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Phase diagrams of new aqueous phase systems composed of aliphatic alcohols, salts and water
Fluid Phase Equilibria, 62 (1991) 53-63
53
Elsevier Science Publishers B.V., Amsterdam
PHASE DIAGRAMS OF NEW AQUEOUS PHASE SYSTEMS COMPOSED OF ALIPHATIC ALCOHOLS, SALTS AND WATER AREND GREVE and MARIA-REGINA
KULA *
Institute of Enzyme Technologv, University of Diirseldorf, Forschungszentrum P.O. Box 20 SO, D-51 70 Jiilich (F. R.G.)
Jiilich,
(Received April 23,199O; accepted in final form August 27, 1990)
ABSTRACT Greve, A. and Kula, M.-R., 1991. Phase diagrams of new aqueous phase systems composed of aliphatic alcohols, salts and water. Fluid Phase Equilibria, 62: 53-63. Aqueous solutions of aliphatic alcohols and salt are not miscible at all concentrations and will often yield two immiscible liquids. The alcohol-rich top phases can be utilized to extract salts from complex mixtures. Detailed data are presented for the ternary systems: ethanolphosphate-water; ethanol-sulfate-water; ethanol-citrate-water; l-butanol-phosphatewater; l-propanol-phosphate-water; 2-propanol-phosphate-water; 2-methyl-2-propanolphosphate-water; at pH 7, 20 o C, 10’ Pa.
INTRODUCTION
two-phase systems resulting from the incompatibility of hydrophilic polymers in water and from the salting out of polyethylene glycol by phosphates, sulfates or citrates have been described (Albertsson, 1986; Vemau and Kula, 1990). These systems form a gentle environment for enzymes and other biologically active proteins, and have been applied in their extraction from cell homogenates and cell culture supematants (Albertsson, 1986; Vemau and Kula, 1990; Hustedt et al., 1985; Kula et al., 1982). This extraction technology offers the advantages of high capacity, high activity yields and being easy to scale up. For large-scale processes, methods for recycling chemicals have been developed (Hustedt, 1986; Greve and Kula, 1990). In this context we recently noted (Greve and Kula, 1990) that lower aliphatic alcohols also form aqueous two-phase systems in the presence of inorganic salts. We Aqueous
* Author to whom correspondence 0378-3812/91/$03.50
should be addressed.
@ 1991 - Elsevier Science Publishers B.V.
54
exploited these two-phase systems for the extraction of salt from the primary bottom phase of a protein extraction process in polyethylene glycol-salt systems. In the following we describe the phase diagrams of the aqueous alcohol-salt systems in some detail to provide a database for theoretical calculations based on the thermodynamics of aqueous two-phase systems (Haynes et al., 1989a, b; Kang and Sandler, 1988; Cabezas and Wag, 1989). MATERIALS
Methanol, ethanol, 1-propanol, 2-propanol, 1-butanol and 2-methyl-2propanol were purchased from E. Merck, Darmstadt, FRG. K,HPO, and KH,PO, were from Fluka, Buchs, Switzerland; sodium sulfate, sodium citrate, NaOH, KOH and HCl were obtained from E. Merck, Darmstadt, F.R.G. All chemicals were of high purity and used without further purification. METHOD
Alcohol determinations The concentrations of the various alcohols were determined by gas chromatography employing a GC 9A chromatograph (Shimadzu, Dtisseldorf, F.R.G.) equipped with a 60 ml column, diameter 3.2 mm, packed with PORAPAC QSO/lOO 75 CC (Macherey and Nagel, Diiren, F.R.G.). The column was thermostatted at 180°C. Helium with a flow rate of 50 ml
1.4
/&e
;.:
P
o,ly
: : ;E+i;;Tl
0.4..
0
10
20
JO
40
50
concentration
60
70
80
90
loo
x (w/w)
Fig. 1. Calibration curves for the analysis of various aliphatic alcohols by gas chromatography.
