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 filtration Chromatography:
When size-exclusion Chromatography is performed using the aqueous mobile phase it is known as the Gel Filtration Chromatography.
Gel Permeation
Chromatography:
When size exclusion Chromatography is performed using the organic
mobile phase it 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 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 phase.
The choice of the mobile phase is to depend 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 perform within the broad PH. The buffer should not be cause 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
Low quantity of the sample required for size exclusion
Chromatography
Disadvantages
Sensitive equipment
Expensive
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