What Is Gel Chromatography?
Chromatography is an excellent science of separation. In many fermentation processes, chromatographic techniques are used to isolate and purify relatively low concentrations of metabolic products. Chromatography is basically concerned with the passage and separation of different solutes as liquid is passed through the column. Chromatographic techniques have great advantages in purification of various products.
Chromatography can be classified in various types, depending upon the mechanism of separation. These different types of the chromatography are-
1. Adsorption chromatography
2. Ion-exchange chromatography
3. Gel permeation chromatography
4. Affinity chromatography
5. Reverse phase chromatography
6. High performance liquid chromatography
Gel chromatography separates bimolecular on the basis of size. This technique uses a molecular sieve in the form of polysaccharide gel that comprises spherical beads. As these beads are tiny and porous, so they allow only small molecules to enter the gel, while the larger molecules remain outside these beads. Therefore, when the column is eluted with an appropriate buffer, larger molecules pass out through the column. At a faster rate then smaller ones. This technique is also called as size exclusive chromatography.
Phases of chromatography
The components to be separated are distributed between two phases: a stationary phase and a mobile phase, which percolates through a stationary bed. The stationary phase basically comprises a column field with porous beads.
Spherical porous beads of defined sizes are used to pack the chromatography column. The porous gel may comprise cross-linked dextrans (sephadex), cross-linked poly-acrylamide (bio- gel P) or cross-linked agarose (sepharose). When columns are created they are packed with porous beads with a specific pore size so that they are most accurate at separating molecules with similar to the pore size.
A sample comprising a mixture of proteins is dissolved in the same solvent that is running through the column. The various molecules of this mixture get flushed through the column at different rates. The separation of these molecules depends on whether they can fit with in the pore size of packing material.
As a molecule flow through the column, it passes by a no. of porous beads. Small molecules enters the porous beads and hence, their rate of passage through the gel is retarded as compared to that of the larger molecule that can not fit into the pores and continue moving along the solvent. So, molecule with the larger size reaches the end of the column before the molecule with smaller size do. Small amount of solvent eluted through the gel are collected in different tubes and the protein present in each sample are analyzed.
Applications of the gel chromatography
Gel chromatography is a very efficient method for the separation, analyzing and characterization of the mixture of macro-molecules.
1. In nay chemical or bioprocessing industry, separation and purification of a product from complex mixture is very important. Gel chromatography can separate complex mixtures with great precision.
2. Chromatography is well suited to a variety of uses in the field of biotechnology, such as separating mixture of proteins.