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HPLC Columns - Hydrophobic Interaction FAQ's

The following are Hydrophobic Interaction Chromatography HPLC column FAQ's. Please click on the corresponding tab below for the questions and answers.

  1. How does HIC work?

  2. What factors affect the protein binding in HIC?

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

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

  5. How stable are the TSKgel HIC resins?

  6. What endfitting do I use with my particular TSKgel HIC column?  

 

1. How does HIC work?

In HIC, proteins bind to a hydrophobic ligand on the surface of a porousl methacrylate support resin under high salt concentration conditions in the mobile phase (typically >1 mol/L 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 µg 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.

 

6. What endfitting do I use with my particular TSKgel HIC column?

Please visit the HPLC Columns Accessories section of the product catalog for the available fittings to be used with your TSKgel column.  Question #10 within the General FAQ category includes additional information on fittings and connectors.