KAIST

Chem. & Biomol. Eng.

Biosepartaion engineering
Ch.6: Extraction

Hyun Gyu Park

Ch. 6: Extraction

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Ch. 6. Extraction

Chem. & Biomol. Eng.

Extraction E i Two phases come into contact with the objective of transferring a solute or particle from one phase to the other For the separation of biological products, the phases are most commonly immiscible liquids and the solute is in soluble form In certain instances, one phage is a liquid and the other phase is a solid the extraction of caffeine from coffee beans An A organic solvent i often used as the extracting liquid when the solute to be extracted is stable in i l is f d h i li id h h l b di bl i the organic solvent low Mw antibiotics Usually not feasible to extract proteins with organic solvents often denatured or degraded as a result of contact with the organic solvent esu t o co tact w t t e o ga c so ve t Proteins can often be successfully extracted by means of two immiscible liquid phases that consist of solutions of two water-soluble but incompatible polymers, or one polymer plus a high concentration of certain salts Usually U ll comes early in the purification process for a bioproduct & typically would precede a high l i th ifi ti f bi d t t i ll ld d hi h resolution step such as chromatography Advantageous bring about a significant reduction in volume Extraction of interest in the purification of biotechnological & pharmaceutical p p g p products are mainly y liquid-to-liquid

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6.1 Instructional Objectives

Chem. & Biomol. Eng.

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 Perform scaling calculations for reciprocating-plate and centrifugal extractors f li l l i f i i l d if l

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6.2 Extraction Principles

Chem. & Biomol. Eng.

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 Aqueous two-phage extraction is a nondenaturing and nondegrading technique for a number of biomolecules 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: the distribution of a solute at equilibrium between two liquid phases - Desirable to have K as large as possible

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6.2.1 Phase Separation and Partitioning Equilibria Chem. & Biomol. Eng.

Partition coefficient Depend on many parameters Size of the molecule being extracted, pH, types of solvent, temperature, concentration & Mw of polymers (or salts) in the phases 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

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6.2.1 Phase Separation and Partitioning Equilibria Chem. & Biomol. Eng.

Brnsted model developed to describe the partitioning of biomolecules B d d ld l d d ib h ii i f bi l l

Aqueous two-phase extraction systems Made by M d b combining two water-soluble polymers or a polymer & a salt i water, above a critical bi i l bl l l l in b ii l concentration These systems separate into two immiscible liquid phases, one of them enriched in one polymer and t e ot e e c ed t e ot e po y e o sa t 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

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6.2.1 Phase Separation and Partitioning Equilibria Chem. & Biomol. Eng.

Aqueous two phase extraction systems two-phase 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: dextran & PEG or PEG & potassium phosphate The PEG rich phase is less dense than either the dextran-rich or salt-rich phase PEG-rich dextran rich salt rich lighter or top phase Typical concentrations: 10% PEG & 15% dextran or 15% PEG & 15% salt

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6.2.1 Phase Separation and Partitioning Equilibria Chem. & Biomol. Eng.

Aqueous two-phase extraction systems A h i 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) p , p g y yp ( )

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6.2.1 Phase Separation and Partitioning Equilibria Chem. & Biomol. Eng.

Aqueous two phase extraction systems two-phase 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. (eqn. 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 tie line - Data are necessary for the protein surface charge as a function of pH

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6.2.1 Phase Separation and Partitioning Equilibria Chem. & Biomol. Eng.

Aqueous two phase extraction systems two-phase 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 proteins pI point to minimize any effects of protein charge

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6.2.1 Phase Separation and Partitioning Equilibria Chem. & Biomol. Eng.

Aqueous two-phase extraction systems two phase 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 - P i i coefficient is greatly affected by the salt type Partition ffi i i l ff db h l

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6.2.1 Phase Separation and Partitioning Equilibria Chem. & Biomol. Eng.

Aqueous two-phase extraction systems 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 f i d from the reactive dyes: Gibacron bl Procion red, Procion yellow h i d ib blue, i d i ll 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 li d f the proteins binding site, causing the protein to be released from the li d b d ligand for h i bi di i i h i b l df h ligandpolymer and shift to the bottom phase

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6.2.1 Phase Separation and Partitioning Equilibria Chem. & Biomol. Eng.

Aqueous two-phase extraction systems Affinity partitioning of enzymes in a PEG-dextran system w/ & w/o Procion yellow-PEG

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6.2.2 Countercurrent Stage Calculations

Chem. & Biomol. Eng.

High bi Hi h bioproduct recovery use more than one extraction stage d h i Countercurrent flow is preferable to cocurrent flow because of the greater difference of solute concentration between the raffinate and extraction phases Graphical and mathematical analysis for equilibrium in each stage of contacting determine the balance between the number of stages and the ratio of the extraction solvent flow rate to the feed flow rate & the extraction efficiency of pilot plant and plant scale extractor Countercurrent extraction cascade (Fig. 6.7) - Th streams l i each stage are in equilibrium The leaving h i ilib i - The streams are numbered according to the stage they are leaving - Once the feed has entered the cascade, it is called the raffinate

Three key assumptions for the mathematical calculation 1. Two solvents are immiscible no solvent or polymer is exchanged between phases the extraction solvent is selected to minimize its solubility in the raffinate 2. The solute concentrations are sufficiently low flow rates of raffinate & extract are constant typically holds for the extraction of bioproducts (less than 10 g/liter or 1 g/liter) 3. 3 Equilibrium is achieved in each stage
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6.2.2 Countercurrent Stage Calculations

Chem. & Biomol. Eng.

