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

Abstract

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 C(18) columns; ether-linked phenyl column with polar endcapping) compared with n-alkyl (C(18), C(8)) 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 N(α)-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<Ala<Val<Ile in order of increasing hydrophobicity), alkyl versus aromatic side-chains (Phe, Tyr) and a side-chain also with polar character (Tyr). Peptide mixtures were run on all columns at pH 2 (linear gradients of ACN or methanol in aqueous mobile phases). Our initial results clearly demonstrate the useful complementarity of different RP high-performance liquid chromatography packing materials, with peptide mixtures showing profound selectivity differences between n-alkyl versus polar-embedded and polar endcapped C(18) stationary phases. In addition, changing the polarity of the RP matrix in combination with changing the polarity of the mobile phase (ACN versus methanol) is shown to be synergistic to maximize selectivity of peptide separations.