Affinity chromatography is a method used to separate biomolecules from a mixture based on specific interactions between the molecules and a ligand attached to a chromatography matrix. It was developed in the 1930s and relies on reversible interactions like enzyme-substrate or antibody-antigen binding. The target molecule binds to the ligand while unbound molecules are washed away. Then, conditions are altered to dissociate the bound molecule from the ligand, eluting it from the column in a purified form. Affinity chromatography is widely used to purify proteins, nucleic acids, and other biomolecules.

1. AFFINITY
CHROMATOGRAPHY
Mr. A. A. Alman
HOD,
Pharmaceutical Chemistry
SGMCP, Mahagaon
Instrumental Methods of
Analysis

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

3. 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.

4. INTRODUCTION
• Affinity Chromatography is essentially a sample
purification technique, used primarily for biological
molecules such as proteins.
• It is a method of separating a mixture of proteins or nucleic
acids (molecules) by specific interactions of those molecules
with a component known as a ligand, which is immobilized
on a support. If a solution of, say, a mixture of proteins is
passed over (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).
• The other proteins in the solution wash through the column
because they were not able to bind to the ligand.

6. PRINCIPLE
• 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 based on highly specific biological interactions between
two molecules such as interactions between enzyme and
substrate,receptor and ligand,or antibody and antigen.
• These interactions which are typically revesible are used for
purification by placing one of the interacting molecules
referred to as affinity ligand onto a solid matrix to create a
stationary phase while a target molecule is in the mobile
phase.
• Many of the commonly used ligands coupled to
affinity matrices are now commercially available and
are ready to use.

8. EXAMPLES
• Antigen
• Antibod
y
• Substrat
e
• DNA
• Hormon
e
Antibod
y
Antigen
Enzyme
Histon
Binding
Protein/Receptor

9. 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)

10. CHROMATOGRAPHIC MEDIA
• A matrix in its use here is a substance,usually in bead form to
which a specific ligand is covalently bound.
• 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.
• In order to for the matrix to be effective it must have certain
characters:
• It must be insoluble in solvents and buffers employed in
the process
• It must be chemically and mechanically stable..
• It must be easily coupled to a ligand or spacer arm onto
which the ligand can be attached.
• It must exhibit good flow properties and have a
relatively large surface area for attachment

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

12. IMMOBILIZED LIGAND
• The ligand can be selected only after the nature of
the macromolecule to be isolated is known.
• When a hormone receptor protein is to be purified
by affinity chromatography, the hormone itself is
an ideal candidate for the ligand.
• For antibody isolation ,an antigen or hapten 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.

13. • 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.

15. ATTACHMENT OF LIGAND TO MATRIX
• Several procedures have been developed for the
covalent attachment of the ligand to the stationary
phase.all procedures for gel modification proceed
in two separate chemical steps:
• 1)Activation of the functional groups on the matrix
and
• 2)Joining of the ligand to the functional group on
the matrix.
• A wide variety of activated gels is now commercially
available.

17. Most widely used are described in the following:
CYANOGEN BROMIDE-ACTIVATED AGAROSE
• This gel is especially versatile because all ligands
containing primary amino groups are easily attached to
the agarose.since the gel is extremely reactive,very
gentle conditions may be used to couple the ligand.
6-AMINOHEXANOIC ACID(CH)-AGAROSE AND 1,6-
DIAMINOHEXANE(AH)-AGAROSE
• These activated gels overcome the steric interference
problems by positioning a six carbon spacer arm
between the ligand and the matrix.
• Ligands with free primary amino groups can be
covalently attatched to CH-agarose,whereas ligands with
free carboxyl groups can be coupled to AH-agarose.

