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

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    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