55
min-’ was used as a carrier gas. The injection port and the FID detector were heated to 200°C. The FID was connected to a C-R 3A integrator (Shimadzu). The system was calibrated by injecting known amounts of the alcohols to be investigated. Calibration curves are shown in Fig. 1. For analysis, 1 g of sample was mixed with 1 g of internal standard and subsequently diluted 1: 10 with water. Aliquots of 1 ~1 of the diluted solution were injected into the gas chromatograph and analyzed as described. Methanol was used as internal standard for the determination of ethanol, while ethanol served as internal standard for all the other alcohols. Three dilutions were prepared independently and samples were analyzed in duplicate to generate the phase diagrams. Potassium phosphate determination
Potassium phosphate with a pH of 7 was prepared from K,HPO, and KH,PO, by mixing them in the ratio of 1.82 : 1. Phosphate concentration was determined by inflection point titration using an autotitrator (Videotitrator VIT 90, Autoburette ABU 91 and sample station ‘M’A 80, Radiometer, Copenhagen, Denmark). 1 ml of sample was placed in the titration beaker and the pH adjusted to 3 by the addition of concentrated HCl. The sample was subsequently titrated with 1 M KOH up to pH 11. During titration the pH was followed as a function of KOH addition. The first derivative of the pH value versus KOH addition yields the plot shown in Fig. 2. The phosphate concentration is determined from the distance between the two maxima. To convert phosphate concentration into salt concentration, the molecular weights of the different potassium phosphate
0t
0
0.2
0.4
a.6 VOiu#
0.6 Of
4
1 1 U KOH
1.2
1.4
1.6
1.6 ml
Fig. 2. Titration of phosphate. First derivative of pH versus volume of titrant (1 M KOH).
56
salts have to be considered according to:
and the molar ratio converted
into mass (M)
msalt = 158.5 - M PO, p-l The density, p, was measured in an oscillating device, DMA 35 (A. Paar, Graz, Austria) with a precision of 0.001 g cme3. Sodium sulfate determination To remove alcohol, which interferes with the sulfate determination, the samples were first dried in an oven for 24 h at 120 OC. Two grams of dry sample were dissolved in 10 ml of water and then mixed with 4 g of 30% BaCl, solution. The resulting BaSO, precipitate was collected after 10 min by centrifugation for 5 min at 2.5OOg and the mass of the sediment determined by weight after washing with dilute HCl and drying for 24 h at 110 OC. The sodium sulfate concentration in the original sample was calculated from the molecular weights of BaSO, (233.36 g mol-‘) and Na,SO, (142.04 g mol-‘). Sodium citrate determination Citrate was determined Kula (1990).
by HPLC analysis as described by Vemau and
Determination of binodals and tie-lines Potassium phosphate was dissolved in water at various concentrations and a known amount placed in a beaker on a table-top balance (Sartorius, GSttingen, F.R.G.). Alcohol was slowly added with stirring until the solution became turbid. Turbidity is a sensitive indicator for the presence of a second phase. Alcohol was added to potassium phosphate solutions to be independent from the dissolution of solid potassium phosphates. For the determination of the tie-lines alcohol, salt and water were mixed to give a defined point in the two-phase region of the phase diagram. The mixture was stirred and then allowed to settle in a closed vessel overnight. Phases were separated with care and analyzed for alcohol and salt concentration. The final water concentration in the different phases was calculated from the mass balance. RESULTS AND DISCUSSION
The solubility of an aliphatic alcohol in water and the mutual miscibility depend on the chain length, and decrease with increasing number of carbon
57 TABLE 1 Ternary phase system 1-butanol-water-potassium
phosphate, pH 7, at 25 ’ C and lo5 Pa
Sample on binodal
Potassium phosphate concentration (wt.%)
1-Butanol concentration (wt.%)
Water concentration (wt.%)
1 2 3 4 5 6 7
0.00 3.79 9.70 19.68 27.09 0.00 0.10
6.70 5.37 2.96 1.62 0.66 82.20 99.90
93.30 90.84 87.34 78.71 72.22 17.80 0.00
atoms in the chain. At room temperature, l-butanol is not completely miscible with water. Water-saturated 1-butanol is reported to dissolve phosphoric acid (Moroto and Watanabe, 1968). Therefore, the ternary system 1-butanol-potassium phosphate-water was investigated in some detail. Only traces of potassium phosphate were found in the butanol-rich phase (Table 1) and only a smalI region of miscibility was observed in the ternary system at pH 7 as shown in Fig. 3. In the phase diagram for 2-methyl-2-propanolpotassium phosphate-water, the binodal is displaced (Fig. 3), indicating the higher polarity of 2-methyl-2-propanol, which leads to improved miscibility and solubility of water and potassium phosphate (Table 2) (Lovrien et al., 1987; Pike and Dennison, 1989). Data for the system 2-propanol-potassium phosphate-water are summarized in Table 3. Reducing the number of carbon atoms in the chain from 4 to 3 improves miscibility, as expected. The effect of the higher polarity of 2-propanol as compared with 1-propanol (Fig. 4) is visible from the shift in
0
5
X (w) lb
Potassium 1'5 20Pho$ate
’
pZ 7
Fig. 3. Binodals of various aqueous alcohol-phosphate Pa.
