Hplc Of Peptides And Proteins, Methods And Protocols

METHODS IN MOLECULAR BIOLOGY TM TM Volume 251 HPLC of Peptides and Proteins Methods and Protocols Edited by Marie-Isabel Aguilar CH01,1-8,8pgs 10/30/03 7:00 PM Page 3 1 HPLC of Peptides and Proteins Basic Theory and Methodology Marie-Isabel Aguilar 1. Introduction High-performance liquid chromatography (HPLC) is now firmly established as the premier technique for the analysis and purification of a wide range of molecules. In particular, HPLC in its various modes has become the central technique in the characterization of peptides and proteins and has, therefore, played a critical role in the rapid advances in the biological and biomedical sciences over the last 10 years. The enormous success of HPLC can be attributed to a number of inherent features associated with reproducibility, ease of selectivity manipulation, and generally high recoveries. The most significant feature is the excellent resolution that can be achieved under a wide range of conditions for very closely related molecules, as well as structurally quite distinct molecules. This arises from the fact that all interactive modes of chromatography are based on recognition forces that can be subtly manipulated through changes in the elution conditions that are specific for the particular mode of chromatography. Peptides and proteins interact with the chromatographic surface in an orientationspecific manner, in which their retention time is determined by the molecular composition of specific contact regions. For larger polypeptides and proteins that adopt a significant degree of secondary and tertiary structure, the chromatographic contact regi