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What Is Hydrophobic Interaction Chromatography?

Written by The Protein Man | Feb 29, 2024 2:37:11 PM

What Is Hydrophobic Interaction Chromatography? 

Hydrophobic Interaction Chromatography (HIC) is a type of column chromatography used to separate proteins and other biomolecules based on their hydrophobicity or the degree of interaction with a hydrophobic stationary phase. In addition, it effectively removes unwanted product-aggregates from the sample solution in the process. 

In this method, proteins containing both hydrophilic and hydrophobic regions are applied to an HIC column under a high salt-containing buffer. Generally, hydrophobic molecules will preferentially interact with hydrophobic ligands attached to the stationary phase of the column, while more hydrophilic molecules or regions will pass through the column more quickly.

Working Principle 

The hydrophobic stationary phase of HIC columns is typically made up of a nonpolar ligand such as phenyl, octyl, or butyl groups covalently bound to a solid support matrix, such as silica or agarose. 

Samples are applied to the column under conditions of high salt concentration, typically with ammonium sulfate or other chaotropic agents, to minimize the non-specific binding of proteins to the column. As the salt concentration gradually decreases, the exposed hydrophobic regions of proteins begin to interact with the stationary phase, causing them to elute more slowly from the column. Mild organic detergents or modifiers can also be added to the elution buffer to facilitate sample elution. 

In general, HIC is a useful technique for separating proteins and other biomolecules with similar hydrophobicity and is often used in combination with other chromatographic methods to achieve high-purity separation and isolation of target molecules.

Factors Affecting Hydrophobic Interaction Chromatography

Listed below are the factors affecting the effectiveness of HIC in separating proteins and biomolecules.

Type of hydrophobic ligand and solid support

In general, the choice of ligand determines the types of proteins that bind to the column. Due to their weaker hydrophobicity, butyl ligands are less selective than phenyl or octyl ligands while phenyl and octyl ligands are more hydrophobic and can be more selective, particularly for proteins with exposed hydrophobic patches. 

Additionally, the length of the alkyl chain and charge distribution in the ligand also impact the selectivity of the column. Longer alkyl chains provide more hydrophobic interactions, leading to stronger binding of proteins with the stationary phase. However, there is a limit to how long the alkyl chain can be before steric hindrance or aggregation becomes problematic.

Ligands with more evenly distributed charges exhibit better selectivity than those with a localized charge distribution. For example, ligands with multiple phenyl groups tend to have a more uniform charge distribution than butyl ligands, making them more selective in separating certain proteins.

 

Matrix 

The matrix used in HIC columns can be made of a variety of materials, such as silica, agarose, cellulose, or polymers, each with its own unique physical and chemical properties. Thus, to ensure optimal selectivity, resolution, and efficiency of the separation; need to consider the hydrophobicity, pore size, and charge before selecting a matrix.

Concentration and type of salt

High salt concentrations are used in the initial stages of HIC to minimize the non-specific binding of proteins to the column. High ionic strength can enhance hydrophobic interactions, resulting in a stronger interaction between the hydrophobic ligands and the sample proteins. However, if the salt concentration is too high, the column may lose its hydrophobicity, leading to non-specific binding.

The type of salt used can also affect the separation process. For instance, potassium chloride is more effective than sodium chloride to promote protein interactions with the stationary phase. 

Temperature and pH 

Higher temperatures can enhance hydrophobic interactions, forming a stronger bond between the hydrophobic ligands and sample proteins. At the same time, the pH of the mobile phase can affect the degree of ionization of the stationary phase and alter the selectivity of the column. Generally, the higher the pH, the weaker the interaction between the proteins and the media. 

Size and shape of molecules being separated.

The separated molecules' size and shape can affect how they interact with the stationary phase. For example, large proteins may interact more strongly with the stationary phase than smaller ones, leading to slower elution times. Similarly, molecules with a compact shape may interact more strongly with the stationary phase than more extended ones.

Amount of sample

The amount of sample applied to the column also affects the separation process. Applying too much can lead to non-specific binding and poor separation, while using too little can result in low yield.

Overall, HIC is a valuable tool in the biochemist's arsenal for separating proteins and other biomolecules based on their hydrophobicity properties. It is a useful technique for purifying proteins and biomolecules in downstream bioprocessing applications such as antibody purification, vaccine production, and enzyme production. However, as with any chromatographic technique, careful optimization of experimental conditions is crucial to achieving optimal separation and purification.





Hydrophobic Interaction Chromatography (HIC): https://www.gbiosciences.com/Hydrophobic-Interaction-Chromatography

Phenyl: https://www.gbiosciences.com/Protein-Research/Purification-Chromatography/Hydrophobic-Interaction-Chromatography/G-Trap_Phenyl_Agarose_6_Fast_Flow_High_Sub

Butyl ligands: https://www.gbiosciences.com/Protein-Research/Purification-Chromatography/G-Sep-Butyl-Agarose-Fast-Flow

 

Image: Hydrophobic Interaction Chromatography (source: https://www.openpr.com/news/1243304/latest-report-on-hydrophobic-interaction-chromatography-market-2018-2022-worldwide-top-key-players-bio-rad-lab-sartorius-ag-thermo-fisher-scientific-ge-tosoh-waters-geno-technology-sepax-tech-jnc.html)

                             

 

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