phase systems, pH 7, at 25 OC and lo5
58 TABLE 2 Ternary phase system 2-methyl-2-propanol-water-potassium lo5 Pa
phosphate, pH 7, at 25 o C and
Sample on binodal
Potassium phosphate concentration (wt.%)
2-Methyl-2-propanol concentration (wt.%)
Water concentration (vJt%)
1 2 3 4
2.44 8.42 17.71 26.27
38.97 15.79 6.45 3.67
58.59 75.79 75.85 70.06
TABLE 3 Ternary phase system 2-propanol-water-potassium
phosphate, pH 7, at 25 o C and lo5 Pa
Sample on binodal
Potassium phosphate concentration (wt.%)
2-Propanol concentration (wt.%)
Water wncentration (wt%)
1 2 3 4
2.25 7.30 17.10 23.84
40.47 25.85 12.30 6.52
57.28 66.85 70.61 69.64
the binodal (Fig. 3) and from a comparison of Tables 3 and 4. As expected, the binodal is displaced even further in the ternary system ethanol-potassium phosphate-water (Fig. 5), yielding aqueous two-phase systems containing an appreciable amount of phosphate in the ethanol-rich phase (Table 5).
z
Two Phase System
loo*=
60
40
20
0 0
10
20
30 X (w)
40
Potassium
50
Phospha61:
Fig. 4. Phase diagram of 1-propanol-potassium 10’ Pa.
pH
;
phosphate-water
system, pH 7, at 25 o C and
59
TABLE 4 Ternary phase system l-propanol-water-potassium
phosphate, pH 7, at 25 o C and lo5 Pa
Potassium phosphate concentration (wt.%)
l-Propanol concentration (wt.%)
Water concentration (wt.%)
1 2 3 4 5 6
2.27 8.10 13.61 18.79 22.96 25.78
43.31 18.62 9.23 6.06 4.14 3.35
54.42 73.28 77.15 75.15 72.69 70.87
8/t 8/b 9/t 9/b 10/t 10/b 11/t 11/b 12/t 12/b 13/t 13/b 14/t 14/b 15/t 15/b 16/t
1.17 12.60 0.68 18.34 0.42 23.73 0.20 28.69 0.09 46.66 0.09 52.15 0.02 58.77 0.02 65.19 0.02
50.87 11.63 54.08 7.07 62.80 4.50 68.40 2.54 84.24 0.21 85.38 0.06 90.91 97.73 98.94
47.94 76.67 45.24 74.59 36.78 71.77 31.40 68.77 15.67 53.13 14.53 47.79 9.07 41.23 2.25 34.81 1.04
Sample
t = top phase; b = bottom phase.
Two
20
Phase
30 X (w)
System
40
Potassium
50
60
Phosphate
Fig. 5. Phase diagram of ethanol-potassium Pa.
ptt
phosphate-water
system, pH 7, at 25 ’ C and lo5
60 TABLE 5 Ternary phase system ethanol-water-potassium Sample
phosphate, pH 7, at 25’ C and lo5 Pa
Potassium phosphate concentration
Ethanol concentration
Water concentration (wt.%
(wt.%)
Mt.%>
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
< 0.01 c 0.02 1.34 2.17 3.73 5.32 7.08 8.79 10.55 12.39 14.32 16.25 18.33 20.43 22.53 23.91
96.99 89.61 47.49 42.86 35.88 31.82 27.73 25.44 23.37 21.32 19.38 17.65 15.79 14.15 12.55 11.48
3.00 10.37 51.17 54.97 60.39 62.86 65.19 65.77 66.08 66.29 66.30 66.10 65.88 65.42 64.92 64.61
17/t 17/b 18/t 18/b 19/t 19/b
0.22 52.36 0.08 57.41 < 0.04 61.54
63.74 0.39 75.53 0.41 85.12 0.22
36.04 47.25 24.39 42.17 14.84 38.24
t = top phase; b = bottom phase.
ETHANOL
Fig. 6. Ternary phase system ethanol-sodium
citrate-water,
pH 7.5, at 25OC and lo5 Pa.
61 TABLE 6 Ternary
phase system ethanol-water-sodium
Sample
Sodium sulfate concentration (wt.%)
(wt.%)
Water concentration (wt.%)
1 2 3 4 5 6 7 8 9 10
0.20 0.93 3.11 5.61 9.22 12.88 17.12 21.61 26.14 31.71
47.58 43.70 34.07 28.66 22.14 17.95 13.36 9.06 5.60 0.79
52.22 55.37 62.82 65.73 68.64 69.16 69.52 69.33 68.25 67.49
11/t 11/b 12/t 12/b 13/t 13/b 14/t 14/b
3.79 27.48 3.00 28.50 1.80 29.12 32.00 0.01
33.00 4.20 35.00 3.40 40.20 2.88 40.20 59.89
63.21 68.32 62.00 68.10 64.61 68.00 64.61 40.10
t = top phase; b = bottom
sulfate at 25 o C and lo5 Pa Ethanol concentration
phase.
