liquid-liquid partitioning methods for bioseparations...

Liquid-Liquid Partitioning Methods for Bioseparations," Chapter 7 in the Handbook of Bioseparations, A. Ahuja, Ed., Academic Press, New York, Vol. 2, p. 329-364 (2000).

extraction which is used for the separations of many metabolites from fermentation such as alcohols, carbox~lic acids, amino acids, and anti- b io t i c~ . ' . ~ The other is the aqueous two-phase partitioning using water soluble polymers such as polyethylene glycol (PEG) and dextran, and salts such as potassium phosphate. The latter method is very attractive for the separation of biomolecules such as proteins and peptides including many e n ~ y m e s ~ - ~ that may be denatured by solvents. As the scale of bioseparation processes goes up, liquid-liquid partitioning becomes more and more com- petitive because it is easy to scale up and it permits continuous steady-state pera at ion.^ The cost for liquid-liquid partitioning is much lower than other more sophisticated bioseparations method, such as liquid chromatography.

II. SOLVENT EXTRACTION FOR BIOSEPARATIONS

The International Union of Pure and Applied Chemistry (IUPAC) recom- mends the use of liquid-liquid distribution over the traditional term solvent extraction." However, solvent extraction is still used prevalently in the literature. Solvent extraction utilizes the partition of a solute between two practically immiscible liquid phases: one solvent phase and the other aqueous phase.1' A separatory funnel can be used in a lab to carry out solvent extraction. Of course, a simple test tube can also be used in conjunction with a glass pipette. The organic phase (solvent ~ h a s e ) is usually the top phase and the aqueous phase bottom phase. However, some organic solvents are heavier than water (for example, methylene chloride's specific gravity is 1.33 at 20°C) and in such cases the organic phase becomes the bottom phase.

Thousands of papers and dozens of books and book chapters have been published on solvent extraction. Most of them deal with extraction compounds that are not biologically derived. The chemistry of solvent extraction is extensively investigated in a book edited by Sekine and ~asegawa" entitled "Solvent Extraction Chemistry: Fundamentals and Application." Many operational aspects of extraction are investigated in a book edited by Rydberg, Musikas, and Choppin13 entitled "Principles and Practices of Sol- vents Extraction." Various extraction equipment and their operations are discussed in a book edited by Godfrey and Slater14 entitled "Liquid-Liquid Extraction Equipment." For practitioners, the "Handbook of Solvent Extrac- tion" by Baird et al.ls should be very helpful. Schiigerl' wrote a book entitled "Solvent Extraction in Biotechnology: Recovery of Primary and Secondary Metabolites." This is a book exclusively dealing with solvent extraction for bioseparations. There are some book chapters dealing with solvent extraction for the separation of biomolecules. Thornton's book of "Science and Practice of Liquid-Liquid Extraction" contains a chapter dealing with solvent extraction of pharmaceuticals (such as antibiotics) and a chapter on extraction of food products.2 Aires-Barros and Cabrall' contributed a chapter on liquid-liquid extraction to a book edited by Kennedy and Cabral17 entitled "Recovery Processes for Biological Materials." Weatherley18 discussed the operational consideration of extraction of fermentation broth in a chapter of the book entitled "Downstream Processing of Natural Products"

LIQUID- LIQUID PARTITIONING 33 1

edited by Verrall.19 wheelwright2' summarized solvent extraction briefly in his downstream protein purification book. Some theoretical and practical aspects of solvent extraction were discussed by Scopes2' in his book on protein purification. Belter et a1.22 discussed the basics of solvent extraction in an easy to read chapter in their bioseparations textbook. They studied the following factors in solvent extraction.

A. Solvent Selection

The partition coefficient K is defined as follows:

in which x and y are the solute concentrations in the lighter phase (usually the organic phase) and the solute concentration in the heavier phase (usually the water phase), respectively. Table 1 shows the partition coefficient of some b iom~lecu l e s .~~ We can also express K in terms of chemical potentials in standard reference states for the lighter phase [ pO(L)] and heavier phase [ $(H)I:

TABLE I Partition Coefficients of Some Biomolecules in Solvent Extraction

Category Solute Solvent

-

K Comments

Amino acids Glycine Alanine Lysine Glutamic acid a-aminohutyic acid a-aminocaproic acid

Antibiotics Celesticetin Cycloheximide Erythromycin

Lmcomyc~n Gramicidin

Novohiocin

Penicillin F

Penicillin K

Proteins Glucose isomerase

Fumarase

Catalase

n-Butanol n-Butanol n-Butanol n-Butanol n-Butanol n-Butanol n-Butanol Methylene chloride Amyl acetate

n-Butanol Benzene Chloroform-methanol Butyl acetate

Amyl acetate

Amyl acetate

PEG 1550-potassium phosphate

PEG 1550-potassium phosphate

PEG-crude dextran

Source: Reprinted with permission from Belter et ~ 1 . ' ~ Copyright 1988 John Wiley Rr Sons, Inc.

In Eq. (21, R is the universal gas constant and T is the absolute temperature in degrees Kelvin. The concept of solubility parameter can be used to predict the partition coefficient qualitatively in the selection of solvent^.'^ We can write K as

in V with different subscripts indicates partial molar volumes of lighter phase (subscript L), heavier phase (subscript H ) and solute A (subscript A); S indicates the solubility parameter. Table 2 shows the solubility parameter values for some solvent^.^'

Obviously, some solvents are better than others for the extraction of a particular compound. A solvent system should provide a suitable partition coefficient, preferably much greater than unity or much less than unity, depending on whether you want the solute to migrate to the solvent phase or to stay in the aqueous phase. The following factors must be considered during the selection of a suitable solvent

1. There must be no irreversible reaction with solutes. 2. The solvent phase after extraction must allow ready recovery of the

solutes from it. 3. The interfacial tension should be appropriate for solvent-water con-

tact. High values for interfacial tension result in an increased energy requirement to maintain sufficient contact time, while low interfacial tension result in the formation of stable emulsions that make separation considerably more difficult.

TABLE 2 Solubility Parameters for Some Solvents

Solvent Solubility parameter (ca~ ' I 2 - cm ' I 1 )

Amyl acetate Benzene Butanol Butyl acetate Carbon disulfide Carbon tetrachloride Chloroform Cyclohexane Hexanol Acetone Pentane Perfluorohexane Toluene Water

Source: Reprinted with permission from Relter rt al." Copyright 1988 John Wiley & Sons, Inc.

LIQUID- LIQUID PARTITIONING 333

4. The density difference between the solvent and water should be sufficiently large.

5. The solvent should be immiscible with water or has a very limited s o h bility.

6. The solvent should be readily available at a reasonable cost.

Above all, the solvent system must provide a selectivity good enough for practical applications. Additional factors such as volatility and toxicity also require attention. The "Handbook of Organic Solvent Properties" by Smal- I w o o d 2 ~ o n t a i n s information on many solvents. In the extraction of fragile biomolecules such as peptides and proteins, one must know whether the solvent system will denature the peptide or the protein. As a rule of thumb, the larger a peptide or a protein, the more complex its stearic structure and the higher possibility 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.

B. Effect of pH

p H is an important parameter in extraction that is often adjusted to achieve desirable r e s ~ l t s . ~ ~ , ~ ~ The partition coefficient of many solutes in solvent-water systems can be altered by changing pH. If the solute is a weak acid, the following relationshipL2 can be used to calculate partition coefficient K at a given p H value of the aqueous phase,

where K, is the intrinsic partition coefficient (not dependent on pH) defined as the ratio of the acid concentration in the solvent phase to that in the water phase.

