Gel filtration

chromatography

known as molecular exclusion

chromatography, or Molecular Sieve
chromatography or permeation
chromatography
Gel filtration chromatography

 This technique separates proteins based on size

and shape
 does not rely on any chemical interaction with
the protein, rather it is based on
a physical property of the protein - that being
the effective molecular radius (which relates to
mass for most typical globular proteins).
 This is the only chromatographic technique which
does not involve binding of the protein to a
support
 The solution mixture containing molecules of
different sizes( say protein) is applied to the column
and eluted with a buffer.

 The larger molecules can not pass through the pores

of a gel and therefore move faster.

 The smaller molecules enter the gel beads and are

left behind which come out slowly. By selecting the
gel beads of different porosity, the molecules can be
separated.
 Separates molecules on basis
of size and shape.

Consist of stationary phase

made up of polysaccharide
like dextran cross linked to
form three dimensional
network in shape of beads.

Smaller molecules spend more

time inside beads, thus eluting
later than larger molecules
which bypass beads and
reaches bottom faster.
 Principle
Principle
Separation of biological molecules is achieved on the basis of SIZE and SHAPE

Small molecules are “included” – can diffuse into

the pores and elute later.

Intermediate size molecules move partially inside

the pores of the bead.

Large molecules are “excluded” from the pores

and travel through the column fastest.
Gel filtration chromatography-An
Overview

Larger particles come out first, Biochemistry

while smaller particles come in later fractions
of Medics 8
 Stationary phases commonly used for exclusion
chromatography

1. Dextran Sephadex G-10,G-25, G-50,G-100, G200

2. Dextran, cross-linked Sephacryl S-100 S-200 S-300 S-400

3. Agarose Sepharose 6B, 4B,2B

4. Polyacrylamide Bio-Gel
Trade
 Cross linked dextrans, agarose
(sepharose),polyacrylamide and porous glass gels
are used as column packing material
 Gel should provide uniform packing density to
avoid brand broadening
 Resolution is directly proportional to square root
of length of column
 Elution time is proportional to length of column
 Sample should not be viscous
 Lower flow rate of eluent are preferable for
better resolution
 After use gel can be washed repeatedly with
eluent buffer and can be stored .
• If the analyte is large and completely excluded
from the mobile phase within the particle, Kd
= 0, whereas, if the analyte is sufficiently small
to gain complete access to the inner mobile
phase, Kd =1.
• hence Kd values vary between 0 and 1
 Applications
 Can be used in determination of protein structure
particularly protein tertiary structure as it gives idea
about hydrodynamic radius of protein
 In desalting of various bio-macromolecules
 Purifications of enzymes and other proteins.
 Viruses, enzymes, hormones, antibodies, nucleic acids
and polysaccharides have all been separated and
purified by use of appropriate gels or glass granules
 Estimation of molecular sizes of proteins
 A polishing step in multistep complex purification
scheme applied particularly in industrial purification of
recombinant proteins
• the elution volume of proteins from gel
filtration columns depends on their mass.
Thus, if we determine the elution volumes of
proteins of known mass on a given gel
filtration column, we can easily determine the
mass of a protein of unknown.
 Advantages
 Good separation of large and small molecules using
minimal volume of eluate
 Doesn’t affect biological activity of particles to be
separated
 No sample loss altogether as solute interact very less
with stationary phase.
 Doesn’t depends upon a particular pH, temp., ionic
strength and buffer composition, so can be carried out in
any conditions required. so elution is carried
out  isocratically.
 High resolution can be achieved
 A wide range of porous gels are available commercially.
 Disadvantages
 For good resolution there has to be a 10% difference
in molecular mass of solutes.

 Limited number of bands can be accommodated as

time scale of chromatogram is short.

 Good expertise is required to achieve best results.

 Standards are needed.
 High Investment cost