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Downstream Processes

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

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

  2. 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

  3. 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)

  4. 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%

  5. 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

  6. 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

  7. Aqueous Two-phase Extraction

  8. Below the line is a single phase • On the line is two phases • The tie-line connect compositions that are in equilibrium

  9. Typical concentrations of systems: • PEG – Dextran10% w/w PEG, 15% Dextran • PEG – Salt 15% w/w PEG, 15% Salt

  10. Effect of Salt Concentration

  11. Effect of protein molecular weight

  12. Typical process flow diagram

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