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What is Gel filtration?
Gel filtration (GF) is a chromatography technique that separates molecules according to size.
Within the fractionation range chosen, molecules are eluted in order of decreasing size.
The separation mechanism is non-adsorptive and independent on the eluent system used and thus very gentle. Recoveries are very high both on the basis of mass and biological activity.
Since GF is non-adsorptive and eluted under isocratic conditions (no gradients or steps) the sample is not concentrated during the separation, but slightly diluted, a fact that limits the applicable sample volume.
The fact that the separation depends solely on molecular size makes the selectivity unique and high overall resolutions are obtained when gel filtration is combined with other LC techniques.
Since large molecules elute earlier than small ones, proteins and peptides will elute in the buffer used to equilibrate the gel filtration column, regardless of the original sample solvent.
GF can thus be used for desalting, buffer exchange and removal of low Mr contaminants.
Compared to other LC techniques the resolution in GF may be judged as relatively low. However, its unique selectivity allows dimers and larger polymeric forms of the target molecule to be separated from monomers and denatured forms may be separated from native ones.
All this together makes GF especially well-suited to so- called polishing.
Gel filtration is commonly used in two different modes:
1. High resolution mode to separate large molecules like proteins, peptides oligonucleotides, polysaccharides etc (Figures. 1 and 2).
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Fig 1. High resolution mode -
Peptides separated on Superdex Peptide
Fig 2. High resolution mode for polishing -
Removal of polymers.
2. Group separation mode (Fig 3) to separate large molecules (Mr >5000) as a group from small molecules like salts and buffer ions.
Fig 3. Group separation mode -
Desalting a protein on a PD-10 column.
Group separation mode accepts much larger sample volumes than high resolution mode and can be performed at considerably higher flow rates.
Simple small gravity-driven columns are normally used to desalt samples with volumes ~1ml. So-called spin columns, used e.g. for post-PCR clean-ups or for clean up after labelling oligos are examples on chromatography driven by centrifugation. The great advantage with centrifugation-driven columns is that a very large number of samples can be dealt with in parallel. |
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