Proteomics/Protein Separations - Chromatography/Hydrophobic Interaction Chromatography (HIC)

Chapter written by: Laura Grell and Alexander Butarbutar
Contact llg3875@rit.edu or nbb3924@rit.edu for contributions
Chapter modified by Kai Burnett and Dalia Ghoneim
Contact kab9783@rit.edu or dxg6098@rit.edu

Hydrophobic Interaction Chromatography (HIC)

Figure 1. Common HIC Stationary Phase Molecules RIT Ion Exchange Site

   Hydrophobic Interaction Chromatography is a separation technique that uses the properties of hydrophobicity to separate proteins from one another. In this type of chromatography, hydrophobic groups such as phenyl, octyl, or butyl, are attached to the stationary column. Proteins that pass through the column that have hydrophobic amino acid side chains on their surfaces are able to interact with and bind to the hydrophobic groups on the column.

   HIC separations are often designed using the opposite conditions of those used in ion exchange chromatography. In this separation, a buffer with a high ionic strength, usually ammonium sulfate, is initially applied to the column. The salt in the buffer reduces the solvation of sample solutes thus as solvation decreases, hydrophobic regions that become exposed are adsorbed by the medium.

Figure 2. The diagram illustrates how an increase in salt concentration can result in enforced hydrophobic interactions

(Res1). The more hydrophobic the molecule, the less salt needed to promote binding (Res1). To elute the proteins, the salt concentration is gradually decreased, eluting bound species in order of increasing hydrophobicity. Additionally, elution can also be achieved through the use of mild organic modifiers or detergent (Res1).

The stationary phase is designed to form hydrophobic interactions with other molecules. These interactions are too weak in water. However, addition of salts to the buffer result in hydrophobic interactions. The following is a list of salts that increase hydrophobic interactions in the order of their ability to enhance interactions:

1. Na₂SO₄
2. K₂SO₄
3. (NH₄)₂SO₄
4. NaCl
5. NH₄Cl
6. NaBr
7. NaSCN

Although reversed phase chromatography and hydrophobic interaction chromatography are very similar, the ligands in reversed phase chromatography are much more hydrophobic than the ligands in hydrophobic interaction chromatography. This enables hydrophobic interaction chromatography to make use of more moderate elution conditions, which do not disrupt the sample nearly as much.

< Previous Page | Next Page >

Resources

1. Bio-Rad Hydrophobic Interaction & Products
2. GE Healthcare Hydrophobic Interaction Chromatography Animation || Introduction to Hydrophobic Interaction & Products