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GE Healthcare Life Sciences Part of GE Healthcare
labsep
Education Centre
About the purification of biomolecules
Purpose of purification
Developing purification protocols
How to combine purification steps
Purification development - summary
LC techniques
Affinity Chromatography
Desalting & Gel Filtration
Hydrophobic interaction chromatography
Ion exchange chromatography
Reversed phase chromatography
Animation
Basic principles
The Separation Mechanism
Elution modes
The typical RPC experiment
The mobile phase
The stationary phase
Resolution in RPC
Optimisation of RPC experiments
RPC in practice
Technique Profile
What is RPC?
Protein Purifier software
BioProcess™ Glossary
The typical reversed phase chromatography experiment

Gradient mode is commonly used for the separation of proteins and peptides by RPC.
For low Mr organic molecules with their shallower desorption curves, isocratic experiments dominate. However, since bio-macromolecules are our main concern here, gradient experiment is described below.
Explanation of symbols

Symbolic representation of a section of an RPC bead.


The least hydrophobic sample component.

Sample component with intermediate hydrophobicity.

The most hydrophobic sample component.

Very hydrophobic contaminant.
.
1. Equilibration
Buffer A is pumped through the column until conditions matches those intended.
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 their respective Hydrophobicities.

Elution order:
4. Regeneration
Remaining hydrophobic contaminants are washed out.