Preparative separation of peptide and protein samples by high-performance liquid chromatography with gradient...

Craig simulations of mass-overloaded gradient elution reported in the preceding paper have been extended to the case of non-Langmuir isotherms. Isotherms were selected that appear to be characteristic of peptide and protein samples in reversed-phase high-performance liquid chromatography. The dependence of bandwidth on sample size and gradient conditions was examined by Craig simulation and compared with experimental data for 13 different experimental systems involving four different proteins. There is a good correspondence between simulations and experimental data, and it seems possible to quantitatively predict bandwidth and resolution as a function of small-sample retention data, experimental conditions, and sample size. A systematic approach for designing the preparative or process-scale separation of protein mixtures by reversed-phase gradient elution is proposed.