Hydrophobic Interaction Chromatography: Understanding its principle and usage

Discover the fascinating principle behind hydrophobic interaction chromatography and how it is used in scientific research and analysis to separate biomolecules based on their hydrophobic properties. 

What is hydrophobic interaction chromatography?

Hydrophobic Interaction Chromatography (HIC) is a technique used in biochemistry and analytical chemistry to separate and purify proteins based on their hydrophobic properties. Unlike other chromatographic methods that rely on charge or size differences, HIC exploits the interaction between hydrophobic regions of proteins and hydrophobic ligands attached to the stationary phase of the chromatography column.

In HIC, proteins are usually loaded onto the column under conditions of a high salt concentration, which promotes the exposure of hydrophobic regions and increased hydrophobic interactions. As the sample is applied, proteins with higher hydrophobicity tend to bind more strongly to the hydrophobic ligands. In contrast, less hydrophobic proteins will bind less strongly, and proteins with the least hydrophobicity will even pass through the column.

To elute the bound proteins, a decreased salt gradient is typically applied, which reduces the hydrophobicity of the proteins and weakens their interaction with the hydrophobic ligands, allowing them to be eluted from the column due to the decreasing hydrophobicity.

HIC is particularly useful for separating proteins with similar charge or size, as it provides an additional dimension of separation based on their hydrophobic characteristics. It can also be used to purify proteins from complex mixtures, such as cell lysates or culture supernatants, or to remove contaminants from protein samples.

The principle behind hydrophobic interaction chromatography

The principle behind hydrophobic interaction chromatography is the tendency of hydrophobic regions in proteins to interact with hydrophobic surfaces or ligands.

Proteins have hydrophobic regions and hydrophilic regions due to their composition of amino acids. The hydrophobic regions contain hydrophobic or nonpolar amino acids such as alanine, phenylalanine, tryptophan, methionine, etc. These nonpolar amino acids are usually buried within the protein structure in the aqueous surroundings. However, some nonpolar amino acids are found on the surface of the protein as hydrophobic patches, which are important for maintaining the structure and biological functions of the protein molecules. The extent of hydrophobicity of proteins varies depending on the number, size, and distribution of these hydrophobic patches on the surface, which are special characteristics of each individual protein.

In the absence of salt, a dense layer of water molecules surrounds the protein, shielding its hydrophobicity, while a thick layer of water is formed on the stationary phase of HIC, which also shields its hydrophobic ligands. These layers of water molecules prevent the protein from interacting with the stationary phase. Introducing a high salt concentration environment results in the disassembling of the polar water layers around the hydrophobic patches of the proteins and hydrophobic ligands. When the water layers are interrupted by the high salt concentration, the hydrophobic patches are exposed, and the hydrophobic interaction between the protein and the ligands is formed. This process is thermodynamically favored since the formation of these interactions allows the decrease of the total energy of the system.

Thus, in HIC, when a protein sample is applied to the column under high salt concentration, proteins with hydrophobic regions will interact with the hydrophobic ligands, resulting in their retention on the column. The degree of retention depends on the strength of the hydrophobic interaction, which is influenced by factors such as salt concentration, types of salt, pH, and temperature.

By manipulating these conditions, it's possible to modulate the strength of the hydrophobic interaction and achieve selective separation of proteins based on their hydrophobicity. All this makes HIC a versatile and powerful tool in protein purification and analysis. The types of hydrophobic ligands, the degree of substitution, and the types of solid support matrix determine the selectivities of different HIC resins, which create more potential for an HIC resin to be engineered and customized for various applications and biomolecules.

Use and application of hydrophobic interaction chromatography

Hydrophobic interaction chromatography is widely used in various fields of research and industry. Some common applications include:

  • Protein purification: HIC can effectively separate and purify proteins from complex mixtures, such as cell lysates or culture supernatants. It can be used as a standalone purification technique or as a complementary method in combination with other chromatographic methods.
  • Protein characterization: HIC can provide valuable information about the hydrophobicity of proteins, which is important for understanding their structure, stability, and function. It can be used to determine the number of hydrophobic regions in a protein or to assess the effect of mutations or chemical modifications on its hydrophobic properties.
  • Vaccine development: HIC can be used to purify and characterize antigens or vaccine candidates, helping in the development of vaccines against various diseases.
  • Drug discovery: HIC can be employed in the purification and analysis of drug targets and in screening small molecule libraries for potential drug candidates.
  • Food and dietary supplement development: HIC can also be applied in the field of food and dietary supplement processing to separate and purify proteins and peptides in order to improve the nutritional value and taste. 

These are a few examples of the wide range of applications of HIC. Its versatility and effectiveness make it a valuable tool in many areas of scientific research and biotechnological fields.

Advantages and limitations of hydrophobic interaction chromatography

Hydrophobic interaction chromatography offers several advantages that make it a popular choice in protein purification and analysis:

  • Selective separation: HIC can separate proteins and other biomolecules based on their hydrophobicity, providing an additional dimension of separation compared to other chromatographic methods that rely on charge or size differences.
  • Gentle purification conditions: HIC can be performed under mild conditions, preserving the stability and functionality of the proteins being purified.
  • High resolution: HIC can achieve high-resolution separations, allowing for the isolation of closely related protein variants or isoforms.
  • Versatility: HIC can be used with various types of proteins, including antibodies, enzymes, membrane proteins, recombinant proteins, peptides, oligonucleotides, and plasmids.

However, there are also some limitations to consider when using HIC:

  • Sample preparation: HIC requires careful sample preparation, including the removal of contaminants that may interfere with the hydrophobic interaction or cause non-specific binding.
  • Salt sensitivity: HIC is sensitive to salt concentration, which makes choosing the appropriate salt conditions a crucial part of achieving optimal separation.
  • Ligand specificity: Different hydrophobic ligands may have different selectivity towards proteins, and the choice of ligand should be carefully considered based on the specific requirements of the purification or analysis.
  • Scale-up challenges: Scaling up HIC for large-scale protein purification can be challenging due to the limitations of column packing and the need for higher sample volumes.

Despite these limitations, hydrophobic interaction chromatography remains a valuable technique in protein and biomolecule purification and analysis, offering unique advantages and the ability to separate biomolecules based on their hydrophobic properties.

 

References 

  1. Brendan F O'Connor, Philip M Cummins, Hydrophobic Interaction Chromatography, Methods Mol Biol. 2017:1485:355-363. doi: 10.1007/978-1-4939-6412-3_18.
  2. Herbert P Jennissen,Hydrophobic Interaction Chromatography, https://doi.org/10.1002/9780470015902.a0002678.pub4
  3. Kennedy, Robert M. Hydrophobic-interaction Chromatography, Current protocols in protein science, volume (1)-Jun 1, 1995
  4. Introduction to hydrophobic interaction chromatography, https://www.youtube.com/chnnel/UCB6sVUWE8d7_Oc_9brp7vTw/videos

 

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