The pK, values for some biomolecules arc listed in Table 3.12 They are also available from biochemistry reference books. Similarly, for weak bases,

Based on Eq. (41, the selectivity between solutes A and H can be obtained via the following relationship kL2 :

The dramatic effect of p H on K for the extraction of some antibiotics can be seen in Table 1.

TINGYUE GU

TABLE 3 The pK, Values of Some Biomolecules

Simple acids and bases Acetic acid Propionic acid H3PO4 H,PO; HPOi- NH: CII,NH;

Amino acids Glycine Leucine Glutamine Aspartic acid Glutamic acid Histidine Cysteine Tyrosine Lysine Arginine

Peptides Gly-Gly Gly-Gly-Gly Ala Ala-Ala-Ala-Ala Gly-Asp Ala-Ala-Lys-Ala

Antibiotics Celesticetins Cephalosporin C Lincomvcin (free base)

pK, values 7.7, 9.8 3.9, 5.3, 10.5 7.6

pK (R Group)

3.86 4.25 6.0

10.78 10.07 10.53 12.48

pK , (R Group)

4.45 10.58

Monensin icarboxyl) 6.65 (in 66% dimethylformamide) Novohiocin ienol, phenolic) 4.3, 9.1 Penicillamin (carboxyl) 1.8 Rifamycin B 2, 1, 6.7 Spectinomycin 6.95, 8.70

Source: Reprinted with permission from Belter et al." Copyright 1988 John Wiley & Sons, Inc.

C. Effect of 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. For example, sodium acetate can increase the solubility of tetrabutylammonium cation in chloroform by 10-fold. Some common counterions for ion-pair extractions are acetate, butyrate, tetrabutylammonium, perfluorooctanoate, dodecanoate, linoleate, cholate, and tetraphenyl- b ~ r i d e . ~ ~ Schiigerl' systematically studied various applications of solvent extraction in his book on solvent extraction of biological metabolites. He discussed the solvent extraction of alcohols, carboxylic acids, amino acids, and antibiotics in fermentation broths. Operational strategy and equipment were also investigated. There is a growing interest in whole broth ex-

LIQUID - LIQUID PARTITIONING 335

traction because of its economical and environmental benefits according to Weatherley.18

D. Solvent Extraction of Amino Acids

Amino acids are zwitterions with amino and carboxyl groups. They act as cations at low pH values, as anions at high pH values and are dipolar at intermediate pH values. Their solubility in nonpolar solvents are very low thus extraction with nonpolar solvents is not feasible. Partition coefficients of amino acids in solvent-water systems are usually very small. An organic carrier, for example trioctylmethylammonium chloride (TOMAC), can be used to enhance e~t rac t ion .~ ' Amino acids are usually extracted with phos- phorus-bonded, oxygen-bearing solvents or with large quaternary aliphatic amines. Not much was done for extraction at low pH values due to excessive coextraction of other inorganic cations. TOMAC is used in xylene to extract amino acids from an aqueous phase. Table 4 lists the isoelectric points and extraction equilibrium constants of several amino acids with TOMAC at high pH values.'

The separation of amino acids from each other is not an easy task according to Schiigerl' who reviewed the following cases. Isoleucine and leucine can be separated from lysine that has a positive charge at pH 6 with TOMAC and 6% decanol in methylcyclohexane at pH 10 to 11. Isoleucine and leucine are transferred to the organic phase because they have nonpolar side chains. After reextraction with HCI or NaCl solution, isoleucine and leucine are separated by extraction using cyclohexane with the addition of an anion exchanger (such as tetra-docylammoniumbromide). Isoleucine will stay in the aqueous phase while leucine migrates to the organic phase. An alternate method is to use 6% decanol in xylene at pH 12 to separated

TABLE 4 Extraction Equilibrium Constants K and lsoelectric Points of Some Amino Acids

Amino acid PI K

Glycine 6.0 0.036 Alanine 6.0 0.038 Valine 6.0 0.089 Leucine 6.0 0.29 Isoleucine 6.0 0.24 Methonine 5.7 0.21 Phenylalanine 5.5 0.97 Tryptophan 5.9 8.89 Tyrosine 5.7 0.40 Histidine 7.6 0.083 Arginine 10.8 0.062 Serine 5.7 0.049 Threonine 6.2 0.017

Source: Reprinted with permission from Schiigerl.' Copyright 1994 Springer Verlag.

isoleucine and leucine. Tryptophan and tyrosine, two aromatic amino acids, can be extracted into the organic phase from the aqueous phase with 15 m M TOMAC in xylene at pH 10 to 11. They can then reextracted with 1.5 M NaCl solution. Tyrosine migrates to the aqueous phase while tryptophan stays in the organic phase because tryptophan's side chain is nonpolar unlike tyrosine's polar side chain. To separate arginine or lysine from aspartic acid, 0.3 M TOMAC in xylene at pH 12 is used. Aspartic acid migrates to the solvent phase while arginine and lysine stay in the aqueous phase. The extractions of some other amino acids are covered by several European patents. '

E. Solvent Extraction of Alcohols from Fermentation Broths

Ethanol and butanol are the most important alcohols produced by fermentation. They are also produced from petroleum. Ethanol and butanol from fermentation are considered renewable resources since they can be produced with renewable agriculture feed stocks. Because of product inhibition in yeast fermentation, ethanol concentration is limited to 5 to 10% (v/v) in the broth.' According to Essien and ~ ~ l e , ~ ~ solvent extraction of ethanol is about 60% higher in capital-related costs than distillation. This more than offsets any advantage in energy and utility even without considering the cost of the solvent. Distillation is the preferred method. Emphasis has been on in situ extractive fermentation to reduce product inhibiti~n. '~. '' It is difficult to find a suitable solvent with a high partition coefficient, that does not form stable emulsion to hamper extraction, and that does not have harmful effects on cells.' The following solvent systems have been reported in the literature: dodecanol, higher iso-alcohols, higher n-alcohols, tributylphosphate, di- butylphthalate, dodecane, fluorocarbons, and higher iso-acids.' Aqueous two-phase systems can also be used for alcohol recoveries."

F. Solvent Extraction of Aliphatic Carboxylic Acids

Kertes and in^^^ reviewed the extraction chemistry of carboxylic acids from fermentation broths. The following methods were reviewed by Kertes and King:

Extraction with carbon-bonded oxygen-bearing extractants Extraction with phosphorous-bonded oxygen-bearing extractants Extraction by proton transfer or by ion-pair formation using high molecular weight aliphatic amines

Table 5 shows the partition coefficient of some common aliphatic carboxylic acids produced from f e r m e n t a t i ~ n . ~ ~ Aromatic carboxylic acids are not produced from fermentation because they are rarely formed by micro- organisms. One noteworthy exception is the production of salicylic acid formed by Pseudomonas aeruginosa from naphthalene through biotransfor- mation.' The extraction of salicylic acid with and without amine in xylene was reviewed by ~chiigerl' in detail including equipment and operational parameters.


