1. Extraction Isolation of Product

2. Introduction An extraction process makes use of the partitioning of a solute between two immiscible or partially miscible phases. When the extraction takes place from one liquid medium to another, the process is referred to as liquid-liquid extraction. When a liquid is used to extract solutes from a solid material, the process is referred to as solid-liquid extraction or leaching. When a supercritical fluid is used as an extracting solvent, the process is referred to as supercritical fluid extraction (SFE).

3. Design Considerations Thermodynamic equilibrium Rate of mass transfer between phases Hydrodynamics of the contacting fluids Choice of solvent Processing equipment Process Economics

4. Non-polar solvents (DC < 15)

5. Polar aprotic solvent

6. Polar protic solvent

7. Impurities Solute Light phase (Extractant) Heavy phase (Feed / Initial solvent)

8. Applications of Extraction in Bioprocessing 1.Purification of antibiotics 2.Purification of alkaloids 3.Protein purification using aqueous two- phase systems 4.Purification of peptides and small proteins 5.Purification of lipids 6.Purification of DNA

9. Liquid-liquid extraction Liquid-liquid extraction involves transfer of solute from one liquid phase to another. The three basic steps common to all liquid-liquid extraction processes are: –Mixing or contacting –Phase separation or settling –Collection of phases

10. Main Categories in Liquid-liquid extraction Conventional solvent extraction –Used for the separations of many metabolites from fermentation such as alcohols, carboxylic acids, amino acids, and antibiotics Aqueous two-phase extraction –Using water soluble polymers such as PEG and dextran, and salts such as potassium phosphate –Attractive for the separation of biomolecules such as proteins and peptides (including enzymes) that may be denatured by solvents

11. Phase diagram for partially miscible solvent systems The concentration of the components are usually expressed in mole fraction or mass fraction The composition of the mixture represented by point H on the diagram is such that content of A is proportional to HL, content of B is proportional to HJ and content of C is proportional to HK. The curve is called the binodal solubility curve. The area under the curve represents the two-phase region. Ternary phase diagram for a solute A, its initial solvent B and its extracting solvent C

12. Phase diagram for partially miscible solvent systems Any mixture represented by a point within this region will split up into two phases in equilibrium with each other. For a mixture having an overall composition H, the composition of the two phases are represented by points P and Q which are obtained from the points of intersection of the binodal solubility curve with the tie line PQ which passes through H. The tie lines are straight lines which connect together the compositions of the two phases, which are in equilibrium with one another. Ternary phase diagram for a solute A, its initial solvent B and its extracting solvent C

13. Phase diagram for partially miscible solvent systems The point F on the binodal solubility curve is called the plait point. The area above the binodal solubility curve represents the single phase region where all three components in the system are mutually miscible. Ternary phase diagram for a solute A, its initial solvent B and its extracting solvent C

14. Conventional Solvent Extraction

15. Theory of Solvent Extraction

16. Partition Coefficient Function of: Composition Temperature pH Ionic species and strength

17. Theory of Solvent Extraction

19. From the above equation, it is evident that the greater the difference between the chemical potentials at standard reference state, the better is the extraction The equation also indicates that solute transfer is favored at lower temperatures.

20. How to change the Chemical Potential The partition coefficient can be written as : Where the are the partial molar volumes and are the corresponding solubility parameters

21. Selection of solvent There must be reversible reaction with solutes The solvent phase after extraction must allow ready recovery of the solutes from it The interfacial tension should be appropriate for solvent-water contact The density difference between the solvent and water should be sufficiently large The solvent should be immiscible with water The solvent should be readily available at a reasonable cost

22. Good solvent should be: Inexpensive Readily available Have a high affinity for the product Highly selective for the product Immiscible with the aqueous feed phase Substantially different density from water Low viscosity Low vapor pressure Non degrading to the product Non-toxic Non-corrosive Non-flammable Non-explosive Environmentally benign Easily recoverable from products

23. Common Solvents for antibiotic extractions Alcohols (n-butanol) Ketones (methyl isobutyl ketone) Acetates (butyl and pentyl acetate)

