Gel Permeation

8/3/2019 Gel Permeation

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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

Gel Permeation

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