Affinity Chromatography

  Affinity chromatography: Definition

Affinity chromatography is a technique used to separate and the purification of the biochemical mixture. Affinity column Chromatography is a highly specific technique.

Affinity chromatography technique depends on the specific affinity between stationary phase and the analyte (Ligand.)

Affinity chromatography is a reversible biochemical reaction.

Affinity chromatography Principle:

Affinity chromatography involves the covalent attachment of an immobilized biochemical (called an affinity ligand) to a solid support.

When the sample is passed through the column, only the solute that selectively binds to the complementary ligand is retained. The other sample components elute without retention.

The interaction between the ligand attached to the solid support of the stationary phase and the target molecule in the analyte is possible because of electrostatic, hydrophobic reaction, or hydrogen bonding.

Nature of interaction between the target molecule and the ligand to help determine the selection of proper stationary phase and the mobile phase.

The separation is well expressed by lock and key binding.

The retained solute(s) can be eluted from the column by changing the mobile phase conditions.

Diagram of Affinity chromatography:

Affinity Chromatography

Stationary Phase and mobile Phase in Affinity chromatography:

Stationary phase in Affinity Chromatography:

The stationary phase is made of a solid matrix and ligand one more important component of the stationary phase in Affinity Chromatography.

Component of stationary Phase in Affinity Chromatography.

Ligand:

Ligand is an immobilized chemically insoluble matrix supported by the stationary matrix. It reversibly adsorbs molecule species (affine components or target molecules) from a mixture of Analyte.

  Ligand is attached to the solid support. It exploits the unique property of extremely specific reversible biological interaction to achieve separation and purification.

The separation exploits the “Lock n Key” binding that prevalent in a biological system. Ligands are coupled to affinity matrix are now commercially available and ready to use.

  Different types of ligand that bind to the substance which needs to purify:

  Antibody: This can be monoclonal or polyclonal. Highly specified and large binding constant

DNA: Can be used for polymerase, DNA –binding proteins helicases, and restriction enzyme.

Biometric dyes: used for protein

Peptides: these are used for biomolecule

Spacer Arm:

A spacer arm is the chain of carbon and/or other atoms that position a functional group away from the solid matrix to which it is covalently bound and makes it more available to a ligand and less restricted by steric hindrance by the matrix.

Matrix:

Solid Matrix or stationary phase is to provide the support to the ligand Solid matrix maybe porous or non-porous

It should be of adequate size and shape i.e increase in particle size reduces the flow resistance and separation power & decrease in size leads to increase flow resistance and clogged.

·        Irregular shape leads to unequal path lengths for substance and band broadening. So spherical shape is best suited.

·        Mechanically and chemically stable and resistant against microorganisms.

·        It should be inert to prevent analyte from interacting non-specifically with matrix itself.

·        Economical

·        Eg. Agarose

           Cellulose dextran

     Sepharose is a bead formed of agarose gel.

Mobile Phase in Affinity chromatography:

The nonpolar or polar mobile phase used as per the requirement of the separation of the compound. 

Instrumentation:

Once the column is prepared the separation is conducted in four basic step Introduction of the sample, Adsorption of the analyte, removal o impurity and Elution of sample

Following are the basic part of the Affinity chromatography:

The injection – pump system:

In Affinity Chromatography Sample is introduced onto the high performance (rigid ) bio affinity matrix by an automated delivery system.

The affinity Column:

The column is used for separation or the purification of the component. Ligand, spacer, solid matrix is part of the columns.

On line monitoring:

The effluent is monitored by online & assayed robotically for specific biological properties.

Post collection monitoring.

  Last step is elution and collection of the analyte.

Example of affinity chromatography

The separation or Purification of Antibody by using of the Affinity chromatography this technique is known by the Immunoaffinity Chromatography.

To purify and separation of the immune biochemical mixture Purify antigens, Modification using Epitopes.

Antigen is immobilized in the stationary phase as ligand and stationary phase with antigen ligand used to isolate and purify the appropriate antibody. Purification of the antibody from the blood serum is one of an example of the antibody separation purification.

Advantages of Affinity Chromatography

·        The major advantage of this technique is its tremendous specificity, which permits rapid isolation with a good yield in a single step.

·        This interaction is reversible, Rapid process, Faster separation time.

·        Better efficiency resolution.

·        Higher pressure stability.

·        Better defined column condition.

·        Simpler detection & analysis.

·          Amenable to smaller samples.

·        No. of purification steps are reduced.

·        No loss of protein due to denaturation.

·        Suitable for large scale purification processes.

  Disadvantages of Affinity Chromatography

·        High expense

·        Time-consuming method

·        Greater irreversible adsorption

·        Greater impact on slow kinetics