Gel permeation or size exclusion chromatography: Principle, procedure & applications
How does Size exclusion chromatography work
Size exclusion chromatography is a type of column chromatography. It has a little modification in that the column is filled or packed with a stationary phase which can act as molecular sieve.
A porous material is used as stationary phase while a liquid as mobile phase. The diameters of pore are in the order few armstrongs similar to that of molecules.
Size exclusion chromatography Principle: Here the principle involved is that molecules move through the column under the influence of mobile phase based on their relative physical size or mass.Those with smaller size or mass move through the all possible pores due to their small size and travel a longer path and get eluted last. But large molecules travel only through few possible pores due to their size and pass out of the column first as they travel shorter path.
The technique is also called as molecular sieve filtration, gel filtration, exclusion chromatography.
Procedure & Applications
1. The dry powder is allowed to swell in the solvent which is used as mobile phase.
2. Then the gel is packed in the column to a sufficient height.
3. The mixture to be separated is mixed in the mobile phase and then poured over the top of column.
4. The outlet elute is collected in different beakers based on suitable time intervals to obtain size differentiated elutes separately.
♣ It is a general purification process for any kind of sample.
♣ It is efficiently used to separate organics like sugars, polypeptides, polystyrenes etc.
♣ It helps in molecular weight determination.
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