Download hydrophobic interaction chromatography.

Hydrophobic Interaction
Dr. Nikhat Siddiqi
1
• Soluble proteins require a solvation shell of
water on their surface to maintain solubility in
aqueous solution.
• This water masks hydrophobic groups that
also exist on the protein surface. In the
presence of reagents that are capable of
binding water from the solvation shell (e.g.
(NH4)2SO4), it is possible to disrupt solvation
and expose these hydrophobic groups.
Dr. Nikhat Siddiqi
2
• Using a hydrophobic stationary phase such as
provided by bonded octyl (strongly
hydrophobic) or phenyl (more weakly
hydrophobic) groups, it is possible to promote
hydrophobic interactions between protein and
such a bonded phase. This is the basis of the
adsorption chromatography technique called
hydrophobic interaction chromatography.
Dr. Nikhat Siddiqi
3
• Protein samples are applied to columns containing octyl or
phenyl groups in the presence of a large concentration
(approx. 2 M) of a highly polar solvent such as (NH4)2SO4.
• In such solvents, hydrophobic interactions are strongly
promoted between proteins and the stationary phase.
• Applying a decreasing gradient of solvent polarity, (e.g. 20M (NH4)2SO4) gradually disrupts hydrophobic
interactions, thus separating proteins (with different net
hydrophobicity) from each other.
• Alternatively, elution may be achieved by the use of a
hydrophobic displacer molecule such as a nonionic
detergent (e.g. Triton X-100), an aliphatic amine (e.g.
butylamine) or an aliphatic alcohol (e.g. butanol).
Dr. Nikhat Siddiqi
4
• Since (NH4)2SO4 precipitation is frequently an early
stage in protein purification, this type of
chromatography may be conveniently used subsequent
to this step in a protein purification strategy.
• Important experimental variations in this technique
include; using stationary phases of differing
hydrophobicities (e.g. phenyl or octyl), different buffer
concentrations and composition, varying pH (lower pH
favours binding) and temperature (higher temperature
favours binding).
Dr. Nikhat Siddiqi
5
Dr. Nikhat Siddiqi
6