Affinity chromatography: Principle, Procedure, Applications, Advantage

Affinity chromatography
Affinity chromatography

Affinity chromatography is a liquid chromatography technique that employs biospecific interactions to separate compounds. The molecule that needs to be purified is selectively and irreversibly adsorbed to a certain ligand.

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History of Affinity chromatography

Immunochemists were the ones who created the early types of affinity chromatography. Antigen-cellulose columns were created in 1951 by Campbell, Luescher, and Lerman for the isolation of specific antibodies. This separation was only possible by utilizing the antibody’s affinity for a nhy6-specific antigen. Immune adsorbents were created by conjugating an antigen to a solid matrix. This idea was extended to the isolation of enzymes by Cuatrecasas, Wilchek, and Anfinsen in 1968, and introduced the term”affinity chromatography.”

Fundamental principles of affinity chromatography

The purification of particular biomolecules, such as proteins, can be accomplished with the use of affinity chromatography. The fundamental idea is that a biospecific ligand is immobilized to a solid support or resin of a column matrix, such as cellulose, agarose, or polyacrylamide.

Affinity chromatography
Affinity chromatography

Affinity chromatography relies on reversible interactions between the protein to be purified and the affinity ligand attached to the chromatographic matrix to separate it. Most proteins naturally contain a recognition site that can be employed to choose the best affinity ligand. The chosen ligand and the target protein must bind specifically and irreversibly.

The procedure of affinity chromatography

A solution containing the protein of interest is run through the column, allowing the proteins to interact with the ligand while washing away other proteins. Ligands are frequently based on biological functional pairs, including enzymes and substrates, antigens and antibodies, receptors and hormones. The target protein is attached to the specified ligand, and all other molecules are eliminated.

Procedure of affinity chromatography
source: Protein Affinity Chromatography

The bound protein can be recovered by altering the experimental setup to promote desorption. In a particular buffer, the protein is eluted either through a shift in pH and/or ionic strength or through competitive displacement. Since it depends on the biological specificity of your target protein, such as the affinities of an enzyme for a substrate, this purification technique is very effective.

Components of affinity medium

The support (matrix), spacer arms, and ligand must all be taken into consideration when using affinity chromatography to purify and separate large macromolecules from the mixtures.


To enhance the surface area to which the molecule can bind, the matrix merely provides a structure. It must be activated for the ligand to bind to it. Additionally, it still being able to retain its own activation toward the target molecule.

Traditionally, porous support materials like agarose, polymethacrylate, polyacrylamide, cellulose, and silica have been used as support materials. These support materials are all readily available on the market and are offered in a variety of particle and pore sizes. Apart from porous material non porous supports are also used. Nonporous beads with diameters between one and three micrometers make up nonporous support materials. Although these supports enable quick purifications, they have smaller surface areas than conventional porous supports. Similar to nonporous beads, affinity chromatography membranes also lack diffusion pores, which reduces surface area yet enables quick separations.

Spacer arms

A spacer arm is frequently placed between the matrix and ligand to aid efficient binding and provide a more effective and better binding environment because the target molecule’s binding sites can occasionally be deeply positioned and challenging to access due to steric hindrance.


A covalently attachable biospecific ligand to a chromatographic matrix is necessary for successful affinity purification. After removing unbound material, the coupled ligand must still have a specified binding affinity for the target molecules. Additionally, the binding between the coupled ligand and the target molecule must be reversible to enable the removal of the target molecules in an active state. To purify its specific binding partner, any element can be utilized as a ligand.

Because of their excellent selectivity and relatively high binding constants, antibodies are also utilized as ligands. In this chromatographic technique, monoclonal antibodies are frequently preferred over polyclonal antibodies because of their lack of variability, which enables the production of more uniform affinity support. Additionally, DNA also can function as an affinity ligand. It can be applied to the purification of restriction enzymes, helicases, polymerases, DNA-binding proteins, and DNA repair proteins.