24. Effect of pH The partition coefficient of many solutes in solvent-water systems can be altered by changing pH If the solute is a weak acid –As the pH of aqueous phase is lowered, K will increase If the solute is a weak base –As the pH of aqueous phase is increased, K will increase

25. Changes in Solute (via ion pairs) If the solute to be separated is ionic, counterions from organic soluble salts (greasy salts) may be used to increase the solubility of the solute in the organic phase greatly. The counter ion can be changed without changing the solute itself The counterions can be replaced with those which are much more soluble in the extraction solvent

26. Example: Extraction of tetrabutyl ammonium If the chloride of this cation is extracted with chloroform If sodium acetate is added to this chloride solution

28. Effect of pH and ionic strength In the solubilization step at pH 9 with 0.1 M KCl, cytochrome c, and lysozyme were transferred to the organic phase with ribonuclease-a remained in the aqueous phase Cytochrome c was then back extracted to a 0.5 M KCl aqueous phase

29. Solvent Extraction Equipment Batch Extraction Continuous Staged Extraction Differential Extraction Fractional Extraction

30. Batch Extraction In a batch extraction process a batch of feed solution is mixed with a batch of extracting solvent in an appropriate vessel. The solute distributes between the two phases depending on its partition coefficient. The rate at which the transfer of solute takes place from the feed to the extracting solvent depends on the mixing rate. Once equilibrium is attained, the mixing is stopped and the extract and raffinate phases are allowed to separate.

32. Batch Extraction The mixer unit must be able to : –generate high interfacial area, –provide high solute mass transfer coefficient and –cause low entrainment of air bubbles. The settler unit must: – have a low aspect ratio, i.e. be of flat geometry, –must allow easy coalescence and phase separation, and –Must allow for easy collection of raffnate and extract as separate streams.

33. Application of batch extraction The antibiotic penicillin partitions favorably in an organic solvent from an aqueous fermentation media at acidic conditions. However, at a neutral pH, the partitioning from organic phase to aqueous phase is favoured. Thus the antibiotic could be purified by sequential reversed batch extraction, where the antibiotic is moved from aqueous to organic phase and back again. This sequence is usually repeated a few times in order to obtain highly pure antibiotic.

34. Batch Extraction If a batch of feed containing R amount of initial solvent and an initial solute concentration of C m is mixed with E amount of extracting solvent, the concentration distribution in the extract and the raffinate at equilibrium is given by: C E = KC R Where C E = solute concentration in extract (kg/m 3 ) C R = solute concentration in raffinate (kg/m 3 )

36. Example

37. Solution

38. Continuous Staged Extraction Staged Extractions are repeated batch extraction Two Types –Multistage cascade constructed from single-stage mixer-settlers –Sieve tray-multistage towers

40. The Device of Continuous Countercurrent Extraction.

41. Staged counter-current extraction Using this scheme the concentration driving force is maintained more or less uniform in all the stages comprising the cascade.

44. Varying Partition Coefficient

45. Example Penicillin is to be extracted from filtered fermentation media (concentration = 1 g/1) to MIBK using staged counter-current extraction. The feed flow rate is 550 1/h while the extracting solvent flow rate is 80 1/h. The equilibrium relationship is given by C E = {{25C R ) I (1+ C R )), where C R and C E are in g/l. Determine the number of theoretical stages needed for 90% extraction of the antibiotic.

47. Differential Extraction Occurs when a heavy liquid and a light liquid continuously flow past one another Solute is transferred from one phase to the other, but never fast enough to reach equilibrium May be carried out in: –Spray column –Packed column –Rotary disc columns –Centrifuge-type extractors

49. Fractional Extraction The same as that used for continuous stage extraction except that solute is introduced at an intermediate point in the system

50. Aqueous Two- Phase Extraction

51. Aqueous two-phase system An aqueous two-phase system is an aqueous, liquid– liquid, biphasic system which is obtained either by mixture of aqueous solution of two polymers, or a polymer and a salt. Generally, the former is comprised of PEG and polymers like dextran, starch, polyvinylalcohol  High Cost, easy to scale up In contrast, the latter is composed of PEG and phosphate or sulphate salts. This polymer-salt system results in higher selectivity in protein partitioning, leading to an enriched product with high yields in the first extraction step  Inexpensive but might caused denaturation The heavy phase will generally be Polyethylene glycol (PEG), and the light phase is generally a polysaccharide