TABLE 5 Partition Coefficients of Some Carboxylic Acids at 25°C

Partition Partition Acid and solvent coefficient Acid and solvent coefficient

Propanoic acid n-Hexane Cyclohexane Benzene Toluene Xylene Carbon tetrachloride Chloroform Nitrobenzene Diethyl ether Diisopropyl ether Methylisobutyl ketone Cyclohexanone n-Butanol n-Pentanol

Lactic acid Diethyl ether Diisopropyl ether Methylisobutyl ketone n-Butanol Isobutanol n-Pentanol n-Hexanol n-Octanol

Pyruvic acid Diethyl ether

Succinic a c ~ d Diethyl ether Methylisobutyl ketone n-Butanol Isobutanol n-Pentanol n-Octanol

Fumaric acid Diethyl ether Methylisobutyl ketone n-Butanol Isobutanol

Maleic acid Diethyl ether Methylisobutyl ketone Isobutanol

Malic acid Diethyl ether Methylisobutyl ketone Isobutanol

Itaconic acid Diethyl ether Methylisobutyl ketone Isobutanol

Tartaric acid Diethyl ether Methylisobutyl ketone n-Butanol

Citric acid Diethyl ether Methylisobutyl ketone n-Butanol Isobutanol

Source: Reprinted with permission from Kertes and in^.'^ Copyright 1986 John Wiley & Sons, Inc.

G. Solvent Extraction of Antibiotics

Antibiotics are microbial compounds that inhibit and even destroy other organisms.' Antibiotics are usually secreted by microbial cells. After clarifica- tion using centrifugation o r microfiltration, solvent extraction can be used to extract antibiotics from the clarified broth. The extract is further purified using reextraction, precipitation, and ion-exchange chromatography, or crys- tallization.' If the antibiotic is a weak acid with a low p K , value, the p H used in the extraction should be lowered to below the p K , value to obtain the antibiotic in its free acid form. One the other hand, if the antibiotic is a weak base with a high pK, value, the pH should be increased to above the p K , value to obtain a free base after extraction. If the antibiotic is highly soluble in water, the broth must be saturated with salt to enhance extraction.' Table

TABLE 6 Solvent Extraction of Some Antibiotics

Product Medium Solvent PH

Actinomycin Adrianimycin Bacitracin Chloramphenicol Clavulanic acid Cyloheximid Erythromycin Fusidic a c ~ d Griseofulvin Macrolides Nisin

Oxytetracycline Penicillin G Salinomycin Tetracycline Tylosin Virginiamycin

Cake Cake Broth Broth Broth Broth Broth Broth Cake Broth Broth Mash Broth Broth Cake Broth Broth Broth

1 MeOH + 2 methylene chloride Acetone n-Butanol Ethylacetate n-Butanol Methylene chloride Amylacetate Methylisobutylketone Butylacetate, methylene chloride Methylisobutylketone, ethylacetate CHC1, + sec-0ct.alc. CHCI, I-Butanol Butylacetate, amylacetate Butylacetate 1 -Butanol Butylacetate, amylacetate Methylisobutylketone

2.5 Acidic 7.0 N-alkalme 2.0 3.5-5.5 Alkaline 6.8 Neutral Alkaline 4.5 2.0

Acidic

Source: Reprinted with permission from Schiigerl.' Copyright 1994 Springer Verlag.

6 is a list of antibiotics extracted with different solvent systems.' Table 1 also contains some antibiotics purified using solvent extraction.12

Solvent extraction of penicillin from fermentation broths has been well documented in the literature. Penicillin G and penicillin V can be efficiently extracted with amyl acetate or butyl acetate at p H 2.5-3.0 and at 0" to 3°C.33 Schiigerll systematically reviewed solvent extraction of different forms of penicillin from fermentation broths. Figure 1 shows an integrated process for the extraction of penicillin G from clarified broth of Penicillium chryso- genum fermentation.' Penicillin G is converted to 6-amino penicillanic acid and phenylacetic acid a t p H 8 m a 1 0 L Kiihni extractor by penicillin G-amidase immobilized in an emulsion liquid membrane. The 6-amino penicillanic acid is subsequently converted to ampicillin at pH 6 and the enzyme is recycled.

A book edited by V a r ~ d a m m e ~ ~ entitled "Biotechnology of Industrial Antibiotics" studied the production of many antibiotics. The recovery and purification of the majority of them involved a step using solvent extraction. erra all" also reviewed solvent extraction of some antibiotics. The antibiotic clavulanic acid is a fused bicyclic p-lactam which is produced naturally by Streptomyces calvuligerus. Clarified broth was first extracted with n- butanol:HCI (3/4 vol.) a t pH 2 and at 5°C. It was reextracted back to aqueous phase with 1 /20 vol. of water a t pH 7 and 20% (w/v) aqueous N a O H . Ion-exchange chromatography was then used for further p~rification.~' The antibiotic tetracycline is a polyketide produced by Strep- tomyces aureofaciens. Tetracycline was extracted from an acid or alkaline medium by 1-butanol. There are also other methods such as extraction with a methylalkyl ketone.37 Nowadays tetracycline is purified by precipitation

LIQUID - LIQUID PARTITIONING

U-FREE BROTH I \ t----C-L, ELECTROCOALESCENCE UNIT

MEMBRANE INNER PHASE

0 0 PH ,. ADJUSTMENT

STERILE FILTER 0

FERMENTOR O O O

EMULSION PREPARATION

CELL-FREE BROTH

FIGURE I A n integrated process for the recovery of penicillin G. (Reprinted with permission

from Schiigerl.' Copyright 1994 Springer Verlag.)

replacing thc old extraction method.' Thc antibiotic erythromyci~~ i s a macrolide secreted by Saccharopolyspora erythraea." Figure 2 depicts an industrial process for the recovery of erythromycin from fermentation." It involves three extraction steps. Gramicidin D is a linear-chain peptide antibiotic produced from Bacillus bre~is .~' Filtered broth is adjusted to pH 4.5-4.8 to precipitate a mixture of gramicidins. The dried solid mass is extracted with alcohol. Saline was added to precipitate the gramicidins again. Further extraction with an acetone-ether mixture produces a neutral fraction containing the linear gramicidins. A system with benzene:chloroform: methano1:water = 15:15:23:7 (v/v) can separate gramicidin D from gramicidins A, B, and C in a countercurrent e~tract ion.~ '

The antibiotic cycloheximide is an effective protein synthesis inhibitor used as an agricultural fungicide. It was discovered in a streptomycin-yielding culture of Streptomyces griseus.40 Several purification schemes involving solvent extraction were reviewed by Jost el aL4" Ethyl acetate, cyclohcxane, amyl acetate, and methylene chloride were used as solvents in those schemes. Fusidic acid is a steroid antibiotic particularly useful in treating staphylococcal infections. It is produced by fermentation of the fungus Fusrdium coc- c i n e ~ m . ~ ' It belongs to a group of tetracyclic triterpenoic acids. Von Daehne et al.41 described an industrial recovery process for fusidic acid. It involved sequential extractions using methylisobutylketone (MIBK), benzene, and acetone. Antimycin A is an antifungal and piscicidal antibiotic produced intracel- lularly by Streptomyces kitasawaensis and S. griseus." 11s recovery involved extractions with CH,CI and CH,CI,.~' Chloramphenicol is an antibacterial agent produced by Streptomyces ~lenezuelae. It is a rather simple aromatic compound. It is easily extracted from a clarified broth by a solvent such as

Broth 40 m' (preheated/cooled to 30°C in Fermenter) I Concentration 5WJ-6@)0 flml

K,Fe CN)&O kg To precipitate Beniol40 kg proteins

Ammonium Chloride

10% NaOH 1501

pH 7.0

Dicalite Filter Aid 800 kg

Agitate (vd~ume 41 m')

I Drum Filter (Dicalite precoat 400 kg)-wash

I . Wet mycelium cake 4000 kg First filtrite 37.4 m'

10% NaOH 8001

Second filtrate 22.4 m' Filter press

Disti I spent filtrate 6Om'

Primary extraction Fresh butyl acetate 15 m3

Rich butyl acetate IS m'

