ABSTRACT
This chapter describes a number of applications of inverse gradient reversed-phase chromatography (RPC) for preparing subnanomole quantities of protein for microsequence analysis. It is not generally recognized that a skewed U-shaped, or bimodal, dependency exists between retention times and concentration of organic solvent during RPC of low-Mr organic compounds, peptides and small proteins. Interestingly, those reversed-phase packings which best exhibit this behavior for proteins are characterized by large surface areas and high carbon content. It is now well-established that sample preparation is one of the major limitations to obtaining amino acid sequence information from subnanomole quantities of peptides and proteins. Although RPC is an established tool for purifying a large range of proteins and peptides, it does have limitations with certain classes of proteins. Moreover, inverse-gradient RPC provides the potential for larger-scale fractionation of proteins at high organic solvent concentrations.
