | 11/20/2019

Why Control pH?

When an acid is more than 2 pH units above or below its pKa, it will be >99% ionised or non ionised, respectively. Bases are ionised below their pKa and non ionised above. The non ionised form will be less polar (more hydrophobic), and so more strongly retained in a reversed-phase system. As a result, at low pH, acids will be more retained, whereas bases will be more retained at high pH.

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