The chromatographic method works on the basis of four different separation mechanisms, such as partition, adsorption, size exclusion, and ion exchange.
What is size exclusion chromatography?
Size exclusion chromatography is a type of chromatography also known as gel chromatography. It is a powerful separation technique based on the molecular size of the components, which is used to separate the components, especially the high-molecular-mass space. In this method, the porous material is used as a stationary phase and liquid is used as a mobile phase. Separation is achieved through the differential exclusion of the sample components as they pass through a bed of porous particles from the pores of the packing material.
Separation mechanism of size exclusion chromatography:
The column bed of size exclusion has three practical components i.e. pore volume, void volume, and matrix volume.
The pore volume: The pore volume corresponds to the space of pore-lumen inside the particles.
The void volume: It refers to the excluded volume, that is, the space amongst the particles.
The matrix volume: It refers to the solid component of the particles which fill the bed of the column.
The analytes larger than the size of the pore cannot penetrate the hole and are separated together as the first peak in the chromatogram. Analytes that can enter the pores will have an average residence time in their particles which depends on the size and shape of the analytes. Therefore, different analytics have dissimilar total travel time through chromatographic columns. Analytes have small in size and reside the longest on the column, which eluted together in the form the last peak in the chromatogram.
Different types of size exclusion chromatography:
There are two basic types of size exclusion chromatography are gel permeation chromatography (GPC) and gel filtration chromatography (GFC).
Gel permeation chromatography: In this type of size exclusion chromatography, uses a hydrophobic column packing material and a non-aqueous organic solvent uses to determine the molecular weight distribution of the molecule.
Gel filtration chromatography: In this type of size exclusion chromatography uses a hydrophilic packing material of columns and an aqueous mobile phase to separate and determines the molecular weight distribution of water-soluble molecules.
Principle of size exclusion chromatography:
The selection of analytes based on their molecular size and shape uses the molecular sieve properties of a diversity of porous materials. Possibly the most widely used of these materials is a group of polymeric organic compounds that have a three-dimensional pores network that gives them gel properties. The basic principle of exclusion chromatography is quite easy. The gel particle of the column is in equilibrium with an effective solvent or mobile phase for separating the molecules. Large size components that are excluded from the pores can pass through the interstitial spaces, as smaller size components are distributed amongst the mobile phase inside and outside the molecular sieve and after that pass at a lower rate through the column.
Commonly asked questions on chromatography are as follows.
What is the size exclusion chromatography used for?
Size exclusion chromatography is mainly used for separating compounds according to their molecular size. It is also used for purification, molecular weight determination, desalting, and protein binding studies.
What is the stationary phase in size exclusion chromatography?
The stationary phase if size exclusion chromatography consists of a column which is commercially available with a group of various packing material pore sizes. The column of the SEC is packed with fine, porous beads, made of dextran polymer, agarose, or polyacrylamide.
Why is size exclusion chromatography important?
The size exclusion chromatography is an important technique in the fractionation of proteins, determining the fine structure of analytes, fractionation of water-soluble polymers, as well as the relative amount of certain analytes in foodstuffs.
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