TABLE 7 Ternary
phase system ethanol-water-sodium
Sample
Sodium citrate concentration (wt.%)
Ethanol concentration (wt.%)
Water concentration
< 0.1
96.0 86.4 76.6 67.0 57.0 47.2 36.9 26.0 16.1
4.0 13.6 23.15 32.75 42.0 51.0 59.1 64.4 67.5
< 0.1 0.25 0.25 1.0 1.75 4.0 9.6 16.4
citrate, pH 7.5, at 25OC and lo5 Pa
(wt.%)
62
Similar results were obtained with the ternary system ethanol-sodium sulfate-water (Table 6). The ternary system ethanol-sodium citrate-water, however, is completely different (Fig. 6 and Table 7). Only a solubility limit is observed, and the solution is in equilibrium with a solid phase of sodium citrate. Preliminary experiments also indicated that the ternary system methanol-potassium phosphate-water yields aqueous two-phase systems; these, however, have not been analyzed in detail. Because of the molecular weight distribution of polyethylene glycol samples, the ternary systems composed of alcohol-salt-water described above are better defined than aqueous polyethylene glycol-salt two-phase systems. These better defined systems are also more amenable to thermodynamic treatment for describing phase separation and partitioning of third components in such systems on the basis of molecular interactions. Such a treatment is, however, beyond the scope of the present publication. ACKNOWLEDGEMENTS
A.G. was supported by a fellowship awarded within the BMFT program “Angewandte Biologie und Biotechnologie” by DECHEMA, Frankfurt. We thank Fonds der Chemie for financial support and J. Vemau for his help with the graphs. REFERENCES Albertsson, P.A., 1986. Partition of Cell Particles and Macromolecules, 3rd edn. Wiley, New York. Cabezas, H. and &lag, D., 1989. Theory of phase formation and ion partitioning in two polymer aqueous systems. Paper presented at the 6th Int. Conf. on Partitioning in Aqueous Phase Systems, Assmarmshausen, F.R.G. Greve, A. and Kula, M.-R., 1990. Recycling of salt from the primary bottom phase of a protein extraction process. J. Chem. Technol. Biotechnol., in press. Haynes, C.A., Beynon, R.A., King, RX, Blanch, H.W. and Prausnitz, J.M., 1989a. Thermodynamic properties of aqueous polymer solutions: polyethylene glycol/dextran. J. Phys. Chem., 93: 5612-5617. Haynes, CA., Blanch, H.W. and Prausnitz, J.M., 1989b. Separation of protein mixtures by extraction: thermodynamic properties of aqueous two-phase polymer systems containing salts and proteins. Fluid Phase Equilibria, 53: 463-474. Hustedt, H., 1986. Extractive enzyme recovery with simple recycling of phase forming chemicals. Biotechnol. Lett., 8: 791-796. Hustedt, H., Kroner, K.H. and Kula, M.-R., 1985. Application of Phase Partitioning in Biotechnology. In: Walter, H., Brook, D.E. and Fisher, D. (I%.), Partioning in Aqueous Two-Phase Systems Academic Press, New York, pp. 529-587. Kang, C.H. and Sandler, S.I., 1988. A thermodynamic model for two-phase aqueous polymer systems. Biotechnol. Bioeng., 32: 1158-1164.
63 Kula, M.-R., Kroner, K.H. and Hustedt, H., 1982. Purification of Enzymes by Liquid-Liquid Extraction. In: Fiechter, A. (Ed.), Advances in Biochemical Engineering. Springer, Berlin, pp. 73-118. Lovrien, R, Goldensoph, C., Anderson, P.C. and Odegaard, B., 1987. Three Phase Partitioning (TPP) via t-Butanol: Enzyme Separation from Crudes. In: R. Burgess (Ed.), Protein Purification: Micro to Macro. Alan R. Liss, Inc., Vol. 24, New York, pp. 131-148. Moroto, S. and Watanabe, A., 1968. Liquid-liquid equilibria of the system phosphoric acid-organic solvent-water and selectivity of phosphoric acid. Nagoya Kogyo Gijutsu Shikensho Hokoku, 17: 96-102. Pike, R.N. and Dennison, C., 1989. Protein fractionation by three phase partitioning (TPP) in aqueous/t-butanol mixtures. Biotechnol. Bioeng., 33:221-228. Vemau, J. and Kula, M.-R., 1990. Extraction of proteins from biological raw material using aqueous PEG/citrate phase systems. Biotechnol. Appl. B&hem., 12: 397-404.
