2. 2
Introduction
A goal of biochemistry is to separate and identify chemical compounds.
chromatography is one of the most effective techniques for accomplishing
this.
In chromatography, substances are placed in a system consisting of two
physically distinguishable components -a mobile phase and a stationary
phase-and molecular species separate because they differ (many of them
only slightly) in their distribution between these two phase.
There are many kinds of chromatography:
Adsorption
Partition
Ion-Exchange
Molecular Sieve
Affinity
Column, paper, thin-layer and gas chromatography.
3. Affinity Chromatography
The technique offers high selectivity, hence high
resolution, and usually high capacity for the proteins of
interest.
Purification that would otherwise be time-consuming,
difficult or even impossible using other techniques can
often be easily achieved with affinity chromatography.
The technique can be used to separate active
biomolecules from denatured or functionally different
forms, to isolate pure substances present at low
concentration in large volumes of crude sample and also
to remove specific contaminants.
3
5. • Affinity chromatography is a method of separating biochemical
mixtures based on a highly specific interaction between antigen
and antibody, enzyme and substrate, or receptor and ligand. It is a
type of chromatographic laboratory technique used for purifying
biological molecules within a mixture by exploiting molecular
properties. Biological macromolecules such as enzymes and other
proteins, interact with other molecules with high specificity
through several different types of bonds and interaction. Such
interactions including hydrogen bonding, ionic interaction,
disulfide bridges, hydrophobic interaction, and more. The high
selectivity of affinity chromatography is caused by allowing the
desired molecule to interact with the stationary phase and be
bound within the column in order to be separated from the
undesired material which will not interact and elute first.[1] The
molecules no longer needed are first washed away with a buffer
while the desired proteins are let go in the presence of the eluting
solvent (of higher salt concentration). This process creates a
competitive interaction between the desired protein and the
immobilized stationary molecules, which eventually lets the now
highly purified proteins be released.[2]
5
6. Uses
• Affinity chromatography can be used to:
• Purify and concentrate a substance from a
mixture into a buffering solution
• Reduce the amount of unwanted substances
in a mixture
• Discern what biological compounds bind to a
particular substance
• Purify and concentrate an enzyme solution.
6
8. 8
Affinity Chromatography
Affinity chromatography separates
proteins on the basis of a reversible
interaction between a protein and a
specific ligand coupled to a
chromatography matrix.
The kinds of Elution
pH Elution
Ionic Strength Elution
Reduced Polarity of Eluent
Competitive Elution
Chemotropic Eluents
15. • Binding: buffer conditions are optimized to ensure that the target molecules interact
effectively with the ligand and are retained by the affinity medium as all other molecules
wash through the column.
• Elution: buffer conditions are changed to reverse (weaken) the interaction between the
target molecules and the ligand so that the target molecules can be eluted from the
column.
• Wash: buffer conditions that wash unbound substances from the column without eluting
the target molecules or that re-equilibrate the column back to the starting conditions (in
most cases the binding buffer is used as a wash buffer).
• Ligand coupling: covalent attachment of a ligand to a suitable pre-activated matrix to
create an affinity medium.
• Pre-activated matrices: matrices which have been chemically modified to facilitate
the coupling of specific types of ligand.
15
17. 17
Proper selection of a matrix or carrier for the ligands is of decisive
importance for the successful application of stereospecific adsorption.
1. Insolubility
2. Sufficient permeability
3. High rigidity and suitable particle form
4. Zero adsorption capacity
5. Chemical stability under the conditions required for adsorption, desorption
and regeneration
6. Chemical reactivity allowing ligands and spacers to be introduced
7. Resistance toward microbial and enzymatic attack
8. Good flow properties for rapid separation
9. An open pore structure ensures high capacity binding even for large
biomolecules.
Matrix
18. 18
No matrix support is ideal in all these respects.
Porous glass
Cellulose
Polyacrylamide
Agarose
19. 19
Ligand
The selection of the ligand for affinity chromatography is influenced
by two factors:
o the ligand must exhibit specific and reversible binding affinity for the target substance
o and it must have chemically modifiable groups that allow it to be attached to the matrix
without destroying binding activity.
The dissociation constant (kD) for the ligand-target complex should
ideally be in the range 10-4
to 10-8
M.
20. 20
Ligand Immobilization
Sience 1970, a large number of methods have been developed for
coupling ligands to matrix materials. The most common
procedure is to link a coupling agent to the matrix material and
then add the ligand.
Coupling Step:
Activation Step:
It is important to mention that, after coupling of the desired ligand, reactive
Y
groups may still be present. Deactivation may occur by spontaneous
hydrolysis but, if this is not the case, coupling with a low molecular weight
substance. Glycine, neutral dipeptides, and ethanolamine are deactivating
substances that should be considered.
23. 23
1. Cyanogen Bromide Coupling
1,2-Diols are especially liable to react with cyanogen halides to
form cyclic imino carbonates. In the coupling step a substance
containing amino groups will form at least three products.
Activation Step:
Coupling Step:
24. 24
This reaction is extremely useful in coupling enzymes, coenzymes,
inhibitors, antigen, antibodies, nucleic acids and most proteins to
agarose.
Although most applications of cyanogen bromide coupling have
involved agarose and cross-linked agarose, other hydroxyl-
containing polymers may also be converted to biospecific
adsorbents by coupling of suitable ligands in the same manner.
25. 25
2. Bisoxirane Coupling
Bisoxiranes (bisepoxides) are particularly useful reagents for
introducing low molecular weight ligands through amino or
hydroxyl groups.
26. 26
3. Divinylsulfone Coupling
The vinyl groups introduced into the matrix are more reactive than are
the oxirane groups.
They will thus couple to amines, alcohols, and phenols at lower
temperatures and at lower pH than the oxirane.
27. 27
Spacer Arm
The binding site of a target protein is often located deep within the
molecule and an affinity medium prepared by coupling small ligands,
directly to matrix may exhibit low binding capacity due to steric
interference i.e. the ligand is unable to access the binding site of the
target molecule.
* The length of the spacer arm is critical.
* when using small ligands (Mr < 5 000) there is a risk of steric
hindrance between the ligand and the matrix that restricts the
binding of target molecules. In this case, select a pre-
activated matrix with a spacer arm. For ligands with Mr > 5
000 no spacer arm is necessary.