18. CARBONYLDIMIDAZOLE(CDI)-ACTIVATED SUPPORTS
• Reaction with CDI produces gels that contain
uncharged N-alkylcarbamate groups.
EPOXY-ACTIVATED AGAROSE
• This gel provides for the attachment of ligands
containing hydroxyl,thiol,or amino groups.
GROUP SPECIFIC ADSORBENTS
• Group specific adsorbents contains ligands that have
affinity for a class of biochemically related
substances.
• For example cibracron blue-agarose is an adsorbent
which would react withenzymes that have nucleotide
cofactors(DNA Polymerase, kinase and serum
albumin.)

19. EXPERIMENTAL PROCEDURE
• IS MATRIX LIGAND AVAILABLE
• SELECT GEL AND LIGAND SWELL GEL IN BUFFER
• COUPLE LIGAND
• PREPARE GEL FOR COLUMN
• PACK GEL IN GLASS COLUMN
• AND SET-UP COLUMN EQUIPMENT
• EQUILIBERATE COLUMN WITH BUFFER
• APPLY SAMPLE
• WASH COLUMN TO REMOVE
• UNBOUND MOLECULES
ELUTE BOUND MOLECULES
COLLECT AND ANALYZE ELUENT
• REGENERATE AND STORE GEL

20.  Hi-Trap Heparin HP (High performance)
 Column size: 5 × 1 mm 1 × 5 mm 5 × 5 mm
 Average particle diameter : 34μm
 Maximum operating flow rate: 4 ml/min 20
ml/min.

21.  At 2-8 °C in an upright position with both caps
in place.
 Thio-mersal may be added for long term
storage.
 DO NOT FREEZE
 Application areas : purification, isolation or
removal of the following substances: Anti-
thrombin III and other coagulation factors,
lipoproteins, lipases, protein synthesis factors

22. SELECTION OF A GEL OR LIGAND
• Many type of matrix-ligand systems are
commercially available and cost are reasonable so
time can be saved by purchasing preactivated gel for
direct attachment of ligand.

23. BUFFER
• Buffer is used for formation of complex between a matrix
and ligand.as slight change in ionic concentration weakens
the interactions between them.
AFFINITY ELLUTION
• In this method a selective substance added to the buffer
causes selective elution of bound macromolecule-ligand
complex.resulting in elution of desired macromolecule.
CHAOTROPIC AGENTS
• If gentle and selective elution methods do not release
the bound macromolecule then mild denaturing agents
can be added to the buffer.the most powerful agents are
urea,guanidine

24.  :Step-1 Attach ligand to column matrix
 Binding of the selected ligand to the
matrix requires that a covalent bond be
formed between the two.
 This is facilitated by derivatization of
the sugar residues' hydroxyl groups.
 It is important to realize that the substrate
might not be able to reach the ligand
active site if it is hidden deep within the
ligand.
 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.

25.  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

26.  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.

27.  Finally to collect isolate,
which is still bound to the
ligand-matrix in the gel, a
stronger second wash is run
through the column.

28.  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.

29.  The protein is then
free to run through
the gel and be
collected.

30. 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.

31. 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.

32. ADVANTAGES
• 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.

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

34. APPLICATIONS
• It is used for isolation and purification of all biological
macromolecule.
• It is used to purify nucleic acid, antibodies, enzymes.etc
• To notice which biological compounds bind to a particular
substance.
• to reduce a amount of substance in a mixture
• Used in Genetic Engineering - nucleic acid purification
• Production of Vaccines - antibody purification from blood
serum
• Basic Metabolic Research - protein or enzyme purification
from cell free extracts

35. 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.

37. TYPES OF CHROMATOGRAPHY

38. REFERENCES
[1]http://en.wikipedia.org/wiki/Affinity_chromatography
[2]www.apsu.edu/reedr/.../Affinity%20Chromatography%2
01.ppt [3] www.rpi.edu/dept/chem-
eng/WWW/faculty/.../Lecture%2001.pdf
[4]www.chemistryinnovation.co.uk/.../Technology%20Area
%20Affinity
%20Chromatography.pdf -