ERT 320 BIOSEPARATION ENGINEERING

PRODUCT ISOLATION & CONCENTRATION The product isolation and concentration using different types of unit operation like: Solvent extraction (Chap 6) Aqueous two phase extraction (Chap 6) Ultrafiltration (Chap 4) Precipitation (Chap 8) Sorption (Chap 7) advantages and limitations, basic principles and parameters involved in product isolation and concentration units. Scale-up and design of selected product isolation units.

ERT 320 BIOSEPARATION ENGINEERING SOLVENT EXTRACTION

Definition of Extraction Liquid-Liquid extraction is a mass transfer operation in which a liquid solution (the feed) is contacted with an immiscible or nearly immiscible liquid (solvent) that exhibits preferential affinity or selectivity towards one or more of the components in the feed.

Purpose of Extraction Usually extraction will be carried out early in the purification process for bioproducts. To give a significant reduction in volume To separate closed-boiling point mixture Mixture that cannot withstand high temperature of distillation Example: - recovery of penicillin from fermentation broth solvent: amyl acetate - recovery of acetic acid from dilute aqueous solutions solvent: ethyl-acetate

Principle of Extraction Feed liquid (contains a component i to be removed) + solvent (immiscible with feed phase, but component i is soluble in both phases) Some of solute (component i) is transferred from feed phase to solvent phase. After extraction, layers to be settled and separated. Extract – the layer of solvent + extracted solute Raffinate – the layer from which solute has been removed Extract may be lighter or heavier than raffinate.

Principle of Extraction component i is then separated from the extract phase by a technique such as distillation. The solvent is regenerated. Further extractions may be carried out to extract more component i.

Extractants The efficiency of a liquid liquid extraction can be enhanced by adding one or more extractants to the solvent phase. The extractant interacts with component i, increasing the capacity of the solvent for i. To recover the solute from the extract phase the extractant- solute complex has to be degraded (dicyclohexano-18-crown-6)

Principle of Extraction Separation of biomolecules in liquid-liquid extraction depends on the partitioning of the biomolecules between the liquid phases. Design of the extraction process depends on the miscibility of the two liquid phases in each other, and the rate of equilibrium of the biomolecules between the two phases. Usually not feasible to extract proteins with organic solvent – protein often denatured and degraded. Aqueous two-phase extraction is a nondenaturing and nondegrading technique for a number of biomolecules.

Explain the factors that affect the partitioning of biomolecules. Instructional Objectives: Define and use key constants such as the partition coefficient, solvent to feed ratio, and extraction factor. Explain the factors that affect the partitioning of biomolecules. Construct a phase diagram for aqueous two-phase systems and understand their applications to the extraction of proteins. Calculate solute concentrations in multistage countercurrent extraction cascades. Draw equilibrium and operating lines and use them to calculate equilibrium stages in countercurrent extraction.

6.2.1 Phase Separation and Partitioning Equilibria Single-stage extraction process (Fig. 6.1) One feed stream contacts one extraction solvent stream The mixture divides into equilibrium extract and raffinate phases Partition coefficient (K): the distribution of a solute at equilibrium between two liquid phases desirable to have K as large as possible K = partition coefficient y = concentration of the solute in the extract phase x = concentration of the solute in the raffinate phase

6.2.1 Phase Separation and Partitioning Equilibria Example - Penicillin G 6-aminopenicillanic acid (6-APA) is manufactured by GSK in Irvine. It is used to manufacture amoxicillin and ‘Augmentin’. Fermentation products (penicillin G broth) are filtered (microfiltration) and extracted at low pH with amyl acetate or methyl isobutyl ketone. The penicillin G is then extracted further at a higher pH into an aqueous phosphate buffer.

Partition Coefficient Depends on many parameters: Size of the molecule being extracted pH types of solvent temperature concentration & Mw of polymers (or salts) in the phases. CX-l Penicillin G

Dependence of K on pH for penicillin G and acidic impurity (Fig. 6.2). Below pH 4, the extraction of penicillin G into the solvent phase is favored over that of the acidic impurity CX-1. CX-1 Penicillin G CX-l Penicillin G Figure 6.2: Dependence of the partition coefficient on pH for penicilin G and acidic impurity CX-1: an organic solvent was used for extraction from filtered fermentation broth.

Bronsted model developed to describe the partitioning of biomolecules (K):   k = Boltzmann constant M = molecular weight of the molecular being partitioned T = absolute temperature λ = constant lumped parameter that includes characteristics of both the phase system and the partitioning substance

AQUEOUS TWO PHASE EXTRACTION

Aqueous two-phase extraction systems Made by combining two water-soluble polymers or a polymer & a salt in water, above a “critical concentration” These systems separate into two immiscible liquid phases, one of them enriched in one polymer and the other enriched in the other polymer or salt. Recognized as a nondenaturing & nondegrading technique for the separation of a number of biological entities such as proteins, enzymes, viruses, cells, and cell organelles.

Typical concentrations: - 10% PEG & 15% dextran or Phase diagram (Fig. 6.3) At concentrations below the curve in Fig. 6.3, there is only one liquid phase On the curve, there are two liquid phases and tie lines connect the compositions of the phases that are in equilibrium The phase enriched in PEG can contain almost no dextran Most common system: PEG & dextran or PEG & potassium phosphate The PEG-rich phase is less dense than either the dextran-rich or salt- rich phase (lighter or top phase) Typical concentrations: - 10% PEG & 15% dextran or - 15% PEG & 15% salt PEG % w/w Dextran (% w/w) DEXTRAN (% w/w) Figure 6.3 Phase diagram for a PEG 6000-dextran D48 system ai 20°C. (Data from P.-A. Albertsson Partition of Cell Particles and Macromolecules, 3rd ed., Wiley, New York, 1986.)

