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Gel Filtration
Gel permeation chromatography
Size exclusion chromatography
Separation of molecules on the basisof size (and shape)
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Theory
Column matrix
Porous beads
Large molecules are excluded from the pores
and travel through the column fastest
Small molecules are included can diffuse
into the pores and elute later
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Theory
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Column Parameters
Vs= volume of
solvent held in
the pores. This isnormally
approximated to
Vt-Vo = volume
of beads
Vo = void
volume
Vt = total
volume
Vo = Elution volume of a large totally
excluded molecule such as blue
dextran
Vt = Physical volume of column
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Calculation of Ve
For a molecule that can partially enter the
pores:
Ve = Vo + Kd (Vs)or Ve = Vo + Kav (Vt-Vo)
Kav = proportion of pores available to the
molecule.
Totally exclude Kav = 0 and Ve = Vo
Totally included Kav = 1 and Ve = Vt
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Behaviour of Molecule on any
Column
Kav = Ve VoVt - Vo
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Resolution
Resolution
0
0.5
1
1.5
2
2.5
1 2 3 4 5 6 7 8 9 10 11 12 13
Fraction number
Amoun
Resolution proportional to square root of column
length. Also affected by rate at which column is
run
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Design of Column
Column size
Analytical or preparative
Solvent
Inert matrix most solvents OK
Matrix
Most important consideration
Many different types Material
Pore size
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Matrix Types
Material Sephacryl
dextran
Sephadex
dextran
Sepherose
agarose
Superdex
mixture
Sephacryl Protein
(kD)
Dextrans
(kD)
S-100 1-100 NS
S-200 5-250 1-80
S-300 10-1500 2-400
S-400 20-8000 10-2000
S-500 NS 40-20,000
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Running the column
Sample size / Fraction size
0.5 5% of total bed volume (Vt).
Concentration limited by viscosity
Running time
Determined by trial and error
Slow rates allow efficient partitioning into pores and
thus increase resolution
Slow rates increase diffusion of sample on column
thus increasing peak width and reducing resolution.
Protein about 5mL cm-2. h-1
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Types of Column Systems
Liquid Chromatography
High Performance Liquid
Chromatography (HPLC)
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Determination of Molecular Weight
Calibrate column with known standards
Plot Kav against lg Mol Wt
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
K
av
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OtherTypes of Column
Chromatography Ion-Exchange Chromatography
Separation on basis of charge
DEAE- sephadex
Hydrophobic Interaction Chromatography
Separation on basis of hydrophobicity
Phenyl-sepherose
Affinity Chromatography
Affinity of enzyme for substrate or other ligand