01 Ion Exchange Chromatography

download 01 Ion Exchange Chromatography

of 17

  • date post

    04-Jun-2018
  • Category

    Documents

  • view

    251
  • download

    6

Embed Size (px)

Transcript of 01 Ion Exchange Chromatography

  • 8/13/2019 01 Ion Exchange Chromatography

    1/17

  • 8/13/2019 01 Ion Exchange Chromatography

    2/17

    Separation of molecules that involves twophases, the mobile phaseand thestationary phase.

    Choice of mobile and stationary phasesdetermines extent of separation

    Common criteria for separation:

    > Polarity> Size

    > Charge

  • 8/13/2019 01 Ion Exchange Chromatography

    3/17

    Where solute molecules are dissolved

    Can be liquidor gaseous

    Carries solute across the stationary phase

  • 8/13/2019 01 Ion Exchange Chromatography

    4/17

    Matrix where separation occurs

    Interactions of molecules with stationary

    phase reduces movement speedthrough matrix

    Can be paperor column type

  • 8/13/2019 01 Ion Exchange Chromatography

    5/17

    Separates molecules based on charge

    Solute is bound on the matrix due to

    electrostatic interactions withimmobilized ionic groups

    Binding is reversible

    Became popular in biochemistry becauseit issimple, controllable, has highresolving powerand capacity

  • 8/13/2019 01 Ion Exchange Chromatography

    6/17

    Stationary phase: ion exchange resins

    Mobile phase : buffer solution

  • 8/13/2019 01 Ion Exchange Chromatography

    7/17

    Protein Detection

    Absorbance Bradford reagent

    Regeneration

    Washing column and re-equilibrating with buffer

    Elution

    Gradient: 0.1 M, 0.2 M, 0.3 M, 0.5 M, 1.0 M KCl in buffer

    Loading Samples

    Sample: 5 mg/ml each of invertase, albumin and casein

    Column and Gel Preparation

    Anionic: DEAE-cellulose Cationic: Dowex 50W

  • 8/13/2019 01 Ion Exchange Chromatography

    8/17

    Has ionic groups covalently bound onsurface

    Attracts oppositely charged molecules Can be cationicor anionic

    Synthetic resins are unsuitable for

    biomolecules Cellulose, agarose, and dextran became

    popular

  • 8/13/2019 01 Ion Exchange Chromatography

    9/17

    Source: Voet, Voet. Biochemistry4thed.

  • 8/13/2019 01 Ion Exchange Chromatography

    10/17

    Source: H. U. Khan. The Role of Ion Exchange Chromatography in Purification andCharacterization of Molecules

    Stable below pI: anionic exchanger

    Stable above pI: cationic exchanger

  • 8/13/2019 01 Ion Exchange Chromatography

    11/17

    Components must have same charge asmatrix

    If not, it will take part in ion exchange pH also important (dependent of pKa)

  • 8/13/2019 01 Ion Exchange Chromatography

    12/17

    Source: D. Voet, J. Voet. Biochemistry4thedition

  • 8/13/2019 01 Ion Exchange Chromatography

    13/17

    -0.03

    -0.02

    -0.01

    0

    0.01

    0.02

    0.03

    0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

    Absorbance

    [KCl], molL-1

    Absorbance versus salt

    concentration for DEAE-cellulose ~0.2 M KCl

    Source of Error: pH of the

    Buffer

    InstrumentalError

  • 8/13/2019 01 Ion Exchange Chromatography

    14/17

    -0.03

    -0.02

    -0.01

    0

    0.01

    0.02

    0.03

    0.04

    0.05

    0 0.2 0.4 0.6 0.8 1 1.2

    Absorbance

    [KCl], molL-1

    Absorbance vs salt

    concentration ~0.3 M KCl

    Source of Error: pH of the Buffer

    InstrumentalError

  • 8/13/2019 01 Ion Exchange Chromatography

    15/17

    Dowex 50 fractions with Bradford reagent (start from left)

  • 8/13/2019 01 Ion Exchange Chromatography

    16/17

    Separation was not achieved using bothexchangers

    Possible sources of error:

    > pH of buffer

    > Instrument

  • 8/13/2019 01 Ion Exchange Chromatography

    17/17

    Use only DEAE-cellulose for IEC of proteinsamples with pI below 7

    Prepare reagents (esp. buffers) on theday of the experiment

    Spectrophotometric assay must be done

    on the same day