Principles of Chromatography

Buddhadeb Mallik1, Bulbul Chakravarti1, Deb N. Chakravarti1

1 The City University of New York, Queens, New York
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 6.1
DOI:  10.1002/cpet.7
Online Posting Date:  November, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Chromatography in its many different forms is an indispensable separation technique for purification or analysis of one or multiple components from a mixture that may contain a variety of simple as well as complex molecules. This unit is focused on a specific type of chromatography known as liquid chromatography (LC), with particular reference to separation of proteins. The theory of chromatography is discussed in the context of partition of the components of a mixture between a solid stationary phase and a liquid mobile phase. Although very useful for chromatographic separation of small molecules, considerable deviation is observed in the case of separation of proteins. The principles of three commonly used chromatographic techniques for separation of proteins, such as size exclusion, ion exchange, and affinity chromatography, are discussed. The unit is expected to be useful for biologists and biochemists, as well as engineers involved in downstream bioprocessing. © 2016 by John Wiley & Sons, Inc.

Keywords: chromatography; liquid chromatography; theory of chromatography; plate model; protein purification; size exclusion chromatography; gel filtration; ion exchange chromatography; affinity chromatography

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Overview and Principles
  • Theory of Chromatography
  • Common Types of Chromatography for Purification of Proteins and Underlying Principles
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Andrews, P. 1965. The gel‐filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96:595‐606.
  Axén, R., Porath, J., and Ernback, S. 1967. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214:1302‐1304.
  Chakravarti, B., Mallik, B., and Chakravarti, D.N. 2016. Column chromatography. Curr. Protoc. Essen. Lab. Tech. 13:6.2.1‐6.2.15.
  Gallagher, S.R. 2014. Overview of electrophoresis. Curr. Protoc. Essen. Lab. Tech. 7:7.1.1‐7.1.7.
  Giddings, J.C. 1965. Dynamics of Chromatography. Part 1. Principles and Theory. Marcel Dekker, New York.
  Hirs, C.H.W., Stein, W.H., and Moore, S. 1951. Chromatography of proteins. Ribonuclease. J. Am. Chem. Soc. 73:1893.
  Hjerten, S. 1962. Chromatographic separation according to size of macromolecules and cell particles on columns of agarose suspensions. Arch. Biochem. Biophys. 99:466‐475.
  Hjerten, S. and Mosbach, R. 1962. Molecular‐sieve chromatography of proteins on columns of cross‐linked polyacrylamide. Anal. Biochem. 3:109‐118.
  Hjerten, S., Levin, O., and Tiselius, A. 1956. Protein chromatography on calcium phosphate columns. Arch. Biochem. Biophys. 65:132‐155.
  Janson, J.C. and Jönsson, J.Å. 1989. Introduction to chromatography. In Protein Purification (J.C. Janson and L. Ryden, eds.) pp. 35‐62. VCH Publishers, New York.
  Lindsay, S. 1992. High Performance Liquid Chromatography, 2nd ed. John Wiley & Sons, London.
  Liu, H.F., Ma, J., Winter, C. and Bayer, R. 2010. Recovery and purification process development for monoclonal antibody production. MAbs. 2:480‐499.
  Martin, A.J.P. and Synge, R.L.M. 1941. A new form of chromatography employing two liquid phases: A theory of chromatography. 2. Application to the micro‐determination of the higher monoamino‐acids in proteins. Biochem. J. 35:1358‐1368.
  Paléus, S. and Neilands, J.B. 1950. Preparation of cytochrome c with the aid of ion exchange resin. Acta Chem. Scand. 4:1024‐1030.
  Peterson, E.A. and Sober, H.A. 1956. Chromatography of proteins. 1. Cellulose ion‐exchange adsorbents. J. Am.Chem. Soc. 78:751‐755.
  Porath, J. and Flodin, P. 1959. Gel filtration: A method for desalting and group separation. Nature 183:1675‐1679.
  Regnier, F.E. and Gooding, K.M. 1980. High‐performance liquid‐chromatography of proteins. Anal. Biochem. 103:1‐25.
  Scopes, R.K. 1982. Protein Purification: Principles and Practice. Springer‐Verlag, New York.
  Willard, H.H., Merritt, L.L., and Dean, J.A. 1974. Instrumental Methods of Analysis, 5th ed. D. Van Nostrand Company, New York.
  Wilson, K. and Walker, J. 1994. Principles and Techniques of Practical Biochemistry, 4th ed. Cambridge University Press, Cambridge.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library