Accepted Manuscript Experimental study and thermodynamic modelling of penicillin-G extraction using PEG 6000 and K2HPO4 aqueous two-phase system Alireza Rabieenezhad, Aliakbar Roosta PII: DOI: Reference:
S0021-9614(18)30011-9 https://doi.org/10.1016/j.jct.2018.01.010 YJCHT 5303
To appear in:
J. Chem. Thermodynamics
Received Date: Revised Date: Accepted Date:
17 November 2017 13 January 2018 13 January 2018
Please cite this article as: A. Rabieenezhad, A. Roosta, Experimental study and thermodynamic modelling of penicillin-G extraction using PEG 6000 and K2HPO4 aqueous two-phase system, J. Chem. Thermodynamics (2018), doi: https://doi.org/10.1016/j.jct.2018.01.010
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Experimental study and thermodynamic modelling of penicillin-G extraction using PEG 6000 and K2HPO4 aqueous two-phase system Alireza Rabieenezhad, Aliakbar Roosta* Department of Chemical, Petroleum and Gas Engineering, Shiraz University of Technology, Shiraz, Iran Email:
[email protected] Tel: +98-7137354520 Fax: +98-7137354520
Abstract The use of extractive fermentation in penicillin production can overcome the low yield of penicillin. In this study, the liquid-liquid equilibrium (LLE) data of the ternary aqueous twophase system (ATPS) of (polyethylene glycol 6000 + dipotassium hydrogen phosphate + water) at 298.2 K were measured. Then, penicillin G was added to the ternary ATPS and the LLE of the quaternary system at 298.2 K was measured. The maximum distribution coefficient of 15.63 was experimentally observed for penicillin G that indicates the good performance of the system in separating penicillin G from the fermentation broth. The experimental LLE data were modelled with the NRTL model with average mass fraction deviations of 0.004 for both ternary and quaternary systems. The model is employed to investigate the effect of mixture composition on the distribution coefficient of penicillin G. The results show that the decrease of water in the mixture increases significantly the distribution coefficient of penicillin G, while increasing K2HPO4 has a small positive effect on the distribution coefficient. Keywords: ATPS; LLE; penicillin G; NRTL
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1. Introduction Penicillin G or Benzyl penicillin is one of the first antibiotics that has been effectively used against different bacterial infections, such as pneumonia, diphtheria, syphilis, strep throat, necrotizing enterocolitis, leptospirosis, cellulitis, gas gangrene, and tetanus [1]. Antibiotics are usually produced as the secondary metabolites in fermentation processes, which inhibit the growth of their creator microorganisms by blocking their cell wall synthesis. The extraction of produced antibiotics from the fermentation medium during the production, which is known as extractive fermentation, can increase the production yield [2]. There are different methods for separation of biological materials from the fermentation medium, such as adsorption, crystallization, precipitation, column chromatography, membrane separation, organic solvent extraction, and aqueous two-phase extraction [3,4]. Aqueous two-phase systems (ATPSs) are the mixture of two hydrophilic components in water, which form two aqueous phases. The hydrophilic components can include two polymers, a salt and a polymer, an ionic liquid and a polymer, an ionic liquid and a salt, or a polymer and an alcohol. The major part of both phases is formed from water, which is compatible with the fermentation medium. Thus, ATPSs are considered as appropriate alternatives for the organic solvents. ATPS was firstly introduced by Beijerinck [5] and then has been used by researchers to selectively separate desired components, especially biomolecules, from aqueous solutions. In this regards, the ATPSs have been used in separation and purification of enzymes [6–9], antibiotics [10–13], DNA [14– 17], proteins [9,18,19], and purine alkaloids [20,21]. Among the studies conducted, some researches have been focused on the separation of penicillin G. Roosta et al. [10] used the ATPS of (PEG6000 + Na2SO4 + water) to separate penicillin G. The maximum distribution coefficient of 3.47 for penicillin G was observed. Liu et al. [22] studied the distribution coefficient of 2
