|Selectivity and mixed-mode retention mechanism of small-molecules in liquid chromatography using a reversed-phase / weak-anion-exchange stationary phase
|Fundamentals in Separation Science & Sample Prep
|Mr José Luís Dores-Sousa
|Prof Sebastiaan Eeltink
|Department of Chemical Engineering, Vrije Universiteit Brussel
Abstract Information :
Mixed-mode chromatography columns combining two or more surface chemistries have been introduced about twenty five years ago and are considered a valuable tool to separate sample mixtures containing a broad range of polar and apolar constituents. Due to presence of multimode retention mechanisms, method development is troublesome and time consuming. The current study comprises a systematic investigation to assess retention properties and selectivity of a stationary phase that features both reversed-phase (RP) and weak-anion-exchange (WAX) selectivity. The retention behavior was investigated for different compound classes including aromatic hydrocarbons, halogenated aromatic hydrocarbons, phenols, and carboxylic acids, which vary in hydrophobicity, Van der Waals surface area, and charge, while changing the organic content of the mobile phase, the pH, the salt concentration, and the column temperature. Using the linear solvent-strength model to study the effect of the organic content on retention factors, it was demonstrated that the kw values change proportional to the log P value for non-charged analytes, when varying the ACN content in the mobile phase. The lower S values (factor 2) observed for phenols and aromatic acids compared to neutral analytes suggest the presence of a dual reversed-phase weak-anion-exchange retention mechanism. To assess the existence of an ion-exchange retention behavior when varying the salt concentration in the mobile phase, the stoichiometric displacement net-charge model was applied to describe analyte retention. The retention as function of salt concentration of charged analytes was only slightly affected in the presence of RP-interactions, suggesting a mixed-mode retention mechanism. The IEX capacity of the mixed-mode column was also evaluated and at pH 6 anionic analytes exhibited maximum retention. Finally, the effect of temperature (10-50°C) on retention was evaluated using the Van't Hoff equation. Van't Hoff plots indicated that temperature significantly affect analyte retention in case of RP partitioning (aromatic hydrocarbons and halogenated aromatic hydrocarbons) and also in the case of a mixed-mode retention behavior (for aromatic acids).