Detailed introduction of (Chromatography and Affinity Chromatography) and its theory, principle ,working ,application and limitation of Affinity Chromatography . This chromatography technique is also useful for GPAT ,UGC NET , GATE, DBT aspirants.

1. AFFINITY CHROMATOGRAPHY
Guided by- Dr.Vijaya Kumar Sir
Presented by- Shiv Kumar
M.Pharma 1st year (1st sem)
Pharmaceutical Analysis
Department Of Pharmaceutical Sciences.
Babasaheb Bhimrao Ambedkar University Lucknow (A Central University),U.P.

2. Content
• Introduction to Chromatography and Affinity
Chromatography
• Principle
• Typical biological interactions used in affinity
chromatography
• Components of affinity chromatography
• Components of affinity chromatography
• Steps of Affinity Chromatography
• Elution methods of Affinity Chromatography
• Types of Affinity Chromatography
• Application of Affinity Chromatography
• Disadvantages of Affinity Chromatography

3. Chromatography :-
• Chromatography – Chromo – “Color” and Graphein – “to write” - derived
from Greek words.
• Chromatography is an important biophysical technique that enables the
separation, identification, and purification of the components of a mixture
for qualitative and quantitative analysis.
• The Russian botanist Mikhail Tswett coined the term chromatography in
1906.
• The first analytical use of chromatography was described by James and
Martin in 1952, for the use of gas chromatography for the analysis of fatty
acid mixtures.
• A wide range of chromatographic procedures makes use of differences in
size, binding affinities, charge, and other properties to separate materials.
Chromatography may be preparative or analytical.
Preparative chromatography is to separate the components of a mixture
for later use, and is thus a form of purification. It should also be noted that
this process is associated with higher costs due to its mode of production.
Analytical chromatography is done normally with smaller amounts of
material and is for establishing the presence or measuring the relative
proportions of analytes in a mixture.

4. Affinity chromatography was conceived and first developed by Pedro
Cuatrecasas and Meir Wilchek.
Affinity chromatography is a separation method based on a specific binding
interaction between an immobilized ligand and its binding partner. Examples
include antibody/antigen, enzyme/substrate, and enzyme/inhibitor
interactions. The degree of purification can be quite high depending on the
specificity of the interaction.
Affinity chromatography techniques are comprised of a stationary phase
(solid phase) and the mobile phase. Mobile phase refers to your cells lysate,
or any other mixture that is made up of biomolecules. A ligand which binds to
the targeted molecule is covalently attached with the solid. The interactions
between the solid and mobile phase is exploited through affinity
chromatography to produce the desired substance in pure form
• Why Use Affinity Chromatography?
Affinity chromatography offers high selectivity, resolution, and capacity in
most protein purification schemes. It has the advantage of utilizing a
protein's biological structure or function for purification. As a result,
purifications that would otherwise be time consuming and complicated, can
often be easily achieved with affinity chromatography.

5. Principle of Affinity Chromatography :-
• Affinity chromatography takes advantage of specific binding
interactions between the analyte of interest (normally dissolved in
the mobile phase), and a binding partner or ligand (immobilized on
the stationary phase). In a typical affinity chromatography experiment,
the ligand is attached to a solid, insoluble matrix—usually
a polymer such as agarose or polyacrylamide—chemically modified to
introduce reactive functional groups with which the ligand can react,
forming stable covalent bonds.
• The basis of affinity chromatography is the fact that its stationary
component is made up of a medium (e.g. cellulose beads) where the
substrate (or often coenzyme) has been covalently bound and in such
a way that the reactive elements that are necessary for binding to
enzymes are exposed. When the mix of proteins is filtered across the
chromatography columns the proteins that possess an affinity site that
is immobilized will attach onto the stationary part and all otter
proteins will be eliminated in the empty space that is the column.

6. • Typical biological interactions used in affinity chromatography
Sr. no Types of ligand Target molecule
1 Substrate analogue Enzymes
2 Antibody Antigen
3 Lectin Polysaccharide
4 Nucleic acid Complementary base sequence
5 Hormone Receptor
6 Avidin Biotin/Biotin-conjugated molecule
7 Calmodulin Calmodulin binding partner
8 Glutathione GST fusion protein
9 Proteins A and G Immunoglobulins
10 Metal ions Poly-histidine fusion protein

7. • Components of affinity chromatography
1. Matrix
• The matrix acts as an inert structure that a ligand may be connected directly
or indirectly. The most efficient matrix materials are polyacrylamide and
agarose.
2. Spacer arm
• It prevents the ligand from attaching to the matrix, which could interfere with
its ability to bond to macromolecules. The optimal length is 6-10 carbon
atoms or equivalent. Most commonly, it is utilized for smaller immobilized
ligands. Examples of Spacer arms are 1,6-diamino Hexane and 6-amino
Hexanoic Acid.
• 3. Ligand
• The ligand is a molecule which binds reversibly with an individual chemical or
group of molecules, making it possible to purify the sample using affinity
chromatography. The choice of a specific ligand to be used for affinity
chromatography can be influenced by two aspects:
• the ligand has to show a an irreversible and specific binding affinity for the
targeted substance(s)
• It should possess chemically modifiable groups that permit it to be bonded to
the matrix without damaging the binding function.

