Affinity chromatography: Definition
Affinity
chromatography is a technique used to separate and the purification of
the biochemical mixture. Affinity column Chromatography is a highly specific
technique.
Affinity
chromatography technique depends on the specific affinity between stationary
phase and the analyte (Ligand.)
Affinity
chromatography is a reversible biochemical reaction.
Affinity chromatography Principle:
Affinity chromatography involves the covalent
attachment of an immobilized biochemical (called an affinity ligand) to a solid
support.
When the sample is passed through the column,
only the solute that selectively binds to the complementary ligand is retained.
The other sample components elute without retention.
The interaction between the ligand attached to
the solid support of the stationary phase and the target molecule in the analyte is
possible because of electrostatic, hydrophobic reaction, or hydrogen bonding.
Nature of interaction
between the target molecule and the ligand to help determine the selection of
proper stationary phase and the mobile phase.
The separation is well expressed by lock and key
binding.
The retained solute(s) can be eluted from the
column by changing the mobile phase conditions.
Diagram of Affinity chromatography:
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| Affinity Chromatography |
Stationary Phase and mobile Phase in Affinity chromatography:
Stationary
phase in Affinity Chromatography:
The stationary phase is made of a solid matrix and
ligand one more important component of the stationary phase in Affinity
Chromatography.
Component
of stationary Phase in Affinity Chromatography.
Ligand:
Ligand is an immobilized chemically insoluble matrix supported by the
stationary matrix. It reversibly adsorbs molecule species (affine components or
target molecules) from a mixture of Analyte.
Ligand is attached to the solid support. It exploits the unique property of extremely specific reversible biological interaction to
achieve separation and purification.
The separation exploits the “Lock n Key” binding that prevalent in a biological system. Ligands
are coupled to affinity matrix are now commercially available and ready to use.
Different types of ligand
that bind to the substance which needs to purify:
Antibody: This can be monoclonal or
polyclonal. Highly specified and large binding constant
DNA: Can be used for
polymerase, DNA –binding proteins helicases, and restriction enzyme.
Biometric dyes: used for
protein
Peptides: these are used
for biomolecule
Spacer Arm:
A spacer arm is the chain of carbon and/or other
atoms that position a functional group away from the solid matrix to which it
is covalently bound and makes it more available to a ligand and less
restricted by steric hindrance by the matrix.
Matrix:
Solid Matrix
or stationary phase is to provide the support to the ligand Solid matrix maybe
porous or non-porous
It should be of adequate size and shape i.e increase in particle size
reduces the flow resistance and separation power & decrease in size leads
to increase flow resistance and clogged.
·
Irregular
shape leads to unequal path lengths for substance and band broadening. So
spherical shape is best suited.
·
Mechanically
and chemically stable and resistant against microorganisms.
·
It should
be inert to prevent analyte from interacting non-specifically with matrix
itself.
·
Economical
·
Eg.
Agarose
Cellulose dextran
Sepharose is a bead formed of
agarose gel.
Mobile Phase in Affinity chromatography:
The nonpolar or polar mobile phase used as per the requirement of the separation
of the compound.
Instrumentation:
Once the column is prepared the separation is conducted in four basic step Introduction
of the sample, Adsorption of the analyte, removal o impurity and Elution of sample
Following
are the basic part of the Affinity chromatography:
The injection – pump system:
In
Affinity Chromatography Sample is
introduced onto the high performance (rigid ) bio affinity matrix by an
automated delivery system.
The affinity Column:
The column is used for separation or the purification of the component.
Ligand, spacer, solid matrix is part of the columns.
On line monitoring:
The effluent is monitored by online & assayed robotically for
specific biological properties.
Post collection monitoring.
Last
step is elution and collection of the analyte.
Example of affinity
chromatography
The separation or Purification of Antibody
by using of the Affinity chromatography this technique is known by the Immunoaffinity
Chromatography.
To purify and separation of the
immune biochemical mixture Purify
antigens, Modification using Epitopes.
Antigen
is immobilized in the stationary phase as ligand and stationary phase with antigen
ligand used to isolate and purify the appropriate antibody. Purification of the
antibody from the blood serum is one of an example of the antibody separation purification.
Advantages of Affinity Chromatography
·
The major advantage of this technique
is its tremendous specificity, which permits rapid isolation with a good yield
in a single step.
·
This interaction is reversible, Rapid process, Faster separation time.
·
Better efficiency resolution.
·
Higher pressure stability.
·
Better defined column condition.
·
Simpler detection & analysis.
·
Amenable to smaller samples.
·
No. of purification steps are
reduced.
·
No loss of protein due to
denaturation.
·
Suitable for large scale purification
processes.
Disadvantages of
Affinity Chromatography
·
High expense
·
Time-consuming method
·
Greater irreversible adsorption
·
Greater impact on slow kinetics
