Interaction properties and separation potential of zwitterionic stationary phases in hydrophilic interaction liquid chromatography

Two commercially available zwitterionic columns, one diol-based column and two newly developed experimental zwitterionic columns for hydrophilic interaction liquid chromatography (HILIC) were characterized and compared in terms of their interaction abilities. The novel HILIC columns offer mixed-mode cation-exchange and anion-exchange interactions together with minor hydrophobic contribution of alkyl spacers between the units.

Tanaka testing protocol, which provides complex information about column retentivity, hydrophobicity, hydrophilicity and cation/anion-exchange characters was performed. The results revealed the differences between individual zwitterionic stationary phases and clear predominance of cation-exchange character over anion-exchange character was observed for all tested columns. The measurements at different pH at various buffer salt concentrations revealed that the predominance of negative charge on the stationary phase surface occurs at pH 4.7 and higher (probably due to the dissociation of residual silanols). At lower pH (pH 3.0) the anion-exchange mechanism also applies. The cation-exchange character (applying across the whole pH range) has been further studied using other simple chromatographic tests and compared with >20 other commercially available columns. The application potential of all the tested columns was verified for analysis of tryptophan and its derivatives and also for cytochrome C tryptic digests.