This document discusses gel filtration chromatography, also known as size exclusion chromatography. It separates molecules based on size - larger molecules elute faster while smaller molecules can enter the pores of the stationary phase gel beads and elute later. Various types of gels are described like Sephadex, Sepharose, polyacrylamide, agarose, and controlled pore glass. Parameters affecting separation like pore size, molecular weight range, flow rate, and column preparation are covered. Applications like purification of biomolecules, desalting, and molecular weight determination are also summarized.

1. Gisha G P
MSc Biotechnology
Mahatma Gandhi
University, Kottayam

2. Introduction
• Gel filtration
(chromatography), is
also known as
molecular sieve /
molecular exclusion
chromatography.
• Gel filtration
chromatography
separates molecules
according to their size
and shape.
Advantages
1. Gentleness of the
techniques permits
separation of liable
molecular species
2. 100% solute
recovery
3. Excellent
reproducibility
4. Comparatively short
time & relatively

3. Principle
• The stationary phase
consists of gel beads
containing pores that
span a relatively narrow
size range.
• Separation is achieved by
differential distribution of
sample component b/w
the stationary solvent
within the pores of a gel
& the mobile eluting
solvent out side the
pores.
• travel through the column
faster but small
molecules are “included”
– can diffuse into the
pores and elute later

4. • Degree of retardation
of molecules is a
function of the
molecules size and
pore diameter.
Exclusion limit –
molecular weight of
the smallest molecule
incapable of entering
the gel pores.

5. Column Parameters
Vs= volume of solvent held in the pores.
This is normally approximated to
Vt-Vo = volume of beads
Vo = Elution volume of a large “totally
excluded” molecule such as blue
dextran
Vt = Physical volume of column

6. Ve = Vo + KdVt
where Ve = effluent
volume
Vo = void
volume
Kd =
distribution
coefficient
Vt = volume of
solvent
Vt = α Wr
α = dry wt. of gel
Wr= water
regaining capacity
Kd = 0, for large
molecules
Kd = 1 , for small
molecules
Kd = -1 , for intermediate
molecules

7. Types of gel
Characteristics of gel
material
• Chemically inert
• Wide choice of pore
and particle size
• Uniform particle and
pore size
Types
• Sephadex (cross
linked dextran)
• Sepharose /Bio-Gel A (
agarose )
• Bio-Gel P (
polyacrylamide)
• Bio Glass /Porasil (
Porous glass&
silica
granules )
• Styragel / BioBeads S

8. Sephadex
• Popular gel for
biomolecule separation .
• A 1-6-polymer of glucose
is prepared by microbial
fermentation of sucrose
(glucose + fructose)
• The resulting glucose
provides the required α1-
6 glucosan polymer
called dextran
• The resulting dextran is
treated with
epichlorohydrin to give
Sucrose
Microbial fermentation
Specific PH
Glucose + fructose
Dertan (a-1-6 glucosan polymer)
Gl-Gl-Gl
O
CH2
CH
CH2
OH
O
Gl-Gl-Gl n
Gl-Gl-Gl-
OH
Gl-Gl-Gl-
OH
+
CH2Cl
HOHC
CH2Cl
Sephadex

9. • Pore size controlled
by molecular wt. of
the dextran & amount
of epichlorohydrin
used.
• By controlling cross
linking reaction ,
various class of gel
beads with exclusion
limits b/w 1 –
200,000 Da can be
Characters of sephadex
1- highly stable gels
2- stable at PH 2-12
3- their particles are free
from ions
4- insoluble in water and
organic solvent
5- they swell in water and
other
hydrophillic solvent
6- they require bactericidal
such as

11. Polyacrylamide
• Can become
compressed in the
column, cause slow
flow rates.
• Insoluble in water and
common organic
solvents , pH 2-11.
• Polymerized
acrylamide into bead
form.
• Numbered like P-10
, P-100.
• Number multiplied by
factor of 1000 indicate
exclusion limit in Da.
• Used to separate
molecules up to
300,000 Da .
• Large pored gels lack
NH
O O
NH
O
NH
NH
O
NH
O
H
N
O
n

12. Gel type Fractionation range in
Molecular wt units
Hydrated bed volume
ml/g of dry gel
Water regain
ml/g of dry gel
Bio Gel P-2 200-2,000 3.8 1.6
P-4 500- 4,000 6.1 2.6
P-6 1,000 – 5,000 7.4 3.2
P-10 5,000- 17,000 12 5.1
P-60 30,000 – 70,000 18 6.8
P-100 40,000 – 100,000 22 7.5
P-200 80,000 – 300,000 47 13.5

13. Agarose
• Use for study of
viruses , nucleic acids
and polysaccharides.
• Stable at pH 4-10
• Freezing temperature
and temperature
above 30 C cause
alterations in gel
structure
• Chromatography
performed b/w
0 C & 30 C
• Produced from agar.
• Linear
polysaccharides of
alternating residues of
D- galactose & 3,6-
anhydro-L- galactose.
• Hydrophilic , free of
charged
groups, completely
inert.
• High porosity, use to
separate
biomolecules of

