Affinity chromatography is a method that separates molecules based on a highly specific non-covalent biological interaction between the target molecule and an immobilized ligand. The process involves passing a sample mixture over a column containing the ligand, where the desired molecule binds selectively while unbound molecules pass through. The bound molecule can then be eluted and collected by changing conditions like pH or introducing a competitive ligand. Affinity chromatography has various applications like antibody purification, enzyme purification, and nucleic acid separation. It provides high selectivity and purity but ligands can be expensive and columns have a limited lifetime.

1. Affinity
Chromatography
Maryam Yekefallah
Fall 2017

2. Content
 Introduction of chromatography
 Different aspects of affinity chromatography
 The principle of Affinity Chromatography
 Procedure
 Application
 Advantages & Disadvantages

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

5. 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

6. Introduction
 Affinity chromatography is one of the most diverse and powerful
chromatographic methods for purification of a specific molecule or a
group of molecules from complex mixtures.
 It is a method of separating a mixture of proteins or nucleic acids by
specific interactions of those molecules with a component known as a
ligand, which is immobilized on a support. Mixture of proteins is
passed 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).

7. Introduction
 The other proteins in the solution wash through the
column because they were not able to bind to the
ligand.

8. Specificity of Affinity
Chromatography
Specificity is based on three aspects of affinity
Matrix
Spacer arm
Ligand

9. 1- Matrix
 The matrix simply provides a structure to increase the surface
area to which the molecule can bind.
 Amino, hydroxyl and carbonyl groups located with the matrix
serve as ligand binding sites.
 Matrix are made up of agarose and other polysaccharides

10. Matrix
For having an effective matrix, it must have certain characters:
 1)It must be insoluble in solvents and buffers employed in
the process
 2)It must be chemically and mechanically stable.
 3)It must be easily coupled to a ligand or spacer arm onto
which the ligand can be attached.
 4)It must exhibit good flow properties and have a relatively
large surface area for attachment

11. Types of Matrix used
 Cellulose : used for DNA affinity chromatography.
 Agarose : DNA or proteins
 Tris- acryl
• It is having higher separation ability.
• It has small particle size 40-80 micrometer.

12. 2- Spacer arm
 The stationary phase is typically a gel matrix, to prevent steric
interference or overlap during the binding process of the target
molecule to the ligand, an inhibitor containing a hydrocarbon chain is
first attached to the agarose bead (solid support). This inhibitor with a
hydrocarbon chain is commonly known as the spacer between the
agarose bead and the target molecule.

14. 3- Ligand
 The Ligand binds only to the desired molecule within the solution
 It attaches to the matrix which is made up of an inert substance
 It 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

15. Ligand
 The ligand can be selected only after the nature of the macromolecule
to be isolated is known.
 Examples:
 For antibody isolation ,an antigen may be used as ligand.
 If an enzyme is to be purified, a substrate analog, inhibitor, cofactor,
or effector may be used as a the immobilized ligand.

17. The principle of Affinity
Chromatography
 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

19. We have 3 main part:

21. Procedure
– Step-1 Attach ligand to column matrix
 Binding of the selected ligand to the matrix requires that a
covalent bond be formed between them.
 Most ligands are attached first to spacer arms which are
then bonded to the matrix. The ligand-matrix gel is then
loaded into an elution column.

22. Step 2: Load protein mixture onto
column
 Once the column has been prepared, the mixture containing
isolate is poured into the elution column.
 Gravity pulls the solution through the gel, because most of the
proteins do not bind to the ligand-matrix complex.
 When ligand is recognized substrate passes through the gel, it
binds to the ligand-matrix complex, halting its passage through
the gel.
 Some of the impurities flow through the gel due to gravity, but
most remain, unbound, in the gel column

23. Step 3: Proteins bind to ligand
 In order to remove these 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 the
impurities are removed.
 Once the impurities are washed-out, the only remaining
part of the protein mixture should be the desired isolates.

24. Step 4: Wash column to remove unwanted
material
In ally to collect isolate, which is still
bound to the ligand-matrix in the gel,
a stronger second wash is run through
the column.

25. Step 5: Wash off proteins that bind
loosely
– This second wash relies on the
reversible binding properties of the
ligand, which allows the bound
protein to dissociate from its ligand
in the presence of this stronger
wash.

26. Step 6: Elute Proteins that bind tightly to ligand
and collect purified protein of interest
The protein is then free to run
through the gel and be collected.

28. Application
 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

29. Nucleic acid separation using immobilized
metal affinity chromatography (IMAC)
 The method can be used to purify compounds containing
purine or pyrimidine moieties where the purine and
pyrimidine moieties are shielded from interaction with the
column matrix from compounds containing a non-shielded
purine or pyrimidine moiety or group.

30. Industrial Application
– 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.

31. Antibody affinity (Immune
affinity Chromatography)
Used to purify antibody against a specific
antigen Ex : Immunoglobulins

32. Others
Pregnancy test
Allergy test
Immune assay
 Kinetic studies
 Qualitative measurement of substrate.

33. 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

34. Disadvantages of affinity
chromatography
 Expensive ligands
 Leakage of ligand
 Degradation of the solid support
 Limited lifetime
 Non-specific adsorption

35. Reference
1- Cuatrecasas, Pedro (June 25, 1970). "Protein Purification by Affinity
Chromatography"(PDF). JBC. Retrieved November 22, 2017.
2- Uhlén M (2008). "Affinity as a tool in life science". Biotechniques. 44 (5): 649–
54. doi:10.2144/000112803. PMID 18474040.
3- Hage, David (May 1999). "Affinity Chromatography: A Review of Clinical
Applications" (PDF). Clinical Chemistry. 45 (5): 593–615.