First description of DryLab 4-5 (the ancestor of the binary isocratic reversed phase module of DryLab for Windows) as applied to a mixture of nitro-aromatic compounds.

A general model has recently been proposed for the separation of peptides and proteins using reverse-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 an optimum flow rate for the separation of a given sample. It is important in this approach to identify which bands in the various separations correspond to the same peptide. Various peak-tracking procedures are discussed and illustrated.

Summary of the basic model that underlies DryLab G applied to large molecules.

Application of the model described in J. Chromatogr., 327, 27 (1985) to the separation of protein/peptide mixtures by reversed-phase gradient elution.

A previously reported model describes retention and band width as functions of experimental conditions, for the reversed-phase gradient elution separation of peptides and proteins. The model begins by relating separation in gradient elution to corresponding separations by isocratic elution (same high-performance liquid chromatographic system). The use of certain semi-empirical relationships then allows band width to be predicted for samples for which isocratic data are unavailable. This in turn allows the prediction of band width, peak capacity and relative peak height (or peak volume) as a function of experimental conditions such as column dimensions, gradient conditions, mobile phase flow-rate, sample molecular weight, etc. The next paper [J. Chromatogr., 327 (1985) 93] demonstrates the practical value of this approach for optimizing the separation of peptide and protein mixtures.In the present paper it is first shown that isocratic data for desamido-insulin (6000 daltons) allow the accurate prediction of band widths as a function of experimental conditions in related gradient separations. It is further shown that the present model accurately predicts how band widths vary with experimental conditions for peptides and proteins having molecular weights in the range 600–80 000 daltons.