10% C H C O O H 3501

+Distill lean secondary : x t ~ n Water 5 m3 ~ u t y ~ aceetate 15 m' + d j

Rich h f e r

10% NaOH 501

Lean buffer 5 m3 Fresh butyl acetate 2.5 m'

Rich butyl acetate

20% NaSCN 220 1 10% CH3COOH

pH 7.0

Mother liquor A Wet EMT 250 k

Wet bu. ac. 2.5 mf Vacuum 176 kg dry (358"~

FIGURE 2 Recovery of erythromycin from fermentation. (Reprinted from err all'^ by permission of Oxford University Press.)

ethyl acetate at a slightly alkaline pH.43 The antibiotic virginiamycin is produced by Streptomyces drginiae. It consists of two synergistic compo- nents, factors M and S which are two cyclic lactone peptolides. B i ~ t ~ ~ reported that virginiamycin was produced from fernlentation broth using a three-stage extraction process with MIBK. Addition of hcxane to the MlKK

LIQUID- LIQUID PARTITIONING 34 1

precipitated virginiamycin which was further separated by centrifugation under a positive CO, pressure to prevent fire. ~ i o t ~ ~ presented a flowchart of the process.

H. Liquid-Liquid Extraction with Reversed Micelles

Organic solvents have the tendency to denature larger peptides and proteins irreversibly and render them biologically inactive. To overcome this draw- back of solvent extraction, liquid-liquid extraction with reversed micelles is used. Reversed micelles are formed when surfactant molecules in a nonpolar solvent aggregate in such way that the polar head groups of the surfactant molecules turn inward to form a polar inner core.45 Water and host molecules such as proteins can be solubilized inside the micellar cores. Riomolecules in an aqueous phase such as a fermentation broth are first transferred to an organic mjcellar phase and then reextracted back into a new aqueous phase.4" Figure 3 illustrates the transfer of a protein from a fermentation broth to an organic micellar phase.46 Figure 4 is a typical flowchart of liquid-liquid extraction with reversed rnicelle~.~"

Experimental results suggest that hydrophillic proteins tend to be solubilized within the water core of the reversed micelles, while lipophilic biomolecules can either stay in the interface or even partially exposed to the organic pha~e.~"ecause of the protection offered by the reversed micelles, proteins were shown to maintain their functional properties.47 The retention of bioactivity depends strongly on the solvent system and it is usually not 100%.4~

The most commonly used anionic surfactant in reversed micelles for the extraction of proteins is AOT [sodium bis(2-ethylhexyl)sulphosuccinate]

FIGURE 3 Protein transfer between an aqueous phase and an organic micellar phase. (Reprinted with permission from Rahaman et Copyright 1988 American Chemical Society and American Institute of Chemical Engineers.)

reversed m~cellar phase c m~xer 1 settler 1

FORWARD

EXTRACTION

mlxer 2 settler 2

B A C K

EXTRACTION

FIGURE 4 Combined forward and back extraction involving reversed micelles. (From M. Dekker, K. Van't Riet. S. R. Weijers, 1. W . Baltussen. C. Laane, and B. H. Bijisterboch. Enzyme recovery by liquid-liquid extraction using reversed micelles. Chern. Eng. 1. 33. 827-33, 1986, with permission from Elsevier Science.)

which aggregates spontaneously in hydrocarbon solven;~ such as iso-octane, forming tiny water ~ o o l s with radii more than 170 A . ~ ' , ~ ~ TOMAC is a commonly used cationic surfactant. When a cationic surfactant is used, a cosurfactant usually an aliphatic alcohol such as octanol is needed to stabilize the reversed micelles by partitioning between the micelles and the continuous phase.4F

The driving forces in the transfer of proteins into reversed micelles are electrostatic interactions because proteins are dipolar molecules. The phase transfer depends on the isoelectric point, size and shape, hydrophobicity, and charge distribution on the protein surface. It also depends on the pH, ionic strength, type of electrolyte, and surfactant concentration o f the two-phase system.4s Figures 5 and 6 show that pH and ionic strength have a dramatic effect on the transfer of cytochrome c, lysozyme, and ribonuclease-a to the reversed m i ~ e l l e s . ~ ~ The advantage of reversed micelles in solvent extraction is the selective solubilization of proteins inside reversed micelles which provides a logical aqueous environment protecting the bioactivities of the proteins. The disadvantage is mainly the often necessary need to reextract the proteins into a aqueous phase. In some cases, the yield of reextraction is very low and the transfer of proteins into the organic phase seems to be irreversible.

Hu and Gulariso studied the reverse micelle system of sodium di-2-ethylhexyl phosphate (NaDEHP, an anionic surfactant) for the extracton of two aminoglycoside antibiotics, namely neomycin and gentamycin. The antibiotic molecules are first transferred from the aqueous phase to the polar core of reverse micelles. During back extraction to the aqueous phase, the antibiotics in the micelle phase are released back to an aqueous phase by breaking up the reverse micelles using divalent cation solutions, such as Ca2+ solutions. Cabral and Ai res -~ar ros~" showed a double extraction process (Fig. 4) for the continuous extraction of a-amylase using TOMAC in iso-octane. Goklen and Hatton4' described an experimental procedure to separate a ternary mixture of ribonuclease-a, cytochrome c, and lysozyme with 50 m M AOT in iso-oc-


tane. In the solubilization step at pH 9 with 0.1 M KCl, cytochrome c, and lysozyme were transferred to the organic phase while ribonuclease-a re- mained in the aqueous phase. Cytochrome c was then back extracted to a 0.5 M KC1 aqueous phase. Lysozyme was subsequently transferred to a 2.0 M KC1 solution at pH 11.5. Recovery of intracellular enzymes directly from bacterial cells using reversed micelles was investigated by Giovenco et al."

FIGURE 6 Effect of ionic strength on protein solubilization in AOT-iso-octane system with no pH control: (0) cytochrome c. ( 0 ) lysozyme. ( A ) ribonuclease-a (from Goklen and at ton^^ by courtesy of Marcel Dekker, Inc.).

When cells are disrupted by surfactants, intracellular enzymes are transferred to the reversed micelles directly. This may provide a more efficient alternative to the conventional multistage purification method.4F

I. Electrically Enhanced Solvent Extraction

Electrical fields have been applied t o solvent extraction. The fullowing mass transfer enhancements were mentioned by Weatherley et al.":

Small oscillating droplets are generated with a high interfacial area. Dispersion phase transfer is greatly irriproved due to sustained droplet oscillation. The continuous phase film mass transfer rate can be increased by electrostatic acceleration of charged droplets of the dispersed phase in the continuous phase.

Improved mass transfer leads to smaller extraction equipment with shorter residence times. Weatherley et aLi2 studied ethanol extraction with decanol under a range of electrically enhanced experimental conditions.

J. Solvent Extraction Equipment and Operational Considerations

Figure 7 shows four common single-stage devices used in solvent extraction." The most common laboratory device for extraction is the separatory funnel. Craig extraction, also known as fractional extractions with a stationary phase, can be used to extract a solute out of an aqueous phase (or an organic phase) repeatedly with a solvent (or water) for a very a high recovery yield.LL Figure 8 is an illustration of the extraction scheme.22 The net effect of Craig extraction resembles that of elution chromatography. Figure 9 shows Craig extraction tubes. These tubes are tipped between two positions: at the left to mix the two phases till a n equilibrium is reached and at the right to empty

Liquld-Liquid Dispersion

I / Separatory Funnel Vmyl Acetate-Acetic Ac~d

Mlxer

, Llght Phase

Vmyl Acetate Product

Water Settler Product

Light Phase

L Dspersion Band "

Heavy Phase 7

FIGURE 7 Separatory funnel and mixer-settlers for extraction. (Reprinted with permission f rom

Better et Copyright 1988 John Wiley & Sons. Inc.)


