Analyte Ionization Acids, Bases, Zwitterions

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

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

For HA a weak acid the products of the dissociation are hydrogen ion (H ) and an anion (A ), which is the conjugate base. Equilibrium constants for acids can be written in the following form  

Using the definition for the pH (Hendersen-Hasselbalch form), one can rewrite 

Weak acids and bases exist in equilibrium with their ions, and their equilibrium constants (Ka) are small. The position of equilibrium is measured by the equilibrium constant [equation (4-10)] and Kt. [equation (4-12)]. The dis- 

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The ion-exchange constant does vary from one pair of ions to another therefore selectivity can be controlled by appropriate choice of the counter ion. However, selectivity is more conveniently manipulated by controlling the pH of the mobile phase and taking advantage of differences in the values of the analytes to be separated. In contrast with reversed-phase liquid chromatography (see section 3.6.2.1) the retention of weak acids and weak bases will reach a maximum when the compounds are in their ionized forms. Zwitterionic compounds such as amino acids, peptides and proteins can be separated on anion exchangers or cation exchangers. 

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