Affinity Chromatography – How Does it Work?

Affinity chromatography is a separation process used to purify molecules or a group of molecules that are in a biochemical mixture. It employs two phases; a stationary phase and a mobile phase. Specific molecules from the moving phase will bond to the stationary phase based on their properties whilst the rest of the solution passing through unaffected.

The process is often used to purify biomolecules such as enzymes, antibodies, and recombinant proteins. It can also help to remove harmful substances such as pathogens through the same principles.

For example, a protein in a solution can be purified by passing it through a column that has another molecule attached to it (the ligand) that has an affinity for the protein. When they bind, this will allow the unreactive, non-bound solution to pass through the column. The target molecule is then eluted from the ligand by a change made in the buffer conditions so that the protein can be removed from that surface.

The process usually involves sets of molecules that interact with their cognate partner  such as enzymes and substrates, antigens and antibodies, and ligands and receptors. Thus the purification requires knowledge of how the two different sets of molecules are likely to interact.

Stationary phase

Scientists have several options for the stationary phase:

  • Porous supports –  materials used include agarose, cellulose, silica, polymethacrylate which can be obtained in different pore sizes
  • Non porous supports – these tend to have lower surface areas in comparison to porous although they can lead to faster purification
  • Monolithic supports – these combine both large and small flow-through pores
  • Membranes – these can be used for quicker purifications but they have a reduced surface area due to a lack of porosity
  • Expanded-bed absorbents – these aim to prevent the chromatography column from being clogged up
  • Perfusion media (flow-through beads) –  these have different sized pores

The particle size of the support matters. Smaller molecules can result in greater surface area but there is a greater chance of contaminant accumulation and unpleasant odors with these. Larger particles can counter these difficulties and so are often used as an alternative.

It is important that the chosen stationary phase is not attractive to any molecule in the solution other than the one required for purification. It needs to be chemically stable and have some inability to bind to various types of solutions that will be passed through it such as enzymes, cleaning agents and elution buffers. The structure itself needs to be strong to withstand the many purification procedures that are likely to be performed.


There are different options for ligands that bind to the substance which needs to be purified:

  • Antibodies – these can be monoclonal or polyclonal. The benefit of them is their high specificity and large binding constant
  • DNA – this can be used for polymerases, DNA-binding proteins, helicases and restriction enzymes
  • Chlorotriazine polysulfanated aromatic molecules – these are used for molecules such as lyases, hydrolases, glycotic enzymes, oxidoreductases
  • Biometric dye ligands – these can be used for protein purification
  • Peptide – these are used for biomolecules

Companies such as AMSBIO have a range of different materials to help with affinity chromatography.


  • Affinity Chromatography: Principles and applications:
  • US National Library of Medicine, National Institutes of Health, Affinity Chromatography: general methods:

Further Reading

  • All Chromatography Content
  • Chromatography Overview
  • Gas Chromatography-Mass Spectrometry (GC-MS) Applications
  • High Performance Liquid Chromatography (HPLC)
  • Liquid Chromatography-Mass Spectrometry (LC-MS) Applications

Last Updated: Feb 26, 2019

Written by

Deborah Fields

Deborah holds a B.Sc. degree in Chemistry from the University of Birmingham and a Postgraduate Diploma in Journalism qualification from Cardiff University. She enjoys writing about the latest innovations. Previously she has worked as an editor of scientific patent information, an education journalist and in communications for innovative healthcare, pharmaceutical and technology organisations. She also loves books and has run a book group for several years. Her enjoyment of fiction extends to writing her own stories for pleasure.

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