2. INVENTED BY
• 1903 – Tswett, a Russian
botanist coined the term
chromatography.
3. 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.
4. TERMINOLOGY
• An immobilized phase is a stationary
phase which is immobilized on the support
particles, or on the inner wall of the column tubing.
5. • 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.
6. • 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.
7. • Elution
- washing of the mixture
• Eluent
- additional solvents used for elution
• Residency
- time spent on column
9. 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.
11. 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)
12. 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.
13. 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.
14. 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.
15. 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.
16. 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.
17. 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.
18. 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.
19. 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
20. 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.
21. DISADVANTAGES OF AFFINITY
CHROMATOGRAPHY
• Expensive ligands
• Leakage of ligand
• Degradation of the solid support
• Limited lifetime
• Non-specific adsorption
• Relatively low productivity
