Principle and Procedure of Affinity Chromatography

What is Affinity Chromatography?

Affinity chromatography is a form of liquid chromatography used to isolate and purify analytes in a specific way. It uses a reversible biological interaction called affinity that refers to the forced attraction used between the atoms in various degrees that cause them to remain in combination.

Affinity Chromatography Principle

Affinity chromatography is one of the most versatile and effective chromatographic methods for separating a complex mixture of a single or a group of components. It is based on very precise biological interactions amongst two components, such as enzyme-substrate interactions, antibodies, and antigens, or receptor and ligand. These typically reversible interactions are used for purification, in which molecules known as a molecule are placed on a solid matrix to form a stationary phase while components are in the mobile phase. Many of the widely used ligands are associated with matrices which are now commercially available and ready to use.The stationary phase consists of a support medium to which the substratum is sympathetically bound, to reveal the reactive groups necessary to binding of the target molecule. As the crude sample mixture of molecules is traveled through the column, in the stationary phase, molecules with a binding site to the stationary phase bind while all other substances are eluted in the void volume of the chromatographic column. Once other molecules are eluted from the column, the bound target molecules can be overcome by including a competing ligand in the mobile phase, or by changing pH, ionic strength, or polarity state.

Affinity Chromatography Procedure

Before beginning an affinity chromatography experiment, we must recognize the various components essential to perform the process.

Matrix: It is passive support that can be directly or indirectly attached to a ligand.

Spacer arm: By eliminating any satirical hindrance effect it is used to enhance the binding between ligand and target molecule.

Ligand: It refers to a component that binds opposite to a particular target molecule.

Experimental Procedure of Affinity Chromatography

Column Preparation:

1.  Flush the column and stationary phase with the buffer solution to prepare the ligand for binding.

2.    Fill the column with solid supports such as agarose, safarose, and cellulose, etc. 

3.    Select the ligand according to the desired separation. 

4.    Attach the spacer arm between the ligand and the solid support.

Sample Loading: 

1.    Pour a sample mixture into a column and allow it to run at a controlled rate.

Elution of Ligand-Molecule:

2.  The desired component is recovered by adjusting favorable conditions for bound molecules to adhere.

Affinity Chromatography Applications

  • For all biological macromolecule separation and purification, affinity chromatography is used. 
  • It is used to purify antibodies, nucleic acids, and enzymes.
  • It is also used to decrease the amount of a substance in a complex mixture.
  • Affinity chromatography is used to notice what biological compounds bind to a given substance.
  • The affinity chromatography is used in clinical application.
  • Another application of affinity chromatography in analytical chemistry is a tool to study the kinetics of biological interaction.

The Advantages of Affinity Chromatography are as follows.

  • Target molecules can be obtained by affinity chromatography in a highly purified state. 
  • It can use to extract particular contaminants. 
  • Affinity chromatography has higher sensitivity than FID and FCD.
  • Affinity chromatography is used in vaccine production.
  • Affinity chromatography provides a high degree of purity.
  • It is used in the production of vaccines at pharmaceutical manufacturing to improve product quality.
  • It is doesn't depend on the temperature, buffer composition, pH, and ionic strength.
  • To improve the solubility of the affinity chromatography used.

The Disadvantages of Affinity Chromatography are as follows. 

  • It takes a lot of skill to do that.
  • This is a non-specific for adsorption compared to other chromatography techniques.
  • The ligands used in this chromatography are expensive.
  • Sometimes ligand leaks occur in affinity chromatography.
  • Metal-ion transfer and leakage lead to protein loss.

Commonly asked questions on affinity chromatography are as follows.

What is affinity chromatography?Affinity chromatography is a technique of separating and analyzing biomolecules based on their structures or biological functions. It is a significant and useful separation method in biochemistry, biotechnology, environmental science, and pharmaceutical science.


What is the basic principle of affinitychromatography? The basic principle of affinity chromatography is that a bio-specific ligand is immobilized to a stationary phase or resin of the column matrix, such as agarose, or cellulose.


What type of chromatography is an affinity chromatography?Affinity chromatography is a type of liquid chromatography used for the purification of specific biomolecules, including proteins.
What is the major advantage of affinity chromatography?The major advantage of affinity chromatography is that it can achieve very high specificity and a high degree of purity.


What is the major difference between Affinity and Ion Exchange Chromatography?The major difference between affinity and ion-exchange chromatography is that affinity chromatography is used to separate charged or uncharged molecules, while ion-exchange chromatography is used to separate charged molecules.

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