|Probing selectivity of mixed-mode reversed-phase / weak-anion-exchange columns for small-molecule separations in liquid chromatography
|Fundamentals in Separation Science (KVCV)
|Mr José Luís Dores-Sousa
|Prof Sebastiaan Eeltink
|Department of Chemical Engineering, Vrije Universiteit Brussel
Abstract Information :
Mixed-mode chromatography has been used in life science research for a long time, however a good understanding of the effects of mobile-phase composition on small molecules retention has still not been established. 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 mixed-mode reversed-phase / weak-anion-exchange (RP/WAX) stationary phase. Retention 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. kw values of the linear solvent-strength model changed proportional to the log P value for non-charged analytes, when varying the ACN content in the mobile phase. Significantly lower S values (factor 2) were observed for phenols and aromatic acids compared to neutral analytes, suggesting the presence of a dual RP/WAX retention mechanism. The stoichiometric displacement net-charge model was applied to describe ion-exchange retention behavior when varying the salt concentration in the mobile phase. The IEX capacity of the mixed-mode column was also evaluated and at pH 6 anionic analytes exhibited maximum retention. 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).