14. Gel type Fractionation range in
Molecular weight units
Agarose 0.5m (10%) 10,000 to 250,000
1.0m(8%) 25,000 to 700,000
2.0m(6%) 50,000 to 2,000,000
15.0m(4%) 200,000 to 15,000,000
50.0m 100,000 to 50,000,000
150m 500,000 to 150,000,000

15. Styragel
• Hydrophobic gel used for
complete aqueous
separation.
• Polymerized styrene
, cross linked by divinyl
benzene.
• Swells in organic solvent
, rigid than hydrophilic
gels.
• Unaffected by high
temperatures up to 150 C
• Solvents –
tetrahydrofuran
Type Fractionation range
in
Mol.wt. Units
Approximate
exclusion limit in
mol.wt. Units
(Average porosity in
A°)
60 styragel 800 1,600
100 2,000 4,000
400 8,000 16,000
1x10 3 20,000 40,000
5x103 100,000 200,000
10x103 200,000 400,000
30x10 3 600,000 1,200,000
1x105 2,000,000 4,000,000
3x105 6,000,000 12,000,000
5x10 5 10,000,000 20,000,000
10x10 5 20,000,000 40,000,000

16. Controlled pore glass
beads
• Fine glass spheres of
porosilicate glass
• Large no. of narrow
sized pores.
• High flow rate , high
rigidity.
• Adsorb significant
amount of protein on
their surface
• To avoid this –treat
with
hexamethyldisilazane.

17. • Sephacryl HR:
Sephacryl High
Resolution (HR) is a
composite gel
prepared by
covalently cross-
linking dextran with N,
N'-methylene
bisacrylamide to form
a hydrophilic matrix of
high mechanical
strength.
• Superdex:
It is based on highly
cross-linked porous
agarose beads to
which dextran has
been covalently
bonded.

18. Column preparation
• Gel must be swollen
, attain equilibrium.
• Greater porosity
much time for
equilibration.
• Previously swollen gel
is added in form of
slurry & allowed to
settle.
• Air bubble should not
be formed.
• Equilibrate the

19. Sample application
• Considerable care must be taken to avoid
disturbing the bed surface.
• 1) Close the outlet and remove most of the
buffer above the gel surface by suction.
• 2) Layer the sample on top of the bed.
• 3) Open the column outlet and allow the sample
to drain into the bed
• 4) Wash the sample which remains on the bed
surface and on the column wall into the bed
with a small amount of eluent.
• 5) Refill the column with eluent and reconnect
to a Mariotte flask or pump.

20. Elution & flow rates
• Samples are eluted
from column using a
single buffer .
• Resolution decreases
as flow rate increases
• Allow time for
molecules to diffuse
in & out of matrix b/w
mobile phase &
stationary phase in
order to achieve a
good separation .

21. Precautions
• Preparing the gel
from too thin a
suspension or
packing the column
in stages, often
results in a badly
packed bed.
• Avoid disturbing the
bed surface , an
uneven bed surface
leads to uneven
separated bands and
• Damaging of matrix
affect separation
process , since the
fractionation is based
on pore size.
• Buffer and matrix
should be degassed
, air bubbles entering
the column can lead
to poor resolution.
• Experimental set up
should be maintained

22. Thin layer gel chromatography
• First done by
Determann
, Johanson , & Rymo.
• Used for clinical
studies.
• Small sample volume.
• Hydrated gel is
applied to the plate
, placed on an air tight
container at an angle
of 20 .
• Plate is connected to
• Equilibration carried
out for
at- least 12 hrs.
• Sample applied either
as spot or
as a band.
• The plate is then
developed and
separated
components detected
by suitable methods .

23. Applications
1- separation of large
molecular weight
compound as
protein, carbohydrate, pe
ptides, nucleic acids
2- desalting of colloids
3- molecular weight
determination
(A linear relationship exists
between the logarithm of
the molecular weight and
the elution volume)
Separation of large
molecular weight
compounds
• Chief use of gel filtration
• Ultimate purification
• Protein , enzymes ,
hormones , antibodies,
nucleic acids ,
polysaccharides and
even viruses can be
separated
• Low molecular weight
compounds such as
amino acids , small

24. Desalting of
colloids
• Removal of salt&
small molecules from
macromolecules.
• Easily performed in
gel filtration ,
distribution coefficient
of salt molecules
differ from
macromolecules
Molecular weight
determination
• Distribution coefficient
of a given
macromolecule is a
function of size &
shape.
• Ve = Vo + KdVt
• Vt = α Wr
• Kd = Ve - Vo
α Wr

25. • Distribution coefficients
of standard proteins of
known molecular wt.
are plotted against log
of their mol.wt.
• shape of proteins vary
– error in mol.wt.
determination
• Solvent confers
identical shapes –
guanidium chloride.(6M
, pH 6)
Calibration curve
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
4 4.5 5 5.5 6
Lg Mol Wt
Kav

26. References
• Gel filtration
,principles and
methods , Amersham
Biosciences .
• Biophysics ,
Vasudeva Pattabi,
N. Gautham
• High Resolution
Chromatography , A
Practical Approach ,
Edited By P.A Millner
• Separation and
Purification
• www. edvoteck/
Principles of Gel
filtration
Chromatography/
• www.sigmaaldrich.co
m
• Gel filtration
chromatography ,
Lave Fischer ,
Elsevier / north –
Holland Biochemical
press 1980