Affinity chromatography (Animated) | How to separate specific molecules by affinity chromatography?

Affinity chromatography video explains how to separate specific compounds from a mixture using affinity column. Affinity chromatography technique follows the same instrumentation as that of HPLC. The principle of affinity chromatography and the separation process is explained. The various steps in the separation process includes equilibration, sample application, elution and re-equilibration. Elution process can be done specifically by using specific competing ligands or specific competing molecules. Elution process can also be done non-specifically by changing the pH, polarity, composition and salt concentration. The advantages of affinity chromatography, disadvantages of affinity chromatography and applications of affinity chromatography is also discussed.
Affinity chromatography
You will be able to
elucidate the principle of affinity chromatography.
explain the steps involved in the separation of specific molecules.
discuss the elution process specifically by competitive ligands and non-specifically by changing pH, salt concentration, polarity etc.
list out the advantages, disadvantages and applications of this technique.
Principle of Affinity chromatography
Affinity chromatography is a technique used to separate the specific group of molecules present in a complex mixture by the specific biological interaction between the stationary phase (immobilized ligand) and its binding partner in the mobile phase.
Stationary phase
It is an insoluble solid matrix (porous or non-porous) covalently bonded to an immobilized ligand separated by a spacer arm
Solid matrix (support material)
Natural : cellulose, agarose, dextrose
Synthetic : polymethacrylate, polystyrene, polyacrylamide etc.
Inorganic : porous silica, glass etc.
Affinity ligand
It is a molecule that binds to specific target molecule reversibly.
Spacer arm
Creates space between matrix and ligand
provide better interaction between the ligand and target molecule by overcoming steric hindrance
Mobile phase – buffer solution
Binding buffer (Equilibration and sample application) – Optimize the conditions to ensure the binding of target molecules to the specific ligand.
Elution buffer (Elution) – Conditions are reversed to weaken the interaction between the target molecule and ligand to elute the target molecules.
Wash buffer (Washing) – Conditions suitable to wash the unbound molecule without eluting the target molecules.
Elution of target molecules specifically
by using competing molecules
by using competing ligands
Elution of target molecules non-specifically
by altering the buffer composition and concentration
by altering the buffer pH
by changing the buffer polarity
Elution by competing ligand
Competing ligand binds to the target molecule.
Target molecules along with the unbound competing ligands are eluted out of the column.
Elution by changing the composition of the buffer
Target molecules are eluted out of the column.
Process of Affinity chromatography/ Procedure of Affinity chromatography/Working of Affinity chromatography
Equilibration – To equilibrate the stationary phase to the required conditions by choosing the proper binding buffer (the immobilized ligands are bound to the pre-modified solid matrix)
Sample application – To bind the target molecules with the ligand and wash out the unwanted molecules from the column.
Elution– To elute the target molecules specifically by using competing molecules/ligands or non-specifically by altering the buffer composition, pH, polarity etc.
Re-equilibration – To wash out the tightly bound molecules by using binding buffer. The affinity system is available for next run with full capacity.
Advantages of Affinity chromatography
Highly specific
One step purification
Highly pure and high yield product (target molecules) obtained
Solid matrix can be easily washed and reused
Disadvantages of Affinity chromatography
Laborious and time consuming process
Immobilized ligands are expensive
Large quantity of solvent is required
Several buffers are required
Applications of Affinity chromatography
Separation of specific compounds present in a mixture
Purification of specific molecules or group of molecules such as protein, amino acids, nucleic acids etc.
Removal of specific impurities in purification process.
Investigation of binding sites in biological molecules.
Isolation of antibodies from blood serum in vaccine preparation.
Related videos
High Performance Liquid chromatography - https://youtu.be/rsKeg0RMdhE
Gas chromatography -https://youtu.be/dlBUrrSB0_o
Column chromatography -https://youtu.be/QZy6Yrys9yU
Paper chromatography -https://youtu.be/eoSydE1ITJE
Thin layer chromatography - https://youtu.be/Av3o0-YYTh0
Ion-exchange chromatography - https://youtu.be/pd9jSAkbFHc

Видео Affinity chromatography (Animated) | How to separate specific molecules by affinity chromatography? канала Revathi Purushothaman