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

•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.