AFFINITY CHROMATOGRAPHY

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 .

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 antigen.
* These interactions which are typically substrate,receptor revesible and ligand,or antibody
and 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.

CHROMATOGRAPHIC MEDIA
• A matrix in its use here is a substance,usually in bead form to which a
specific ligand is covalently bound.
• In order to for the matrix to be effective 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

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.

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.the
most widely used are described in the following:

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.

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.
•
•
•
• 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 with enzymes that
have
nucleotide cofactors(DNA Polymerase, kinase and serum albumin.)

EXPERIMENTAL PROCEDURE

• SELECT GEL AND COUPLE LIGAND SWELL GEL IN BUFFER

• 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

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

APPLICATIONS
1)It is used for isolation and purification of all biological
macromolecule.
2)It is used to purify nucleic acid,antibodies,enzymes.etc
3)To notice which biological compounds bind to a particular
substance.
4)to reduce a amount of substance in a mixture

ELEMENTS OF HPLC
 The apparatus consists of a container of the mobile
phase, a pump capable of pressures up to 4000 psi or greater, a valve for injecting the sample
(usually 10 to 500 μL volumes), the column (sometimes thermostatted), a detector, electronics
associated with the detector, and a recorder. A schematic of the HPLC instrument can be seen
in Figure. This instrument in this lab used a C18 column.
Elements present in the above Schematic Diagram of a High-Performance Liquid
Chromatograph.
(1) Solvent reservoirs,
(2) Solvent degasser,
(3) Gradient valve,
(4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump,
(6) Switching valve in "inject position", (6') Switching valve in "load position",
(7) Sample injection loop,
(8) Pre-column or guard column,
(9) Analytical column,
(10) Detector (i.e. IR, UV),
(11) Data acquisition,
(12) Waste or fraction collector.

UV-visible absorbance is the most commonly used mode of detection. Such detectors enable
the component (or effluent) from the column to flow through an 8 to 10 μL spectrophotometric
cell for detection of compounds at a particular wavelength (often in the ultraviolet, < 400nm,
where many organic molecules absorb). Electrochemical and fluorescence detectors often are
used to achieve lower detection limits.
WORKING
In order to separate mixture components, HPLC takes advantages of partitioning between a
mobile and stationary phase under a
uniform pressure that is typically between 500 to 5000 psi. High pressure is required to obtain a
reasonable flow rate through the
column. The process begins when a small amount of liquid sample is injected into the column
that has a stream of liquid flowing
through (which is known as the mobile phase). In partition chromatography, the column is
packed with particles that are coated with
the stationary phase. The polarity of the component and the type of HPLC being performed
determines which phase the component is
more attracted to. If the component is more attracted to the mobile phase, it will flow out of the
column and have a shorter retention
time. If the component is more attracted to the stationary phase, the component will be retained
and will, therefore, have a longer
retention time. Similar to Capillary Electrophoresis (CE) or Gas Chromatography (GC), these
retention times can be used to determine
components. Selecting the mobile phase (or solvent) is one of the most important steps when
performing HPLC and is selected based
on polarity. Solvent polarity relates to the ability of the components to partition into that phase.
The polarity scale for different solvents
can be found in Table below. These solvents can be used exclusively or mixed to achieve the
desired polarity.