Column Chromatography

Bulbul Chakravarti1, Buddhadeb Mallik2, Deb N. Chakravarti1

1 Department of Chemistry, York College, The City University of New York, Queens, New York, 2 Department of Chemistry and Chemical Technology, Bronx Community College, The City University of New York, Bronx, New York
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 6.2
DOI:  10.1002/cpet.6
Online Posting Date:  November, 2016
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Chromatography is a widely used technique of separation which depends on the differential interaction of molecules between a stationary phase and a mobile phase. In most modern life science laboratories, purification of proteins plays an important role. Almost all types of laboratory‐based liquid chromatography techniques used for purification of proteins are carried out with the stationary phase contained in tubular columns. Although individual methods for sample loading or elution of these columns are usually different depending on the specific technique used, this general experimental procedure that uses tubular columns is known as column chromatography. This unit is focused on setting up laboratory‐based column chromatography methods for separation of proteins using three commonly used chromatographic techniques for separation of proteins, such as size exclusion, ion exchange, and affinity chromatography. © 2016 by John Wiley & Sons, Inc.

Keywords: chromatography; liquid chromatography; column chromatography; size exclusion chromatography; gel filtration chromatography; ion exchange chromatography; affinity chromatography

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Table of Contents

  • Overview
  • Choice of Chromatography Column
  • Frits, Stopcocks, Tubing, and Other Accessories
  • Buffer Systems
  • Planning and Running the Column Chromatography Experiment
  • Detectors and Recorders
  • Fraction Collectors
  • Pooling Fractions
  • Prepacked Columns and Kits
  • Literature Cited
  • Figures
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Literature Cited

Literature Cited
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  Chakravarti, D.N., Chakravarti, B., and Mallik, B. 2014. Reagent preparation: Theoretical and practical discussions. Curr. Protoc. Essen. Lab. Tech. 9:3.1.1‐3.1.14. doi: 10.1002/9780470089941.et0301s9.
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  Mallik, B., Chakravarti, B., and Chakravarti, D.N. 2016. Principles of chromatography. Curr. Protoc. Essen. Lab. Tech. 13:6.1.1‐6.1.23.
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  Stein, W.H. and Moore, S. 1948. Chromatography of amino acids on starch columns: Separation of phenylalanine, leucine, isoleucine, methionine, tyrosine and valine. J. Biol. Chem. 176:337‐365.
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