2. Size
Exclusion
Chromatography
Gel Filtration
For proteins and
Water-Soluble
Polymers using
aqueous mobile
phase
Gel Permeation
Chromatography
For
polar and Organic-
Soluble Polymers
using organic
mobile phase
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3. Definition:
Gel filtration is a technique of partition
chromatography in which the partitioning is
based on the molecular size of the substances to
be separated.
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4. Gel filtration is well suited for:
biomolecules that may be sensitive to changes in
pH,
concentration of metal ions or cofactors and
hard environmental conditions.
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5. Principle of gel filtration (cont.):
Porous matrix spherical particles chemically
and physically stable and inert (no reactivity and
adsorptive properties).
The packed bed is equilibrated with buffer which
fills the pores of the matrix and the space in
between the particles.
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6. Principle of gel filtration (cont.):
The liquid inside the pores is referred to as the
stationary phase and
This liquid is in equilibrium with the liquid
outside the particles, referred to as the mobile
phase.
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8. Principle of gel filtration (cont.):
It should be noted that samples are eluted
isocratically, i.e. there is no need to use
different buffers during the separation.
However, a wash step using the running buffer
is usually included at the end of a separation to
facilitate the removal of any molecules that may
have been retained on the column and to
prepare the column for a new run.
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9. Principle of gel filtration (cont.):
Separations can be performed in the presence of:
Essential ions or cofactors
Detergents
Urea
Guanidine hydrochloride
At high or low ionic strength
At 37 °C or in the cold room according to the
requirements of the experiment
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10. Characterization of solute behavior:
Vt or total column volume:
Refers to total volume occupied by the gel in the
column
Vo or void volume:
space outside the granules
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Some definitions:
11. Characterization of solute behavior (cont.):
Volume of gel matrix:
Vt – Vo = volume occupied by gel space, including
gel matrix (Vgel)
Stationary phase:
(Vs) = Vt-Vo-Vgel
This is difficult to measure
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12. Size of column:
Choice depends on your purpose - preparative
or analytical.
What is the volume of your sample?
2.5 x 33 cm column - about 5 ml sample
5.0 x 100 cm column - about 15-20 ml sample
For better resolving power: a longer column is
recommended
For analytical purpose:
2.5 diameter column - satisfactory
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13. Samples for Separation:
Nature of samples:
Ensure sample free from particles & precipitates
that can cause:
surface blockage flow rate irregular
sample flow
Sample viscosity must not drastically deviate
from that of the eluent otherwise distorted sample
zones will form. Avoid viscosities larger than 4 cP
or protein concentrations above 5%.
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14. Types of Gels:
It depends on the size of the proteins to be separated.
Name Type Fractionation range
Sephadex G25 Dextran 1-5
Sephadex G100 Dextran 4-150
Sephadex G200 Dextran 5-600
Bio-Gel P30 Polyacrylamide 2.4 –40
Bio-Gel P300 Polyacrylamide 60-400
Sepharose 6B Agarose 10-4,000
Sepharose 4B Agarose 60-20,000
15. How to Increase Resolution:
Keep within the optimum fractionation range.
Reduce the sample volume.
Change to a gel with smaller beads.
Connect two columns in series.
Reduce the flow rate.
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17. Additional Considerations:
Non-specific adsorption rarely occurs with agarose
or dextran-based GF media. However, if peaks are
unexpectedly retarded, show tailing or if poor
recovery is experienced, ionic or hydrophobic
interactions may be suspected.
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18. How to Solve this problem:
Buffer should contain > 0.05M NaCl to avoid
ionic interactions.
Hydrophobic interactions may be avoided by
adding 25% acetonitrile; EtOH or isopropopanol.
Alternatively add 10% ethyleneglycol.
19. General uses of gel filtration procedures:
Desalting: removing small molecular weight
compounds from protein samples.
Preparative separation: fractionation of
protein.
Analytical use: estimation of molecular size of
proteins.
20. 1) Desalting (group separation):
Separates the target protein from low-molecular
mass contaminants.
The pore size of the matrix
exclude the protein of
interest
allow maximum
permeation of the
contaminants
21. Chromatogram illustrating desalting of proteins by gel filtration.
A 4 × 85–cm column packed with Sephadex G-25 is used. The
sample consists of 400 ml hemoglobin (protein peak; solid line)
in NaCl (salt peak; broken line)
22. 2) Protein fractionation:
Refers to the
separation of
proteins of
similar
molecular size
for purification
purposes.
The objective may
be to separate
dimer and higher
aggregates from a
pharmaceutically
active protein or
peptide.
23. Chromatogram illustrating protein fractionation by gel filtration. A
Superdex 75 HR 10/30 column is used. The sample consists of
recombinant human growth hormone.
24. 3) Determination of molecular size:
In simple manual columns: eluent is collected in
constant volumes fractions of the same size
can’t be detected separately More advanced
columns overcome this problem by constantly
monitoring the eluent.
25. Determination of molecular size (cont.):
Columns are often calibrated using 4-5 standard
samples (e.g., folded proteins of known
molecular weight), and a sample of the very
large molecule blue dextran to determine the
void volume.
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26. Determination of molecular size (cont.):
The elution volumes
of the standards are
divided by the
elution volume of the
blue dextran (Ve/Vo)
and plotted against
the log of the
standards' molecular
weights.
