Downstream processing (Concentration)

Question 1

Name commonly used techniques used for concentrating biological products?

Answer 1

1. Evaporation
2. Liquid-liquid extraction
3. Membrane filtration
4. Precipitation
5. Adsorption.

Question 2

Name the 4 types of evaporators

Answer 2

1. plate evaporators
2. falling film evaporators
3. forced film evaporators
4. centrifugal forced film evaporators

Question 3

Explain liquid-liquid extraction

Answer 3

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.

Question 4

Besides for concentration, what else can liquid-liquid extraction be used for?

Answer 4

Partial purification

Question 5

What does the efficiency of liquid-liquid extraction depend on?

Answer 5

partition coefficient i.e. the relative
distribution of a substance between the two liquid phases.

Question 6

Name the types of liquid-liquid extraction used for low molecular weight (hydrophobic) compounds

Answer 6

1. physical extraction
2. dissociative extraction
3. reactive extraction
4. supercritical fluid extraction

Question 7

State the principle of physical extraction

Answer 7

The compound gets itself distributed between two liquid phases based on the physical properties. This technique is used for extraction of non-ionising compounds.

Question 8

State the principle of supercritical fluid extraction.

Answer 8

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).

Question 8

State the principle of dissociative extraction

Answer 8

This technique is suitable for the extraction of ionisable compounds. Certain antibiotics can be extracted by this procedure.

Question 8

State the principle of reactive extraction

Answer 8

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.

Question 9

Name liquid-liquid extraction techniques used for the extraction of high molecular weight compounds.

Answer 9

1. Aqueous two-phase systems
2. Reverse micellar systems

Question 10

State the principle of aqueous two-phase systems.

Answer 10

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.

Question 11

Suggest a phase for aqueous two phase systems

Answer 11

polymer + salt solution (polyethylene glycol + ammonium sulfate)

polymer + polymer

Question 12

In aqueous two-phase systems, what is the distribution of the desired product is based on?

Answer 12

ionic character and the nature of phases

Question 13

State the principle of reverse micellar systems

Answer 13

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.

Question 14

What could be extracted through reverse micelles system?

Answer 14

Enzymes

Question 15

What could be extracted through dissociative extraction?

Answer 15

antibodies

Question 16

Suggest materials that membranes used in membrane filtration could be made of?

Answer 16

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.

Question 17

Name membrane filtration techniques

Answer 17

1. Membrane adsorption (affinity ligands and/or ion exchange groups)
2. Pervaporation (permeation through a membrane and vaporization)
3. Perstraction (filtration and solvent extraction)

Question 18

Name a limitation of pervaporation

Answer 18

High cost –> can’t be done in large scale

Question 19

What types of products is perstraction used for?

Answer 19

hydrophobic

Question 20

Why are SCFs not widely used?

Answer 20

expensive

Question 21

What could be used for precipitation?

Answer 21

1. Neutral salts
2. Organic solvents
3. Ionic polymers
4. Non-ionic polymers
5. Increase in temperature
6. Changes in pH
7. Affinity precipitation
8. Precipitation by ligands

Question 22

What is the principle of precipitation by neutral salts?

Answer 22

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.

Question 23

What factors does precipitation by neutral salts depend on?

Answer 23

Temperature, pH, protein concentration

Question 24

What is the principle of precipitation by organic solvents?

Answer 24

Organic solvents reduce dielectric constant and increase electrostatic interaction between proteins.

Question 25

Name some organic solvents that could be used for precipitation

Answer 25

ethanol, propanol and acetone.

They can denature proteins thus precipitation has to be carried out below 0C.

Question 26

What is the principle of precipitation by ionic polymers?

Answer 26

Ionic polymers from complexes with oppositely charged protein molecules and lead to neutralisation.

Question 27

Name some ionic polymers that could be used for protein precipitation

Answer 27

Polyethyleneimine and polyacrylic acid

Question 28

What is the principle of precipitation by non-ionic polymers?

Answer 28

Non-ionic polymers reduce the quantity of water available for protein solvation.

Question 29

Name non-ionic polymers that could be used for protein precipitation. Also state the advantage

Answer 29

Polyethylene glycol (PEG)

It is non-toxic and does not denature protein

Question 30

Name some materials that could be used for protein adsorption

Answer 30

-Activated charcoal
-cellulose based adsorbents

For low MW:
-methacrylate based matrices
-acrylate based matrices
polystyrene