Size Exclusion Chromatography: Principle, Definition and types of chromatography

Size Exclusion Chromatography (SEC): Definition

Size Exclusion chromatography is one of HPLC technique which separates the component base on their size and in some case by the molecular weight and the shape of the molecules.

Size Exclusion Chromatography is generally used for the Separation of a large molecule or the macromolecule.

It also is known as the gel permeation chromatography and gel filtration chromatography depends on the Mobile phase used in Size exclusion Chromatography.

Gel Permeation Chromatography:

When size exclusion Chromatography is performed using the aqueous mobile phase is known as the Gel Filtration Chromatography.

Gel Permeation Chromatography:

When size exclusion Chromatography is performed using the organic the mobile phase is known as the Gel Filtration Chromatography.

Size Exclusion Chromatography Principle

The Basic principle of the Size Exclusion Chromatography is Smaller the particle of the analyte is trapped into the Column bed. The column bed act as sieve which trapped the smaller molecule of the analyte easily, Hence Smaller the molecule of the analyte, it retains for more period time in SEC column temporarily

Large molecules are not trapped in the column bed. Large molecule passes around the bed and excluded from the column bed. Larger the molecule quickly pass through the column

Large molecules are eluted first from the column and then small molecules are eluted

In other terms, we can say that large molecule has less retention time while the small molecule has more retention time.

Component of Size Exclusion Chromatography:

Stationary Phase:

Chromatography column contain Stationary phase in the form of sphere or bead, It is also known as a column bed. The beads of Stationary phase are semipermeable, porous cross link polymer.

Smaller pore size for rapid desalting of protein or for protein purification. Intermediate pore size is used to separation of a relatively small protein. Large size is used for separation of biological molecular

E,g. Dextran (Sephadex),, Poly Acrylamide ( Sephacryl or BioGel P)., Agarose (Sepharose),

The stationary phase is chemically inert and stable; it does not react with the mobile phase or analyte to separate the component. ‘

Mobile Phase:

Aqueous and non-aqueous both are used as mobile phases.

The choice of the mobile phase is depending on the type of separation to be achieved and material needs to be separated.

How it Works:

Polymer solution (Analyte) is dissolved on the mobile phase pass through the column. That solution passes through the column, The sample molecule passes through the column which contains the stationary phase in the form of a bead. Which also known as the column bed.

The sample molecule enters into the column, it moves along the column. The larger molecule excluded from the pores and travels through the column and eluted first while the small molecule enters into the pore and temporarily traps with the stationary phase (column Bed). Thus a molecule separated from each other. measured by one or more detectors when they elute from the column.

Buffer is used in Size exclusion chromatography. Separation should be performed within the broad PH. The buffer should not because of the inactivation of or precipitation. It should maintain the stability of the analyte.

Advantages of Size Exclusion Chromatography

The sample is not decomposed in Size exclusion chromatography

The low quantity of the sample required for size exclusion Chromatography

Disadvantages

Sensitive equipment

Expensive