Protocol

Protein Purification Protocols

Volume 244 of the series Methods in Molecular Biology pp 253-258

Fast Protein Liquid Chromatography

  • David SheehanAffiliated withDepartment of Biochemistry and Analytical and Biological Chemistry Research Facility, University College Cork
  • , Siobhan O’SullivanAffiliated withDepartment of Biochemistry and Analytical and Biological Chemistry Research Facility, University College Cork

* Final gross prices may vary according to local VAT.

Get Access

Abstract

High-resolution protein separation techniques critically depend on the availability of column packings of small average particle size. This gives a minimum of peak broadening on the column owing to the direct relationship between the theoretical plate height parameter, H, and particle size (lowest values of H give the highest resolution). High-performance liquid chromatography (HPLC) procedures exploit column packings with average diameters of as small as 5–40 μm. However, these are used in high-pressure systems (up to 400 bar) often with organic solvents and are generally limited to rather low sample loadings (1). To provide a more biocompatible high-resolution separation of biopolymers, including (although not exclusive to) proteins, Pharmacia (Uppsala, Sweden; now known as, Amersham Biosciences) developed fast protein liquid chromatography (FPLC) in 1982 (2). More recently, Regnier and his colleagues developed “perfusive” packings that contain large through-pores (3). These allow high-resolution chromatography with ion-exchange groups similar to those of Amersham Biosciences columns but at much faster flow rates, giving remarkably short separation times (e.g., 5–7 min). Perfusion chromatography columns are also compatible with the FPLC system.