HPLC - Hydrophobic Interaction

1. How does HIC work?

In HIC, proteins bind to a hydrophobic ligand on the surface of a porous methacrylate support resin under high salt concentration conditions in the mobile phase (typically >1 M ammonium sulfate). The salt promotes the hydrophobic interaction between the protein and the solid support. To desorb the protein, the salt concentration is lowered via a decreasing salt gradient which diminishes the hydrophobic interaction.

2. What factors affect the protein binding in HIC?

A number of factors are involved in retention and elution of proteins by HIC, including the type of ligand and ligand density on the support resin, the hydrophobic nature of the protein, and the type and concentration of salt used. Thus, the analyst has several options when developing a HIC separation strategy.

3. What is the order of hydrophobicity of TSKgel HIC columns?

Butyl-NPR (non-porous resin) < Ether-5PW < Phenyl-5PW

4. What are the sample capacities of TSKgel HIC columns?

Resolution and peak width are dependent in part on how much sample is loaded on the HIC column. In general the following guidelines for sample loading can be applied:

  • Ether-5PW 5 -10 mg of sample protein
  • Phenyl-5PW 5 -10 mg of sample protein
  • Butyl-NPR 100 ug of crude protein

5. How stable are the TSKgel HIC resins?

The G5000PW base material used as the support matrix for Ether and Phenyl 5-PW HIC columns is physically and chemically stable in water (pH 2-12) and in up to 50% polar organic solvents such as methanol, ethanol, and acetonitrile.

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