All tubes except Transfer

the zeroth contain only heavy solvent.

The light in the One Transfer

zeroth tube is moved to the first, and replaced with lresh light.

Two Transfers The light phases are again moved. and new light added.

Three Transfers After each step. equihbrlum IS

allowed to occur. 1:;:: 3 6 3/16 6 3/16 [:I:: H ti Four Transfers

The result is a solute peak moving through the tubes

FIGURE 8 Craig extraction scheme. (Reprinted with permission from Belter et 0 1 . ~ ~ Copyright 1988 john Wiley & Sons. Inc.)

the two phases.22 Various laboratory and industrial devices and equipment for liquid-liquid extracted were discussed in a book entitled "Liquid-Liquid Extraction Equipment" edited by Godfrey and slater.14 They include plate and packed columns, mixers, gravity settlers, centrifugal extractors, and so on. Figure 10 shows four types of reciprocating plate column." The openings on each plate are designed for countercurrent flow. Figure 1 I is the schematic

FIGURE 9 Craig extraction tubes. (Reprinted with permission from Belter et 0 1 . ~ ~ Copyright 1988 john Wiley & Sons, Inc.)

( a ) KRPC (b) PRPC (c l PRPC (counterphase)

( d l MVDC

FIGURE 10 Some types of reciprocating plate columns. (From Baird et d3 Reproduced by permission of john Wiley & Sons Limited.)

of a Westfalia countercurrent centrifugal extraction decanter." Centrifugal force is used for mixing and separation of the lighter and heavier phases.

Various operational topics including mass transfer and extraction equipment in solvent extraction of fermentation broth were discussed by Weather- ley.'' In his mass transfer textbook, Treybal'4 devoted one chapter to various industrial extraction equipment and calculations. ~ i n g ' s " textbook on separation processes is also a good source for solvent extraction calculations. Figure 12 shows a multistage extraction process with simple individual mixers and settlers in series.54 Three extraction towers are illustrated in Fig. 1 3 . ~ ~ The left one is a packed bed extractor. The middle one is Mixco Lightnin CMC contactor with flat-blade turbine impellers to disperse and mix

Separating disc Scroll

phase

extract phase

FIGURE I I Westfalia countercurrent extraction decanter. (Bakerperkins technical literature. Reprinted from verral13' by permission of Oxford University Press.)

LIQUID- LIQUID PARTITIONING

Stoqe Stage I 2

Miser Settler

----A Solvent

FIGURE I 2 Three-stage countercurrent mixer-settler extraction cascade. Reprinted from 1reybals4 with permission of The McGraw-Hill Companies.

the two phases and horizontal compartmenting plates to reduce axial mixing. The right one is a rotating-disk contactor. Figure 14 is a sieve-tray extraction tower arranged for light liquid dispersed."4 These extractors are multistage extractors. Multistage extraction is required for extraction systems with partition coefficients close to unity or when the partition coefficients are close for the product and impurities. The calculations of multistage extraction are well documented in chemical engineering textbooks such as McCabe et al.'ss6 unit operations textbook and Wankat's "' staged separations textbook, apart from Treybal's and Kings textbooks on mass transfer and separation~.~~*'"elter et presented calculation methods for extractions of biological compounds such as antibiotics in their textbook on bioseparations. Mass transfer calculations and operational parameters for various extractors were investigated in a book on extraction equipment edited by Godfrey and Slater.I4 The selection, design, pilot testing, and scale-up of

FIGURE 13 Differential extractors. Reprinted from ~ r e y b a l ' ~ with permission of The McGraw-Hill Companies.

fLiqh1 l iqu~d out

gP

Heovy liquid out C

FIGURE 14 Sieve-tray extraction tower arranged for ~ r e y b a l ' ~ with permission of The McGraw-Hill Companies.

light liquid dispersed. Reprinted from

various extraction equipment were investigated by Pratt and stevens." They classified extractors into about 20 types. A comprehensive extractor selection chart was presented.

Ill. AQUEOUS TWO-PHASE PARTITIONING FOR BIOSEPARATIONS

According to Albertsson3 aqueous two-phase partitioning systems were first reported by Beijerinck in 1896 who described that gelatin, agar, and water mixture within a certain concentration range separated into two aqueous phases, the top phase being gelatin rich, and the bottom phase, agar rich. As a matter of fact, for polymer mixtures miscibility of different aqueous phases is an exception rather than the rule.' Dobry and Boyer-Kawenoki" systematically tested 3.5 pairs of polymers soluble in solvents, only 4 gave homogeneous solutions. The remaining palrs all exhibited phase separation$.' Similar results were obtained for water soluble polymers.h"


A popular aqueous two-phase system is the PEG-dextran-water system. For example,' a 5 wt % dextran 500 and 3.5% PEG 6000 water solution partitions into two aqueous phases at 20°C. The top phase contains 4.9% PEG, 1.8% dextran, and 93.3% H 2 0 , and the bottom phase 2.6% PEG, 7.3% dextran, and 90.1% H 2 0 . Solutes can have different solubilities in the two phases, thus providing a basis for separation. The high water content in aqueous two-phase systems, typically greater than 70% (w/w), provides a biocompatibility with biomolecules not attained in solvent e x t r a ~ t i o n . ~ ' Aqueous two-phase systems have been used to separate biomolecules and bioparticles such as proteins including enzymes, peptides, nucleic acids, and viral and cell particles.3'4 Several thousand research papers have been published on this subject. A comprehensive 49-page bibliography on aqueous two-phase systerns was provided by Sutherland and Fisherh2 covcring 1956 to 198.5. There are also several monographs dealing theories and applications of aqueous two-phase systems. The most prominent monograph, now in its third edition is the one by Albertsson-3 entitled "Partition of Cell Particles and Macromolecules." It is a comprehensive and most cited work on aqueous two-phase systems. ~ lber t s son" presented a systematic mechanistic study of aqueous two-phase partitioning. He also attached more than 5 0 phase diagrams of PEG-dextran-water, methylcellulose-dextran-water, and pol yvin ylalcohol-dextran-water systems for practical applications. More recently, ZaslavskyX authored a monograph entitled "Aqueous Two-Phase Partitioning-Physical Chemistry and Bioanalytica! Applications." It contains more than 160 phase diagrams of PEG-dextran-water, PEG-polyvinyl- methylether-water, PEG-salt-water, Polyvinylpyrrolidone-dextran-water, polyvinylalcohol-dextran-water, and Ficoll-dextran-water systems. Both monographs have sections dealing with separations of biomolecules.

Aqueous two-phase systems are routinely used for enzyme purification. Walter and Johansson7 edited a comprehensive book on the subject entitled "Aqueous Two-Phase Systems" as Vol. 228 of the series Methods in Enzy- mology published by Academic Press. Another very useful book is the one edited by Walter, Brooks, and Fisherh entitled "Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotech- nology" which deals with separations of a variety of biomolecules and bioparticles.

