Solvent selectivity and strength in reversed-phase liquid chromatography separation of peptides

A set of tryptic peptides was analyzed in reversed-phase liquid chromatography using gradient elution with acetonitrile, methanol, or isopropanol. We used these retention data as training sets to develop retention prediction models of peptides for the three organic eluents used. The coefficients of determination, R2, between predicted and observed data were approximately 0.95 for all systems. Retention coefficient values of twenty amino acids calculated from a model were utilized to investigate differences in separation selectivity between acetonitrile, methanol, or isopropanol eluents. The experimentally observed difference in separation selectivity appears to be a complex interplay of multiple amino acids, each contributing to a different degree to overall peptide retention. While retention contribution of hydrophilic amino acids was higher in methanol than acetonitrile, peptides containing aromatic amino acids (tyrosine, phenylalanine, tryptophan) exhibit relatively lower retention in methanol compared to acetonitrile. The differences between acetonitrile and isopropanol eluents were less pronounced. We also compared the relative elution strength of the three organic eluents for peptides. The relationship between the elution strength of two solvents is not linear, rather it was best fitted by a cubic polynomial function. Three solvents can be arranged in the order of increasing elution power as methanol < acetonitrile < isopropanol. The equations for relative solvent strength conversion were proposed.