Hydrophobic interaction chromatography (HIC) is a powerful technique used for the purification of proteins in analytical and preparatory applications.
HIC separates and purifies protein molecules on the basis of their hydrophobicity – it is more popular than other chromatography techniques for the separation of proteins as it employs a less denaturing environment compared to them. Therefore the biological activity of the proteins is kept intact.
HIC can also be effectively used to remove impurities or product aggregate species in aqueous solutions as it exploits the difference in hydrophobic properties of the aggregates and the target molecules. It is often used in combination with techniques such as ion exchange or gel filtration chromatography.
The Principle of Hydrophobic Interaction Chromatography
In HIC, the sample protein molecules are introduced into the column containing a high-salt buffer. The salt promotes interaction between the hydrophilic and hydrophobic regions of the protein and the medium by reducing the solvation of sample molecules and exposing their hydrophobic regions.
The amount of salt needed to promote binding is inversely proportional to the hydrophobicity of the molecules. Hence the sample molecules can be eluted out in the order of increasing hydrophobicity using a decreasing salt gradient. Bound protein molecules can be effectively desorbed by washing with a dilute buffer or water.
The Steps in Hydrophobic Interaction Chromatography
- HIC media are composed of alkyl or aryl ligands coupled to an inert, porous matrix which are then packed into a chromatography column as a packed bed arrangement.
- A moderately high salt buffer is used to fill the pores and space between particles in the matrix. . Typical salts used are 1–2M ammonium sulfate or 3M sodium chloride which are selected to promote the key interaction between the protein sample and the medium whilst minimizing that of other less hydrophobic proteins (impurities).
- The column is washed to remove non-bound proteins.
- The salt concentration is gradually lowered to start eluting proteins. The manipulation of salt gradients allows differential elution of the proteins - Proteins having the lowest hydrophobicity are eluted first.
- A final wash using salt-free buffer helps remove tightly-bound proteins. Additives in the buffer can further promote desorption of the bound proteins. Additives may include water-miscible alcohols, chaotropic (‘‘salting-in’’) salt solutions and detergents.
- Rarely, harsher conditions such as 0.5–1.0M sodium hydroxide, 70% ethanol, or 30% isopropanol are required to remove all bound proteins.
Crucial Factors that Affect Hydrophobic Interactions
Ligand - The type of ligand used determines the adsorption behavior of proteins e.g. straight chain alkyl ligands is hydrophobic while aryl ligands show both aromatic and hydrophobic interactions.
Matrix - Hydrophilic carbohydrates such as cross-linked agarose and synthetic copolymer materials are the most commonly used supports. Different supports will vary in their selectivity even with the same ligand.
Degree of substitution - The binding capacity of a protein is directly proportional to the degree of substitution of the ligand. However, high levels of ligand substitution increase the strength of the interaction and make it difficult to elute the proteins.
Temperature – There is a direct and positive correlation between temperate and the affinity of hydrophobic interactions. High temperature also influences the structure and solubility of proteins. Temperature is seldom used to modulate elution of molecules in HIC.
pH – The mobile phases used in HIC mostly have a neutral pH range of 5 – 7. The effect of pH on protein-medium interactions varies from protein to protein. Generally, the hydrophobic interaction between media and protein decreases with increase in pH as the protein charge tends to increase. Whilst pH can have an effect on the degree of protein binding, it is not considered significant enough to use pH gradients for the elution of solute molecules.
Salt concentration – Salt addition to the buffer and sample promotes ligand-protein binding but there is a risk of protein precipitation at high salt concentrations. Sodium, ammonium, or potassium sulfates are known to produce higher precipitation effects though they are very effective in promoting interaction between the ligand and the protein.