HPLC OF PEPTIDES AND PROTEINS

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The three major areas under investigation are: (1) development of novel separation methods of peptides/proteins; (2) multiple and simultaneous peptide purification by sample displacement chromatography (SDC); and (3) utility of reversed-phase chromatography (RPC) to monitor stability, folding and conformation of peptides. The ever-increasing demand for synthetic peptides has fueled a constant demand for efficient analytical and preparative purification techniques. Novel purification protocols, such as mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEC) with the development of volatile mobile phases, will rival RPC. Tailored RPC stationary phases will allow the advantageous manipulation of the elution patterns of peptide mixtures. A requirement for multiple peptide synthesis will be met by the development of a rapid, low pressure and simultaneous purification technique based on a combination of solid-phase extraction technology coupled with the novel SDC approach, allowing up to 100 peptides at a time to be purified in the absence of organic modifier. Combination of the sample displacement technique with microbore RPC to separate and concentrate synthetic impurities prior to direct elution into LC-MS will also allow at least a 100-fold increase in sensitivity over present methods to judge peptide homogeneity. RPC is a potent probe of polypeptide structural characteristics and it will be possible to correlate elution behaviour with amphipathicity and hydrophobicity of antimicrobial alpha-helical and cyclic beta-sheet peptides. Such work has major implications for the de novo design of more effective antimicrobial agents.