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

The Craig model (assuming a Langmuir isotherm) has been used by us previously to successfully simulate isocratic high-performance liquid chromatographic (HPLC) separation in a mass-overload mode. Here we have extended this approach to the case of gradient elution for large samples. These simulations support our earlier conclusion that so-called "corresponding" isocratic and gradient separations provide similar sample resolution when the sample size is the same. "Corresponding" separations refer to the case where isocratic retention k' is equal to average gradient retention k, and where other conditions (column, flow-rate, etc.) are the same. Craig simulations reported here also provide further insight into the factors that affect preparative HPLC separations under mass-overload conditions.