ABSTRACT
A number of researchers have compared the resolution of peptides and/or proteins obtained on standard reversed-phase chromatography (RPC) analytical columns with that on narrower-bore columns. This chapter reviews that the literature on separations of peptides and proteins on narrowbore and microbore columns is that assessment of the performance of these columns, as well as comparing the effectiveness of columns of different diameters, is generally carried out using mixtures of unrelated peptides or proteins. Varying flow- and gradient-rates can be very effective in optimizing peptide separations, with the selection of optimal conditions on a specific column and on a specific liquid chromatography (LC) instrument depending on the individual researcher's requirements. The instrumentation must have an efficient micromixer for preparing gradients, and the post-column volume, including detector cell volume, must be minimized to prevent post-column mixing and band broadening. Thus, the LC instrument must be able to generate gradients accurately and reproducibly at flow-rates of 10 to 100 µl/min.
