Chromatography is a technique which is widely used for the separation of mixtures based on the physio-chemical differences between the stationary and the mobile phase of the chromatographic apparatus. The sample to be separated is dissolved in the mobile phase, which passes over the stationary phase (the immobile/fixed surface) and carries out the process of separation of molecules. 
See the source image

Most chromatographic processes employ and rely on these physio-chemical differences between the stationary and mobile phases for the process of separation of molecules. For example, gel permeation chromatography, ion exchange chromatography etc.

However, an exception is affinity chromatography. Unlike gel permeation and ion exchange chromatography, affinity chromatography exploits the tendency or capacity of certain biomolecules to bind specifically and non-covalently to other molecules called ligands (i.e., “bio-specificity of molecules)


One of the most familiar concepts for anyone involved in the studies of biochemistry is “bio-specificity”. 

Specificity is a molecular recognition mechanism which operates through structural and conformational complementarity between the biomolecule and its substrate. It is known that a given enzyme only bind/react with its specific group of substrates and not with any other. 

This bio-specificity may not always be limited only to enzymes. Other examples of this kind of biomolecular interactions include;

  • A given hormone binds only to its specific glycoprotein (known as the hormone receptor).
  • Antibodies specifically bind to only a given antigen which is shaped/has a confirmation specific to that antibody.See the source image

To understand this principle better with respect to affinity chromatography, let us suppose that an enzyme is to be purified from a mixture of thousands of proteins.

  •  Let the substrate analogue (molecule resembling the substrate but not capable of reaction) specific for this enzyme (which is to be separated) be coupled to the stationary phase (let’s say the stationary phase for this particular example be e.g., agarose). 
  • All other molecules, which have no specificity for the ligand and are incapable of binding will pass down and out of the column, (as seen in the second diagram in the picture.)
  • Lastly, once the other substances are eluted, the bound target molecules can be eluted by methods such as including a competing ligand in the mobile phase or changing the pH, ionic strength, or polarity conditions which would completely alter the strength of binding of the enzyme to the substrate and help in the elution of the desired product.


There are some complications which arise during affinity chromatography, they may be due to the nonspecific adsorption of sample components other than the desired one on to the matrix. Usually, when this happens, ionic and hydrophobic interactions are involved. 

This complication may be taken care of by judicious choice of operating conditions (e.g., pH, temperature, or ionic strength) in such a way that the physical conditions exclusively favour the binding of the desired molecule to the substrate and not any other undesired molecule.  

Another type of complication arises when one uses ligands, which interact with more than one macromolecule present in a given mixture.


  1. The type of matrix that is used.
  2. The type of ligand used.
  3. The conditions of binding and elution of the sample from the matrix.

A general discussion of these points has been given below:

Properties of the supporting matrix:

  1. The matrix that is used in the process must be chemically inert to other molecules in order to minimize the rate of non-specific adsorption.
  2. The matrix should possess good flow properties.
  3. The matrix should be able to remain stable even at varying levels of pH and temperature, ionic strengths and denaturing conditions.
  4. It should be highly porous to provide a large surface area for the attachment of ligand.

Ligand Selection:

  1. The ligand used should be capable of forming moderately strong interactions with the desired macromolecule. 
  2. The ligand that is to be bound must possess functional groups that may be modified to form covalent linkages with the supporting matrix. 

Ligand attachment:

  • The covalent coupling of the ligand to the supporting matrix occurs in two steps;
  1. Activation of the matrix functional groups, and
  2. Covalent attachment of the ligand to the matrix.
  • The chemical methods that are used must be relatively mild so as to ensure no/minimal damage to the ligand or the matrix.


  1. Affinity chromatography has been widely used for the purification of varied number of macromolecules which are capable of showing ligand specific interactions like enzymes, hormones, antibodies, nucleic acids, membrane receptors etc.
  2. Affinity chromatography has also been used for the purification of cells as this technique is known to affect the viability of cells less than other chromatographic techniques. Cells that have been purified using this technique include fat cells, T and B lymphocytes, spleen cells, lymph node cells etc.
  3. An extension of affinity chromatographic principles is using magnetic gels. The gel beads contain a magnetic core that is chemically coupled to a protein ligand. The suspended cells are then allowed to interact with the microspheres. When a magnetic field is passed, the cells of interest move towards the poles of the magnets thereby getting separated. The cells can be collected by removing the magnetic field. 

Immunoglobulin negative thymocytes and neuroblastoma cells are separated using this technique.


Biophysical Chemistry principles and techniques – Upadhyay and Nath


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