UNIT – 5: Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography is the liquid chromatography that

used for the separation of molecules based on their surface hydrophobicity.
It is the useful separation technique for purifying the biomolecules by their
hydrophobic interaction with the hydrophobic ligands coupled to the porous
media and maintaining the biological activity due to the use of conditions and
matrices.

It was described for the first time by Shepard & Tiselius in 1949 using the term
“salting-out chromatography”. Finally, Hjerten in 1973 described this
technique as the “hydrophobic interaction chromatography” based on the
retention of proteins on weakly hydrophobic matrices in presence of salt.

Principle of Hydrophobic Interaction Chromatography

Proteins with the different degrees of surface hydrophobicity can be separated
by using the hydrophobic interaction chromatography.
In this chromatography, the protein sample is introduced into the column
containing the high-salt buffer.
The proteins are bound to the hydrophobic ligand on the resin in the binding
buffer with high salt concentration.
The salt promotes the interaction between the hydrophilic and hydrophobic
regions of protein and media by reducing the solvation of protein molecules and
exposing their hydrophobic regions.
The amount of salt required to promote the binding interaction is inversely
proportional to the hydrophobicity of molecules.
The binding interaction is reversed when the ionic strength of buffer is reduced.
Therefore, the protein with the lowest degree of hydrophobicity is eluted first
and the most hydrophobic protein elutes last when decreasing the salt gradient.
Steps in Hydrophobic Interaction Chromatography
The media are composed of alkyl ligands or aryl ligands coupled to the porous
matrix.
The matrix is then packed into the chromatography column.
The moderately high salt buffer is used to fill the pores and space between the
particles in matrix.
Typical salts used are 1–2 M ammonium sulfate or 3 M sodium chloride which
are selected to promote the binding interaction between the protein sample and
media.
The column is washed to remove the non-bound proteins.
The salt concentration is gradually decreased to start eluting the proteins.
Proteins having the lowest hydrophobicity are eluted first.
The final wash using salt-free buffer helps to remove all the tightly-bound
proteins.

Crucial factors that affect Hydrophobic interactions

The factors which should be considered for the hydrophobic interaction
chromatography are:
Choice of ligand type: The adsorption behavior of proteins is determined by
the type of immobilized ligand.
In general, the straight chain alkyl ligands demonstrate the hydrophobic
character while the aryl ligands show the both aromatic and hydrophobic
interactions.
Degree of ligand substitution: The protein binding capacity increases with the
increased degree of substitution of the immobilized ligand.
The protein binding capacity is directly proportional to the degree of ligand
substitution. However, the high levels of ligand substitution increase the
interaction strength and make it difficult to elute the proteins.
Matrix: The most widely used supports are the hydrophilic carbohydrates:
cross-linked agarose and synthetic copolymer materials.
Salt concentration: The interaction between the hydrophobic proteins and resin
is greatly influenced by the running buffer.
The addition of salts is to the equilibration the buffer and to promote the ligand-
protein interactions in Hydrophobic interaction chromatography.
The high salt concentration enhances the interaction. Lowering the salt
concentration decreases the interaction.
pH of mobile phase: The mobile phases used in Hydrophobic interaction
chromatography mostly have the neutral pH range of 5 – 7 and buffered with
either sodium phosphate or potassium phosphate.
The effect of pH on protein-media interactions is varies from protein to protein.
Generally, the hydrophobic interaction between the protein and media decreases
with increase in pH as the protein charge tends to increase.
Therefore, pH can impact the level of protein binding and the media selectivity.
Temperature: There is a direct and positive correlation between the affinity of
hydrophobic interactions and temperate. The affinity of hydrophobic
interactions increases with the temperature.
High temperature also influences the protein structure, solubility, and the
interaction with the Hydrophobic interaction chromatography matrix.

Applications of Hydrophobic interaction chromatography

 Purification of proteins and enzymes
 Purification of protein aggregates
 Protein folding
 Antibodies purification
 Proteomics
 Applicability of HIC as a plasma fractionation method
 Analysis of protein interaction networks
 Affinity chromatography
Affinity chromatography has been specially developed for protein purification.
It is the only technique that separates the proteins based on their biological
functions rather than individual physical properties or chemical properties.
The basis of the selectivity of affinity chromatography is the specific interaction
between the affinity ligand and protein of interest.
These affinity ligands such as antibody, enzyme inhibitor and lectin can
bioselectively bind to the complementary analyte molecules in the mobile
phase.
Since the strong interaction between the target protein molecules and affinity
ligands, the affinity chromatography often requires the harsh conditions to
release the bound protein molecules from affinity ligands, such as extreme low
or high pH (acid or alkaline elution) and using an eluting agent (competing
agent).
This may cause the less recovery of protein during the elution.
Furthermore, using of competing agent requires additional separation steps to
remove such a competing component from the purified antibody.

Figure: Affinity chromatography

Figure: Ion exchange chromatography