downstream processes bie/cee 5930/6930 spring 2010
Post on 20-Dec-2015
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TRANSCRIPT
Aqueous Two-phase Extraction
• Made by combining two water-soluble polymers or a polymer and salt in water
• Above a “critical concentration” phase separation occurs – Two immiscible liquid phases are formed– One phase is enriched in one polymer and other phase is
enriched in other polymer or salt
• Can be used to separate proteins, cells, cell organelles, nucleic acids, polysaccharides.
• Examples– Polymer/polymer: Poly-ethylene glycol (PEG)/Dextran– Polymer/salt: PEG/(NH4)2SO4 (ammonium sulfate)
Aqueous Two-phase Extraction• PEG-water phase is
typically lighter and floats
• Water-salt or water-dextran phases are heavier
• Typical concentrations– PEG/Dextran: 10%:15%– PEG/salt: 15%:15%
Factors that Affect Protein Partitioning
• Protein Molecular Weight• Protein charge and surface properties• Polymer(s) molecular weight• Phase composition of the system• Salt effects• Affinity ligands attached to the polymers
Aqueous Two-phase Extraction• Cells and cell debris settle to the bottom phase or stay at
the interface• Protein can stay in either phase• Partition coefficient is a function of protein size and
properties
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re temperatuabsolute theis
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where
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• Below the line is a single phase
• On the line is two phases
• The tie-line connect compositions that are in equilibrium
Typical concentrations of systems:
• PEG – Dextran 10% w/w PEG, 15% Dextran
• PEG – Salt 15% w/w PEG, 15% Salt