Hydrophobic interaction chromatography [HIC] Theory and Principle

Hydrophobic Interaction
Chromatography [HIC]
Theory & Principle
Bio-Resource
www.technologyinscience.blogspot.com

Hydrophobic Interaction Chromatography

- Introduction
• HIC is a powerful technique for separation and purification of
bio molecules.
• This technique was initially termed as “Salting Out”.
• HIC is widely used for Protein purification, isolating protein
complexes and studying protein folding and unfolding.

• Sample molecules(Proteins) containing hydrophobic and hydrophilic
regions (amino acids) are applied to an HIC column in a high-salt buffer.
• The salt in the buffer reduces the solvation of sample solutes. As solvation
decreases, hydrophobic regions that become exposed are adsorbed by the
media.
• The more hydrophobic the molecule, the less salt is needed to promote
binding.
• Usually a decreasing salt gradient is used to elute samples from the
column in order of increasing hydrophobicity.
• Sample elution may also be assisted by the addition of mild organic
modifiers or detergents to the elution buffer.

- Theory
• Salting Out and Hydrophobic Interaction.
• Thermodynamic Theory.
• Surface Tension, van der Waals Forces.

Salting Out and HIC
• Precipitation by Salting out and HIC are based on same
phenomena.
• In HIC, hydrophobic interaction happens at much lower salt
concentration than that happens in the precipitation (Salting
out).

hofmeister series salt

Salting in – Salting out

HIC – Salting out

Thermodynamic Theory - HIC
• Thermodynamic theory of HIC directly relates to Gibb’s Free
Energy equation:
ΔG = ΔH – TΔS
ΔG – Gibbs Free Energy change
ΔH – Change in enthalpy or heat
T –Temperature in Kelvin
ΔS – change is disorder or randomness

HIC Thermodynamics
• When a Hydrophobe immersed in water, it will be surrounded
by water molecules. An ordered shell of water molecule will
be formed around the hydrophobe.
• When salt is added, the water molecules forms hydrogen
bonding with the salt causing the disruption of water shell.
• When protein is introduced into such system, to minimize
entropy hydrophobic regions of the proteins are forced to
merge to the hydrophobe of the HIC media.

HIC Hydrophobe - Protein Interaction
Mechanism
Hydrophobe water shell around
hydrophobe
Disordered water shell
after addition of salt
To minimize entropy
protein is forced to
merge towards the
hydrophobe

Surface Tension, van der Waals Forces
• A third theory suggests that van der Waals forces are
responsible for the hydrophobic interaction in HIC.
• This is supported by the fact that these forces should be
increased as the order of water increases in the presence of
salt.

HIC Media / Matrix
In HIC, media is described based on
• Ligand
• Ligand Density
• Available Capacity: It is the actual amount of protein that can
bind to the media. If flow rate is included in the
defined conditions then it is called as Dynamic Binding
Capacity.

Dynamic Binding Capacity
Dynamic Binding Capacity is based on these factors:
• Salt Concentration
• Flow Rate
• Temperature
• pH to a lesser extent

HIC Media – Dynamic Bindin Capacity

HIC Matrix - Characteristics
Characteristics of good HIC Matrix
• High Binding Capacity - Larger Area will results in high
capacity binding
• Physical Stability
• Chemical Stability
• Matrix should be Inert

Salts Used in HIC
Most commonly used salts in HIC are
• Ammonium Sulfate,
• Sodium Sulfate,
• Sodium Chloride,
• Potassium Chloride, and
• Ammonium acetate.

HIC Ligands
• Ligands Used in Hydrophobic Interaction Chromatography meida has Alkyl
or Aryl Groups.
• Alkyl - Butyl, Octyl, Ether, Isopropyl - Eg: Butyl S
• Aryl - Phenyl - Eg: Phenyl 650
• Alkyl shows pure hydrophobic character while Aryl shows mixed
behaviour.
• Ligand attachment to the matrix in HIC is done through glycidyl ether.

Binding and Elution in HIC
Binding is done at high salt concentration
• 1 – 2 M Ammonium Sulfate or
• 3M Nacl
Elution is performed by reducing the salt concentration
• Elution of proteins from the HIC will be in the increasing order
of hydrophobicity.

References
• Protein Purification Technical Resources, GE Amersham
• Protein Purification Technical Resources, Biorad
• Protein Purification Technical Resources, TOSOH Biosciences
• Hydrophobic Interaction Chromatography:Fundamentals and Applications in
Biomedical Engineering, Andrea Mahn
• Hydrophobic Interaction Chromatography, Encyclopedia of Bioprocess Technology
• Calibration and Optimization of Hydrophobic Interaction Chromatography , Alex
Olsson

Bio-Resource
http://www.technologyinscience.blogspot.com/2013/07/things-
to-look-for-in-hydrophobic.html

Hydrophobic interaction chromatography [HIC] Theory and Principle

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Hydrophobic interaction chromatography [HIC] Theory and Principle