AFFINITY CHROMATOGRAPHY

PRESENTED BY:-

RAJPAL CHOUDHARYREG. NO.- 161103004

M.SC. 1ST YEAR

INVENTED BY

• 1903 – Tswett, a Russian botanist coined the term chromatography.

CHROMATOGRAPHY

Chromatography 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.

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.

• The analyte is the substance that is to be separated during chromatography.

• The sample is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components.

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

TYPES OF CHROMATOGRAPHY

HISTORY OF AFFINITY CHROMATOGRAPHY

• 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.

EXAMPLES

• Antigen Antibody• Antibody Antigen• Substrate Enzyme• DNA Histon• Hormone Binding Protein/Receptor

SPECIFICITY OF AFFINITY CHROMATOGRAPHY

• Specificity is based on three aspect of affinity:-

Matrix: for ligand attachmentSpacer arm: used to bind ligand to

matrixLigand: molecule that binds

reversibly to a specific target molecule(site of interaction)

PROCEDURE• The Sample is injected into the equilibrated

affinity chromatography column.• Only the substance with affinity for the ligand

are retained on the column.• The substance with no affinity to the ligand will

elute off.• The substances retained in the column can be

eluted off by changing the pH of salt or organic solvent concentration of the eluent.

MATRIX• The matrix simply provides a structure to

increase the surface area to which the molecule can bin.• The matrix must be activated for the ligand to

bind to it but still able to retain it’s own activation towards the target molecule.

MATRIX• Amino, hydroxyl, carbonyl and thio groups

located with the matrix serve as ligand binding sites.• Matrix are made up of agarose and other

polysaccharides.

LIGAND• The Ligand binds only to the desired molecule within the

solution.• The ligand attaches to the matrix which is made up of an

inert substance.• The ligand should only interact with the desired molecule

and form a temporary bond.• The ligand/molecule complex will remain in the column,

eluting everything else off.• The ligand/molecule complex dissociates by changing the

pH.

ANTIBODY AFFINITY(IMMUNOAFFINITY CHROMATOGRAPHY)• Used to purify antibody against a specific antigen.

Ex: Immunoglobulins • Purification of IgG, IgG fragments and subclasses have the high affinity of protein A and protein G for the Fc region of polyclonal and monoclonal IgG-type antibodies.

PROTEIN A AND PROTEIN G• Protein A and protein G are bacterial cell surface

proteins (from Staphylococcus aureus and Streptococcus respectively).• Recombinant protein A is available;• Engineered to include a C-terminal.• Results in an enhanced binding capacity.

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.

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

ADVANTAGES OF AFFINITY CHROMATOGRAPHY

• Extremely high specificity.• High degrees of purity can be obtained.• The process is very reproducible.• The binding sites of biological molecules

can be simply investigated.

DISADVANTAGES OF AFFINITY CHROMATOGRAPHY• Expensive ligands• Leakage of ligand• Degradation of the solid support• Limited lifetime• Non-specific adsorption• Relatively low productivity

REFERENCES[1]http://en.wikipedia.org/wiki/Affinity_chromatography[2]www.apsu.edu/reedr/.../Affinity%20Chromatography

%201.ppt

[3] www.rpi.edu/dept/chem-eng/WWW/faculty/.../Lecture%2001.pdf

[4]www.chemistryinnovation.co.uk/.../Technology%20Area%20Affinity%20Chromatography.pdf -

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