A material balance on the solute around the feed end of the cascade down to stage n-1

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6.2.2 Countercurrent Stage Calculations

Chem. & Biomol. Eng.

McCabe-Thiele-type di M C b Thi l diagram (Fi 6.8) (Fig. 6 8) - Calculation of the number of stages required to reduce the concentration of solute in the feed stream to xn - The cascade operating line of slope F/S intersects the axis at point (xn, ys) Assume that ys=0 extraction solvent is usually solute free - Th equilibrium curve (K / ) originates at x=y=0 The ilib i (K=y/x) i i t t 0 - K may not be constant for the range of concentrations that apply The stepping off of a equilibrium stages starts on the operating line where the feed enters and the extract exits the cascade (xf, y1) - H i t l line drawn from (xf, y1) equilibrium curve at (x1, y1) Horizontal li d f ( ilib i t( - Vertical line drawn from (x1, y1) operating line at (x1, y2) - Continue until ys is reached

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6.2.2 Countercurrent Stage Calculations

Chem. & Biomol. Eng.

For isothermal, dilute solutions, K is often constant Extraction factor slope of equilibrium line/ slope of operating line

Material balance on the solute for the nth stage, assuming no solute in the entering extraction solvent (ys=0)

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6.2.2 Countercurrent Stage Calculations

Chem. & Biomol. Eng.

1. E>1 & n y g g extract almost all the solute with a very large # of stages 2. E=1, 3. E<1.0 & n ,

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Example 6.1

6.2.2 Countercurrent Stage Calculations

Chem. & Biomol. Eng.

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Example 6.2

6.2.2 Countercurrent Stage Calculations

Chem. & Biomol. Eng.

xn/xf = 0.05 & by substituting the values for xn/xf, K, and F, and various values of S in this equation plot of n versus S
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6.2.2 Countercurrent Stage Calculations

Chem. & Biomol. Eng.

Example 6.2 62 Below an extract flow rate of 30 liters/min, the number of ideal stages appears to increase exponentially Above a flow rate of about 80 liters/min, the change in the number of stages with the flow rate becomes relatively small Economic optimum will likely be in the butyl acetate flow rate range of 30-80 liters/min At a pH of 10.0, the tertiary amine is essentially unprotonated, and the antibiotics is thus more readily extracted into the more hydrophobic butyl acetate phase than if it were protonaed

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6.3 Scaleup and Design of Extracts

Chem. & Biomol. Eng.

Most commonly used extractors in biotechnology (Table 6.2) 6 2) Mechanically agitated extraction column Reciprocating-plate column Centrifugal extractor Podbielniak centrifugal extractor widely used in the recovery of antibiotics

Two useful concepts in scaleup & design 1. Overall stage efficiency 2. HETS (height equivalent to a theoretical stage) Design goals are to maximize stage efficiency & minimize HETS

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6.3.1 Rciprocating-plate extraction columns

Chem. & Biomol. Eng.

Most i M t important feature: ability to handle liquids with suspended solids and mixtures that emulsify t tf t bilit t h dl li id ith d d lid d i t th t l if easily Interdispersion is achieved by reciprocating or vibrating plates The column consists of a stack of perforated plates, which are reciprocated vertically p p , p y Amplitude of reciprocation: 3-50 mm Reciprocation frequency is variable up to 1000 strokes/min

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6.3.1 Rciprocating-plate extraction columns

Chem. & Biomol. Eng.

This l Thi column obeys the relationship for the number of theoretical stages in a counter-current staged b th l ti hi f th b f th ti l t i t t t d extractor Scaled on the basis of column diameter D & rate of reciprocation of the plate, SPM (strokes per min) Based on the performance data obtained for columns having diameters of 25, 76, 305, and 914 mm, p g , , , , the following equations have been developed for the scaleup of reciprocating-plate extractors where 1 and 2 refer to the small- and large-scale columns

Column volumetric efficiency is defined as follows (Total volumetric throughput is defined as the sum of the feed flow rate and the solvent flow rate, S + F)

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6.3.2 Centrifugal extractors

Chem. & Biomol. Eng.

Most popular in antibiotics production, where the large volumes of fermentation broth that must be production processed are easily emulsified Key advantages: ability to avoid emulsions & to separate liquid phases with small density differences ( (as small as 0.01 g/cm3) g Consist of several concentric perforated cylinders that rotate around a shaft Principle feed the heavy phase near the axis of the rotating cylinder, while the light phase is fed close to the periphery Podbielniak centrifugal extractor (fig. 6.10) (fig 6 10) Centrifugal force applied causes the two phases to move radially in a countercurrent mode, such that the heavy phase moves to the periphery and the light phase to the axis of rotation

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