Molecules interact with each other differently depending on their structure. These interactions are important in many areas including purification and chromatography. One important interaction is how molecules interact with water.
Hydrophobic materials are “water fearing” and do not mix with water. There are also materials which are the opposite, or hydrophilic, which are “water loving”.
You can sometimes tell if a material is hydrophobic or hydrophilic based on how it reacts when water is poured on it. For example, if you drip water on a hydrophilic surface the water will spread out evenly. In contrast, when you drip water on a hydrophobic it repels the water and causes it to form droplets.
In addition to describing solid surfaces, hydrophobic and hydrophilic can be used to describe molecules. Different molecules will be more or less hydrophobic based on their structure. Molecules with a greater number of carbon atoms, like fats, will be more hydrophobic.
Depending what amino acids proteins are composed of, they fold in different ways. This folding causes hydrophobic and hydrophilic regions that interact with water in different ways.
When Hydrophobic molecules are in a water medium they tend to clump together to limit the surface interaction between the water molecules and hydrophobic molecules.
An example of this is oil droplets in water. They clump together to avoid interacting with the water droplets around.
Mobile Phase: The solution flowing through the stationary phase
Stationary Phase: A solid that the mobile phase flows through, typically made up of tiny resin beads
Elute: Travel through the chromatography column
Hydrophobic interaction chromatography (HIC) separates molecules based on hydrophobicity. It is used to separate and purify proteins depending on their surface hydrophobicity. This process is used frequently in biologics purification, like in Skyrizi, because the protein is not denatured (when it unfolds) which means that the biologic activity is maintained.
The stationary phase possesses hydrophobic groups that interact with the hydrophobic regions of the proteins. This promotes bonding between the stationary phase and proteins in the mobile phase.
Proteins, due to their composition, have areas of hydrophobicity and hydrophilicity. When proteins are situated in an aqueous environment, in the absence of a salt, a dense layer of water molecules surround the protein which shield the hydrophobic patches. This same dense layer surrounds the stationary phase coating the hydrophobic areas. These water molecules prevent the protein from interacting with the stationary phase.
Introducing a salt concentration results in the disassembly of the water layer around the stationary phase and proteins. The water molecules now interact with the salt leaving the protein to interact with the stationary phase.
Once the proteins are attached to the stationary phase, they can be separated by gradually reducing the salt concentration. Lowering the salt concentration results in the hydrophobic interactions between the stationary and mobile phases to be destabilized. Due to the lack of salt to interact with, the layer of water will gradually reform around the protein causing proteins with lower hydrophobicity to elute first.
