Retention of peptides in RP LC and HILIC can be predicted by model, which take in account a retention contribution of each amino acids present in the peptide sequence. We have previously shown that the values of amino acid retention coefficients (RC) contain the information about LC retention mechanism. In this sense the method is similar to LFER or LSER methods also utilized to probe the stationary phase-solute interactions. We have previously shown that RC are useful for illustrating column and mobile phase selectivity differences in RP LC (Gilar et al., Anal. Chem. 2010, 82, 265-275) and in HILIC mode (Gilar et al., J. Chromatogr. A, 2011, 1218, 8890-8896). For example the RC values of charged amino acids (Arg, Lys, His) are strongly increasing when ion-pairing agents are added to mobile phase in RP LC.
More recently we have investigated the impact of mobile phase modifiers such as acetonitrile, methanol and isopropanol on peptides retention selectivity. Similarly as in previous studies, the retention of peptides was converted into RCs, and their values were used to investigate selectivity differences between modifiers. It was found that the apparent selectivity changes in peptide separation is a complex interplay of multiple RCs. In addition, the elution strength of different modifiers was expressed as the percentage of organic solvent at point of peptide elution. It appears that the relationship between elution strength of methanol, acetonitrile, or isopropanol is not described by linear or quadratic function and sometimes used in literature, but it is more complex, best fitted by polynomial expression.