Characterization and comparison of mixed-mode and reversed-phase columns; interaction abilities and applicability for peptide separation

• Published in: Journal of Chromatography A 2021-07, Vol.1648, p.462182, Article 462182
• Main Authors: Kadlecová, Zuzana, Kozlík, Petr, Tesařová, Eva, Gilar, Martin, Kalíková, Květa
• Format: Article
• Language: English
• Subjects: Mixed-mode chromatography; Mixed-mode columns; Peptide separation; Reversed-phase column; RP/anion-exchange mechanism
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2021.462182

•Characterization and comparison of three columns providing RP/AEX mechanism.•Effects of buffer pH and concentration on retention and peak shape were evaluated.•Contribution of ionic interactions strongly depends on the buffer pH and column type.•Fast, efficient peptides separation needs appropriate buffer pH and concentration.•Fourteen peptides were baseline separated on all the columns using buffer of pH 3.0. In this work, two mixed-mode columns from a different manufacturers and one marketed as a reversed-phase column were characterized and compared in the terms of their interaction abilities, retentivity, peak symmetry, and applicability for peptide separation. All the tested columns contain octadecyl ligand and positively charged modifier, i.e. pyridyl group for the reversed-phase column XSelect CSH C18, quaternary alkylamine for mixed-mode column Atlantis PREMIER BEH C18 AX, and permanently charged moiety (details not available from the manufacturer) for mixed-mode column Luna Omega PS C18. For detailed characterization and comparison of their interaction potential, several approaches were used. First, a simple Walters test was performed to estimate hydrophobic and silanophilic interactions of the tested columns. The highest values of both parameters were observed for column Atlantis PREMIER BEH C18 AX. To investigate the effect of pH and buffer concentration on retention, mobile phases composed of acetonitrile and buffer (ammonium formate, pH 3.0; ammonium acetate pH 4.7 and pH 6.9) in various concentrations (5mM; 10mM; 15mM and 20mM) were used. The analysis of permanently charged compounds was used to describe the electrostatic interaction abilities of the stationary phases. The most significant contribution of electrostatic interactions to the retention was observed for Atlantis PREMIER BEH C18 AX column in the mobile phase with buffer of pH 3.0. A set of ten dipeptides, three pentapeptides and one octapeptide was used to investigate the effects of pH and buffer concentration on retention and peak symmetry. Each of the tested columns provides the optimal peak shape under different buffer pH and concentration. The gradient separation of the 14 tested peptides was used to verify the application potential of the tested columns for peptide separation. The best separation was achieved within 4 minutes on column Atlantis PREMIER BEH C18 AX.