Steady-migration retention characteristics of peptides under gradient elution: application towards a dynamic separation method for minor-adjustments of the retention of peptides in RPLC

Minor-adjustment of the retention of peptides, induced by varying the mobile phase flow-rate (MPF-R), is a new dynamic separation method for simultaneously and rapidly identifying and improving the selectivity of hidden and overlapping peptide peaks. It can also-stabilize the reverse elution order of some pair-peaks under gradient elution in reverse phase liquid chromatography. The retention characteristics of peptides under gradient elution in RPLC was firstly found to be dominated by two variables of the steady region (SR) and migration region (MR). The changes in peptide retention induced by varying the MPF-R can be attributed to changes in the rate of bond breaking of multiple molecular interactions of peptides from the SR and of the mass transfer of peptides from the stationary phase to the mobile phase in the MR. The two dynamic variables were also found to independently depend on the type of peptide. Desirable results were obtained using six standard oligopeptides and a real sample of trypsin-digested lysozyme. It is expected that the quality control of peptide drugs, high dispersion of peptide peaks in peptide mapping and “bottom-up MS” in proteomics will be improved by this method, even enabling peptide purification on a preparative scale in industry.