The most popular aqueous two-phase systems in use today are the PEG-dextran-water system and the PEG-potassium phosphate-water sys-

Both PEG and dextran are fully water soluble, yet the two polymers are incompatible and separate into two aqueous phases in certain concentration ranges. Table 7 is a list of various aqueous two-phase partitioning systems compiled recently by ~ a s l a v s k ~ . ~

Figure 1.5 is a phase diagram of PEG 3400-dextran 500-Water4 in which the average molecular weight of PEG is 3400 and that of dextran 500 is 500,000. At point A, the system is a 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 PEG-rich phase is the top phase and the dextran-rich phase is the bottom phase. The four straight lines above the phase envelop

350 TINGYUE GU

TABLE 7 Aqueous Two-Phase Systems

. Nonionic polymer-nonionic polymer-water Polypropylene glycol Methoxypolyethylene glycol

Polyethylene glycol Polyvinyl alcohol Hydroxypropyldextran Dextran Polyvinyl alcohol Polyvinylpyrrolidone

Polyethylene glycol

Dcxtran Arabinogalactan Hydroxypropyl starch Ficoll

Polyvinyl alcohol Methylcellulose Hydroxypropyldextran Dextran

Polyvinylpyrrolidone Methylcellulose Maltodextrin Dextran

Methy lcellulose Hydroxypropyldextran Dextran

Ethylhydroxyethylcellulose Dextran Hydroxypropyldextran Dextran Ficoll Dextran

B. Polymer-low molecular weight component-water Polypropylene glycol Potassium phosphate

Glycerol Methoxypolyethylene glycol Potassium phosphate Polyethylene glycol Inorganic salts, e.g., Kt , Na ', Lit , NH:, etc. and

PO:-, SO:-, etc. Glucose, maltose, cellobiose, iso-maltose,

maltotriose, iso-maltotriose, P-cyclodextrin

Polyvinylpyrrolidone Butylcellosolve Potassium phosphate

Polyvinyl alcohol Butylcellosolve Dextran Butylcellosolve

Propyl alcohol, isopropyl alcohol Na dextran sulfate Sodium chloride (PC)

C. Polyelectrolyte-nonionic polymer-water Na dextran sulfate Polypropylene glycol

Methoxypolyethylene glycol NaCl Polyethylene glycol NaCl Polyvinyl alcohol NaCl Polyvinylpyrrolidone NaCl Methylcellulose NaCl Ethylhydroxyethylcellulose NaCl Hydroxypropyldextran NaCl Dextran NaCl Polypropylene glycol NaCl Po\yethylene glycol Li2S0, Polyvinyl alcohol Methylcellulose

(Continues)

DEAE dextran HCI


TABLE 7 (Continued)

C. Polyelectrolyte-nonionic polymer-water Casein Dextran

Pectin Ficoll Amilopectin

Na carboxymethyldextran Methoxypolyethylene glycol NaCl Polyethylene glycol NaCl Polyvinyl alcohol NaCl Polyvinylpyrrolidone NaCl Methylcellulose NaCl Ethylhydroxyethylcellulose NaCl Hydroxypropyldextran NaCl

Na carboxymethylcellulose Polypropylene glycol NaCl Methoxypolyethylene glycol NaCl Polyethylene glycol NaCI Polyvinyl alcohol NaCl Polyvinylpyrrolidone NaCl Methylcellulose NaCl Ethylhydroxyethylcellulose NaCl Hydroxypropyldextran NaCl

D. Polyelectro~yte-polyelectrolyte-water Na dextran sulfate Na carboxymethyldextran

DEAE dextran HCI NaCl Na carboxvmethvlcellulose

Na carboxymethyldextran Na ~arboxymeth~lcellulose Casein Sodium alginate, 0.1 M NaOH

Na carboxymethylcellulose, 0.1 M NaOI-I Ovalbumin (pH 6.6) Soybean globulins

Ovalbumin thermotropic aggregates Casein

Source: Reprinted from Zaslavskyn by courtesy of Marcel Dekker, Inc.

15 PEG 34OO/Dextron T-SOO/Water I

C 0 5 10 15 20 2 5

Dextron %(w/w)

FIGURE 15 PEG 3400 - dextran SO0 - water phase diagram at 4°C. (Reprinted with permission from Diamond and H S U . ~ Copyright 1992 Springer Verlag.)

are tie lines just like those in phase diagrams for solvent extraction. If the solution is mixed with compositions a t point B, two phases will result at equilibrium. The top phase has PEG and dextran concentrations at point C and the bottom phase a t point D. Like in other phase diagrams, the lever rule applies. 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. The distances can be measured eithcr using the points' x axis readings or y axis readings. A complete experimental procedure on how to obtain the binodial curve, the tie lines and the critical point was described by Albertsson.' The procedure is based on observing turbidity change, while adding a polyn~er solution to another polymer solution dropwise.

The partition coefficient K for a solute in aqueous two-phase partitioning is defined as

in which C, and Ch are concentrations of the solute in the top and bottom phases, respectively.5 The partition coefficients for proteins generally fall within the range of 0.1 t o 10. For large molecules (such as high molecular weight DNA and RNA) and particles (such as cells and viral particles), partition coefficients > 100 to < 0.01 are observed.', ' Small ions tend to partition equally between the two phases.

According to Albertsson,' the mechanism for aqueous liquid-liquid partitioning is complicated and largely unknown. Hydrogen, ionic, hydrophobic, and other weak forces may be involved. The following equation was proposed by Albertsson' to describe the various influences on partition coefficient K :

in which subscripts el, hfob, biosp, size, and conf denote the electrochemical, hydrophobic, biospecific, size, and conformational contributions to the partition coefficient and KO lumps all other factors. The ~riinsted' ' equation may be used to describe the partition coefficient K qualitatively [S]:

in which M is the molecular weight of the solute, k is the Boltzmann constant, T is the absolute temperature, and A is a parameter characteristic of the aqueous two-phase system and its interaction with the solute. Among the many factors that affect the partitioning of biomolecules and bioparticles, there are the polymers and their molecular weights and salts used in the system, concentrations of the polymers and salts, ionic strength, pH, and tempera t~re .~ . 5 3 4 F

A. Effect of Polymer Molecular Weight and Concentration

A l b e r t s ~ o n ~ ~ found that the molecular weight of the polymers strongly affects the partitioning of a protein in PEG-dextran-water systems. If it is desirable


TABLE 8 Effect of Molecular Weight of Dextran on Partition Coefiicient of Proteins with Different Molecular weightsa

Molecular Protein weight Dex 40 Dex 70

Cytochrome Ovalhumin Bovine serum albumin Lactic dehydrogenasc Catalase Phycoerythrin P-galactosidase Phosphofructokinase Ribulose diphosphate

carboxy lase

Dextran

Dex 220 Dex 500

0.15 0.17 0.74 0.78 0.3 1 0.34 0.09 0.16 0.40 0.79 - 12

1.38 1.59 0.0 1 0.02 0.15 0.28

Dex 2000

Source: Reprinted with permission from ~ l b e r t s s o n . ~ Copyright 1986 John Wiley & Sons, Inc.

'1n phase systems with 6 % (w/w) PEG 6000 and 8%) dcxtran having different molecular weights; 10 mM sodium phosphate at pH 6.8.

to have a higher partition coefficient, lowering the average PEG molecular weight may h e l p . V h e effect of polymer molecular weight depends on molecular weight of the solute as demonstrated by Tables 8 and 9.' Higher molecular weights and higher concentrations for the polymers usually bring higher viscosities to the liquid solutions. T o provide a discrimination basis, the polymer concentrations in the two phases should differ sufficiently. As expected, the larger the difference between the polymer concentrations of PEG in the two phases, the better the partition coefficient (i.e., deviating farther away from unity) as demonstrated in Fig. 16.' Several factors influ- ence the difference including starting polymer concentrations, temperature, etc. In PEG-dextran-water systems, increased PEG concentration will result

TABLE 9 Effect of PEG Molecular Weight on the Partition Coefficient of Proteins with Different Molecular weightsd

Molecular Protein weight

Cytochrome c 12,384 Ovalbumin 45,000 RSA 69,000 l x t i c dehydrogenase 140,000 Catalase 250,000

Dex 500 (9%), PEG 4000 (7. 1%)

Dex 5000, Dex 500, Dex 500, PEG 600 PEG 20,000 PEG 40,000

Source: Reprinted with permissio~~ from ~lber t sson . ' Copyright 1986 John Wiley Sr Sons, Inc.

