Affinity chromatography

Affinity chromatography is still in the phase of development. This technique is applicable in, for example- purifying enzymes by separating them from inhibitors and other biologically useful macromolecules, separating antigen from the body, etc.

• Bio affinity + chromatography= Affinity Chromatography, a type of liquid chromatography for the separation and purification of components of a mixture and their specific analysis.
• You can also call affinity chromatography as affinity purification which utilizes the specific binding interactions between the molecules of a mixture. 
• This purification gives high resolution, even several thousand folds and that’s why the recovery of the active components is usually high. 

More examples- Lysine can be used in the purification of our RNA, avidin can be used for the purification of biotin-containing enzymes, nickel can be used for the purification of proteins that have a histidine tag, and so on. 

The birth of Affinity chromatography

The first idea of using affinity chromatography is widely held as isolation of α-amylase by using starch by German scientist Emil Starkenstein. 

However, it was first developed by Pedro Cuatrecasas and Meir Wilchek.

Application of Affinity Chromatography

1. The crude sample is incubated with affinity support. It allows the target molecule to be separated from the mixture to bind to the immobilized ligand.
2. Non-bound sample components are washed away from the affinity support. 
3. The target molecule is dissociated and recovered from the immobilized ligand. This process called elution is done by changing the buffer conditions to make sure that the binding interaction between the molecules no longer exists.

Step 1. Column is prepared

• Solid support materials known as affinity support such as cellulose, agarose, Sepharose are added to the column.
• Selection of ligands according to the target molecule.
• Attaching a spacer arm between the affinity support and ligand.

Step 2. Sample loading

• The mixture is transferred into the elution (dissociation and recovery) column and it is enabled to run at a moderated rate.

Step 3. Dissociation and Recovery of Ligand- Molecule Complex

• The target material is recovered by changing the buffer conditions. 

Components of Affinity Chromatography

In the process of using affinity chromatography slideshare to purify and separate large biomolecules from complex mixtures, three things that are essentially considered are the support (matrix), spacer arms, and ligand.

Matrix

• The matrix needs inert- both physically and chemically for the attachment of ligands.
• While coupling the desired ligand, the matrix has a passive role during the separation. 
• It requires porous material for good flow and provides a large surface area for attachment like silica, agarose, cellulose, polymethacrylate, etc.
• An ideal matrix needs to be inexpensive and insoluble in solvents and the buffers being used in the process.
• The support material should easily be coupled to the spacer arm or ligand on which the ligand is supposed to attach.

Spacer Arm

• It helps in overcoming any steric hindrances and thus helps to improve the binding between the target molecule and the ligand.
• To aid in efficient binding, the spacer arm exists between the matric and ligand.

• It is important to consider the length of the spacer arm as too long or too short arms can prevent the binding from happening.
• A spacer arm is ideally at least 3 atoms long to help keep the substance at a necessary distance.

Ligand

• It is referred to as the molecule that binds reversibly to a group of target molecules or a specific target molecule.
• The nature of the target antibody or macromolecule to be isolated is essential to be known to determine the selection of the right ligand.
• An antigen can be used as a ligand in the separation of an antibody. 
• Since these molecules play an important role in the stability of the system, they are necessary for affinity chromatography.
• The ligands can be both specific such as an antibody and an antigen, enzyme and substrate, and general such as Amino acids, coenzymes, lectin, dyes, Protein A and G, etc. 
• There are two types of affinity ligands- biological and synthetic. 

Biological ligands- These are derived from natural sources like nucleotides, antibodies, lectins, coenzymes, RNA, and DNA fragments 

Synthetic ligands- They are obtained by altering existing molecular structures or by de novo synthesis such as non-natural peptides, triazine dyes, etc.

Fundamental Principles of Affinity Chromatography

• The samples are brought in conditions favoring maximum binding with the ligand.
• Unbound substances are removed which allows the desired molecule to remain attached to the affinity support. 
• The desorption method is undertaken to dissociate and recover the desired bound molecules. The methods used to elute the bound molecules include altering the pH, polarity conditions, or ionic strength, or even adding a competing ligand in the mobile phase. 

Conclusion

Affinity chromatography is used to purify molecules in a biochemical mixture and hence it has applicability in various fields like biomedical and pharmaceuticals. This powerful technique can be used in the purification of certain proteins in the human plasma. This rapid, and highly efficient purification method can give several thousand-fold purifications individually.