penicillin G in the aqueous solution of 1-butyl-3-methylimidazolium chloride and Na2H2PO4. They showed that the extraction yield of 93% can be achieved in this system. Pazuki et al. [12] studied four ATPS containing two polymers, i.e. PEG 20000 and PEG 35000, and two salts, i.e. KH2PO4 and C6H5Na3O7·5H2O for separation of penicillin G at temperatures of (301.2, 307.2, and 310.2) K. The results showed that the distribution coefficient of penicillin G varied between 0.951 and 1.054. In this study, the distribution coefficient of penicillin G in the ATPS of PEG 6000 and K2HPO4 at 298.2 K is investigated. In this regard, equilibrium compositions of PEG 6000, K2HPO4, penicillin G are measured at different compositions of feed. Then, the experimental data are modeled with the NRTL model. The model is employed to study the effect feed composition on the distribution coefficient of penicillin G in order to investigate the ability of this system in the separation of penicillin G. 2. Experimental 2.1.Materials The list of chemicals, their purity and source are given in Table 1. As can be seen in this table, all reagents were of analytical grade, thus the chemicals were used as received, without further purification. PEG 6000 and K2HPO4 purchased from Merck Co with mass fraction purities greater than 0.99. Penicillin G sodium salt with the potency more than 1477 units∙mg-1 was purchased from Sigma Aldrich. Double deionized water with the resistivity of 18.2 MΩ∙cm was used in experiments. In order to measure the concentration of phosphate in the aqueous solutions, the reagents of ascorbic acid and ammonium molybdate tetrahydrate with mass fraction purities greater than 0.99 were purchased from Sigma Aldrich.
3
2.2. Methods In this study, the liquid-liquid equilibria (LLE) of the ternary mixture of PEG 6000, K2HPO4 and water, and the quaternary system of PEG 6000, K2HPO4, penicillin G and water were studied at 298.2 K. In order to determine the binodal curve of the ternary system, a mixture consisting of PEG 6000, K2HPO4 and water was prepared by an analytical balance (A&D, HR200) with precision of 0.0001 g so that it formed a two-phase system. Then, a few drops of water (less than 0.1 mL) were added to the mixture. After mixing well, the mixture was centrifuged (Selecta Lab, TL320) at 5000 rpm. At this time, the interface was distinct if the system was still a two-phase system. The process of adding water and centrifuge continued until a single-phase system appeared. The weight of the added water was measured by the difference of the mixture weight before and after the addition of water. Then, the weight fraction of components was calculated to get one of the points of the binodal curve in the K2HPO4- PEG 6000 diagram. The procedure was continued to obtain sufficient points to draw the binodal curve. In order to obtain tie lines, a ternary mixture was prepared using the analytical balance to get a two-phase system. The mixture was well mixed and then was centrifuged. Two phases were separated by a micropipette. The mass and volume of two phases were measured with precise of 0.0001 g and 0.02 mL, respectively. After that, the concentration of phosphate was measured at both phases based on a colorimetric method by a spectrophotometer (Jenway, 7315) [23]. Then, the ends of the tie line were determined on the binodal curve by knowing concentrations of phosphate at both phases. Consequently, the concentration of PEG 6000 was determined by the ends of the tie line on the binodal curve. The material balances of phosphate and PEG 6000 were verified by the concentrations, mass, and volume of both phases. The experiment was valid if the
4
material balances were satisfied with accuracy less than 0.5%, otherwise, the experiment was repeated. The experiments were conducted to obtain more tie lines of the ternary system. In the next part of experiments, the LLE of the quaternary system was studied. Since the concentration of penicillin G in the fermentation broth is less than 800 units∙mL-1 (or less than 0.5 g∙L-1) during the production [24–26], in this study, the concentration of penicillin G in the ATPS was changed between (0.35 and 0.5) g∙L-1. Hekayati et al. [27] and Roosta et al. [10] showed that if the concentration of the extract component in the ATPS is low, the binodal curves of ternary systems are almost unchanged by the extract component. Because the concentration of penicillin G is low compared to other components in the quaternary system of this study, the equilibrium data of quaternary system is obtained with the help of the binodal curve of the ternary system. In this regard, a quaternary mixture was prepared by the analytical balance. The mixture was well mixed and then was centrifuged. Two phases were separated by a micropipette. The mass and volume of two phases were measured with precise of 0.0001 g and 0.02 mL, respectively. The concentration of phosphate was measured based on the colorimetric method [23], and the concentration of penicillin G was measured by a turbidimetric method [10] by using a spectrophotometer (Jenway- 7315). The concentrations of PEG 6000 were determined by the concentrations of phosphate and the binodal curve of ternary system. Finally, the material balances of phosphate, PEG 6000 and penicillin G were verified by the concentrations, mass, and volume of both phases. The experiment was valid if the material balances were satisfied with an accuracy less than 1%. 3. Model
