Column plate numbers, N, were measured for 12 different proteins as a function of mobile phase flow-rate in two gel filtration systems (either denaturing or non-denaturing conditions). These data were used to extend a previous model that predicts bandwidths in reversed-phase and ion-exchange chromatography. Restriction of diffusion of large molecules within column packing pores is now defined more precisely, with a single relationship describing this effect for both reversed-phase and size-exclusion chromatography (SEC) (and presumably other high-performance liquid chromatography systems). Separations by gel filtration (SEC) are now included in our general model. A total of 17 flow-rate studies were carried out, involving different proteins, columns and/or mobile phase conditions (denaturing or non-denaturing). Comparisons of plate numbers predicted by the model with experimental values were satisfactory in 15 out of 17 cases. The remaining two cases appear to represent "non-well-behaved" systems, where experimental bandwidths were higher than predicted values by more than 20%. Initial attempts at understanding the origin of these non-ideal effects are described.

First description of DryLab 1 (the ancestor of the column optimization portion of the present DryLab for Windows) as applied to a steroid sample.

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.