Affinity Chromatography/Ligand/Mobilized

Affinity Chromatography is a highly specific separation technique used to purify biomolecules based on their unique interactions with a ligand. The process involves passing a mixture through a column where the target molecule binds to an immobilized ligand attached to a stationary phase. Unwanted components are washed away, and the bound molecule is then eluted using a buffer that disrupts the specific interaction.

This method is widely used for purifying proteins, antibodies, enzymes, and other biomolecules. Due to its precision, affinity chromatography allows for the efficient isolation of target compounds from complex mixtures, making it a key technique in biochemistry, molecular biology, and pharmaceutical development.

The technique offers high specificity and can often purify molecules in a single step, though it can be costly due to the ligands used and may require careful optimization for different applications.

1. AFFINITY CHROMATOGRAPHY Sweta Y adav

2. CHROMATOGRAPHY Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures.

3. CHROMATOGRAPHY It is a physical method of separation in which the components to be separated are distributed between two phases , one of which is stationary (immobilize phase) while the other (the mobile phase) moves in a definite direction

4. • Can separate complex mixtures composed of many very similar components. • Chromatography is often coupled with analytical instruments to complete analysis. • A single chromatographic analysis can isolate, identify , and quantitate multiple components of mixtures

5. The term comes from Greek χρώμ α: chroma means color and γραφειν: graphein means write

6. INVENTED BY 1903 – Tswett, a Russian botanist coined the term chromatography.

7. TERMINOLOGY An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing. The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC), a gas (GC), or a supercritical fluid (SFC). A better definition:The mobile phase consists of the sample being separated and the solvent that moves the sample through the column.

8. TERMINOLOGY The analyte is the substance that is to be separated during chromatography. The sample is the matter analysed in chromatography. It may consist of a single component or it may be a mixture of components.

9. Elution - washing of the mixture Eluent - additional solvents used for elution Residency - time spent on column

10. Types of Chromatography Techniques

11. Properties of Chromatography 1) Immiscible stationary and mobile phases 2) An arrangement where a mixture is deposited at one end of the Stationary phase 3) Flow of the mobile phase towards the other end of the stationary Phase 4) Different rates of partitioning for each component 5) Means for visualizing the separation of each component

12. AFFINITY CHROMATOGRAPHY

13. Affinity History 1930s, first developed by A.Wilhelm Tiselius-a swedish biochemist, won the Nobel Prize in 1948 Used to study enzymes and other proteins Relies on the affinity of various biochemical compounds with specific properties • Affinity Chromatography is essentially a sample purification technique , used primarily for biological molecules such as proteins.

14. • It is a method of separating a mixture of proteins or nucleic acids (molecules) by specific interactions of those molecules with a component known as a ligand, which is immobilized on a support. If a solution of, say, a mixture of proteins is passed over (through) the column, one of the proteins binds to the ligand on the basis of specificity and high affinity (they fit together like a lock and key). • The other proteins in the solution wash through the column because they were not able to bind to the ligand.

15. The principle of affinity chromatography 1)Inject a sample into an initially equilibrated affinity chromatography column. 2) Only the substances with affinity for the ligand are retained in the column. 3) Other substances with no affinity for the ligand are eluted from the column. 4) The substances retained in the column can be eluted from the column by changing pH or salt or organic solvent concentration of the eluent.

16. Specificity of Affinity Chromatography Specificity is based on three aspect of affinity Matrix: for ligand attachment. Spacer arm: used to bind ligand to matrix Ligand: molecule that binds reversibly to a specific target molecule(site of interaction)

17. Chromatographic Media • A matrix in its use here is a substance, usually in bead form to which a specific ligand is covalently bound. Properties: • It must be insoluble in solvents and buffers employed in the process • It must be chemically and mechanically stable... • It must be easily coupled to a ligand or spacer arm onto which the ligand can be attached. • It must exhibit good flow properties and have a relatively large surface area for attachment

18. Immobilized Ligand • The ligand can be selected only after the nature of the macromolecule to be isolated is known. • When a hormone receptor protein is to be purified by affinity chromatography, the hormone itself is an ideal candidate for the ligand. • For antibody isolation, an antigen or hapten may be used as ligand. • If an enzyme is to be purified, a substrate analog, inhibitor, cofactor, or effector may be used as an immobilized ligand.

19. Examples Antigen Antibody Antibody Antigen Substrate Enzyme DNA Histon Hormone Binding Protein/Receptor

20. Attachment of Ligand to Matrix Several procedures have been developed for the covalent attachment of the ligand to the stationary phase. All procedures for gel modification proceed in two separate C hemical steps: • Activation of the functional groups on the matrix and • Joining of the ligand to the functional group on the matrix.

21. Pre-packed columns Hi-Trap Heparin HP (High performance) Column size: 5 × 1 mm 1 × 5 mm 5 × 5 mm Average particle diameter : 34μm Maximum operating flow rate: 4 ml/min 20 ml/min.

22. Storage of pre-packed columns At 2-8 °C in an upright position with both caps in place. Thio-mersal may be added for long term storage. DO NOT FREEZE Application areas : purification, isolation or removal of the following substances: Anti-thrombin III and other coagulation factors, lipoproteins, lipases, protein synthesis factors

23. AFFINITY CHROMATOGRAPHY Can be used; Purify and concentrate a substance from a mixture into a buffering solution Reduce the amount of a substance in a mixture Discern what biological compounds bind to a particular substance, such as drugs Purify and concentrate an enzyme solution

24. Applications Used in Genetic Engineering - nucleic acid purification Production of Vaccines - antibody purification from blood serum And Basic Metabolic Research - protein or enzyme purification from cell free extracts

25. Industrial Applications Affinity chromatography is widely used in the pharmaceutical industry to purify and extract molecules of interest from complex mixtures . These molecules tend to be enzymes, proteins or amino acids, but other biological species can be selectively retained. Once isolated, these biological species can be selectively amplified to produce larger quantities, although at large concentrations.

26. Therapeutic & clinical application: Hyper-lipidemia : here the sample is made to pass through coloumn containing antibody & plasma LDL so, it can easily be separated out by eluting with glycine hydrochloride buffer (pH 3). Others : Pregnancy test Allergy test Immuno assay Kinetic studies Qualitative measurment of substrate.

27. ADVANTAGES OF AFFINITY CHROMATOGRAPHY Extremely high specific ity High degrees of purity can be obtained The process is reproducible The binding sites of biological molecules can be simply investigated

28. DISADVANTAGES OF AFFINITY CHROMATOGRAPHY 1) Expensive ligands 2) Leakage of ligand 3) Degradation of the solid support 4) Limited lifetime 5) Non-specific adsorption 6) Relatively low productivity

29. REFERENCES [1]http://en.wikipedia.org/wiki/Affinity_chromatography [2] www.apsu.edu/ reedr /.../ Affinity %20 Chromatography %201.ppt [3] www.rpi.edu/dept/chem- eng /WWW/faculty/.../Lecture%2001.pdf - [ 4] www.chemistryinnovation.co.uk/.../Technology%20Area%20 Affinity %20 Chromatography .pdf -

30. THANKS…