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2. What is Affinity Chromatography?
 Chromatographic technique for selective separation/purification of
a molecule from a biochemical mixture.
 PRINCIPLE
 Separation of mixture of protein/nucleic acids/enzymes take place by specific
and reversible interaction of these molecules with a component – Ligand,
which is immobilized on an inert support, and packed in a column
 When a mixture of, say, proteins passes through the column, one of the proteins
binds to the ligand based on its specificity and high affinity
• The protein and the ligand fit together like a lock and key
 Other proteins in the mixture pass through the column since these are unable
to bind to the ligand

3. Components of Affinity Matrix
Matrix
Spacer
Target protein
Affinity Matrix
 Highly selective
Ligand

4. Matrix or Affinity Supports
 Inert support to which ligand is directly or indirectly bound
 Chemically and mechanically stable
 Uniform particle and pore size
 Low non specific adsorption
 Examples: Cellulose, agarose, silica, polystyene, sepharose
(All these are available commercially)

5. Ligand
 Selection based on specific and reversible binding with the target molecule
Carries a group which can couple to matrix without losing binding activity
 Stable in different binding and elution conditions
 Ligand can be bound to matrix by
Covalent bonding
Non-covalent bonding
Adsorption
Biospecific interaction

6. Commonly used Ligands
Ligand Specificity
Blue B Kinases, dehydrogenases, nucleic acid binding
proteins
Orange A Lactate dehydrogenase
Lysine Plasminogen, rRNA, dsDNA
Protein A Fc regions of many IgG subtypes; species
dependent weak interactions with IgA, IgM, IgD
Biotin Streptavidin, avidin
Gelatin Fibronectin
Lectins Glycoproteins, polysaccharides, glycolipids
 Direct binding of ligand to matrix may lead to stearic hindrance resulting
in inefficient binding of target to ligand.
Solution: Spacer

7. Spacer
Overlap between target
molecule and bead leads to
inefficient binding ,i.e., stearic
hindrance
Spacer covalently
bound to matrix
• Allows complete access
of target molecule to
ligand.
• Should be hydrophillic
• Should be of optimum
length
Matrix

8. Steps Involved in Affinity Chromatography
 Matrix beads + Buffer: Beads swell
 Couple the ligand (and spacer, if required) with the matrix
 Filter: Affinity matrix
 Pack in a glass column
 Equilibrate the column with buffer
 Load the sample
 Wash the column to remove unbound molecules
 Elute bound molecules
 Analyze the eluent

9. 1.Binding 2. Washing 3. Elution
Ligand Other proteins
Target protein
Steps Involved in Affinity Chromatography

10. Elution
 Specific Elution
- Competing free ligand
- Competing binding substance
Competitive
ligand in solution
+
Example: Elution of enzymes from Blue Sepharose by free NADH
Competitive binding
substance
+
Example: Elution of Antigens from antibody columns with specific peptides
 Competitive soluble ligands or binding substances can elute the bound target specifically
 Gentler than general methods
Expensive (like specific peptides for antigen elution)

11. Applications
 Purification of substances from biological mixtures.
 Separation of native form of protein from
denatured form.
 Purification and concentration of enzymes in
solution.

12. Gel Chromatography

13.  Liquid chromatography
 PRINCIPLE: Separates molecules in solution by their “effective size” in
solution. Hence, also called size exclusion chromatography.
 Separation is achieved by the differential exclusion or inclusion of solutes as
they pass through porous stationary phase consisting of cross linked
polymeric gel beads.
Gel Chromatography
Also known as gel filtration/gel permeation/size exclusion chromatogaphy
(GFC) (GPC) (SEC)

15. Gel Chromatography
Stationary Phase
Semi permeable, porous polymer gel beads.
Properties of gel beads
• Chemically inert
• Mechanically stable
• Uniform particle and pore size
Examples
• Dextran gel
• Agarose gel
• Polyacrylamide gel

16. Advantages
 Short analysis time
 No sample loss
 Less consumption of mobile phase
Disadvantage
 Molecules with closely related molecular mass show broad peak

17. Ultra High Performance Liquid
Chromatography

18. UHPLC v/s HPLC
Characteristics UHPLC HPLC
Particle size <2 µm 5-7 µm
Pressure 20000 psi 500-6000 psi