pH effect on hplc mobile phase

Mobile phase pH is a primary tool for controlling this selectivity through the change of the analyte ionization state.

Analyte Ionization

A simplistic rule for the retention in reversed-phase HPLC is the more hydrophobic the component the more it retained. By simply following this rule one can conclude that any organic ionizable component will have longer retention in its neutral form then in the ionized form. Ionization is pH dependent process, so we expect a significant effect of the mobile phase pH on the separation of complex organic mixtures containing basic or acidic components.

Ionization of the analyte could be expressed by one of the following equilibria

AH --> A^- + H⁺ for acidic components,

B + H⁺ --> BH⁺ for basic components

Equilibrium constants are usually written in one of the following forms:

(1)

and using the definition for the pH, one can rewrite

(2)

Similar expression could be written for bases.

As we mentioned before, compound in its ionic form is more hydrophilic so it not only tends to have less interaction with hydrophobic stationary phase, it also tends to be more solvated with water molecules. This also causes significant decrease of the retention of ionic components.

Since the pK_a is a characteristic constant of the specific analyte, from the above equation one can conclude that relative amounts of neutral and ionic forms of the analyte could be easily adjusted by varying the mobile phase pH. Moreover, if the pH is about two units away from the component pK_a more then 99% of the analyte will be in either ionic or neutral form, depending upon the direction of the pH shift.