53
Elsevier Science Publishers B.V., Amsterdam
PHASE DIAGRAMS OF NEW AQUEOUS PHASE SYSTEMS COMPOSED OF ALIPHATIC ALCOHOLS, SALTS AND WATER AREND GREVE and MARIA-REGINA
KULA *
Institute of Enzyme Technologv, University of Diirseldorf, Forschungszentrum P.O. Box 20 SO, D-51 70 Jiilich (F. R.G.)
Jiilich,
(Received April 23,199O; accepted in final form August 27, 1990)
ABSTRACT Greve, A. and Kula, M.-R., 1991. Phase diagrams of new aqueous phase systems composed of aliphatic alcohols, salts and water. Fluid Phase Equilibria, 62: 53-63. Aqueous solutions of aliphatic alcohols and salt are not miscible at all concentrations and will often yield two immiscible liquids. The alcohol-rich top phases can be utilized to extract salts from complex mixtures. Detailed data are presented for the ternary systems: ethanolphosphate-water; ethanol-sulfate-water; ethanol-citrate-water; l-butanol-phosphatewater; l-propanol-phosphate-water; 2-propanol-phosphate-water; 2-methyl-2-propanolphosphate-water; at pH 7, 20 o C, 10’ Pa.
INTRODUCTION
two-phase systems resulting from the incompatibility of hydrophilic polymers in water and from the salting out of polyethylene glycol by phosphates, sulfates or citrates have been described (Albertsson, 1986; Vemau and Kula, 1990). These systems form a gentle environment for enzymes and other biologically active proteins, and have been applied in their extraction from cell homogenates and cell culture supematants (Albertsson, 1986; Vemau and Kula, 1990; Hustedt et al., 1985; Kula et al., 1982). This extraction technology offers the advantages of high capacity, high activity yields and being easy to scale up. For large-scale processes, methods for recycling chemicals have been developed (Hustedt, 1986; Greve and Kula, 1990). In this context we recently noted (Greve and Kula, 1990) that lower aliphatic alcohols also form aqueous two-phase systems in the presence of inorganic salts. We Aqueous
* Author to whom correspondence 0378-3812/91/$03.50
should be addressed.
@ 1991 - Elsevier Science Publishers B.V.
54
exploited these two-phase systems for the extraction of salt from the primary bottom phase of a protein extraction process in polyethylene glycol-salt systems. In the following we describe the phase diagrams of the aqueous alcohol-salt systems in some detail to provide a database for theoretical calculations based on the thermodynamics of aqueous two-phase systems (Haynes et al., 1989a, b; Kang and Sandler, 1988; Cabezas and Wag, 1989). MATERIALS
Methanol, ethanol, 1-propanol, 2-propanol, 1-butanol and 2-methyl-2propanol were purchased from E. Merck, Darmstadt, FRG. K,HPO, and KH,PO, were from Fluka, Buchs, Switzerland; sodium sulfate, sodium citrate, NaOH, KOH and HCl were obtained from E. Merck, Darmstadt, F.R.G. All chemicals were of high purity and used without further purification. METHOD
Alcohol determinations The concentrations of the various alcohols were determined by gas chromatography employing a GC 9A chromatograph (Shimadzu, Dtisseldorf, F.R.G.) equipped with a 60 ml column, diameter 3.2 mm, packed with PORAPAC QSO/lOO 75 CC (Macherey and Nagel, Diiren, F.R.G.). The column was thermostatted at 180°C. Helium with a flow rate of 50 ml
1.4
/&e
;.:
P
o,ly
: : ;E+i;;Tl
0.4..
0
10
20
JO
40
50
concentration
60
70
80
90
loo
x (w/w)
Fig. 1. Calibration curves for the analysis of various aliphatic alcohols by gas chromatography.
