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Downstream Processes. BIE/ CEE 5930/6930 Spring 2010. 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

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downstream processes

Downstream Processes

BIE/CEE 5930/6930Spring 2010

aqueous two phase extraction
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
slide3

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 extraction1
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
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 extraction2
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
slide9
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
Typical concentrations of systems:
  • PEG – Dextran10% w/w PEG, 15% Dextran
  • PEG – Salt 15% w/w PEG, 15% Salt