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Size Exclusion Chromatography GE Healthcare Size Exclusion Chromatography Size Exclusion Chromatography – Principles and Methods Size Exclusion Chromatography gelifesciences.com GE, the GE Monogram, ÄKTA, Biacore, BioProcess, HiLoad, HiPrep, HiScale, HiTrap, illustra, MicroSpin, MidiTrap, MiniTrap, MultiTrap, Sephacryl, Sephadex, Sepharose, SPARTAN, SpinTrap, Superdex, Superose, Principles and Methods GE Healthcare Bio-Sciences AB Tricorn, UNICORN, and Whatman are trademarks of GE Healthcare Company. Tween is a trademark of Uniqema Americas LLC. Björkgatan 30 All other third-party trademarks are the property of their respective owners. 751 84 Uppsala IMAC Sepharose products, Ni Sepharose products and Fe Sepharose products: These products are sold under a license from Sigma-Aldrich under patent number EP 1276716 (Metal chelating Sweden compositions) and equivalent patents and patent applications in other countries. © 2000–2018 General Electric Company. 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.............................................................................................................................................................................5 Common acronyms and abbreviations ..............................................................................................................5 Chromatography terminology ................................................................................................................................7 Chapter 1 Introduction ............................................................................................................................................11 Versatility of size exclusion chromatography .............................................................................................. 11 Purification by size exclusion chromatography .......................................................................................... 12 Size exclusion chromatography in theory...................................................................................................... 14 Defining the process ....................................................................................................................................... 14 Selectivity curves and resin selection ..................................................................................................... 17 Resolution ............................................................................................................................................................ 19 Chapter 2 Size exclusion chromatography in practice ......................................................................................21 General advice ............................................................................................................................................................ 21 Columns and resin preparation ................................................................................................................. 21 Scaling up ............................................................................................................................................................ 21 Sample and buffer preparation ................................................................................................................. 22 Filtration ................................................................................................................................................................ 22 Equipment selection ....................................................................................................................................... 23 Setting column pressure limits ................................................................................................................. 23 Maintenance of SEC columns ..................................................................................................................... 24 Optimizing your separation ................................................................................................................................. 24 Resin selection ................................................................................................................................................... 25 Sample volume and column dimensions .............................................................................................. 26 Elution and flow rates .................................................................................................................................... 27 Sample concentration and viscosity ....................................................................................................... 29 Buffer composition .......................................................................................................................................... 31 System configuration ..................................................................................................................................... 33 Troubleshooting .......................................................................................................................................................... 35 Reference ............................................................................................................................................................. 38 Chapter 3 Considerations for analytical SEC ......................................................................................................39
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