(i) Gel permeation or gel filtration : Size exclusion chromatography (SEC) is a cinematographic method in which particles are separated based on their size, or in more technical terms, their hydrodynamic volume. The name gel permeation chromatography is used when an organic solvent is used as a mobile phase. The main application of gel permeation chromatography is used to analyse the molecular weight distribution of organic-soluble polymer.
Theory and method : The underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates. This results in the
separation of a solution of particles based on size, provided that all the particles are loaded simultaneously or near simultaneously.
Particles of the same size should elute together. This is usually achieved with an apparatus called a column which consists of a hollow tube tightly packed with extremely small porous polymer beads designed to have pores of different sizes. These pores may be depressions on the surface or channels through the bead. As the solution travels down the column some particles enter into the pores. Larger particles cannot enter into as many pores. The larger the particles, the less overall volume to traverse over the length of the column and the faster the elution.
The filtered solution that is collected at the end is known as the elute. The void volume includes any particles too large to enter the medium and the solvent volume is known as the column volume.
(ii) Electrophoresis : It is the method of moving charged particles through a medium under the influence on an electric field. It is also used to separate molecules with different physical characteristics using electrical charges.
Types :
• Capillary electrophoresis — commonly used to separate biomolecules by their charge and frictional forces.
• Gel electrophoresis — a technique used by scientists to separate molecules based on physical characteristics such as size, shape, or isoelectric point. It can be used as a preparative technique to partially purify- molecules prior to use of other methods such as mass spectrometry. PCR, cloning, DNA sequencing, or immuno-blotting for further characterization.
Examples of specific techniques include :
• DNA electrophoresis — a specific type of gel electrophoresis used to analyse DNA.
• Protein electrophoresis — a specific type of gel electrophoresis used to analyse proteins.
• Two-dimensional gel electrophoresis — a specific type of gel electrophoresis commonly used to analyse proteins which involves two separation mechanisms to separate molecules.
• SDS PAGE, (sodium dodecyl sulphate polyacrylamide gel electrophoresis) — commonly used to analyse proteins.