55
min-’ was used as a carrier gas. The injection port and the FID detector were heated to 200°C. The FID was connected to a C-R 3A integrator (Shimadzu). The system was calibrated by injecting known amounts of the alcohols to be investigated. Calibration curves are shown in Fig. 1. For analysis, 1 g of sample was mixed with 1 g of internal standard and subsequently diluted 1: 10 with water. Aliquots of 1 ~1 of the diluted solution were injected into the gas chromatograph and analyzed as described. Methanol was used as internal standard for the determination of ethanol, while ethanol served as internal standard for all the other alcohols. Three dilutions were prepared independently and samples were analyzed in duplicate to generate the phase diagrams. Potassium phosphate determination
Potassium phosphate with a pH of 7 was prepared from K,HPO, and KH,PO, by mixing them in the ratio of 1.82 : 1. Phosphate concentration was determined by inflection point titration using an autotitrator (Videotitrator VIT 90, Autoburette ABU 91 and sample station ‘M’A 80, Radiometer, Copenhagen, Denmark). 1 ml of sample was placed in the titration beaker and the pH adjusted to 3 by the addition of concentrated HCl. The sample was subsequently titrated with 1 M KOH up to pH 11. During titration the pH was followed as a function of KOH addition. The first derivative of the pH value versus KOH addition yields the plot shown in Fig. 2. The phosphate concentration is determined from the distance between the two maxima. To convert phosphate concentration into salt concentration, the molecular weights of the different potassium phosphate
0t
0
0.2
0.4
a.6 VOiu#
0.6 Of
4
1 1 U KOH
1.2
1.4
1.6
1.6 ml
Fig. 2. Titration of phosphate. First derivative of pH versus volume of titrant (1 M KOH).
56
salts have to be considered according to:
and the molar ratio converted
into mass (M)
msalt = 158.5 - M PO, p-l The density, p, was measured in an oscillating device, DMA 35 (A. Paar, Graz, Austria) with a precision of 0.001 g cme3. Sodium sulfate determination To remove alcohol, which interferes with the sulfate determination, the samples were first dried in an oven for 24 h at 120 OC. Two grams of dry sample were dissolved in 10 ml of water and then mixed with 4 g of 30% BaCl, solution. The resulting BaSO, precipitate was collected after 10 min by centrifugation for 5 min at 2.5OOg and the mass of the sediment determined by weight after washing with dilute HCl and drying for 24 h at 110 OC. The sodium sulfate concentration in the original sample was calculated from the molecular weights of BaSO, (233.36 g mol-‘) and Na,SO, (142.04 g mol-‘). Sodium citrate determination Citrate was determined Kula (1990).
by HPLC analysis as described by Vemau and
Determination of binodals and tie-lines Potassium phosphate was dissolved in water at various concentrations and a known amount placed in a beaker on a table-top balance (Sartorius, GSttingen, F.R.G.). Alcohol was slowly added with stirring until the solution became turbid. Turbidity is a sensitive indicator for the presence of a second phase. Alcohol was added to potassium phosphate solutions to be independent from the dissolution of solid potassium phosphates. For the determination of the tie-lines alcohol, salt and water were mixed to give a defined point in the two-phase region of the phase diagram. The mixture was stirred and then allowed to settle in a closed vessel overnight. Phases were separated with care and analyzed for alcohol and salt concentration. The final water concentration in the different phases was calculated from the mass balance. RESULTS AND DISCUSSION
The solubility of an aliphatic alcohol in water and the mutual miscibility depend on the chain length, and decrease with increasing number of carbon
57 TABLE 1 Ternary phase system 1-butanol-water-potassium
phosphate, pH 7, at 25 ’ C and lo5 Pa
Sample on binodal
Potassium phosphate concentration (wt.%)
1-Butanol concentration (wt.%)
Water concentration (wt.%)
1 2 3 4 5 6 7
0.00 3.79 9.70 19.68 27.09 0.00 0.10
6.70 5.37 2.96 1.62 0.66 82.20 99.90
93.30 90.84 87.34 78.71 72.22 17.80 0.00
atoms in the chain. At room temperature, l-butanol is not completely miscible with water. Water-saturated 1-butanol is reported to dissolve phosphoric acid (Moroto and Watanabe, 1968). Therefore, the ternary system 1-butanol-potassium phosphate-water was investigated in some detail. Only traces of potassium phosphate were found in the butanol-rich phase (Table 1) and only a smalI region of miscibility was observed in the ternary system at pH 7 as shown in Fig. 3. In the phase diagram for 2-methyl-2-propanolpotassium phosphate-water, the binodal is displaced (Fig. 3), indicating the higher polarity of 2-methyl-2-propanol, which leads to improved miscibility and solubility of water and potassium phosphate (Table 2) (Lovrien et al., 1987; Pike and Dennison, 1989). Data for the system 2-propanol-potassium phosphate-water are summarized in Table 3. Reducing the number of carbon atoms in the chain from 4 to 3 improves miscibility, as expected. The effect of the higher polarity of 2-propanol as compared with 1-propanol (Fig. 4) is visible from the shift in
0
5
X (w) lb
Potassium 1'5 20Pho$ate
’
pZ 7
Fig. 3. Binodals of various aqueous alcohol-phosphate Pa.
