Affinity chromatography

Affinity chromatography
Specificity
Matrix
Spacer arm
Ligand
Process
Advantages
Disadvantages
Applications

1. Presented by:- Machhi Dhruvi Anilkumar 1 st sem M.Pharm . Department of Pharmaceutical Quality Assurance Smt. B. N. B. Swaminarayan Pharmacy College, Salvav -Vapi TOPIC:- AFFINITY CHROMATOGRAPHY Subject Name:- Modern Pharmaceutical Analytical Techniques Subject Code:- M AT101T

2. Contents Introduction Specificity of affinity chromatography Matrix Spacer arm Ligand Process Of Affinity chromatography Advantages Disadvantages Applications References 12/22/2021 Dhruvi Machhi 2

3. Introduction Definition :- It is the purification of a biomolecule with respect to the specific binding of that biomolecule with another molecule to which it can specifically bind. It is also called as Affinity purification. Principle :- The principle of affinity chromatography is based on the property of specific & non-covalent binding of biomolecules to other molecule which are referred as ligand or substrate or cofactors. 12/22/2021 Dhruvi Machhi 3

4. The technique was originally developed for the purification of enzymes, but it has been extended to nucleotides, neucleic acid & immunoglobulins. 12/22/2021 Dhruvi Machhi 4 Molecule Ligand Antigen Antibody Enzyme Substrate Receptor Ligand

5. Specificity of Affinity Chromatography The specificity is based on three aspects : Matrix :- for Ligand attachment Spacer arm :- used to bind ligand to matrix Ligand :- molecule that binds reversibly to a target molecule Only the substance with affinity for the ligand are retained on the column & the substance with no affinity to ligand will elute off. 12/22/2021 Dhruvi Machhi 5 Matrix Spacer arm Ligand

6. Matrix Matrix provides structure to increase the surface area to which the molecule can bind. Amino (-NH 2 ), hydroxyl(-OH) , carboxyl(-CO) , thoi (-SH) groups located within the matrix act as a ligand binding site. Matrix are made up of Agarose & Polysaccharide. Matrix should possess following qualities; does not adsorb molecules Must be coupled without altering its binding properties Should be stable under wide range of experimental conditions 12/22/2021 Dhruvi Machhi 6

7. Examples of matrix & their trade name Matrix Trade name Agarose Sepharose 2B, 4B, 6B Cross-linked dextran Sephadex Polyacrilamide Bio-gel-P Cross linked cellulose Matrix cellufine 12/22/2021 Dhruvi Machhi 7

8. Spacer Arm It is used to prevent attachment of ligand to the matrix by interfering with its ability to bind the macromolecules. Optimum length:- 6 to 10 carbon Ex:- 1,6-diamino hexane & 6-amino hexanoic acid 12/22/2021 Dhruvi Machhi 8 (Matrix)

9. Ligand The ligand binds only to the desired molecule within the solution. It attaches to the matrix which is made up of inert substance. It should only interact with the desired molecule & form a temporary bond. The [ Ligand-Molecule ] complex will remain in the column & eluting everything else off. 12/22/2021 Dhruvi Machhi 9

10. Commonly used ligands Ligand Affinity Heparin Lipase, lipoprotein, DNA, coagulation factors Avidin Biotin containing enzymes Calmodulin Calmodulin containing enzymes 12/22/2021 Dhruvi Machhi 10

11. Process Of Affinity chromatography 12/22/2021 Dhruvi Machhi 11 Immobilized ligand Sample with impurity Complex Ligand Matrix Impurities Purified sample

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14. Step 1 :- Attachment of ligand with column matrix Binding of selected ligand to the matrix requires formation of covalent bond between them. The Ligand-Matrix gel is then loaded into an elution column. 12/22/2021 Dhruvi Machhi 14

15. Step 2 :- Loading of sample mixture into column Once the column has been prepared, the mixture containing sample/ substrate mixture is poured into the elution column. Gravity pulls the solution through the gel, because most of the substances do not bind to the Ligand-Matrix complex. 12/22/2021 Dhruvi Machhi 15

16. Step 3 :- Binding of substrate to ligand Substrate passes through the gel & when the ligand is recognized, the substrate binds to Ligand-Matrix complex, halting its passage through the gel. 12/22/2021 Dhruvi Machhi 16

17. Step 4 :- Washing of Column to remove impurities In order to remove the unbound impurities, a wash of extreme pH, salt concentration or temperature is run through the gel. It is important to use a strong wash, so that all impurities are removed. 12/22/2021 Dhruvi Machhi 17

18. Step 5 :- Washing of Column to remove Substrate Finally to collect the sample/ substrate which is still bound to the Ligand-Matrix complex, a strong 2 nd wash is run through the column. This 2 nd wash relies on the reversible binding properties of the ligand, which allows the bound substrate to dissociate from the ligand. 12/22/2021 Dhruvi Machhi 18

19. Step 6 :- Elution of Substrate from column The substrate is then eluted off from the column & collected as purified sample of interest. 12/22/2021 Dhruvi Machhi 19

20. Advantages Extremely high specificity High degree of purity can be obtained The matrix can be reused  Give purified product with high yield 12/22/2021 Dhruvi Machhi 20 Expensive ligand Time consuming method. matrix can be degraded Leakage of ligand Disad vantages

21. Applications Used in genetic engineering - for Nucleic acid purification Used in production of vaccine - for Antibody purification from blood serum Protein & enzyme purification from free cell extract To purify & concentrate a substance from a mixture into a buffering solution To purify & concentrate an enzyme solution Reduce the amount of particular substance in mixture 12/22/2021 Dhruvi Machhi 21

22. References Anusha, Shyamala, and P. Sirisha. "A overview on affinity chromatography: a review."  Pharmaceutical Research  8.07 (2018). Principles of instrumental analysis- Doglas A. Skoog, F. James Holler, 6e, pp 848 Rodney Boyer; “ Modern Experimental Biochemistry ” ; 3rd edition; pp 99-104 12/22/2021 Dhruvi Machhi 22

23. Thank you 12/22/2021 Dhruvi Machhi 23