Column Chromatography: Gel - filtration or Size - exclusion Chromatography

Column Chromatography: Gel - filtration or Size - exclusion Chromatography


Column Chromatography: Gel - filtration or Size - exclusion Chromatography

Column Chromatography: It refers to the separation of different types of proteins from each other based on two phases -
a) Solid stationary phase
b) mobile phase

Gel - filtration or Size - exclusion Chromatography

It is a chromatographic technique that separates dissolved molecules based on their size by pumping them through column.

Basis of separation -
 differences in shape or mass of proteins or molecules.

Molecules to be separated -
 natural a dsynthetic polymers, biopolymers, proteins and nano particles.

Principle of Gel - filtration Chromatography:

1. The stationary phase consists of gel beads with pores that span relatively narrow size range.
2. The column is packed with gel beads and the smaller molecules are able to enter the pores of the beads and trapped.
3. Large molecules pass the beads and are quickly eluted.
4. The average residence time in the beads depends on the size of the molecules.
The smaller the protein, the longer it is retained within the beads. They are eluted from the column in order of decreasing molecular size.


Methods of Gel - filtration Chromatography:

1. Setting the column and cleaning them with buffer.
2. Pouring the gel beads into the column to form packed bead.
3. Injecting or percolating the protein containing solution in the range of 2-5% of the column volume.
4. Large proteins are eluted and when the protein of interest is pure and present in a small, concentrated volume, they are flushed with an elution buffer.
5. The eluted proteins are collected as a series of fractions.
6. Beads are made of dextran, agarose, vinyl polymers.
The higher the degree of crosslinking, the smaller the average pore size.
7. Buffer: Tris buffer, Phosphate buffer solution.

Application of Gel - filtration Chromatography:

1. Fractionation of proteins and other water-soluble polymers.
2. Used to analyze the molecular weight of organic soluble polymers.
3. Used for the purification of proteins,  nucleic acids and polysaccharides.

Advantages of Gel - filtration Chromatography:

1. Doesn’t depend on temperature, pH, ionic strength,  buffer composition.
2. Very little adsorption.
3. Less zonal spreading  than any other techniques.
4. Elution volume is related to molecular weight.
5. No sample loss become solutes don’t interact with the stationary phase.
6. Various solutions can be applied.

Disadvantages of Gel - filtration Chromatography:

1. Limited sample volume.
2. Filtration must be performed before using the instrument to prevent dust.
3. Time - lengthy.
4. Chance of clogging.
5. Expensive.


"Column Chromatography: Gel - filtration or Size - exclusion Chromatography"
Written By
Sadia Akhtar
Student of Department of Microbiology
Jagannath University.
Email- sadiabd810@yahoo.com

Post a Comment

0 Comments