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HPLC of Peptides and Proteins

Proteins, Peptides and Amino Acids

  1. Tzong-Hsien Lee,
  2. Marie-Isabel Aguilar

Published Online: 15 SEP 2006

DOI: 10.1002/3527600906.mcb.200400113

Reviews in Cell Biology and Molecular Medicine

Reviews in Cell Biology and Molecular Medicine

How to Cite

Lee, T.-H. and Aguilar, M.-I. 2006. HPLC of Peptides and Proteins. Reviews in Cell Biology and Molecular Medicine. .

Author Information

  1. Monash University, Clayton, Australia

Publication History

  1. Published Online: 15 SEP 2006

This is not the most recent version of the article. View current version (5 OCT 2015)


HPLC is extremely versatile for the isolation of peptides and proteins from a wide variety of synthetic or biological sources. The complexity of the mixture to be chromatographed will depend on the nature of the source and the degree of preliminary clean up that can be performed. In the case of synthetic peptides, RPC is generally employed both for the initial analysis and the final large-scale purification. The isolation of proteins from a biological cocktail, however, often requires a combination of techniques to produce a homogenous sample. HPLC techniques are then introduced at the later stages following initial precipitation, clarification and preliminary separations using soft gel. Purification protocols therefore need to be tailored to the specific target molecule.

This chapter deals with the different HPLC techniques that are commonly employed for the analysis and purification of peptides and proteins. A brief overview of the theory of each mode of chromatography will be presented and then discussed in terms of the parameters that control resolution and illustrated with relevant examples. The interested reader is also referred to a number of recent publications that provide a comprehensive theoretical and practical overview of this topic.


  • Capacity Factor;
  • Capillary Column;
  • Gradient Elution;
  • high-performance liquid chromatography (HPLC);
  • Ion Trap Mass Spectrometer;
  • Isocratic Elution;
  • Mobile Phase;
  • Peak Capacity;
  • Peptide Mapping;
  • Resolution;
  • Stationary Phase