The physicochemical properties of an analyte play a significant role in HPLC method development. Chromatographic method development requires studying the physical properties of the drug molecule such as solubility, polarity, pKa, and pH. Both pH and pKa are very important factors in HPLC method development.
The acidity or basicity of a molecule is usually defined by the pH value. Choosing an appropriate pH for an ionizable molecule often produces sharp and symmetric peaks in HPLC. Symmetric and sharp peaks are essential to achieve reproducible retention times, low relative standard deviations between injections, and low detection limits.
The acidity of an aqueous sample solution is determined by the concentration of (H3O+) ions. Hence, the pH of the solution specifies the hydrogen ions concentration in the solution.The pKa is a characteristic of a particular analyte and describes how the compound readily gives up a proton. The buffer has an ability to maintain pH; it relies on the concentration of buffer and the pKa of the buffering agent.
Consider that the most buffering capacity occurs at the pKa value. For HPLC, the rule of thumb is commonly used to adjust the pH of the mobile phase or buffer, the adjustable range is ±1 pH unit for the buffering ion’s pKa. If the components pKa value is very close to the pH value for the aqueous phase of the mobile phase, we get unstable retention times.
The pH of the mobile phase has to be less than the pKa value, this is to suppress ionization there to avoid split peaks.