8. • Steps of Affinity Chromatography
• Step: 1 Preparation of Column
• The column is loaded with solid support such as sepharose, agarose,
cellulose etc.
• Ligand is selected according to the desired isolate.
• Spacer arm is attached between the ligand and solid support.
• Step: 2 Attachment of ligand to column matrix
The binding of the ligand with the matrix requires that a covalent bond is
created between both. This is accomplished through derivatization of the
sugar-based”hydroxyl groups. It is essential to understand that the substrate
may not be able to access the active site of the ligand, in the event that it is
hidden inside the ligand. The majority of ligands are connected to spacer
arms that are then attached onto the matrix.
• Step: 3 Load Protein Mixture onto the Column.
• Solution containing a mixture of substances is poured into the elution
column and allowed to run at a controlled rate.

9. • Step 4: Proteins Binds to the ligand
• To remove these impurities which are not bound the wash must be of
high pH or salt concentration or temperature is passed across the gel.
It is crucial to make the most powerful wash possible to ensure all
impurities are eliminated. After the impurities have been removed
then the only thing left of the protein mixture must be the specific
isolates.
• Step 5: Wash the column to remove the unwanted Materials
• To finally collect the an isolate that is attached to the ligand matrix in the gel
second wash is run over the column.
• Step 6: Wash off the Proteins that are loosely bind
• The second wash is based on the reversible properties of binding of the
ligand. This lets the bound protein separate from its ligand in the presence of
the more powerful wash.
• Step 6: Elute proteins that are tightly bound to ligand and
collect purified protein and interest
• The protein then has the freedom to pass through the gel before being
removed.

10. Process Presentation of Affinity
chromatography

11. Mechanism of Affinity Binding

12. Elution of Proteins

13. • Elution methods of Affinity Chromatography
• 1. pH elution
• A variation in pH alters the amount of ionization by charged groups on the
ligand as well as proteins bound. The alteration could affect site of binding
directly decreasing its affinity or trigger an indirect change in affinity due to
modifications in the conformation.
• 2. Ionic strength elution
• The exact mechanism behind elimination through changes in the strength of
ionic depends on the specific relationship between the ligand and the target
protein. This is a moderate elimination using a buffer that has higher Ionic
strength (usually NaCl), applied in a linear manner as well as in steps.
Enzymes typically elute with a concentration of 1 million NaCl and less.
• 3. Competitive elution
• They are typically used to distinguish substances in an individual medium or
when the attraction of the target protein interactions is quite high. The
eluting agent is competing with the protein of interest or for binding to the
binding ligand. Substances can be eluted by the concentration gradient of

14. • 4. Reduced polarity of eluent
Conditions are employed to reduce the polarity of the eluent
and allow for elution but not inactivating the substances that
are eluted. Dioxane (up to 10 10%) as well as Ethylene glycol (up
to 50 percent) are common to this kind of liquid eluent.
• 5. Chaotropic eluents
If other methods of elution fail the deforming buffers which
alter the protein’s structure can be employed, e.g. Chaotropic
agents like the guanidine hydrochloride and urea. Avoid
chaotropes whenever possible as they can cause to cause
denature of the protein eluted.

15. • Types of Affinity Chromatography
1. Lectin Affinity Chromatography
• Purification of glycoproteins, specifically the membrane-
receptor proteins.
• Lectins are a class of proteins made by animals and plants that
are able to bind glycoproteins and carbohydrates.
• Useful to separate cells into different types, making use of
saccharide component of their outer membranes.
• The most commonly used lectins include: ConcanavalinA
Soyabean lectin, etc.
2. Immuno Affinity chromatography
• It is used in the isolation and elimination of a variety of
proteins such as antigens, membrane proteins that are of viral
origin.
• It is used to purify antibodies.
• Ligands used include the Protein A as well as protein G.

16. • 3. Metal Chelate Chromatography
• Special type of chromatography which immobilised metal ions
such as Cu2+ Zn2+ , Mn2+, Ni2+ etc. are employed.
• It is used to purify proteins that contain imidazole groups or
indole groups.
• Commonly metal ions are immobilised by attachment to an
imino-diacetate or tris(carboxymethyl)ethylenediamine
substituted agarose.
• 4. Dye Ligand Chromatography
• Utilizes a variety of triazine dyes for ligands.
• The most popular color is Cibracron Blue F3G-A.
• It is used to purify interferons and lipoproteins as well as factors
that cause coagulation, etc.
• 5. Covalent Chromatography
• Specially designed to separate proteins containing thiol.
• The most frequently used ligand an adiquate 2′-pyridyl group
• It is used to purify many proteins, however its application is
restricted due to its price and difficult regeneration.