A dye ligand is another kind of affinity ligand that can be used to separate biomolecules from complicated mixtures. In 1971, pyruvate kinase was purified using a Blue Dextran column, which was when the dye-enzyme binding was used for the first time. In biomimetic dye-ligand chromatography, modified dyes are used to mimic the target protein’s native receptor, taking dye-ligand chromatography one step further. One of the most popular modified triazine dyes used for protein purification is Cibacron Blue 3GA. Additionally, DNA can function as an affinity ligand. It can also be applied to the purification of restriction enzymes, helicases, polymerases, DNA-binding proteins, and DNA repair proteins. This chromatographic technique typically employs some common biological interactions, including

  1. Enzyme – substrate analogue, inhibitor, cofactor.
  2. Antibody – antigen, virus, cell.
  3.  Hormone, vitamin – receptor, carrier protein.
  4. Glutathione – glutathione-S-transferase or GST fusion proteins.

Factors should be considered when choosing a support material

Several considerations are also need to be taken into account when selecting a support material for affinity purification, regardless of the type of support used. These include pore size, particle size, chemical stability, chemical inertness, and mechanical stability.

Chemical inertness

The support material necessitates that the affinity support has low or nonspecific binding and only binds the target molecule. While the affinity ligand immobilized on the support plays a role in specificity, the support’s characteristics must be chosen carefully to prevent the non-specific binding of other molecules.

chemical stability

Under typical operating conditions, a support material must be chemically stable.The matrix should be resistant to degradation, elution buffers, and cleaning agents.

Mechanical stability

Mechanical stabilityshould be taken into considerationwhile selecting chromatographic support for affinity chromatography. The supporting materials must be able to resist backpressures experienced during typical separations without compressing.

Particle size

To minimize band widening and mass transfer effects, tiny particle sizes are preferred. Additionally, smaller particle sizes often provide more surface area for the support material and also enable more ligands to be bound on the surface of the support.

Pore size

When using affinity chromatography, it’s also important to take into account the size of the pores because the target biomolecules need to be able to properly interact with the affinity ligand in addition to passing through the column.

Application of affinity chromatography

For purposes like the purification of fusion proteins, antibodies, and glycoproteins, affinity chromatography is frequently used. Several uses are as follows:

1. It is used to separate and purify every biological macromolecule.

2. It is used to purify enzymes, antibodies, nucleic acids, etc.

3. This chromatography can be used to examine the interactions between drugs and proteins.

4. This technique is also used to produce vaccines by purifying antibodies from blood serum.

5.  For the purification of DNA through genetic engineering.

6. For the separation of a chemical combination.

Advantages of affinity chromatography

  1. It has high sensitivity and selectivity.
  2. Preservation of quality and purity, and reproducibility are advantages of affinity chromatography.
  3. It is possible to achieve a high level of purity.
  4. It is also easy to investigate the  binding sites of biological substances.
    The method is fairly reproducible.
  5. It also helps in removing particular impurities.
  6. It also offers a quick separating procedure.

Disadvantages of affinity chromatography

  1. For affinity chromatography to be successfully carried out, great analytical skills and practical experience are needed.
  2. One of the main drawbacks of affinity chromatography is sometimes regarded to be sample or protein loss.
  3. This chromatography also requires expensive ligands.
  4. The majority of the ligands employed in affinity chromatography are proteins (i.e., antibodies), and to preserve their lifespan, they need particular temperature conditions.
  5. When the ligands are antibodies, affinity chromatography shows the finite lifetime of the resins utilized.


  3. Sameh Magdeldin and Annette Moser (2012). Affinity Chromatography: Principles and Applications, Affinity Chromatography, Dr. Sameh Magdeldin (Ed.), ISBN: 978-953-51-0325-7, InTech.

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Kabita Sharma

Kabita Sharma, a Central Department of Chemistry graduate, is a young enthusiast interested in exploring nature's intricate chemistry. Her focus areas include organic chemistry, drug design, chemical biology, computational chemistry, and natural products. Her goal is to improve the comprehension of chemistry among a diverse audience through writing.

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