Reversed-phase HPLC of peptides: Assessing column and solvent selectivity on standard, polar-embedded and polar endcapped columns

We desired to evaluate the chromatographic selectivity for peptides of silica‐based RP high‐performance liquid chromatography stationary phases with various modifications (polar embedding and polar endcapping on C18 columns; ether‐linked phenyl column with polar endcapping) compared with n‐alkyl (C18, C8) and aromatic phenylhexyl columns. Thus, we have designed and synthesized two series of synthetic peptide standards with the sequence Gly–Gly–Leu–Gly–Gly–Ala–Leu–Gly–X–Leu–Lys–Lys–amide, where the N‐terminal either contains a free α‐amino group (AmC series) or is Nα‐acetylated (AcC series) and where position X is substituted by Gly, Ala, Val, Ile, Phe or Tyr. These represent series of peptides with single substitutions of n‐alkyl (Gly