ABSTRACT
Reversed-phase high-performance liquid chromatography (HPLC) has rapidly become the most widely used tool for the separation, purification, and analysis of peptides. In the case of proteins, reversed-phase chromatography (RPC) has suffered from problems associated with denaturation including loss of activity, poor recoveries, broad misshapen peaks, and ghosting. RPC is also a very powerful tool for the micropreparative purification of proteins and peptides. As with small molecule HPLC, there are a large number of reversed-phase columns for use with peptides and proteins. This chapter reviews all of the column variables as well as the extra-column variables in the hope that users will be better able to utilize commercially available RPC columns to solve their current peptide and protein separations problems. The column variables include packing support, bonded phase, pore size, particle size, and column dimensions. The extra-column variables include sample pretreatment, mobile phase composition, pH, temperature, additives, gradient profile, and hardware.
