Gel permeation chromatography – Revolutionary separation of polymers made uncomplicated
Gel permeation chromatography is a type of column chromatography where it is based on the separation of particles purely based on the size of the sample. This technique is also known as Size Exclusion Chromatography(SEC) or Molecular-sieve chromatography.
As the name suggests, gel permeation chromatography means that the sample will pass or permeate through a gel and the separation will be on the basis of the size. This is the reason why it is known as size exclusion chromatography. This mode of separation is not based on the molecular weight, but only on the size of the material being analyzed(usually a polymer).
In-gel permeation chromatography, the solvent is allowed to form two phases namely,
- Stationary Phase
- Mobile Phase.
In the column packed with microporous gel particles, the stationary phase is made up of the part of solvent which is inside the gel. The mobile phase is made up of the flowing part of the solvent which is outside the gel particle. The gel used are hard and incompressible polymer.
In this technique, the column is filled with a gel which acts as a stationary phase. The gel mostly used are microporous glass beads and micro polystyrene gel. The gel is made up of porous beads. The commonly used gel are:
- Polyacrylamide gels.
These all have porous structures.
The total volume occupied by the gel in the column(Vt) can be given as the sum of the volume occupied by the gel beads(Vg) plus the inner volume of the gel beads(Vi) and the free volume that exits outside the beads(Vo). In short, Vt = Vg+Vi+Vo.
Working of gel permeation chromatography
If the sample molecules are very small, they can easily enter the pores of the beads. And if the sample molecules are large they fail to enter the pores of the beads.
The top of the column is attached to the pump, usually, a peristaltic pump, which is continuously pumping the mobile phase in the column. The bottom of the column is attached to the detector. The detector can either be a refractive index or UV detector or an infrared detector. The choice of detector is based on the type of sample.
An amount of polymer in a known volume of solvent is injected into the solvent stream flowing down the gel-packed column. The entry of large size polymer molecules into the gel pores is more restricted due to the small size of the pores and they flow outside the gel beads. Thus spend less time in gel and are eluted faster from the column.
On the contrary, the small size polymer molecules enter the pores of the gel and spend more time in the gel. Therefore they are eluted after a very long time from the gel column.
Thus, the large size molecules are eluted faster from the gel column and small sizes molecules are eluted slower. This technique is called gel permeation chromatography, which allows the fractionation of polymer molecules according to their sizes.
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