GE Healthcare Life Sciences - The HIC experiment

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Education Centre
Education Centre

Purpose of purification
Developing purification protocols
Purification development - summary
How to combine purification steps
LC techniques
Affinity Chromatography
Desalting & Gel Filtration
Hydrophobic interaction chromatography
Animation
Basic principles
The Separation Mechanism
Elution modes
The HIC experiment
Effect of choice of ligand
Effect of eluent composition
Effect of temperature
Resolution in HIC
Optimisation of HIC experiments
HIC in Practice
Technique Profile
What is HIC?

The typical hydrophobic interaction chromatography experiment

Hydrophobic interaction chromatography (HIC) is most frequently used in gradient mode and the experiment consists of four phases as shown below.

Explanation of symbols

Symbolic representation of a section of a HIC bead.	Slightly hydrophobic sample component.	Reasonable hydrophobic sample component.	Quite hydrophobic sample component.	Highly hydrophobic contaminant.

1. Equilibration. Buffer A is pumped through the column until conditions match those of buffer A.	2. Sample application and wash. Sample is added and adsorbed and non-adsorbed components are washed out.	3. Gradient elution. Gradient is started and adsorbed sample components are eluted in order of increasing hydrophobicity. Elution order:	4. Regeneration. Hydrophobic contaminants still remaining are washed out.