Kinetic and thermodynamic insights into novel zwitterionic macrocyclic glycopeptide chiral stationary phases for high-efficiency, ultrafast enantioseparations under different elution conditions

In this work, a novel type of chiral stationary phase (CSP) has been prepared by covalently bonding teicoplanin chiral selector to monodisperse 1.9 µm fully porous particles (FPPs) and - for the first time in chiral chromatography - to 2.0 µm superficially porous particles (SPPs) by means of a bonding protocol that allows to obtain the zwitterionic version of the macrocyclic glycopeptide CSP. Columns in-house packed with these CSPs have been characterized from both a kinetic and a thermodynamic viewpoint towards the enantioseparation of small molecules and amino-acid derivatives comparing chromatographic behavior in RP-LC and HILIC mode. Kinetic performance has been experimentally evaluated through the analysis of van Deemter curves while thermodynamic properties and interactions of molecules with the stationary phase have been investigated by calculating adsorption isotherms (Inverse Method was used for this purpose). Results of this study revealed that the column packed with 2.0 µm SPPs shows outstanding kinetic performance (300’000 plates/m or more) [2]. In general, however, higher efficiencies and flatter van Deemter curves can be obtained when columns are operated in HILIC mode while better resolution and higher selectivity values are typical of RP-LC. This is due to the interplay of different factors including a different retention mechanism, a different contribution of adsorption-desorption kinetics and the absence of surface diffusion in HILIC conditions. These findings suggest that HILIC mode is most suitable for ultrafast enantioseparations while RP-LC should be preferred for the resolution of complex samples containing different pairs of enantiomers and for the challenging separation of racemic mixtures usually poorly resolved. Finally, zwitterionic macrocyclic glycopeptide CSPs have been also excellently used for an application of practical relevance in pharmaceutical analysis that is the simultaneous separation of chiral active pharmaceutical ingredients (APIs) from their counterions in a single chromatographic run. This is possible thanks to the peculiar properties of the zwitterionic version of teicoplanin CSPs to retain both cations and anions, differently from the traditional version of the CSP, where the latter ones are almost unretained due to Donnan repulsion effect.