Separation of peptide mixtures by reversed-phase gradient elution. Use of flow rate changes for controlling band spacing and improving resolution

A general model has recently been proposed for the separation of peptides and proteins using reversed-phase gradient elution liquid chromatography. One application of this model suggests that flow rate, gradient time, or column configuration can be varied for band spacing control in the separation of enzymatic digests of proteins. Here a systematic procedure is described that uses repeated separations with different flow rates to maximize the separation of individual peaks within the chromatogram. From these initial separations it is possible to choose and optimum flow rate for the separation of a given sample.