ABSTRACT
This chapter demonstrates that several different commercially available high-performance liquid chromatography systems can be used to isolate sub-nanomole amounts of peptides from enzymatic digests of proteins. The major theoretical advantage of using a narrow bore instead of a standard bore column results from the fact that when solutes are eluted from both columns at the same linear flow velocity, the flow rate on the narrow bore column will be only 20 to 25% of that on the standard bore column. In addition to decreasing the amount of mobile phase used, the sensitivity of detection is markedly increased since peak volume is directly related to the flow rate when the gradient times are kept constant. In contrast to reversed-phase protein isolations, where column length appears to be relatively unimportant, a direct relationship was found between column length and peptide resolution in the case of peptide separations.
