N-Propyl-N′-2-pyridylurea-modified silica as mixed-mode stationary phase with moderate weak anion exchange capacity and pH-dependent surface charge reversal

• Published in: Journal of Chromatography A 2018-07, Vol.1560, p.45-54
• Main Authors: Bäurer, Stefanie, Polnick, Stefan, Sánchez-Muñoz, Orlando L., Kramer, Markus, Lämmerhofer, Michael
• Format: Article
• Language: English
• Subjects: HILIC; Mixed-mode chromatography; pH-gradient driven umpolung; Stationary phase classification by principal component analysis; Zwitterionic surface; ζ-Potential of modified silica particles
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2018.05.012

•Mixed-mode stationary phase based on a 2-pyridylurea-modified silica with moderate weak anion exchange capacity.•Solid state 13C and 29Si cross-polarization/magic angle spinning NMR of surface.•ζ-potentials at different pH by electrophoretic light scattering measurements.•Column characterization and classification by RP and HILIC tests.•Repulsive charge inducted elution of phosphates through umpolung by pH gradient. Herein, we present a novel silica-based stationary phase modified with N-propyl-N′-2-pyridylurea selector. Due to the weakly basic properties of the pyridine selector and the presence of residual silanols after selector immobilization, a zwitterionic surface with a pI observed at approximately pH 5.5 was measured by electrophoretic light scattering in pH-dependent ζ-potential determinations. The capability of the new N-propyl-N′-2-pyridylurea-modified silica to serve as mixed-mode stationary phase was investigated. For this purpose, it was characterized under RP and HILIC conditions using test mixtures. Subsequent classification of this stationary phase in comparison to in-house and commercial benchmarks was carried by principal component analysis of resultant retention factors from chromatographic tests. The results show a relatively unique mixed-mode character amongst the tested stationary phases. The chromatographic retention characteristics of acidic compounds matched well the ζ-potential determinations. The application of anion-exchange at low pH values (e.g. pH 5) and ion exclusion chromatography at pH 7 for the separation of uridine 5′-mono-, di- and triphosphate demonstrated a pH-dependent umpolung of the stationary phase surface. The combination of these separation principles in a pH gradient from 5 to 7 gave rise to weak anion-exchange selectivity with a charge-inducted elution due to repulsive interactions at higher pH and resulted in a significant faster separation with improved peak shape under mild elution conditions.