5
The distribution coefficient of penicillin G can be affected by different parameters such as the concentration of PEG 6000, K2HPO4 and water in the feed. In order to study the performance of the current ATPS in the separation of penicillin G under different conditions, the experimental data can be correlated with an appropriate model. The complex behavior of partially miscible systems is usually described by thermodynamic models such as the NRTL, UNIQUAC and UNIFAC activity models. According to the literature, the NRTL model can accurately model the behavior of ATPSs [10,27–30]. In this study, the NRTL model is used to model the liquid-liquid equilibria of the ternary and quaternary aqueous two-phase systems. The Rachford-Rice method is used to carry out the LLE calculations [31]. This iterative method combines the equilibrium equations and material balance equations in order to compute the compositions of two equilibrium phases according to Eqs (1) to (4):
ibottom K i top i
for i 1 to N
(1)
z i K i 1 0 i 1 1 EOF K i 1 N
x ibottom
(2)
zi for i 1 to N 1 EOF K i 1
x itop K i x ibottom
(3)
for i 1 to N
(4)
where z, x, K, N, and EOF denote the mole fraction in overall composition, mole fraction in each phase, distribution coefficient, activity coefficient, the number of components, and the molar ratio of top (extract) phase to feed. The activity coefficients (γ) are calculated by the NRTL activity model according to Eqs (5) and (6) [32]:
6
N
ln i
j 1 N
ji
G ji x j
G j 1
ji
xj
N x j G ij N j 1 M G kj x k j k 1
N kj G kj x k k 1 ij N G kj x k k 1
(5)
Gij exp(ij ij ) and ij ji
(6)
where αij and τij are the adjustable parameters of the NRTL model which are related to the nonrandomness of the mixture and the interaction energy between molecules i and j, respectively. The adjustable parameters of the NRTL model are calculated by minimizing the deviation between the experimental and estimated mole fractions by using the optimization method of genetic algorithm combined with the Rachford-Rice method. The average absolute relative deviation, as is shown by Eq. (7), was minimized in calculating the parameters of the NRTL model. AARD %
D N 1 (w Top, Exp. w Top, Calc. ) (w ijBottom, Exp w ijBottom, Calc. ) 100 ij ij 2D (N 1) i 1 j 1 w ijTop, Exp. w ijBottom, Exp.
(7)
where D denotes the number of datasets.
4. Results and Discussion The measured experimental data of the binodal curve of the ternary system of (K2HPO4 + PEG 6000 + water) are in Table 2 and also are illustrated in Figure 1. As can be seen in this figure, on the right side of the binodal curve, the ATPS contains more K2HPO4; consequently, the ratio of bottom (phosphate-rich) phase to top (PEG 6000-rich) phase is higher too. The effect of this issue on the separation of penicillin G is discussed in the next parts of this study. The experimental data of the ternary system, including the weight percentages of feed, top phase and bottom phase for 11 data sets are listed in Table 3. Using the LLE data of the ternary system,
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the binary interaction parameters of the NRTL model are calculated. The best parameters of the model, which led to the minimum deviation between experimental and estimated data, are listed in Table 4. In order to show the accuracy of the model in calculating the LLE of the ternary system, average mass fraction deviations between experimental and estimated data are listed in Table 5, and also a comparison is made in Figure 2 between experimental and calculated LLE data. According to results of Table 4, the overall average deviation of the mass fraction is 0.0038 that indicates the good accuracy of the NRTL model. Furthermore, Figure 2 shows the excellent performance of the NRTL model in estimating the equilibrium mass fractions for both the bottom and top phases. As discussed before, it was assumed that the binodal curve of the ternary system is almost unchanged by the penicillin G, because of low concentration of penicillin G. The assumption is verified in Table 6. As can be seen in this table, the weight percentages of top phase and bottom phase before and after addition of penicillin G are compared for two points. According to the results, the weight percentages of (K2HPO4 + PEG 6000) in both phases do not change by the penicillin G significantly. The experimental data of the quaternary system of (K2HPO4 + PEG 6000 + penicillin G + water) are shown in Table 7. The data include the weight percentages of feed, top phase and bottom phase for 21 data sets, alongside the distribution coefficient of penicillin G which is defined as the ratio of the penicillin G mass fraction in the top phase to that of the bottom phase (w3top/w3bot). As can be seen in this table, the distribution coefficient of penicillin G changes with the composition of the feed. According to results of Table 7, the maximum and minimum distribution coefficients of 15.63 and 2.93 indicate the good performance of the current ATPS in separating penicillin G. However, an accurate model is required to find the effect of feed 8
