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Basic Theory

The basic theory of affinity chromatography has been conveniently consolidated into a single PDF file for easy distribution and note-taking. The document aims to answer the following questions:

  • What is affinity chromatography?
  • What types of biological interactions used in affinity chromatography?
  • What are the steps involved in affinity purification?
  • How do you use affinity purification when a specific ligand for your target molecule is not available?
  • What are the analysis methods for purified biomolecules?

Click here to view a movie demonstrating affinity chromatography.

Click here to view the basic theory PDF file.


Examples of applications of affinity chromatography can be accessed through the link for the applications PDF file. The applications covered in this document are as follows:

  • Purifying a protein of interest based on histidine-nickel interaction
  • Purifying a protein of interest based on inherent ligand-binding specificity
  • Affinity-based chiral separations
  • Study of drug interactions

Click here to view the applications PDF file.


Subscription-Based Reading Material:

Rogue AC, Lowe CR., Affinity chromatography: history, perspectives, limitations and prospects, Methods Mol Biol. 2008;421:1-21.

Collins, MO; Choudhary, J, Mapping multiprotein complexes by affinity purification and mass spectrometry, Current Opinion in Biotechnology (2008), 19(4), 324-330.

Sousa, F; Prazeres, DMF; Queiroz, JA, Affinity chromatography approaches to overcome the challenges of purifying plasmid DNA, Trends in Biotechnology (2008), 26(9), 518-525.

Fang, Xiangming; Zhang, Wei-Wei, Affinity separation and enrichment methods in proteomic analysis, Journal of Proteomics (2008), 71(3), 284-303.

Chong, Shaorong, Edited by Cox, Michael H.; Phillips, George N, Proteins: affinity tags, Handbook of Proteins (2007), 2, 1270-1275.

Kaur-Atwal, Gushinder; Weston, Daniel J.; Bonner, Philip L. R.; Crosland, Susan; Green, Philip S.; Creaser, Colin S., lmmobilized metal affinity chromatography for the analysis of proteins and peptides, Current Analytical Chemistry (2008), 4(2), 127-135.

Hage, David S., Chromatography, affinity, Kirk-Othmer Separation Technology (2nd Edition) (2008), 1, 453-470.

Ngo, That T.; Jogie-Brahim, Sherryline; Narinesingh, Dyer, Affinity Chromatographic Purification of Antibodies, Analytical Letters (2007), 40(15), 2799-2820.

Rogue, Ana C. A.; Silva Claudia S. O.; Taipa, M. Angela, Affinity-based methodologies and ligands for antibody purification: Advances and perspectives, Journal of Chromatography, A (2007), 1160(1-2), 44-55.

Hage, David S., Edited by Seidel, Arza, Chromatography, affinity, Kirk-Othmer Encyclopedia of Chemical Technology (5th Edition) (2004), 6, 390-407.

Gutierrez, R; Martin del Valle, EM; Galan, MA, Immobilized metal-ion affinity chromatography: Status and trends, Separation and Purification Reviews (2007), 36(1), 71-111.

Chan, Nora; Lewis, Darren; Kelly, Michele; Ng, Ella S. M.; Schriemer, David C., Frontal affinity chromatography - mass spectrometry for ligand discovery and characterization, Methods and Principles in Medicinal Chemistry (2007), 36(Mass Spectrometry in Medicinal Chemistry), 217-246.

Okanda, Fred M.; El Rassi, Ziad, Biospecific interaction (affinity) CEC and affinity nano-LC, Electrophoresis (2007), 28(1-2), 89-98.

Mallik, Rangan; Hage, David S., Journal of Separation Science (2006), 29(12),1686-1704.

Varilova, Tereza; Madera, Milan; Pacakova, Vera; Stulik, Karel, Separation media in affinity chromatography of proteins - a critical review, Current Proteomics (2006), 3(1), 55-79.

Pichon, Valerie; Haupt Karsten, Affinity separations on molecularly imprinted polymers with special emphasis on solid-phase extraction, Journal of Liquid Chromatography & Related Technologies, (2006), 29(7&8), 989-1023.

Wang, Guangquan; SaIm, Jeffrey R.; Gurgel, Patrick V.; Carbonell, Ruben G., Edited by Galan, Miguel A.; Martin del Valle, Eva, Small peptide ligands for affinity separations of biological molecules, Chemical Engineering (2005), 63-83.

Cuatrecasas, Pedro; Wilchek, Meir, Edited by Lennarz, William J.; Lane, M. Daniel, Affinity chromatography, Encyclopedia of Biological Chemistry (2004), 1, 51-56.

Open-Source Reading Material:

Affinity Chromatography: Methods and Protocols: Bailon, P; Ehrlich, G.K.; Fung, W.; Berthold, W., Eds.; Methods in Molecular Biology; Humana Press, 2000, Vol. 147.

Affinity Chromatography: Bio-Medicine (2009).

Hemdan, E.S.; Zhao, Y.; Sulkowski, E.; Porath, J. Surface topography of histidine residues: A facile probe by immobilized metal ion affinity chromatography. Proc. Natl. Acad. Sci. USA 1989, 86, 1811-1815.

Jameson, G.W.; Elmore, D.T. Affinity Chromatography of Bovine Trypsin. Biochem. J. 1974, 141, 555-565.

Large-Scale Affinity Chromatography: Genetic Engineering & Biotechnology News (2007).

Affinity Chromatography: Practical and Theoretical Aspects: Mohr, P.; Pommerening, K.; Chromatographic Science Series; CRC Press, 1985. Vol. 33.