phase systems, pH 7, at 25 OC and lo5
58 TABLE 2 Ternary phase system 2-methyl-2-propanol-water-potassium lo5 Pa
phosphate, pH 7, at 25 o C and
Sample on binodal
Potassium phosphate concentration (wt.%)
2-Methyl-2-propanol concentration (wt.%)
Water concentration (vJt%)
1 2 3 4
2.44 8.42 17.71 26.27
38.97 15.79 6.45 3.67
58.59 75.79 75.85 70.06
TABLE 3 Ternary phase system 2-propanol-water-potassium
phosphate, pH 7, at 25 o C and lo5 Pa
Sample on binodal
Potassium phosphate concentration (wt.%)
2-Propanol concentration (wt.%)
Water wncentration (wt%)
1 2 3 4
2.25 7.30 17.10 23.84
40.47 25.85 12.30 6.52
57.28 66.85 70.61 69.64
the binodal (Fig. 3) and from a comparison of Tables 3 and 4. As expected, the binodal is displaced even further in the ternary system ethanol-potassium phosphate-water (Fig. 5), yielding aqueous two-phase systems containing an appreciable amount of phosphate in the ethanol-rich phase (Table 5).
z
Two Phase System
loo*=
60
40
20
0 0
10
20
30 X (w)
40
Potassium
50
Phospha61:
Fig. 4. Phase diagram of 1-propanol-potassium 10’ Pa.
pH
;
phosphate-water
system, pH 7, at 25 o C and
59
TABLE 4 Ternary phase system l-propanol-water-potassium
phosphate, pH 7, at 25 o C and lo5 Pa
Potassium phosphate concentration (wt.%)
l-Propanol concentration (wt.%)
Water concentration (wt.%)
1 2 3 4 5 6
2.27 8.10 13.61 18.79 22.96 25.78
43.31 18.62 9.23 6.06 4.14 3.35
54.42 73.28 77.15 75.15 72.69 70.87
8/t 8/b 9/t 9/b 10/t 10/b 11/t 11/b 12/t 12/b 13/t 13/b 14/t 14/b 15/t 15/b 16/t
1.17 12.60 0.68 18.34 0.42 23.73 0.20 28.69 0.09 46.66 0.09 52.15 0.02 58.77 0.02 65.19 0.02
50.87 11.63 54.08 7.07 62.80 4.50 68.40 2.54 84.24 0.21 85.38 0.06 90.91 97.73 98.94
47.94 76.67 45.24 74.59 36.78 71.77 31.40 68.77 15.67 53.13 14.53 47.79 9.07 41.23 2.25 34.81 1.04
Sample
t = top phase; b = bottom phase.
Two
20
Phase
30 X (w)
System
40
Potassium
50
60
Phosphate
Fig. 5. Phase diagram of ethanol-potassium Pa.
ptt
phosphate-water
system, pH 7, at 25 ’ C and lo5
60 TABLE 5 Ternary phase system ethanol-water-potassium Sample
phosphate, pH 7, at 25’ C and lo5 Pa
Potassium phosphate concentration
Ethanol concentration
Water concentration (wt.%
(wt.%)
Mt.%>
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
< 0.01 c 0.02 1.34 2.17 3.73 5.32 7.08 8.79 10.55 12.39 14.32 16.25 18.33 20.43 22.53 23.91
96.99 89.61 47.49 42.86 35.88 31.82 27.73 25.44 23.37 21.32 19.38 17.65 15.79 14.15 12.55 11.48
3.00 10.37 51.17 54.97 60.39 62.86 65.19 65.77 66.08 66.29 66.30 66.10 65.88 65.42 64.92 64.61
17/t 17/b 18/t 18/b 19/t 19/b
0.22 52.36 0.08 57.41 < 0.04 61.54
63.74 0.39 75.53 0.41 85.12 0.22
36.04 47.25 24.39 42.17 14.84 38.24
t = top phase; b = bottom phase.
ETHANOL
Fig. 6. Ternary phase system ethanol-sodium
citrate-water,
pH 7.5, at 25OC and lo5 Pa.
61 TABLE 6 Ternary
phase system ethanol-water-sodium
Sample
Sodium sulfate concentration (wt.%)
(wt.%)
Water concentration (wt.%)
1 2 3 4 5 6 7 8 9 10
0.20 0.93 3.11 5.61 9.22 12.88 17.12 21.61 26.14 31.71
47.58 43.70 34.07 28.66 22.14 17.95 13.36 9.06 5.60 0.79
52.22 55.37 62.82 65.73 68.64 69.16 69.52 69.33 68.25 67.49
11/t 11/b 12/t 12/b 13/t 13/b 14/t 14/b
3.79 27.48 3.00 28.50 1.80 29.12 32.00 0.01
33.00 4.20 35.00 3.40 40.20 2.88 40.20 59.89
63.21 68.32 62.00 68.10 64.61 68.00 64.61 40.10
t = top phase; b = bottom
sulfate at 25 o C and lo5 Pa Ethanol concentration
phase.
