HPLC of Peptides and Proteins: Standard Operating Conditions

Reinhard I. Boysen1, Milton T.W. Hearn1

1 Monash University, Victoria, Australia
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.13
DOI:  10.1002/0471142727.mb1013s54
Online Posting Date:  May, 2001
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Abstract

The standard operating conditions for the eight basic modes of HPLC are presented in this unit. They include: size‐exclusion chromatography (HP‐SEC), ion‐exchange chromatography (HP‐IEX), normal phase chromatography (HP‐NPC), hydrophobic interaction chromatography (HP‐HIC), reversed‐phase chromatography (RP‐HPLC), hydrophilic interaction chromatography (HP‐HILIC), immobilized metal ion affinity chromatography (HP‐IMAC), and biospecific/biomimetic affinity chromatography (HP‐BAC). In addition, some subsets of these chromatographic modes, e.g., mixed mode chromatography (HP‐MMC), charge transfer chromatography (HP‐CTC), or ligand‐exchange chromatography (HP‐LEC) are described. Procedures for multimodal column switching are also included, as are guidelines for a systematic approach to method development. Example separations help illustrate the procedures.The standard operating conditions for the eight basic modes of HPLC are presented in this unit.

     
 
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Table of Contents

  • Standard Operating Conditions for HP‐SEC
  • Standard Operating Conditions for HP‐NPC
  • Standard Operating Conditions for HP‐HIC
  • Standard Operating Conditions for HP‐IEX
  • Standard Operating Conditions for HP‐HILIC
  • Standard Operating Conditions for HP‐IMAC
  • Standard Operating Conditions for HP‐BAC
  • Standard Operating Conditions for RP‐HPLC
  • Desalting of Peptide and Protein Mixtures by RP‐HPLC Techniques
  • Multimodal HPLC: Column Switching
  • Method Development in RP‐HPLC
  • Systematic Approach to Method Development
  • Determination of Thermodynamic Parameters Associated with Peptide or Protein Interactions with Immobilized Ligands
  • Troubleshooting
  • Terminology
  • Summary
  • Figures
  • Tables
     
 
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Materials

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Literature Cited

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Key References
   Dolan and Snyder, 1989. See above.
   An excellent practical manual.
   Glajch, J.L. and Snyder, L.R. 1990. Computer Assisted Method Development for High‐Performance Liquid Chromatography. pp. 1‐682. Elsevier Science Publishers, Amsterdam.
   An advanced text for experienced scientists.
   Hearn, M.T.W. 1991. HPLC of Peptides, Proteins and Polynucleotides, Fundamental Principles and Contemporary Applications. pp. 1‐776. VCH Publ, Deerfield Beach, Fla.
   A comprehensive text encompassing most applications.
   Hearn, M.T.W. 2000. Physicochemical factors in polypeptide and protein purification by high‐performance liquid chromatography: Current status and future challenges. In Handbook of Bioseparation (S. Ahuja ed.) pp. 1‐652. Academic Press, New York.
   A detailed survey of application and theory.
   Hearn, M.T.W. 2001. RPC and HIC of peptides and proteins. In HPLC of Biological Macromolecules. Methods and Applications (K.M. Gooding and F.E. Regnier, eds.). Marcel Dekker, New York.
   A comprehensive survey of applications.
   Mant, C.T. and Hodges, R.S. 1991. High‐Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis and Conformation. pp. 1‐938. CRC Press, Boca Raton, Fla.
  A general guide to HPLC.
   McMaster, M.C. 1994. HPLC: A Practical User's Guide. pp. 1‐211. VCH Publishers, Inc., New York.
  An excellent advanced text.
   Snyder et al., 1988. See above.
  A useful guide to modern chromatography.
   Vijayalakshmi, M.A. 2001. Theory and Practice of Biochromatography. pp. 1‐721. Harwood Academic Publishers.
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