Predicting retention time shifts associated with variation of the gradient slope in peptide RP-HPLC.

We have developed a sequence-specific model for predicting slopes (S) in the fundamental equation of linear solvent strength theory for the reversed-phase HPLC separation of tryptic peptides detected in a typical bottom-up-proteomics experiment. These slopes control the variation in the separation selectivity observed when the physical parameters of chromatographic separation, such as gradient slope, flow rate, and column size are altered. For example, with the use of an arbitrarily chosen set of tryptic peptides with a 4-times difference in the gradient slope between two experiments, the R(2)-value of correlation between the observed retention times of identical species decreases to ~0.

A model for predicting slopes S in the basic equation for the linear-solvent-strength theory of peptide separation by reversed-phase high-performance liquid chromatography.

A model for predicting the slope (S) in the fundamental equation of linear-solvent-strength theory for peptidic compounds was developed. Our approach is based on the novel assumption that three well-defined molecular descriptors: peptide length (N), charge (Z) and hydrophobicity index (HI) are the major contributors to the value of S. Following the definition of the model's variables, the retention of a number of Arg-terminated synthetic peptides was investigated under isocratic elution conditions (100 A pore size C18 phase, 0.