Effect of pH in reversed-phase chromatography

The pH of the mobile phase in reversed-phase HPLC is an important factor, it can affect the peak shape as well as the retention time of the molecule as it affects the ionization state of the molecule, and therefore the chemistry of the interactions in the HPLC column. It is therefore important that to maintain the correct ph for the compound that may be affected by the pH change of the mobile phase and buffer. The triethylamine and trifluoroacetic acid are common mobile phase modifiers, but they do not maintain pH, you need buffer solution for this. They resist a change in pH when small amounts of acid or alkali are added. A buffer solution contains a mixture of a weak base and its conjugate acid (or a weak acid and its conjugate base).

The effect of the pH of the mobile phase in high-performance liquid chromatography can influence the selectivity, retention time, and method ruggedness.

Selectivity:

Minute changes in the mobile phase pH can also have a dramatic effect on the selectivity of weakly ionizable analytes.

The retention time of the analyte:

When the pH of the mobile phase changes a dramatic change occurs in the retention time of acidic and basic analytes. When the ionization of these molecules is changed it is often the result of different interactions amongst the column and the molecules.

Method Ruggedness:

For best roughness, choose a pH for the buffer or mobile phase where selectivity and RT do not change if the slight changes in pH.

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