Downstream processing (Concentration) Flashcards
Name commonly used techniques used for concentrating biological products?
- Evaporation
- Liquid-liquid extraction
- Membrane filtration
- Precipitation
- Adsorption.
Name the 4 types of evaporators
- plate evaporators
- falling film evaporators
- forced film evaporators
- centrifugal forced film evaporators
Explain liquid-liquid extraction
The concentration of biological products can be achieved by transferring the desired product
(solute) from one liquid phase to another liquid phase, a phenomenon referred to as liquid-
liquid extraction.
Besides for concentration, what else can liquid-liquid extraction be used for?
Partial purification
What does the efficiency of liquid-liquid extraction depend on?
partition coefficient i.e. the relative
distribution of a substance between the two liquid phases.
Name the types of liquid-liquid extraction used for low molecular weight (hydrophobic) compounds
- physical extraction
- dissociative extraction
- reactive extraction
- supercritical fluid extraction
State the principle of physical extraction
The compound gets itself distributed between two liquid phases based on the physical properties. This technique is used for extraction of non-ionising compounds.
State the principle of supercritical fluid extraction.
SCFs are intermediates between gases and liquids and exist as fluids above their critical temperature and pressure. Supercritical CO 2 , with a low critical temperature and pressure is commonly used in the extraction. Supercritical fluid extraction is rather expensive, hence not widely used (SCF has been used for the extraction of caffeine
from coffee beans, and pigments and flavor ingredients from biological materials).
State the principle of dissociative extraction
This technique is suitable for the extraction of ionisable compounds. Certain antibiotics can be extracted by this procedure.
State the principle of reactive extraction
the desired product is made to react with a carrier molecule (e.g., phosphorus compound, aliphatic amine) and extracted into organic solvent.
Highly suitable for compounds that are highly soluble in water such as organic acids.
Name liquid-liquid extraction techniques used for the extraction of high molecular weight compounds.
- Aqueous two-phase systems
- Reverse micellar systems
State the principle of aqueous two-phase systems.
They can be prepared by mixing a polymer (e.g., polyethylene glycol) and a salt solution (ammonium sulfate) or two different polymers. Water is the main component in ATPS, but the two phases are not miscible.
Suggest a phase for aqueous two phase systems
polymer + salt solution (polyethylene glycol + ammonium sulfate)
polymer + polymer
In aqueous two-phase systems, what is the distribution of the desired product is based on?
ionic character and the nature of phases
State the principle of reverse micellar systems
Reverse micelles are stable aggregates of surfactant molecules and water in organic solvents.
The proteins can be extracted from the aqueous medium by forming reverse micelles.
What could be extracted through reverse micelles system?
Enzymes
What could be extracted through dissociative extraction?
antibodies
Suggest materials that membranes used in membrane filtration could be made of?
polymers: polyvinyl di-fluoride, polyethersulfone
In recent years, micro-filters and ultrafiIters composed of ceramics and steel are available.
Cleaning and sterilization of such filters are easy.
Name membrane filtration techniques
- Membrane adsorption (affinity ligands and/or ion exchange groups)
- Pervaporation (permeation through a membrane and vaporization)
- Perstraction (filtration and solvent extraction)
Name a limitation of pervaporation
High cost –> can’t be done in large scale
What types of products is perstraction used for?
hydrophobic
Why are SCFs not widely used?
expensive
What could be used for precipitation?
- Neutral salts
- Organic solvents
- Ionic polymers
- Non-ionic polymers
- Increase in temperature
- Changes in pH
- Affinity precipitation
- Precipitation by ligands
What is the principle of precipitation by neutral salts?
The most commonly used salt is ammonium sulfate, since it is highly soluble, nontoxic to proteins and low-priced.
Ammonium sulfate increases hydrophobic interactions between
protein molecules that result in their precipitation.
What factors does precipitation by neutral salts depend on?
Temperature, pH, protein concentration
What is the principle of precipitation by organic solvents?
Organic solvents reduce dielectric constant and increase electrostatic interaction between proteins.
Name some organic solvents that could be used for precipitation
ethanol, propanol and acetone.
They can denature proteins thus precipitation has to be carried out below 0C.
What is the principle of precipitation by ionic polymers?
Ionic polymers from complexes with oppositely charged protein molecules and lead to neutralisation.
Name some ionic polymers that could be used for protein precipitation
Polyethyleneimine and polyacrylic acid
What is the principle of precipitation by non-ionic polymers?
Non-ionic polymers reduce the quantity of water available for protein solvation.
Name non-ionic polymers that could be used for protein precipitation. Also state the advantage
Polyethylene glycol (PEG)
It is non-toxic and does not denature protein
Name some materials that could be used for protein adsorption
-Activated charcoal
-cellulose based adsorbents
For low MW:
-methacrylate based matrices
-acrylate based matrices
polystyrene
