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Effect of choice of ligand
The possibility to select between ligands of different hydrophobicities is important, since this offers a way to control the salt concentration necessary to adsorb a certain protein.
With low-hydrophobicity ligands the difference between adsorption and precipitation may sometimes be so small that certain proteins may partially precipitate under binding conditions (Fig 4.1).
Fig 4.1 Selecting the appropriate HIC ligand is often a balance
between strong enough binding and precipitation.
On the other hand, quite hydrophobic proteins may bind sufficiently strongly to high-hydrophobicity ligands that harsh, potentially denaturing conditions (e.g. organic solvents) are needed to desorbe them.
Listed below are the most commonly used ligands in order of binding strength:
Ether < Isopropyl < Butyl < Octyl < Phenyl
The difference between the first four ligands is quantitative rather than qualitative.
The phenyl ligand, however, offers another type of selectivity, mainly because of its possibility to form additional bonds.
Figure 4.2 demonstrates some effects of varying the ligand used.
Fig 4.2 Varying the type of HIC ligand has a profound effect on the separation. |
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