composition on the distribution coefficients of penicillin G in more details. In this regard, the LLE data of the quaternary data was modelled by the NRTL model. A part of the model parameters have been calculated by using the LLE data of the ternary system as reported in Table 4. The remaining parameters of the NRTL model for the quaternary system were calculated by using the LLE data of the quaternary system, as listed in Table 8. To assess the accuracy of the NRTL model in estimating the LLE data of the quaternary system, the mass fraction deviations between experimental and calculated data of all components in both phases were calculated and listed in Table 9. It is obtained from Table 9 that the overall average deviation of mass fraction of all components is 0.0039 for the quaternary system. Furthermore, the estimated weight fraction of all components in both phases was plotted against the experimental data and compared with the 45° line in Figure 3. As can be seen in this figure, the superior fit between data points and the 45° line illustrates the good performance of the NRTL model in estimating the LLE data of the quaternary system. In this part of the study, the effect of feed composition on the distribution coefficient of penicillin G is investigated by using the NRTL model. As shown in Figure 4a, three tie lines and some feed points on each tie line have been considered for this purpose. Farther tie line from the plait point (tie line 1 in Figure 4(a)) means less water in the feed. Furthermore, at left-hand sides of tie lines (more PEG 6000 in the feed), the mass ratio of extract (top) phase to the feed (EOF) is higher, vice versa. For instance, EOF for feeds F1 and F2 in Figure 4(a) are 0.7 and 0.3, respectively. Figures 4(b) to 4(d) illustrate the effects of tie line location and EOF on the weight percentages of penicillin G in both phases (w3top and w3 bot), and the distribution coefficient of penicillin G (k3), respectively.
9
For a more efficient separation of penicillin G, it is desirable to increase w3top and decrease w3bot, consequently increase k3. As can be seen in Figure 4(b), w3 top is higher at lower EOFs. In the other words, w3top increases with the increase of K2HPO4 in the feed. In addition, w3top increases as the amount of water in the feed is decreased (tie lines farther from the plait point). Figure 4(c) shows that w3bot decreases with the decrease of both K2HPO4 and water in the feed. A comparison between Figures 4(b) and 4(c) demonstrate that decreasing water in the feed has a positive effect on the separation. However, increasing K2HPO4 leads to increase of both w3top and w3bot which causing contradictory effects on the separation. Figure 4(d) shows the effects of EOF and the amount of water in the feed on k3. As seen in this figure, k3 increases significantly with a decrease of water in the feed, while increasing K2HPO4 has a small positive effect on k3.
5. Conclusions In this study, the applicability of aqueous two-phase system of (PEG 6000 + K2HPO4+ water) in separating penicillin G was studied. The results showed that the distribution coefficient of 15.63 for penicillin G can be achieved in this system. According to results, the current ATPS is an efficient system for separating penicillin G. The NRTL model with a good accuracy estimated the LLE data of the ATPS. The model was employed to study the effect of feed composition on the distribution coefficient of penicillin G. According to the results, the amount of K 2HPO4 and water in the feed can significantly affect the weight fraction of penicillin G in both phases. Although the distribution coefficient of penicillin G is a strong function of the water mass fraction in the feed, it is almost independent of the mass fraction of K2HPO4 in the feed. 10
Acknowledgment The authors are grateful to Dr Payam Parvasi for his help, and also acknowledge the Shiraz University of Technology for supporting this research.
References [1]
G.L. Burci, C.-H. Vignes, World Health Organization, Kluwer Law International, New York, 2004.
[2]
L.F. Gutiérrez, Ó.J. Sánchez, C.A. Cardona, Analysis and Design of Extractive Fermentation Processes Using a Novel Short-Cut Method, Ind. Eng. Chem. Res. 52 (2013) 12915–12926. doi:10.1021/ie301297h.
