PART 6 Flashcards
General Approach
- Separation of insoluble products or components.
- Primary isolation or concentration and removal of water.
- Purification and removal of contaminated chemicals.
- Product preparation.
Factors that impact difficulty and cost of recovery
- Product can be biomass, intracellular or extracellular component.
- Fragile or heat sensitive.
- Concentration or titer in the broth.
- Typically recovery and purification is more than 50% of total manufacturing costs
Insoluble Products or Components
- Filtration
- Centrifugation
- Coagulation and Flocculation
Most Cost-efiectve, most common in industrial biotechnology.
Filtration
_______________: traditional. Penicillin mold.
Rotay Vacuum precoat filter
Cross fow ultrafiltration: _______________
0.02-0.2 um bacterial separations
Cross flow microporous filtration: ___________
0.2-2 um for yeast
- Used to separate solids of size. 0.1 um to 100 um using centrifugal forces.
- Being replaced by microfiltration
Centrifugation
Pretreatment to centrifugation, gravity settling, or filtration to improve separation.
Coagulation and Flocculation
________: formation of small flocs of cells using coagulating agents electrolytes.
Coagulation
________: formation of agglomeration of flocs into settleable particles using flocculating agents, polyelectrolytes, or
CaCl2
Flocculation
Used wastewater treatment processes to improve clarification.
Coagulation and Flocculation
Cell Disruption - Intracellular Products
Mechanical Methods
- Sonication
- Bead beating
- Pressing
Cell Disruption - Intracellular Products
Non-Mechanical methods
- Osmotic shock
- Freeze-thaw
- Enzymatic
_______: disrupts cell membrane. Mostly used at the laboratory scale.
Ultrasound
_________: extrude cell paste at high pressure.
Pressing
__________: grind cells with glass, metal beads.
Bead beating
________ is a problem with all of these methods.
Heat dissipation
____________: salt differences to cause the membrane to rupture. Common.
Osmotic Shock
________: Causes cell membrane to rupture. Common.
Freeze-thaw
_________: Lysozyme attacks the cell wall.
Enzymatic
Separation of Soluble Products
- Liquid-liquid extraction
- Aqueous two phase extraction
- Precipitation
- Adsorption
- Dialysis
- Reverse osmosis
- Ultrafiltration and microfiltration
- Cross-flow filtration and microfiltration
- Chromatography
- Electrophoresis
- Electrodialysis
Separate inhibitory fermentation products from broth.
Based on the solubility difference for the compound between the phases.
Liquid-Liquid Extraction
Solubility reduction at low temperatures (less than -5°C) by adding organic solvents
Precipitation
_____________ - inorganic salts (NH4)2SO4 at high ionic strength
Salting out
Removal of solutes from aqueous phase onto a solid phase.
Chromatography is based on _____________.
Adsorption
Membrane separation used to remove low molecular weight solutes.
Used to remove salts from protein solutions.
Dialysis
Transport occurs due to a concentration gradient driving force.
Dialysis
In ______________, pressure is applied to the salt phase, causing water to move against a concentration gradient.
Salt phase becomes more concentrated.
Reverse Osmosis
Transport of water molecules from a high to a low concentration of pure water to salt water.
Osmosis
Pressure driven molecular sieve to separate molecules of different size.
Ultrafiltration and Microfiltration
______________: retained components accumulate on the filter. Gel layer formed on the filter.
Dead end filtration
______________ : retained components flow tangentially across the filter
Cross flow filtration
Separates mixtures into components by passing the mixture through a bed of adsorbent particles.
Chromatography
Solutes travel at different speeds through the column resulting in the separation of the solutes.
Chromatography
Highly specific interaction between a ligand on the particle and a component in the mixture. Often based on antibodies.
Affinity Chromatography
Separation of molecules based on size and charge in an electric field.
Electrophorensis
Membrane separation to separate charged molecules from a solution.
Electrodialysis
Finishing Steps
- Crystallization
- Drying
