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The affinity chromatography separation mechanism
Common for all types of affinity chromatography is that an affinity ligand specific for a binding site on the target molecule, is coupled to an inert chromatography matrix.
Under suitable binding conditions this affinity matrix will bind molecules according to its specificity only. All other sample components will pass through the medium unadsorbed (Fig.1.1).
Fig 1.1 Affinity chromatography relies upon a reversible highly specific binding reaction.
After a wash step the adsorbed molecules are released and eluted by changing the conditions towards dissociation or by adding an excess of a substance that displaces the target molecule from the affinity ligand.
As for all reversible binding in chromatography, desorption curves (Fig. 1.2) can be used to describe the partitioning of the sample between the mobile phase (the "buffer") and the stationary phase(the affinity matrix).
Fig 1.2 Since only the target protein binds, step elution will
not co-elute other sample components.
However, since no other sample components bind within the partition zone, a simple "on-off" mode of chromatography can be applied by switching abruptly from full binding to complete release conditions.
Thus affinity chromatography fishes out the target molecule by way of highly specific binding and release, rather than removes contaminants by fine-tuned isocratic or gradient elution techniques.
The development of an affinity chromatography method boils down to:
- Finding a ligand specific enough to allow step elution.
- Finding conditions for safe binding and release within the stability window of the target molecule and the ligand.
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