[3]
R.G. Harrison, P. Todd, S.R. Rudge, D.P. Petrides, Bioseparations Science and Engineering, second, Oxford University Press, New York, 2003. doi:10.1017/CBO9781107415324.004.
[4]
A. Kumar, A. Awasthi, Bioseparation Engineering, I.K. International Publishing House Pvt. Ltd., New Dehli, 2009.
[5]
M.W. Beijerinck, Parasiten und Infektionskrankenheiten, Zentralblatt Fur Bakteriol. Parasiten Und Infekt. 2 (1896) 697–699.
[6]
S. Dreyer, U. Kragl, Ionic liquids for aqueous two-phase extraction and stabilization of enzymes, Biotechnol. Bioeng. 99 (2008) 1416–1424. doi:10.1002/bit.21720.
[7]
C.W. Ooi, S.L. Hii, S.M.M. Kamal, A. Ariff, T.C. Ling, Extractive fermentation using aqueous two-phase systems for integrated production and purification of extracellular lipase derived from Burkholderia pseudomallei, Process Biochem. 46 (2011) 68–73. doi:10.1016/j.procbio.2010.07.014.
[8]
J.H.P.M. Santos, J.C.F. Santos, G.P. Meneguetti, C.O. Rangel-Yagui, J.A.P. Coutinho, M. Vitolo, et al., In situ purification of periplasmatic L-asparaginase by aqueous two phase systems with ionic liquids (ILs) as adjuvants, J. Chem. Technol. Biotechnol. (2017). doi:10.1002/jctb.5455.
[9]
B. Perez, L.P. Malpiedi, G. Tubío, B. Nerli, P. de Alcântara Pessôa Filho, Experimental determination and thermodynamic modelling of phase equilibrium and protein partitioning in aqueous two-phase systems containing biodegradable salts, J. Chem. Thermodyn. 56 (2013) 136–143. doi:10.1016/j.jct.2012.07.017.
[10] A. Roosta, F. Jafari, J. Javanmardi, Liquid–Liquid Equilibrium in an Aqueous Two-Phase System of Polyethylene Glycol 6000, Sodium Sulfate, Water, Glucose, and Penicillin-G: Experimental and Thermodynamic Modelling, J. Chem. Eng. Data. 61 (2016) 565–570. doi:10.1021/acs.jced.5b00715. 11
[11] S.G. Doozandeh, G. Pazuki, B. Madadi, A.A. Rohani, Measurement of cephalexin partition coefficients in PEG+K2HPO4+H2O aqueous two-phase systems at 301.15, 306.15 and 311.15K, J. Mol. Liq. 174 (2012) 95–99. doi:10.1016/j.molliq.2012.07.026. [12] G. Pazuki, M. Vossoughi, V. Taghikhani, Partitioning of Penicillin G Acylase in Aqueous Two-Phase Systems of Poly(ethylene glycol) 20000 or 35000 and Potassium Dihydrogen Phosphate or Sodium Citrate, J. Chem. Eng. Data. 55 (2010) 243–248. doi:10.1021/je900319s. [13] J. Han, Y. Wang, C. Chen, W. Kang, Y. Liu, K. Xu, et al., (Liquid+liquid) equilibria and extraction capacity of (imidazolium ionic liquids+potassium tartrate) aqueous two-phase systems, J. Mol. Liq. 193 (2014) 23–28. doi:10.1016/j.molliq.2013.12.022. [14] G.A. Gomes, A.M. Azevedo, M.R. Aires-Barros, D.M.F. Prazeres, Purification of plasmid DNA with aqueous two phase systems of PEG 600 and sodium citrate/ammonium sulfate, Sep. Purif. Technol. 65 (2009) 22–30. doi:10.1016/j.seppur.2008.01.026. [15] F. Luechau, T.C. Ling, A. Lyddiatt, Partition of plasmid DNA in polymer–salt aqueous two-phase systems, Sep. Purif. Technol. 66 (2009) 397–404. doi:10.1016/j.seppur.2008.12.003. [16] H.S.C. Barbosa, A.V. Hine, S. Brocchini, N.K.H. Slater, J.C. Marcos, Dual affinity method for plasmid DNA purification in aqueous two-phase systems, J. Chromatogr. A. 1217 (2010) 1429–1436. doi:10.1016/j.chroma.2009.12.059. [17] T. Matos, H.-O. Johansson, J.A. Queiroz, L. Bulow, Isolation of PCR DNA fragments using aqueous two-phase systems, Sep. Purif. Technol. 