" ~ h a s e systems contain 8% (w/w) dextran and 6% PEG unless rnarkcd otherwise; phosphate, 10 I ~ M sodium phosphate at pH 6.8.

0 2 4 6 8 10 12 14 16 18 APEG, %wt.

FIGURE 16 Effect of PEG concentration difference in the two phases on partition coefficient K in PEG 6000 - dextran 70 -water system containing 0. IS m o l l kg NaCl in 0.0 1 mol I kg sodium phosphate buffer at pH 7.4: ( I ) myoglobin. (2) cytochrome c, (3) human serum albumin. (Reprinted from Zaslavsky8 by courtesy of Marcel Dekker, Inc.)

in compositions of phases deviate from the critical point where the two phases have identical concentrations.' ~ l b e r t s s o n ~ ' found the following fair relationship for proteins:

in which a is a constant depending on the concentration of polymers and M is the molecular weight of the protein. In PEG-dextran-water and PEG- methylcellulose-water systems, most proteins prefer the lower (dextran-rich or methylcellulose-rich) phase.3,64

0. Effect of Temperature

Temperature affects the shape of phase diagrams. When temperature is decreased, phase separation occurs at lower polymer concentrations for PEG-dextran-water systems, meaning less PEG and dextran are needed to achieve phase separation. The opposite is true for PEG-salt-water Temperature also changes the partitioning of biomolecules. Table 10 shows the partition coefficients of lysozyme and catalase and their ratios in different PEG-dextran-water systems at different temperatures.' It seems that a lower temperature tends to provide a better separation of the two proteins. Kula' also pointed out that K values are usually higher at a lower temperature. This trend is consistent with the aforementioned Bronsted equation. Using a lower temperature will cause the viscosity to be higher. The use of hy-


TABLE 10 Partition Coefficient of Lysozyme and Catalase in Four Aqueous Two-Phase Systems

Separation

TcC) K l y r o z y m e K c r t a ~ a r a factor System composition

Source: Reprinted from zaslavsky8 by courtesy of Marcel Dekker, Inc.

drophilic polymers enhances enzyme stability so that room temperature can be used with minimal bioactivity losses. This means chilling aqueous two- phase systems is usually not required,' unless very fragile proteins are involved. Grimonprez and Johanssonh6 achieved enhanced bioactivity for some enzymes, especially phosphofructokinase, from baker's yeast partitioned at subzero temperature with the addition of ethylene glycol in PEG-dextran-water systems.

C. Effect of Salt

Salts at moderate concentrations have only marginal effects on the phase diagram of nonionic polymer-polymer-water systems. However, systems containing polyelectrolytes, such as DEAE-dextran-water systems, are strongly affected. Usually a much lower polymer concentration is required for phase separation when the salt concentration increases." Salt can be used rather effectively to change the partition coefficient of biomolecules. At low salt concentrations (0.1 to 0.2 M), the effects of salt type and concentration can be dramatic for proteins at pH far away from their isoelectric points.4 As a rule of thumb, the decrease of partition coefficient for negatively charged proteins in PEG-dextran-water systems is sulfate > floride > acetate > chloride > bromide > iodide and lithium > ammonium > sodium > potassium. Positively charged proteins follow the opposite trend.4'64 Alberts- son3 reported that increasing NaCl concentration in the range of 0 to 5 M greatly increased the partition coefficient of several proteins (phycocyanin, phycoerythrin, gamma globulin, ceruloplasmin, and serum albumin) in a phase system containing 4.4% PEG 8000 and 7% dextran at pH 6.8. Salt has little effect on proteins close to their isoelectric point.

TABLE I I Affinity Partitioning of Biomolecules in Aqueous Two-Phase Systems

Biomolecule

Trypsin Serum albumin p-Lactoglobulin S-23 myeloma protein Histones 3-Oxosteroid isomerase Formaldehyde deh~drogenase Formate dehydrogenase Colipase Myosin Phosphofructokinase Interferon Pyruvate kinase Glutamate dehydrogenase Glycerol kinase Hexokinase Lactate dehydrogenase Malate dehydrogenase Transaminase a-Fetoprotein Pre-albumin Glucose-6-ph~s~hatedehydrogenase

Glyceraldehyde phosphate dehydrogenase

3-Phosphoglycerate kinase Alcohol dehydrogenase Nitrate reductase Acid proteases Thaumatin IgG Human hemoglobin

Cytochromec Myoglobins Hemoglobins a,-Macroglob~din Tissue plasminogen activator Superoxide dismutase Monoclonal antibodies Membranes from calf brain

synaptosomcs Albumins Thylakoid membranes

Affinity linand

Fatty acid Fatty acid Ihi trophenyl Fatty acid Estradiol NADH NADH/procion red 1.ecithin Fatty acid Triazine dye Phosphate Triazine dye Triazine dye Triazine dye Triazine dye Triazine dye Triazine dye Triazine dye Triazine dye Remazol yellow Triazine dye-triazine dye and charged groups

(DEAE, sulfate) Triazine dye

Triazine dye-ATP Triazine dye Triazine dye Pepstatin Glutathione Protein A Cu(1I)IDA 11-ala-aln-ala Cu(1I)IDA C:u(Il)IDA Cu(1I)LDA Metal-IDA Metal-IDA Metal-IDA Metal-IDA I'rocion yellow

Alcohols Alcohols

HE-

Source: Reprinted with permission from Diamond and H s u . ~ Copyright 1992 Sprmgcr Verlag.

LIQUID- LIQUID PARTITIONING

D. 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 with the ligand preferentially move the biomolecule to the desired phase. T o do so, it is believed that the ligand must be covalently coupled to the target phase Affinity ligands can, in some cases, even reverse the partitioning behavior of certain proteins.x Compared to affinity chromatography, affinity binding does not require an expensive stationary phase and there are no problems such as loss of ligands on the stationary phase, nonspecific binding which are conlmonly seen in affinity chromatography media. Stability of biomolecules is usually increased in an aqueous two-phase system. The drawbacks of adding ligands include the added cost of ligands and coupling of the ligands to the polymers.

The two commonly used types of ligand are fatty acids and t r i a ~ i n e . ~ Metallated iminodiacetic acid (IDA) derivatives of PEG including Cu(1I)IDA- PEG were also used for binding with proteins rich in surface histidines. Table 11 is a list of biomolecules purified using affinity ligands in aqueous two-phase systems.4 The partitioning of penicillin acylase from Escherichia coli, human hemoglobin, myoglobin, cytochrome c, monoclonal antibodies, horseradish peroxidase, porcine lactate dehydrogenase isoenzynle 5 , phosphofructokinase from rate erythrocyte haemolysate, isoenzymes of lactate dehydrogenase from rabbit tissues, human alkaline phosphatasc isoenzymes, and glucose 6 phos phate dehydrogenase from yeast extract were reviewed by Z a ~ l a v s k ~ . ~ Vari- ous polymer-ligands used in aqueous two-phase affinity partitioning were reviewed by Harris and Yalpani." KopperschlagerhR reviewed the pH, temperature, and competition effects in affinity partitioning for the separation of various enzymes using dye ligands.

