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GE Healthcare Life Sciences Part of GE Healthcare
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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
Animation
Basic Principles
Gel filtration in practice
Technique Profile
What is Gel Filtration?
Hydrophobic interaction chromatography
Ion exchange chromatography
Reversed phase chromatography
Protein Purifier software
BioProcess™ Glossary
Gel filtration in practice

Use of gel filtration
High resolution mode
Group separation
Separates macromolecules according to size.Desalting macromolecular samples.
Suitable for polishing in multistep purification protocols.Buffer exchange of macromolecular samples.
Separates macromolecular polymers from monomers.Removal of low and high Mr contaminants.
Separates de-natured forms of proteins from native ones.
Separates conformers.
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Experimental
High resolution mode
Group separation
GF mediumSelect fractionation range to embrace target molecule.

For best resolution select steepest selectivity curve.

For narrow peaks select small bead size.

Use Sephadex G-25 or equivalent.
ColumnUse long and narrow columns. Typical length: 30-100 cm.

Column volume should be 20 - 200 times sample volume.

Column length less important.
Eluent Any eluent may be used provided it is compatible with sample and medium. Any eluent may be used provided it is compatible with stability of sample and medium.
Flow rateUse low flow rates to prevent peak broadening .Accepts flow rates considerabily times higher than those used in high resolution mode.
Sample volumeFor high resolution restrict sample volume to 0,5 - 5% of column volume.Desalting and buffer exchange will accept sample volumes up to 1/4 of the column volume.

Before applying the sample:
  • Check the viscosity.
  • Filter or spin to remove particles.


To increase resolution:
  1. Keep within the optimum fractionation range.
  2. Reduce the sample volume.
  3. Change to a gel with smaller beads.
  4. Connect two columns in series.
  5. Reduce the flow rate.

Sample viscosity must not drastically deviate from that of the eluent or distorted sample zones will form. Avoid viscosities larger than 4 cP or protein concentrations above 5%.


Non-specific adsorption rarely occurs with agarose or dextran-based GF media. However, if peaks are unexpectedly retarded, show tailing or if poor recovery is experienced, ionic or hydrophobic interactions may be suspected.

  • Buffer should contain > 0.05M NaCl or equiv. to avoid ionic interactions
  • Hydrophobic interactions may be avoided by adding 25% acetonitrile; EtOH or isopropopanol. Alternatively add 10% etyleneglycol.