122 (2014) 144–148. doi:10.1016/j.seppur.2013.11.014. [18] B.A. Andrews, A.S. Schmidt, J.A. Asenjo, Correlation for the partition behavior of proteins in aqueous two-phase systems: Effect of surface hydrophobicity and charge, Biotechnol. Bioeng. 90 (2005) 380–390. doi:10.1002/bit.20495. [19] R.K. Desai, M. Streefland, R.H. Wijffels, M. H. M. Eppink, Extraction and stability of selected proteins in ionic liquid based aqueous two phase systems, Green Chem. 16 (2014) 2670–2679. doi:10.1039/C3GC42631A. [20] J. Ren, Z. Li, J. Liu, Y. Pei, H. Wang, J. Wang, Choline derivative ionic liquids-based aqueous two-phase systems: Phase diagrams and partition of purine alkaloids, J. Chem. Thermodyn. 118 (2018) 51–57. doi:10.1016/j.jct.2017.10.017. [21] D. de Araujo Sampaio, L.I. Mafra, C.I. Yamamoto, E.F. de Andrade, M.O. de Souza, M.R. Mafra, et al., Aqueous two-phase (polyethylene glycol+sodium sulfate) system for caffeine extraction: Equilibrium diagrams and partitioning study, J. Chem. Thermodyn. 98 (2016) 86–94. doi:10.1016/j.jct.2016.03.004. [22] Q. Liu, J. Yu, W. Li, X. Hu, H. Xia, H. Liu, et al., Partitioning Behavior of Penicillin G in Aqueous Two Phase System Formed by Ionic Liquids and Phosphate, Sep. Sci. Technol. 41 (2006) 2849–2858. doi:10.1080/01496390600786135. [23] T.G. Towns, Determination of aqueous phosphate by ascorbic acid reduction of phosphomolybdic acid, Anal. Chem. 58 (1986) 223–229. doi:10.1021/ac00292a054. 12
[24] M.J. Johnson, Recent advances in penicillin fermentation., Rend. Ist. Sup. Sanit. 16 (1953) 125–53. http://www.ncbi.nlm.nih.gov/pubmed/13134605. [25] J. Rajendhran, V. Krishnakumar, P. Gunasekaran, Optimization of a fermentation medium for the production of Penicillin G acylase from Bacillus sp., Lett. Appl. Microbiol. 35 (2002) 523–527. doi:10.1046/j.1472-765X.2002.01234.x. [26] B.K. Bhuyan, M.J. Johnson, The effect of medium constituents on penicillin production from natural materials., Appl. Microbiol. 5 (1957) 262–267. [27] J. Hekayati, A. Roosta, J. Javanmardi, Liquid–liquid equilibria in the quinary aqueous two-phase system of poly(ethylene glycol) 6000+sodium sulfate+water in the presence of glucose and ethanol: Experimental investigation and thermodynamic modelling, Thermochim. Acta. 625 (2016) 47–52. doi:10.1016/j.tca.2015.12.013. [28] B. Yan, X. Cao, Phase diagram of novel recycling aqueous two-phase systems composed of two pH-response polymers: Experiment and modelling, Fluid Phase Equilib. 364 (2014) 42–47. doi:10.1016/j.fluid.2013.11.037. [29] C.P. Song, R.N. Ramanan, R. Vijayaraghavan, D.R. MacFarlane, E.-S. Chan, J.A.P. Coutinho, et al., Primary and secondary aqueous two-phase systems composed of thermo switchable polymers and bio-derived ionic liquids, J. Chem. Thermodyn. 115 (2017) 191– 201. doi:10.1016/j.jct.2017.07.028. [30] Y. Lu, T. Hao, Y. Zhou, J. Han, Z. Tan, Y. Yan, Aqueous two-phase systems of polyoxyethylene lauryl ether and potassium gluconate/potassium oxalate/potassium citrate at different temperature-experimental results and modelling of (liquid+liquid) equilibrium data, J. Chem. Thermodyn. 71 (2014) 137–147. doi:10.1016/j.jct.2013.12.005. [31] H.H. Rachford, J.D. Rice, Procedure for Use of Electronic Digital Computers in Calculating Flash Vaporization Hydrocarbon Equilibrium, J. Pet. Technol. 4 (1952) 19–3. doi:10.2118/952327-G. [32] J.M. Prausnitz, R. Lichtenthaler, E. Gomes de Azevedo, Molecular Thermodynamics of Fluid-Phase Equilibria, Third, Prentice-Hall, Inc., New Jersey, 1999.