E. Large-Scale Aqueous Two-Phase Partitioning of Biomolecules

Aqueous two-phase partitioning has been used widely in bioseparations especially separations of proteins. It is an attractive addition or alternative to other bioseparation Table 12 is a list of biomolecules purified at large-scale using aqueous two-phase systems.4 PEG-dextran-water and PEG-salt-water are most commonly used in large-scale applications because they possess a general applicability, relatively suitable viscosity, and density difference and they are nontoxic and biodegradable and are certified in the pharmacopoeias of most ~ o u n t r i e s . ~ The cost of purified dextran is very high (in the lower hundreds of dollars per kilogram). Crude dextran or hydrolyzed crude dextran, and more recently, hydroxypropyl starch have been used to cut Crude dextran causes a rather high viscosity in water. Hydrolyzed dextran reduces the viscosity.'

F. Equipment and Operational Considerations

The operational modes for aqueous two-phase partitioning are similar to those used in solvent extraction. Single-stage partitioning, repeated batch

TABLE I 2 A List of Biornolecules Purified at Large-Scale Using Aqueous Two-Phase Systems

Acyl aryl amidase Alcohol Dehydrogenase a-Amylase Aspartase Aspartate p-decarbox ylase Chlorophyll a/b-protein (LHPC) Chromatophores Formaldehyde dehydrogenase Formate dehydrogenase Furnarase p-Galactosidase Glucose dehydrogenase Glucose isomerase Glucose-6-phosphate dehydrogenase a-Glucosidase Hexakinase D-2-hydroxyisocarproate dehydrogenase L-2-hydroxyisocarproate dehydrogenase Interferon Isoleucyl-tRNA synthetase Isopropanol dehydrogenase D-Lactate dehydrogenase Leucine dehydrogenase NADkinase Pencillin acylase Phosphofructokinase Phospholipase Phosphorylase Pullulanase Staphylococcal protein A-p-galactosidase hybrid Whey proteins

Source: Reprinted with permission from Diamond and H S U . ~ Copyright 1992 Springer Verlag.

extraction, continuous countercurrent extraction using extractors-settlers in series and liquid-liquid column extraction can be used.-l The same equipment used in solvent extraction may be used for aqueous two-phase partitioning. The use of large extraction equipment for PEG-dextran-water systems is limited due to low feed rates owing to the longer time required for partitioning. Phase separation is easier in PEG-salt-water systems because relatively large bubbles are generated during mixing. They disappear faster than tiny bubbles. The relatively large density difference between the two aqueous phases and lower viscosity of the salt phase are also favorable facto~-s.~' Bamberger et ~ 1 . ~ ' discussed details of laboratory preparation of phase systems and measurement of their physiochemical properties.

The time needed for phase separation depends on polymer type and concentration, the volume ratio, the rate of coalescence of droplets, and the presence of particles. Typical times required for PEG-dextran-water systems are 5 to 30 min. Low-speed centrifugation cuts the time down to 1 min.6' In


acceleration field -

FIGURE 17 Section of a centrifugal countercurrent distribution apparatus with several test tubes shown: a, the lower phase; b, the upper phase; c, the outer ring with cavities for the lower phase; d. the inner ring with cavities for the upper phase; e, the lid ring; and f, an O-ring for sealing. (Reprinted with permission from ~lbertsson.' Copyright 1986 John Wiley & Sons, Inc.)

many cases, a single-stage partitioning does not provide an adequate separation. As in solvent extraction, multistage aqueous two-phase partitioning is used. Figure 17 is an anatomy of a centrifugal countercurrent distribution apparatus designed for such an appli~at ion.~ The separation results resemble elution chromatography. Figure 18 shows the partitioning results from the apparatus for the partitioning of enolase from baker's yeast.3 Enolase is known to have three isoenzymes. Two peaks are shown in the figure.

There are several ways in which polymers can be removed after aqueous two-phase partitions. If the desired protein is partitioned to the salt phase in a PEG-salt-water system, the salt can be removed easily using dialysis with an

12 24 36 60 Tube No.

FIGURE 18 Countercurrent distribution of enolase from baker's yeast: (0) experimental data; (0) theoretical: (-) sum of theoretical curves. (From G. Blomquist and S. Wold. Numerical resolution of CCD [counter current distribution] curves. Aba Chem. Scand. 828. 56-60, 1974.)

Loading Stripping

Top PEG-rich phase

P

4 (Cell debris, contaminants) T ~ottom

Disrupted

phosphate-rich phase

FIGURE I 9 Illustration of a two-stage aqueous two-phase partitioning process with integrated phase recycling. (From Huddleston and Lyddiact6' Copyright John Wiley & Sons Limited. Reproduced with permission.)

ultrafiltration membrane. If the protein ends up in the PEG phase, ultrafiltration can be used to remove PEG, which typically has a relatively small molecular weight of a few thousands. Another approach is to add a salt to partition the protein again into a new salt phase.',4r Figure 19 illustrates a process with integrated phase recycling." The majority of the target protein (denoted by stars in the figure) in a disrupted broth is first partitioned to the top PEG-rich phase. This phase is then contacted with a fresh bottom phase first in the second extractor in the stripping extractor. Most of the protein ends up in the bottom phase, which then undergoes an ultrafiltration step to recover the protein. Precipitation of the protein using the salting out method or solvent is also an option. Size exclusion chromatography should be an effective method to separation PEG from the protein. However, it is more expensive and time-consuming. Ion exchange for the PEG protein solution suffers from a high-pressure drop due to the high viscosity of the solution.

I

" ,I

, '"' *'

' ,

IV. SUMMARY

Top phase (producttPEG)

Liquid-liquid partitioning methods including solvent extraction and aqueous two-phase partitioning are very useful tools for bioseparations. This chapter provided the methodology with various examples and principles as guidelines for the liquid-liquid partitioning in a practical application. Although the engineering aspects of equipment design and operation for liquid-liquid partitioning in industrial applications are firmly established owing to the long history of solvent extraction, the science of predicting partition coefficient accurately in solvent extraction and aqueous liquid-liquid partitioning is not fully developed. Trial and error is still a needed approach in many applications to locate an optimal partitioning system and its operating conditions. With rapid advances in biotechnology and a growing emphasis on products

(salt, p ~ , TLL) I

. , 01 , I#

" 1 ,, a # Phosphate

cells PEG+

phosphate "

Ultra- f~ltrat~on

Products -

LIQUID - LIQUID PARTITIONING 36 1

from renewable resources, the need for efficient downstream processing is increasing. Liquid-liquid partitioning methods will become more and more popular because of their low cost, versatility, and facility of large-scale continuous operations. They can be used in research laboratories or in industry as part of an integrated downstream process for the recovery and purification of biomolecules. Whole broth extraction can even be carried out t o cut down the number of stages in downstream processing for a higher overall process yield.

REFERENCES

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3. Albertsson, P. A. (1986). "Partition o f Cell Particles and Macromolecules," 3rd ed. Wiley, New York.

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5. Kula, M.-R. (1979). Extraction and purification of enzymes using aqueous two-phase systems. AppI Biochem. Bioeng. 2, 71 -95.

6. Walter, H., Brooks, D. E., and Fisher, I)., eds. (1985). "Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology." Academic Press, New York.

7. Walter, H., and Johansson, G., eds. (1994). "Methods in Enzymology," Vol. 228. Academic Press, San Diego, CA.

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tion of Amino Acids" (K. Yamada, S. Kinoshita, T. Tsunoda, and K. Aida, eds.), pp. 227-259. Wiley, New York.

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