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Wednesday, September 14, 2011

Gel Filtration Chromatography (GF) / Size Exclusion Chromatography (SEC)


Gel Filtration Chromatography (GF) / Size Exclusion Chromatography (SEC)

Gel Filtration Chromatography (GF) / Size Exclusion Chromatography (SEC)

Gel filtration chromatography (GF) (also known as molecular exclusion chromatography) relies on a stationary phase consisting of spherical gel particles whose size and porosity are carefully controlled during manufacture.

The alternative names for Gel filtration chromatography are molecular sieve chromatography, gel permeation chromatography.

The main application of gel filtration chromatography / size exclusion chromatography is desalting, removal of salts from the sample.
When the mobile phase is passed through a column of this material, molecules are fractionated on the basis of their sizes and shapes. Small molecules are able to diffuse into the pores, while larger molecules are excluded. This occurs repeatedly as the mobile phase moves down the column, and as a result, during the elution process, the small molecules are retarded with respect to the larger molecules. This causes the smaller molecules to appear in the later fractions of the eluate.
Size is not the only factor that influences the rate at which solutes are retarded. For a given pore size, rod-like molecules will tend to be excluded with respect to spherical molecules of the same size. In addition, certain molecules may have some affinity for the stationary phase as a result of non-covalent interactions.

gel filtration chromatography, size exclusion chromatography

The solid support used in gel filtration chromatography is called media or matrix. it is available in different ranges depending upon the nature of impurity need to be removed. GE lifescience has the gel filtration matrix in the name of Sephadex G-10, Sephadex G-25Sephadex G-50Sephadex G-75 etc
G-25 has the molecular sieving ability of 1KDa - 5KDa and the chromatography matrix is available as G-25 coarse,G-25 medium and G-25 fine the classification is based on the particles.

Bio rad has gel filtration matrix in the name of Biogel P10, Biogel P4, Biogel P2, Biogel P30 etc, it is polyacrylamide based, the number denotes the molecular cut off Biogel P4 has molecular cut off range of 800 - 4000 Daltons(0.8 - 4 KDa)

Storage of Gel Filtration Matrix
If the chromatography matrix is not using for long time it can be stored at 4 degree Celsius with 20% Ethanol. while reusing after storage ethanol should be removed by repeated washing in distilled water.


Advantages / Applications of Gel Filtration Chromatography (GF) / Size Exclusion Chromatography (SEC):
  • Separation process is carried out under very mild conditions, from 37 ÂșC. to cold room conditions.
  • High resolution can be achieved.
  • A wide range of buffer systems may be used (buffer molecules and ions are generally excluded).
  • Small molecules or ions that stabilize the biomolecules may be added to the buffer.
  • Separation process is independent of ionic strength, so elution is carried out isocratically(only one buffer is used).
  • Gel filtration can be used for desalting and/or buffer exchange.
  • Gel filtration can be used to obtain an estimate of molecular size.
  • A wide range of porous gels are available commercially.
Precautions to be taken while using Gel Filtration chromatography Matrix
Since the fractionation or separation is based on the pore size of the particle (Matrix) damage in the particle can affect the separation process. 
Buffers and matrix should be degassed, air bubbles entering the column can lead to poor resolution.

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