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Table 1. Source and purity of chemicals. Chemical Name
Source
Polyethylene glycol 6000 Merck Dipotassium hydrogen phosphate Merck Penicillin G sodium salt Sigma-Aldrich Ascorbic acid Sigma-Aldrich Ammonium molybdate tetrahydrate Sigma-Aldrich
14
Mass fraction purity >0.99 >0.99 >1477 units∙mg-1 >0.99 >0.99
Purification method None None None None None
Table 2. Experimental data of the binodal curve of the ternary system of K2HPO4 (1) + PEG 6000 (2) + water (3) at temperature T=298.2 K and pressure p=0.1 MPa.* 100w1† 15.8 13.6 12.4 11.6 10.6 9.5 8.5 7.9 100w2 0.5 1.1 1.8 2.4 3.7 5.7 7.9 9.9 100w1 7.5 6.9 6.2 5.7 5.3 4.4 4.0 3.6 100w2 11.1 13.1 15.9 17.4 19.1 23.5 25.2 27.5 * Expanded uncertainties (confidence of 95 %): Uc(T) = ± 0.1 K, Uc(p) = ± 5 kPa, Uc(w) = ± 0.003. †
w: mass fraction
15
Table 3. Experimental LLE weight percent data for the ternary system of K2HPO4 (1) + PEG 6000 (2) + water (3) at temperature T=298.2 K and pressure p=0.1 MPa.* feed bottom phase no. 100w1 100w2 100w1 100w2 7.2 20.2 16.0 0.6 1 7.4 18.7 15.5 0.5 2 6.8 19.3 14.9 0.5 3 6.7 18.7 14.4 0.6 4 8.7 13.0 13.7 0.8 5 8.3 13.0 13.1 1.1 6 8.6 11.5 12.7 1.4 7 7.8 13.1 12.3 1.7 8 7.5 13.4 12.0 2.0 9 7.4 13.1 11.5 2.6 10 7.6 11.8 10.9 3.4 11 * Expanded uncertainties (confidence of 95 %): 0.003. †
†
top phase 100w1 100w2 3.4 28.5 3.6 27.3 3.9 25.9 4.1 24.8 4.4 23.2 4.7 21.7 5.0 20.7 5.2 19.6 5.4 18.7 5.8 17.2 6.2 15.5 Uc(T) = ± 0.1 K, Uc(p) = ± 5 kPa, Uc(w) = ±
w: mass fraction
16
Table 4. Estimated NRTL parameters (τij and τji) of ternary system (α=0.25). pair i-j K2HPO4-PEG 6000 K2HPO4-H2O PEG6000-H2O
τij 15.167 -3.897 10.018
τji 10.321 8.416 15.655
17
Table 5. Average absolute deviations between experimental and calculated weight fraction in the ternary system of K2HPO4 (1) + PEG 6000 (2) + water (3). top phase bottom phase †
Δw1† 0.002 0.002
Δw2 0.006 0.005
Δw3 0.004 0.003 , w: mass fraction, N: number of data points
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Table 6. The compositions of coexisting phases before and after adding 0.05 (%w/w) of penicillin G to at temperature T=298.2 K and pressure p=0.1 MPa.* top phase 100w1 100w2 Before 4.1 25.1 1 9.1 13.2 After 4.2 24.8 Before 5.4 18.7 2 8.5 11.0 After 5.6 19.0 * Expanded uncertainties (confidence of 95 %): Uc(T) = 0.003, Uc(w3) = ± 0.00002. no.
