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labsep
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
HIC in Practice
Technique Profile
What is HIC?
Hydrophobic interaction chromatography in practice
.
Use of hydrophobic interaction chromatography
High resolution mode
(gradient elution)
Group separation mode
(step elution)
Separates proteins, according to net hydrophobicity.Concentrates dilute samples.
Suitable for capture and intermediate steps in multi-step purification protocols.Suitable for capture steps in multi-step purification protocols.

Especially suitable as the first chromatography step after ammoniun sulfate precipitation.

.

Experimental
High resolution mode
(gradient elution)
Group separation mode
(step elution)
HIC medium
Use media with 10-30 mm beads for purification work.Use media with 30 mm beads or larger to allow higher flow rates.
Column
Typical column lengths are 1-10 cm.

Short columns may prohibit the use of very shallow gradients (large gradient volumes).

Column length is less important. Short and "fat" columns, however, will allow higher flow rates.
Eluents
Buffers in the pH range 4-8 are compatible with the stability of most proteins. The pH in this range has little influence on the selectivity

Gradient salt can be varied quite widely. However most experiments are run with ammonium sulfate.

A buffer concentration of 20-50 mM is usually sufficient.

Buffers in the pH range 4-8 are compatible with the stability of most proteins. The pH in this range has little influence on the selectivity

Gradient salt can be varied quite widely. However most experiments are run with ammonium sulfate.

A buffer concentration of 20-50 mM is usually sufficient.

Sample volume
With a properly designed gradient the target molecule will bind and concentrate at the top of the column.

The sample volume is thus not important.

Sample volume is not important since the sample will bind to the column.
Sample amount
5-10 % of the total loading capacity of the column used can be applied without loss of resolution.Around 40 % of the total loading capacity of the column used can be applied.
.

Buffer preparation tips
  • Adjust pH of start (buffer A) after the salt has been added.
  • Adjust buffer pH at the temperature intended for the experiment.
  • Always test that target protein is stable in start buffer!

Sample preparation tips
  • Filter or spin the sample to remove any particulate matter.
  • Especially with large sample volumes, adjust sample pH and salt content to match those of start buffer.
  • Buffer exchange on Sephadex G-25 is a rapid and mild way to adjust sample conditions.
  • Especially with large sample volumes, adjust sample temperature to match that of start buffer A.
Arriving at optimal results
Choice of HIC medium
Optimising resolution
  1. First test the stability of the target protein in ammonium sulfate up to 1.4 M.
  2. Test binding and recovery using HiTrap HIC test kit or individual resource HIC columns.
  3. Adjust ammonium sulphate of "Buffer A" to make the target protein elute late in the gradient.
  4. If necessary use a larger column applying the condition worked out above.
  1. Screen the media (HIC ligands) for proper selectivity
  2. Optimise type and concentration of salt during adsorption.
  3. Select the steepest gradient providing acceptable resolution.
  4. If sample binding is unsatisfactory, temperature can be used to adjust it (decrease binding by lowering temp. and increase by elevating temp.).


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