Effect of temperature on the retention of ionizable compounds in reversed-phase liquid chromatography: application to method development.
The analysis of pharmaceutical compounds is often a difficult challenge which requires mathematical tools to improve the quality of the separation method. This work is an attempt to rationalize the anomalous variation of the logarithm of the retention factor with temperature in case of ionizable compounds. The effect of temperature on ionizable compounds was studied within a large range of temperature, ranging from 30 to 130 degrees C. The determination of the so-called chromatographic pKa and the study of its variation with temperature allow to explain why the forms of the van't Hoff curves are so different depending on the type of solute, the type of buffer and the type of the mobile phase. A retention model along with a computation procedure is proposed to optimize both temperature and mobile phase composition and to provide good and robust conditions as shown by illustrative examples.