Investigation into reversed phase chromatography peptide separation systems part I: Development of a protocol for column characterisation

•A RPC column characterisation protocol was developed using peptides as probes.•The probes assessed hydrophobic, electrostatic, H-bonding, & aromatic interactions.•Common peptide degradants were included as subtle but relevant probes.•14 columns with different ligands were evaluated at low and mid pH.•Hydrophobic, electrostatic and polar interactions are fundamental for peptide separations.