Related Experiment Video
Updated: Aug 21, 2026

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis
Published on: July 11, 2014
Effect of mobile phase composition, pH and buffer type on the retention of ionizable compounds in reversed-phase
1Laboratoire des Sciences Analytiques (CNRS UMR 5180), Université Claude Bernard, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France. heinisch@univ-lyon1.fr
Abstract:
Optimizing separation of ionizable compounds in order to find robust conditions has become an important part of method development in liquid chromatography. This work is an attempt to explain the observed variations of retention of acid and basic compounds with the organic modifier content in the mobile phase, according to various factors: the type of modifier, the type of buffer, the temperature and of course the type of solute. This is done by considering the variation of the so-called chromatographic pKa which refers to the pH measured in the aqueous medium and is determined from retention data. A procedure is described that accurately relates, from nine experiments, retention to solvent composition and pH. The limits of such a procedure are evaluated and two examples of optimized separations of basic compounds are given.
Related Concept Videos
Ion-Exchange Chromatography
Chromatographic Methods: Terminology
Analyte Adsorption and Distribution
High-Performance Liquid Chromatography: Introduction
In HPLC, two phases play a critical role in the separation process:
Chromatography: Introduction
The phase in which the compounds linger or on which the compounds adsorb is called the stationary phase, whereas the mobile phase is the solvent that carries the solutes to be analyzed. In traditional column chromatography, the mixture flows through the stationary phase, and the compounds partition between the stationary and mobile phases...
High-Performance Liquid Chromatography: Elution Process