52. Rule of thumb The larger a peptide or a protein, the more complex its stearic structure and the higher posssibility for denaturation. As a result, larger peptides and proteins cannot usually be separated using solvent extraction The more biocompatible aqueous two-phase partitioning method is used instead of solvent extraction If the target compound being separated is a protein or enzyme, it is possible to incorporate a ligand to the target into one of the polymer phases  Reactive extraction

53. Gentle operation conditions, normal temperature and pressure. The interfacial tension of two phase is low, magnitude order is 10 -4 N ／ cm, two phase are easy to disperse. The preparation of two phase are changed by operating condition. The density difference of the two phase is little, about 10 g/L. So it is not easy to separate the two phase, at present more research in this area. Easy to continuous operation, big treatment capacity, was fitted for industry. The Merits of Aqueous Two-phase Extraction:

54. Aqueous Two-Phase Partitioning A popular aqueous two-phase system is the PEG-dextran-water system –E.g. A 5 wt% dextran 500, 3.5% PEG 6000 water solution partitions into two aqueous phase at 20 o C. –The top phase contain 4.9% PEG, 1.8% dextran and 93.3% H 2 O –The bottom phase 2.6% PEG, 7.3% dextran, and 90.1% H2O Solutes can have different solubilities in the two phases, thus providing a basis for separation

59. At point A, the system is homogeneous liquid Point P in the phase diagram is the critical point at which the compositions of the two liquid phases are identical Above the phase envelop, the system splits into two separate phases The ratio of the weight of the top phase (point C) to that of the bottom phase (point D) is equal to the ratio of the distance between B and D to that between C and B

62. Bronsted Equation The Bronsted Equation may be used to describe the partition coefficient K In which M is the molecular weight of the solute, k is the Boltzmann constant, T is the absolute temperature, and λ is a parameter characteristic of the aqueous two-phase system and its interaction with the solute

63. Partition Coefficient This coefficient depends on the difference in chemical potential of the protein between top and bottom phases, It is a function of the chemical nature of the polymers, the protein, added electrolytes, and temperature. Consequently, the partition behavior of proteins can be manipulated by changing the concentration of the phase-forming species, their molecular weigh, pH, type and concentration of added salts and by the addition of affinity ligands.

64. Factors that affecting the partitioning of biomolecules and bioparticles Polymer and their molecular weight Salt used in the system Concentrations of the polymers and salts Ionic strength pH temperature

65. Effect of polymer molecular weight and concentration The MW of polymers strongly affects the partition of protein in PEG-dextran-water system

66. The higher the PEG concentration difference in two phases, the higher will be the partition coefficient Higher molecular weight and higher concentrations for the polymers usually bring higher viscosity of the solution To have higher partition coefficient, –lowering the average PEG moleculer weight –increasing the average dextran MW  Not too high or else gelation

68. Effect of Temperature When temperature is decreased, phase separation occurs at lower polymer concentrations for PEG-dextran-water system Less PEG and dextran are needed to achieve phase separation K values are usually higher at lower temperature

69. Effect of Salt The presence of ions in the aqueous solution affects the partition of a solute between two aqueous phases When a salt is introduced in an aqueous mixture, an electrical potential is created in the interface owing to the unequal distribution of cations and anions of the salt This electrical potential ( ψ) is given by:

70. Effect of Salt System containing polyelectrolytes are strongly affected by the presence of salt As a rule of thumb, the decrease of partition coefficient for negatively charged proteins in PEG- dextran-water systems is sulfate>fluoride>acetate>chloride>bromide>iodide and lithium>ammonium> sodium> potassium Salt has little effect on proteins close to their isoelectric point Salt is more soluble in water and thus push the solute from water rich phase to water poor phase

71. Affinity Partitioning Addition of affinity ligands to an aqueous two-phase partitioning system can greatly enhance the partitioning of biomolecules The biospecific binding of the biomolecule to the desired phase The ligand must be covalently coupled to the target phase polymer Drawback: added cost of ligands The two commonly used types of ligand are fatty acids and triazine

72. Temperature Induced