Hydrophobic interaction chromatography

Hydrophobic interaction
Umair Saleem
Methods in protein chemistry
■ Alternatives
• Gel filtration chromatography
• Ion exchange chromatography
• Reverse phase chromatography
Why HIC?
• Different basis of separation
• Weaker interactions
→ Less structural damage
→ Maintain high activity
→ Purpose
→ Principle of HIC
→ Advantages of using
→ What are the factors
affecting HIC
→ Conclusion
Source of protein
Purity &
■ Purpose
• Downstream purification
• Separation of biomolecuoles
• Exploits differences in hydrophobicity.
→ Number of hydrophobic aminoacids.
→ Distribution of these aminoacids.
Separation of substances is based
on their varying strength of
interaction with hydrophobic
groups attached to an uncharged
gel matrix
Hydrophobic groups on proteins
are sufficiently exposed to bind to
the hydrophobic groups on the
• How is this
Source of protein
Purity &
General Concept
Hydrophobic Interaction Chromatography
Experimental Technique
■ Choice of column → XK colums for HIC.
• Column dimensions → Short bed height
(5-15 cm) suitable for HIC
■ Packing of Column: a modern, highly
crosslinked agarose-based gel such as Sepharose
Fast Flow is however easier than packing a gel
filtration column since the bed height required is
much smaller.
■ Sample preparation
• Sample composition
• Sample volume
• Sample viscosity
■ Sample application
■ Batch Separation
Advantages of HIC
Large volume of sample
can be loaded
Samples with high ionic
strength can be used
Well suited to use
before gel filtration,
ion-exchange and
affinity chromatography
Sample eluted with low
Purification steps that
generate large sample
volume can be coupled
with this method
Good for samples after
ammonium sulfate
These techniques may
require pretreatment of
samples (e.g. reducing
ionic strength)
Sample can be used in
ion exchange
chromatography step
Factors affecting HIC
 Type
and concentration of ligand
 Type of base matrix.
 Type and concentration of salt
 pH
 Temperature
 Additives
HIC Ligands
O CH 2 CH CH 2 O
( CH 2) 3
CH 3
O CH 2 CH CH 2 O
( CH 2) 7
CH 3
O CH 2 CH CH 2 O
Effects of ligand density
Degree of substitution
• ‰
Binding capacity of protein to HIC increases with
increased alkyl chain length (A) and increased
degree of substitution of immobilized ligand (B)
• ‰
Caution: protein can bind via multipoint
attachment, thus difficult to elute
Type of base matrix
Important to take note that selectivity will not be exactly the
same even with the same type of ligand if the base matrix is
Two widely used supports are cross-linked agarose and
synthetic copolymer materials
May be necessary to modify adsorption and elution conditions
Influence of salts
Type of salt
 salt effect follow the Hofmeister series.
 Hydrophobic interaction increases at
increased salt concentration
Increasing salting out effect
Anions: PO43- >SO42- >Cl- >Br- >NO3- >ClO4>I- >SCNCations: NH4+ > K+ >Na+ >Li+ >Mg2+
Decreasing surface tension
Increasing chaotropic effect
Effect of pH on HIC is not straight forward.
n general an increase in pH weakens hydrophobic
interactions. It could be due to increased titration of
charged groups leading to an increase in hydrophilicity of
the proteins
Decrease in pH leads to an apparently increase in
hydrophobic interaction
mplication: Important factor to consider for optimization
of HIC interaction. It is observed that proteins which do
not bind to HIC adsorbent at neutral pH, bind at acidic pH.
Visser and Strating (1975): that role of temperature is a
complex issue and differ from observation of Hierten.
Binding of proteins to HIC adsorbents is entropy driven
(Hjerten, 1976), i.e. interaction increases with increase in
Discrepancy in views could be due to differential effects
by temperature on the conformational state of different
proteins and solubility in solution
Practical terms: To be aware that procedure developed
at room temperature may be different if used in the cold
Salts that cause “salting-in” will
weaken protein-ligand interactions.
lcohols and detergents (non-polar
parts) can compete with protein for
HIC absorbent sites and may
displace proteins.
Hydrophobic Interaction Chromatography Overall Conclusions
Very useful technique for mAb purification
Mainly used in the third step as a complementary
technique to protein A and IEC (in-vivo)
HIC can be used in both binding and removal
Can be a useful alternative to SEC for aggregate
HIC is also very useful for purification of
antibodies in 2-step techniques (non-protein A)
for in-vitro applications.
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