†
feed 100w1† 100w2
w: mass fraction
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bottom phase 100w1 100w2 14.4 0.9 14.2 1.0 12.0 2.1 11.8 2.0 ± 0.1 K, Uc(p) = ± 5 kPa, Uc(w) = ±
Table 7. Experimental LLE weight percent data for the quaternary system of K2HPO4 (1) + PEG 6000 (2) + penicillin G (3) + water (4) at temperature T=298.2 K and pressure p=0.1 MPa.* feed top phase † 3 no. 100w1 100w2 100w 100w1 100w2 100w3 1 10.7 6.8 0.046 4.5 22.1 0.145 2 9.0 11.2 0.050 4.5 22.3 0.098 3 7.3 16.3 0.042 4.3 23.5 0.059 4 5.5 21.4 0.039 4.0 25.0 0.046 5 10.2 6.9 0.038 4.9 20.3 0.109 6 8.9 9.9 0.046 5.0 19.6 0.090 7 7.5 13.8 0.043 4.9 20.4 0.063 8 6.1 17.8 0.041 4.7 21.4 0.049 9 9.6 7.3 0.046 5.3 18.2 0.116 10 8.5 9.6 0.046 5.5 17.5 0.084 11 7.5 12.6 0.044 5.3 18.2 0.063 12 6.5 15.7 0.043 5.2 18.9 0.052 13 9.0 7.9 0.037 5.8 16.2 0.078 14 8.2 9.8 0.037 5.9 15.9 0.061 15 7.5 12.1 0.036 5.7 16.6 0.050 16 8.2 9.1 0.037 6.4 14.0 0.060 17 7.8 10.4 0.037 6.2 14.5 0.053 18 8.9 8.0 0.033 5.8 16.1 0.070 19 7.5 11.7 0.036 5.8 16.2 0.050 20 8.3 9.0 0.036 6.3 14.4 0.061 21 7.9 10.2 0.037 6.2 14.7 0.054 * Expanded uncertainties (confidence of 95 %): Uc(T) = ± 0.003, Uc(w3) = ± 0.00002. †
w: mass fraction
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w3top/w3 bottom bottom phase 100w1 100w2 100w3 12.8 1.9 0.014 10.16 12.8 1.8 0.010 10.09 13.4 1.4 0.005 12.15 14.2 1.1 0.003 15.63 12.0 2.5 0.014 7.60 11.6 2.8 0.014 6.55 12.0 2.5 0.009 7.20 12.4 2.1 0.006 8.29 11.1 3.4 0.021 5.57 10.8 3.8 0.018 4.73 11.1 3.4 0.012 5.07 11.4 3.1 0.009 5.52 10.3 4.5 0.020 3.99 10.2 4.7 0.017 3.65 10.4 4.3 0.013 3.88 9.5 5.9 0.021 2.83 9.6 5.5 0.018 2.93 10.2 4.6 0.018 3.88 10.3 4.5 0.014 3.65 9.6 5.6 0.020 2.98 9.7 5.4 0.018 3.03 0.1 K, Uc(p) = ± 5 kPa, Uc(w) = ±
Table 8. Estimated NRTL parameters (τij and τji) between penicillin G and other components of the quaternary system (α=0.25). pair i-j Penicillin G-K2HPO4 Penicillin G - PEG6000 Penicillin G -H2O
τij 5.561 -3.814 -7.249
τji 5.421 0.058 1.303
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Table 9. Average absolute deviations between experimental and calculated weight fractions (Δw) in the quaternary system of K2HPO4 (1) + PEG 6000 (2) + penicillin G (3) +water (4). top phase bottom phase †
Δw1† 0.004 0.001
Δw2 0.013 0.002
Δw3 Δw4 3.4˟10-5 0.009 -6 2.9˟10 0.002 , w: mass fraction, N: number of data points
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Figure 1. Binodal curve of PEG 6000 + K2HPO4 + water system.
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Figure 2. Comparison between experimental and calculated values for the ternary system.
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Figure 3. Calculated vs. experimental values for the quaternary system.
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(b)
(a)
(c) (d) Figure 4. Effects of mass ratio of top phase to feed and distance of tie line from plait point on separation of penicillin G. (a) three given tie lines. (b) effect on w3top. (b) effect on w3 bot. (d) effect on k3.
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Distribution coefficient of penicillin G in an aqueous two-phase system is studied. Maximum experimental distribution coefficient of 15.63 was observed. The amount of water has a significant effect on the distribution coefficient. Ternary and quaternary LLE data are modeled with the NRTL model.
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