5 Steps of Hydrophobic Interaction Chromatography (HIC)

Описание: 0:00-0:44 | Hydrophobic effect simplified
0:44-2:13 | What is hydrophobic interaction chromatography?
2:13-3:24 | Hydrophobic interaction chromatography workflow
3:24-4:11 | Why is hydrophobic interaction chromatography useful?

Hydrophobic interactions describe the relationship between water and hydrophobes, molecules which barely or outright refuse to dissolve in water. For our purposes, the old adage, like attracts like is relevant. Basically hydrophobes abhor water and will band together in order to defend themselves against it! This is of course a simplified explanation, not EXACTLY true to how it works in reality but the same effect occurs there as well. If you are interested in the exact reason why, just search for the hydrophobic effect.

This interaction is utilized in hydrophobic interaction chromatography or HIC for the purpose separating molecules based on their level of hydrophobicity, or in other words, based on how much they hate water. HIC utilizes a reversible interaction between the proteins and the hydrophobic ligand of a HIC resin. This HIC resin is a bead with a hydrophobic surface which therefore attracts hydrophobic proteins. The interaction between the the hydrophobic proteins and HIC resins is largely dependent on the salt concentration of the buffer. A higher salt concentration enhances the interaction while a lower salt concentration weakens the interaction. This is because the salt buffer reduces the solvation (dissolution) of sample solutes and as solvation decreases the hydrophobic regions that become exposed are adsorbed by the media. The more hydrophobic the molecule, the less salt is needed to promote binding. The salt concentration in the buffer, can then gradually be decreased in order to separate our proteins based on how hydrophobic they are. As such the protein with the lowest degree of hydrophobicity is eluted first and the one with the highest degree of hydrophobicity is eluted last, requiring a greater reduction in salt concentration to reverse the interaction.

The workflow is very typical for column chromatography, but with the important difference that we gradually reduce the salt concentration in order to separate the proteins from one another. The column contains HIC resins which are also hydrophobic, therefore attracting and slowing the down the proteins based on their level of hydrophobicity. The separation is carried out in 5 main steps.

1. The sample is added to the column
2. Proteins collect at the top
3. Proteins begin to move through the column, pulled by gravity, but starts separating based to their attraction to the HIC resins, which again is dependent on their level of hydrophobicity (hydrophobicity, can’t believe that’s a word, ENGLISH IS WEEEIRD!) Anyway step
4. We gradually change the salt concentration, ensuring that only the most water hating (hydrophobic) proteins stick and eventually, they also have to give up
5. During this changing salt concentration we can start colleting our proteins, from the least hydrophobic, to the most hydrophobic

Hydrophobic interaction chromatography can be used for the purpose of capturing a protein of interest. HIC can also be used for the intermediate step as well as the final polishing step in protein purification. It is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing or less harmful conditions.