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| Column Chromatography: Gel - filtration or Size - exclusion Chromatography |
Column
Chromatography: Gel - filtration or Size - exclusion Chromatography
Column
Chromatography: It refers to the separation of different types of
proteins from each other based on two phases -
a) Solid
stationary phase
b) mobile
phase
Gel -
filtration or Size - exclusion Chromatography
It is a chromatographic technique that separates
dissolved molecules based on their size by pumping them through column.
Basis of
separation -
differences in shape or mass of proteins or
molecules.
Molecules
to be separated -
natural a dsynthetic polymers, biopolymers,
proteins and nano particles.
Principle
of Gel - filtration Chromatography:
1. The
stationary phase consists of gel beads with pores that span relatively narrow
size range.
2. The
column is packed with gel beads and the smaller molecules are able to enter the
pores of the beads and trapped.
3. Large
molecules pass the beads and are quickly eluted.
4. The
average residence time in the beads depends on the size of the molecules.
The smaller the protein, the longer it is
retained within the beads. They are eluted from the column in order of
decreasing molecular size.
Methods of
Gel - filtration Chromatography:
1. Setting
the column and cleaning them with buffer.
2. Pouring
the gel beads into the column to form packed bead.
3. Injecting
or percolating the protein containing solution in the range of 2-5% of the
column volume.
4. Large
proteins are eluted and when the protein of interest is pure and present in a
small, concentrated volume, they are flushed with an elution buffer.
5. The
eluted proteins are collected as a series of fractions.
6. Beads are
made of dextran, agarose, vinyl polymers.
The higher the degree of crosslinking, the
smaller the average pore size.
7. Buffer:
Tris buffer, Phosphate buffer solution.
Application
of Gel - filtration Chromatography:
1.
Fractionation of proteins and other water-soluble polymers.
2. Used to
analyze the molecular weight of organic soluble polymers.
3. Used for
the purification of proteins, nucleic
acids and polysaccharides.
Advantages
of Gel - filtration Chromatography:
1. Doesn’t
depend on temperature, pH, ionic strength,
buffer composition.
2. Very
little adsorption.
3. Less
zonal spreading than any other techniques.
4. Elution
volume is related to molecular weight.
5. No sample
loss become solutes don’t interact with the stationary phase.
6. Various
solutions can be applied.
Disadvantages
of Gel - filtration Chromatography:
1. Limited
sample volume.
2. Filtration
must be performed before using the instrument to prevent dust.
3. Time -
lengthy.
4. Chance of
clogging.
5.
Expensive.
"Column
Chromatography: Gel - filtration or Size - exclusion Chromatography"
Written By
Sadia
Akhtar
Student of
Department of Microbiology
Jagannath
University.
Email-
sadiabd810@yahoo.com
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