TABLE 7 Ternary
phase system ethanol-water-sodium
Sample
Sodium citrate concentration (wt.%)
Ethanol concentration (wt.%)
Water concentration
< 0.1
96.0 86.4 76.6 67.0 57.0 47.2 36.9 26.0 16.1
4.0 13.6 23.15 32.75 42.0 51.0 59.1 64.4 67.5
< 0.1 0.25 0.25 1.0 1.75 4.0 9.6 16.4
citrate, pH 7.5, at 25OC and lo5 Pa
(wt.%)
62
Similar results were obtained with the ternary system ethanol-sodium sulfate-water (Table 6). The ternary system ethanol-sodium citrate-water, however, is completely different (Fig. 6 and Table 7). Only a solubility limit is observed, and the solution is in equilibrium with a solid phase of sodium citrate. Preliminary experiments also indicated that the ternary system methanol-potassium phosphate-water yields aqueous two-phase systems; these, however, have not been analyzed in detail. Because of the molecular weight distribution of polyethylene glycol samples, the ternary systems composed of alcohol-salt-water described above are better defined than aqueous polyethylene glycol-salt two-phase systems. These better defined systems are also more amenable to thermodynamic treatment for describing phase separation and partitioning of third components in such systems on the basis of molecular interactions. Such a treatment is, however, beyond the scope of the present publication. ACKNOWLEDGEMENTS
A.G. was supported by a fellowship awarded within the BMFT program “Angewandte Biologie und Biotechnologie” by DECHEMA, Frankfurt. We thank Fonds der Chemie for financial support and J. Vemau for his help with the graphs. REFERENCES Albertsson, P.A., 1986. Partition of Cell Particles and Macromolecules, 3rd edn. Wiley, New York. Cabezas, H. and &lag, D., 1989. Theory of phase formation and ion partitioning in two polymer aqueous systems. Paper presented at the 6th Int. Conf. on Partitioning in Aqueous Phase Systems, Assmarmshausen, F.R.G. Greve, A. and Kula, M.-R., 1990. Recycling of salt from the primary bottom phase of a protein extraction process. J. Chem. Technol. Biotechnol., in press. Haynes, C.A., Beynon, R.A., King, RX, Blanch, H.W. and Prausnitz, J.M., 1989a. Thermodynamic properties of aqueous polymer solutions: polyethylene glycol/dextran. J. Phys. Chem., 93: 5612-5617. Haynes, CA., Blanch, H.W. and Prausnitz, J.M., 1989b. Separation of protein mixtures by extraction: thermodynamic properties of aqueous two-phase polymer systems containing salts and proteins. Fluid Phase Equilibria, 53: 463-474. Hustedt, H., 1986. Extractive enzyme recovery with simple recycling of phase forming chemicals. Biotechnol. Lett., 8: 791-796. Hustedt, H., Kroner, K.H. and Kula, M.-R., 1985. Application of Phase Partitioning in Biotechnology. In: Walter, H., Brook, D.E. and Fisher, D. (I%.), Partioning in Aqueous Two-Phase Systems Academic Press, New York, pp. 529-587. Kang, C.H. and Sandler, S.I., 1988. A thermodynamic model for two-phase aqueous polymer systems. Biotechnol. Bioeng., 32: 1158-1164.
63 Kula, M.-R., Kroner, K.H. and Hustedt, H., 1982. Purification of Enzymes by Liquid-Liquid Extraction. In: Fiechter, A. (Ed.), Advances in Biochemical Engineering. Springer, Berlin, pp. 73-118. Lovrien, R, Goldensoph, C., Anderson, P.C. and Odegaard, B., 1987. Three Phase Partitioning (TPP) via t-Butanol: Enzyme Separation from Crudes. In: R. Burgess (Ed.), Protein Purification: Micro to Macro. Alan R. Liss, Inc., Vol. 24, New York, pp. 131-148. Moroto, S. and Watanabe, A., 1968. Liquid-liquid equilibria of the system phosphoric acid-organic solvent-water and selectivity of phosphoric acid. Nagoya Kogyo Gijutsu Shikensho Hokoku, 17: 96-102. Pike, R.N. and Dennison, C., 1989. Protein fractionation by three phase partitioning (TPP) in aqueous/t-butanol mixtures. Biotechnol. Bioeng., 33:221-228. Vemau, J. and Kula, M.-R., 1990. Extraction of proteins from biological raw material using aqueous PEG/citrate phase systems. Biotechnol. Appl. B&hem., 12: 397-404.