In general for aqueous two-phase extraction, contaminants such as cells & cell debris partition to the bottom phase or interface. For proteins, the partitioning is affected by many parameters (Table 6.1) TABLE 6.1: Factors That Affect Protein Partitioning in Two-Phase Aqueous Systems" Protein molecular weight Protein charge, surface properties Polymer(s) molecular weight Phase composition, de-line length Salt effects Affinity ligands attached to polymers ___________________________________ "See reference 3.

Models developed to explain several molecular level mechanisms influencing the partition of proteins in aqueous two-phase extraction: lattice models, virial expansions, scaling-thermodynamic approaches An example of virial expansion model developed by King et al. (Eq. 6.2.3) Virial coefficients measured for each type of polymer and salt by membrane osmometry or by low angle laser light scattering Electrical potential determined as a function of the type of salt and tie-line length Data are necessary for the protein surface charge as a function of pH

(Eq. 6.2.3)  

Influence of protein size on the phase partitioning (Fig. 6.4): ■ Partitioning of the protein to the top phase is weakly favored at lower Mw while bottom phase partitioning is strongly favored for the largest proteins ■ For all data, the pH of the solution was at the protein’s isoelectric point (pI) to minimize any effects of protein charge O s Cl (X 0.05 Protein molecular weighL{x 10 DlW Protein molecular weight (x 10-4 Da) Figure 6.4: Effect of protein molecular weight on partitioning in PEG 6000-dextran 500 system with pH at the isoelectric point (pI) for all proteins.

Partition coefficient is greatly affected by the salt type Effects of type & concentration of the salt in the system and the charge on the protein Partition coefficient for ovalbumin can vary widely depending on the pH (electrostatic effect) All the curves in Fig. 6.5 intersect at the isoelectric pH of the protein Partition coefficient is greatly affected by the salt type n t Figure 6.5: Dependence of ovalbumin partitioning on solution pH and salt type in PEG 600-detran 500 system. Open symbol denote chloride salts, solid symbols, sulphates (square, potassium; circle, sodium; triangle, lithium) L972.)

Enhancement of the selectivity of the extraction for the protein of interest ■ Coupling of a biospecific ligand to one of the polymers ■ PEG: often selected for conjugation, well known & simple & efficient chemical modification ■ Conversion of the PEG terminal -OH to halides, sulfonate esters, or epoxide derivatives couple to a ligand ■ Ligands from the reactive dyes: Gibacron blue, Procion red, Procion yellow - Used for the affinity separation of many proteins Recovery of the protein in free form ■ Adding salt to the top phase, rich in ligand-polymer, yielding two phases and consequent partitioning of the protein to the resulting bottom phase ■ Another approach -> soluble effector was added to a new two-phase system to compete with the bound ligand for the protein’s binding site, causing the protein to be released from the ligand-polymer and shift to the bottom phase

Affinity partitioning of enzymes in a PEG-dextran system w/ & w/o Procion yellow-PEG mo With Procion yellow HE-3G-PEG Glucose 6-phosphate dehydrogenase 3- Phosphoglycerate kinase Total protein Alcohol Without Liganded PEG '"U PEG weight fraction (%) Figure 6.6 Affinity partitioning of enzymes in a PEG 6000-dexiran 500 system with and without Procion yellow-PEG. {Data from G. Johansson and M. Andetsson, “Parameters determining affinity partitioning of yeas! enzymes using polymer-bound triazine dye ligands,” /, Ckramaiogr.t vol. 303, p. 39, 1984.)

Recovery of the protein in free form ■ Adding salt to the top phase, rich in ligand-polymer, yielding two phases and consequent partitioning of the protein to the resulting bottom phase ■ Another approach -> soluble effector was added to a new two-phase system to compete with the bound ligand for the protein’s binding site, causing the protein to be released from the ligand-polymer and shift to the bottom phase

6.2.2 Countercurrent Stage Calculation

6.2.2 Countercurrent Stage Calculation Countercurrent extraction cascade (Fig. 6.7) - The streams leaving each stage are in equilibrium The streams are numbered according to the stage they are leaving Once the feed has entered the cascade, it is called the “raffinate”

6.2.2 Countercurrent Stage Calculation Three key assumptions for the mathematical calculation: Two solvents are immiscible or are already in phase equilibrium. The solute concentrations are sufficiently low, - the flow rates of raffinate & extract are constant - typically holds for the extraction of bioproducts (less than 10 g/liter or 1 g/liter (often)). 3. Equilibrium is achieved in each stage.

6.2.2 Countercurrent Stage Calculation

6.2.2 Countercurrent Stage Calculation

Exercise 1 Batch Extraction Penicillin F is recovered from a dilute aqueous fermentation broth by extraction with amyl acetate, using 6 volumes of solvent per 100 volumes of the aqueous phase. At pH 3.2 the partition coefficient K is 80. What fraction of the penicillin would be recovered in a single ideal stage?

Exercise 2 Countercurrent-Stage Extraction An inlet water solution of 100 kg/h containing 0.010 wt fraction nicotine in water is stripped with a kerosene stream of 200 kg/h containing 0.0005 wt fraction nicotine in a countercurrent-stage extraction tower. It is desired to reduce the concentration of the exit water to 0.0010 wt fraction nicotine. The equilibrium data are as follow, with x the weight fraction of nicotine in water solution and y in the kerosine: x y 0.00101 0.000806 0.00246 0.001959 0.00500 0.00454 0.00746 0.00682 0.00988 0.00904 0.0202 0.01850 a) Calculate the flow rate of the nicotine in both of the exit streams. b) Plot the equilibrium data and graphically determine the number of theoretical steps.