Standard Article

Reversed-Phase High-Performance Liquid Chromatography in Peptide and Protein Analysis

Peptides and Proteins

  1. Dr Marie-Isabel Aguilar

Published Online: 15 SEP 2006

DOI: 10.1002/9780470027318.a1631

Encyclopedia of Analytical Chemistry

Encyclopedia of Analytical Chemistry

How to Cite

Aguilar, M.-I. 2006. Reversed-Phase High-Performance Liquid Chromatography in Peptide and Protein Analysis. Encyclopedia of Analytical Chemistry. .

Author Information

  1. Monash University, Clayton, Australia

Publication History

  1. Published Online: 15 SEP 2006


Reversed-phase high-performance liquid chromatography (RPHPLC) involves the separation of molecules on the basis of hydrophobicity. The separation depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase, i.e. the sorbent. The solute mixture is initially applied to the sorbent in the presence of aqueous buffers, and the solutes are eluted by the addition of organic solvent to the mobile phase. Elution can proceed either under isocratic conditions, where the concentration of organic solvent is constant, or by gradient elution, whereby the amount of organic solvent is increased over a period of time. The solutes are therefore eluted in order of increasing molecular hydrophobicity. RPHPLC is a very powerful technique for the analysis of peptides and proteins because of a number of factors which include (1) the excellent resolution that can be achieved under a wide range of chromatographic conditions for very closely related molecules in addition to structurally distinct molecules, (2) the experimental ease with which chromatographic selectivity can be manipulated through changes in mobile phase characteristics, (3) the generally high recoveries and hence high productivity and (4) the excellent reproducibility of repetitive separations carried out over a long period of time, which is due partly to the stability of the sorbent materials under a wide range of mobile phase conditions. However, RPHPLC can cause the irreversible denaturation of protein samples, thereby reducing the potential recovery of material in a biologically active form.