Protein Elution in Hydrophobic Chromatography

Hydrophobic interaction chromatography (HIC) is a very valuable tool in protein purification technology. After the protein is bound to the medium, it must be eluted and collected to obtain the target protein. In most cases, the elution process separates or removes protein impurities from the target protein. Sometimes protein impurities may not bind so tightly to the media and thus elute before the target protein. However, when protein multimers are separated by hydrophobic chromatography (HIC), the binding of target protein multimers to the filler is more tightly than that of monomers to the filler, and the target protein will still bind to the filler after elution. During elution, the effluent may contain high-purity product or it may contain significant protein impurities. Therefore, when HIC is used for separation and purification, in order to obtain a product of suitable purity, the effluent should be collected in aliquots during the elution step. The elution process (gradient elution) is shown in the figure.

Schematic diagram of gradient elution during HIC separation of protein mixtures

The elution process can be carried out in an isocratic or gradient manner. The 4 most common methods for elution of bound proteins are as follows:

1. Reduce salt concentration (relative to binding conditions)
   The decrease in salt concentration correspondingly weakens the hydrophobic interaction between the protein and the ligand, and the protein is desorbed and eluted from the column.
2. Add organic solvent
   The addition of organic solvents such as ethylene or propylene glycol can alter the polarity of the solution, which can interfere with hydrophobic interactions.
3. Provide salt concentration (using chaotropic salt)
   The addition of chaotropic salts can interfere with hydrophobic interactions.
4. Add detergent
   Detergents can be used as protein displacers, mainly for HIC purification of membrane proteins.

In the elution steps described above, the most common method to elute proteins from HIC media is to reduce the salt concentration. This method is also used first when using HIC to purify new protein fractions. The disadvantage of other methods is that additional substances are required, which may affect the stability of the protein. However, these reagents are still required to elute proteins that are more tightly bound to the media.

The following HIC fillers are available from Welch Materials: