Ion Exchange Chromatography & Chromatofocusing

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Transcript of Ion Exchange Chromatography & Chromatofocusing

  • Ion Exchange Chromatography &

    Chromatofocusing Principles and M

    ethods

    Principles and Methods

    11-0004-21

    Edition AA

    www.gehealthcare.com

    www.chromatography.amershambiosciences.com

    Ion ExchangeChromatography& Chromatofocusing

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  • 1

    Ion Exchange Chromatography

    & ChromatofocusingPrinciples and Methods

  • 2

    ContentsIntroduction ............................................................................................................. 7Symbols ................................................................................................................................................... 8

    Common abbreviations .............................................................................................................................. 9

    Chapter 1Principles of ion exchange ..................................................................................... 11Net surface charge and pH ......................................................................................... 11

    Steps in an IEX separation ......................................................................................... 12

    Resolution ................................................................................................................ 14Efficiency ............................................................................................................................................... 15

    Selectivity ............................................................................................................................................... 17

    Components of ion exchange media ............................................................................ 21Matrix ..................................................................................................................................................... 21

    Functional groups .................................................................................................................................... 23

    Binding capacity and recovery .................................................................................... 25

    Chapter 2Ion exchange in practice ........................................................................................ 26Introduction .............................................................................................................. 26

    Media selection ......................................................................................................... 26Capture .................................................................................................................................................. 26

    Intermediate purification .......................................................................................................................... 27

    Polishing ................................................................................................................................................ 27

    Fast media selection and method development ............................................................. 30Automated media selection, method development and optimization .............................................................. 30

    Manual media selection, method development and optimization ................................................................... 31

    Practical considerations for IEX separation ................................................................... 34pH and ionic strength ............................................................................................................................... 34

    Anion or cation exchanger ........................................................................................................................ 35

    Strong or weak ion exchangers .................................................................................................................. 36

    Buffer selection and preparation ............................................................................................................... 37

    Column and media preparation ................................................................................................................. 39

    Sample preparation .................................................................................................................................. 39

    Sample application .................................................................................................................................. 40

    Sample load ............................................................................................................................................ 40

    Elution ................................................................................................................................................... 42

    Linear gradient elution ............................................................................................................................. 42

    Step elution ............................................................................................................................................ 44

    pH elution ............................................................................................................................................... 46

    Flow rates ............................................................................................................................................... 46

    Flow control ............................................................................................................................................ 47

    Wash and re-equilibration ......................................................................................................................... 48

    Detergents, denaturing agents and other additives ...................................................................................... 48

    Analysis of results and further steps ............................................................................ 50

    Scaling-up ................................................................................................................ 51

    Equipment selection .................................................................................................. 52

    Care of ion exchange media ........................................................................................ 52

    Troubleshooting ......................................................................................................... 53

    BioProcess Media made for bioprocessing ................................................................. 58

    Custom Designed Media ............................................................................................. 58

  • 3

    Chapter 3Ion exchange media ............................................................................................... 59Introduction .............................................................................................................. 59

    MiniBeads: purification or analysis of microgram-milligram quantitieswith highest resolution ............................................................................................... 60Purification options ..............................................................................