High performance hydrophobic interaction chromatography is a very powerful technique
for the isolation of proteins in an analytical and preparative scale. The method is based
on the interaction between moderately hydrophobic ligands, fixed on the chromatographic
support, and the hydrophobic areas located on the surface of proteins. The hydrophobic
feature of a given protein is mainly caused by the amino acids isoleucine, valine,
leucine, and phenylalanine.
The hydrophobic ligands are presumed to interact with hydrophobic side chains of the
protein where an electron donor-acceptor complex is formed. The hydrophobic character
of a protein is promoted by high salt concentrations. The salt, however, not only
affects the molar concentration of water, but changes the conformational structure
of the proteins, the hydrophobic interaction forces and the hydration shell of the
protein (salting out effect).
Elution:
The proteins are desorbed by decreasing concentrations of salt after the hydrophobic
properties of the proteins are reduced during the gradient. The desorption from HIC
columns occur in the order of increasing surface hydrophobicity.
Getting Started Now (Practical hints)
Adequate Buffers
Typically, HIC is carried out with a linear elution from a relatively high ammonium
sulphate concentration to a low ammonium sulphate concentration. Due to different
hydrophobic properties of proteins, the salt concentration for each separation on
hydrophobic media should be tested experimentally. In general, increasing ionic
strength increases hydrophobic interactions. Both anions and cations can be sorted
in a list starting with those that highly favor the interaction to those that will
reduce hydrophobic forces.
For anions, the series is
PO43- > SO42- > CH3COO-
> Cl- > Br- > NO3- > SCN-.
The cations series is
NH42+ > Rb+ > K+ > Na+
> Li+ > Mg+ > Ca2+ > Ba2+.
Strong chaotropic salts disrupt the structure of water resulting in decreased
hydrophobic interactions. Many different buffer systems can be used for hydrophobic
interaction chromatography. Because ammonium sulphate is instable under basic
conditions the pH should be below 8 when working with ammonium sulphate.
Sodium sulphate is suitable as a salting-out agent, but solubility problems
exclude its use at high concentrations. Buffers should be prepared with an
appropriate salt concentration. For example, phosphate buffer (20 - 50mM)
can be used containing 1.5 - 1.8M ammonium sulphate. Also other salts can
be recommended such as various phosphate salts, sodium citrate, or sodium acetate.
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