17. • Types of affinity media used in Affinity Chromatography
• A variety of affinity media are available to serve a range of
applications. Briefly, they are (generalized)
activated/functionalized that work as a functional spacer, support
matrix, and eliminates handling of toxic reagents.
• Amino acid media: It is used in conjunction with a variety of
proteins from serum enzymes, and peptides in addition to dsDNA
and rRNA.
• Avidin biotin media: Avidin biotin media is used to purify the
process of biotin/avidin, as well as their derivatives.
• Carbohydrate bonding is most often used with glycoproteins or
any other carbohydrate-containing substance; carbohydrate is
used with lectins, glycoproteins, or any other carbohydrate
metabolite protein
• The dye ligand medium is nonspecific however it mimics
biological substrates as well as proteins.
• Hydrophobic interaction medium are frequently employed to
attack free carboxyl groups and proteins.

18. • Immuno affinity media uses antigens’ as well as antibodies that have high
specificity to differentiate immobilized metal ions. It uses interactions between
proteins and metal ions (usually specifically labeled) to separate.
Nucleotide/coenzyme which helps separate dehydrogenases, kinases and
transaminases (Used to separation of peptides or proteins according to their
affinity for metal ions).
• Speciality media are made for specific classes or types of co enzyme. This kind of
media can only function to isolate a particular type of protein, or coenzyme.
• Weak affinity chromatography : -
• WAC is a weak affinity technique. (WAC) is an affinity chromatography technique
used for testing affinity for drug development.
• WAC is an affinity-based technique for liquid chromatography which separates
chemical compounds according to their weak affinities with the immobilized
object.
• The greater affinity compound has with the target the longer it will remain within
the separator and this is measured as a longer retention time.
• The measurement of affinity and rank of affinity are accomplished by processing
the retention times of the compounds being studied.
• Affinity chromatography is a part of a wider set of chemoproteomics techniques for
the purpose of identifying drug targets.
• The WAC technology has been demonstrated against various protein targets ,
including proteases chaperones, kinases, along with protein-protein interaction
(PPI) specific targets. WAC has been proven to be more efficient than traditional
methods for screening based on fragments.

19. • Application of Affinity Chromatography
• It is used to isolate and purification of all biological
macromolecules.
• It is used to purify nucleic acid,antibodies,enzymes.etc
• To determine which compounds in the biological world are
bound to a specific substance.
• In in vitro antigen-antibody reactions
• Utilized for Genetic Engineering for nucleic acid purification.
• Utilized for the Production of Vaccines – antibody purification
from blood serum.
• It is used for Basic Metabolic Research such as the purification
of enzymes or proteins from cells free extracts.
• Investigation of binding sites of enzymes
• Detection of Single Nuceotide polymorphisms and mutations
in nucleic acids

20. • Extremely high-specificity
• The purest of levels can be achieved
• The process is highly reproducible.
• The binding sites of biological molecules could be investigated
by simply looking at the binding sites of biological molecules.
• Single-step purification.
• The matrix is reusable in a short time.
• The matrix is solid that is easy to clean and dried.
• Provide purified products with high yield. Removal of impurities
or in purification process.
• Detection of substrates

21. • Disadvantages of Affinity Chromatography
• Expensive ligands
• Leakage of the ligand
• Degradation of the solid support
• Relatively low productivity.
• Non-specific adsorption can not be totally
eliminated, it can only be minimized.
• The limited life span and the high cost for
immobilized ligands.
• Proteins are denatured when the necessary pH is
not maintained.

22. • Keywords
• Analyte – the substance to be separated during chromatography. It is also normally
what is needed from the mixture.
• Column - is a device used in chromatography for the separation of chemical
compounds. A chromatography column contains the stationary phase, allowing the
mobile phase to pass through it.
• Affinity Media – used for purification of proteins or other macromolecules.
• Immobilized phase – a stationary phase that is immobilized (to prevent something
from moving) on the support particles, or on the inner wall of the column tubing.
• Matrix or support - is material to which a biospecific ligand is covalently attached.
• Ligand - having affinity for a target molecule is covalently attached to an insoluble
support and functions as bait for capturing the target from complex solutions.
• Elution – is process of using a solvent to extract an adsorbed substance from a solid
adsorbing medium.
• The removal of antibody from the antigen to which it is attached.
• Eluent - is the "carrier" portion of the mobile phase. It moves the analytes through
the chromatograph.
• Resin – are media used to capture and polish mAbs, antibody fragments, vaccines,
and other biomolecules using a stationary phase. Following are the examples
• Nickel-NTA : for purification of poly-histidine tagged proteins
• Cobalt-NTA : for purification of poly-histidine tagged proteins
• GST : for purification of GST tagged proteins
• Protein A : for purification of antibodies