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Amino acid extraction and mass transfer rate in the reverse micelle system
Enzyme and Microbial Technology 38 (2006) 557–562
Amino acid extraction and mass transfer rate in the reverse micelle system ¨ u Mehmeto˘glu Zuhal D¨ovyap, Emine Bayraktar ∗ , Ulk¨ Ankara University, Faculty of Engineering, Department of Chemical Engineering, Ankara, Turkey Received 25 November 2004; received in revised form 29 June 2005; accepted 19 July 2005
Abstract The mass transfer of l-isoleucine from NaOH solution of pH 12.0 to a reverse micelle phase includes Aliquat-336 as a cationic surfactant, 1-decanol as a co-surfactant and isooctane as an apolar solvent has been investigated in a stirred cell. Interfacial mass transfer coefficients were determined using two film models. The extraction in this system was found to be controlled by interface solubilization and the diffusion of the amino acid in the aqueous phase boundary layer. The total mass transfer coefficient, K0 , increased from 1.77 × 10−5 to 6.02 × 10−5 m s−1 with the increase of Aliquat-336 concentration from 50 to 200 mM for 100 rpm stirring rate. The organic phase coefficient, korg was evaluated using the data of transport of water to the reversed micelle phase. korg increased from 1.50 × 10−5 to 2.00 × 10−5 m s−1 with the increase of stirring rate from 50 to 100 rpm for 50 mM Aliquat-336 concentration. © 2005 Elsevier Inc. All rights reserved. Keywords: Reverse micelle; Cationic surfactant; Mass transfer coefficient; l-Isoleucine; Aliquat-336
1. Introduction Reverse micelles are the nanometer-sized aggregates of surfactant molecules in apolar organic solvents surrounding an inner core of water. These surfactant aggregation structures are known to be thermodynamically stable and have been shown to be capable of solubilizing the bioactive compound such as amino acids, enzymes or protein molecules [1]. The partitioning of bioactive compound between a reversed micelle phase and an aqueous phase depends on several factors, the most important one being electrostatic interactions between the interface of the reverse micelles and the hydrophobic part of the bioactive compound. It has been demonstrated that under certain condition bioactive compounds can be transferred from the aqueous phase to the reversed micelle phase [2–5]. The coexistence of aqueous and reversed micelle phases thus allows the application of liquid–liquid extraction technology to microbial products extraction and separation. The ∗ Corresponding author. Tel.: +90 3122126720x1373; fax: +90 3122121546. E-mail address: [email protected] (E. Bayraktar).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.07.013
transport of the bioactive compounds by the reversed micelles is very complicated. Determination of mass transfer rates are important not only for designing an extraction process using reverse micelle phases, but also gaining a more fundamental understanding of the physical processes occurring the interfacial solubilization of amino acids. In the literature, there are many different studies based on the mass transfer characterization [1,6–10]. Mass transfer of bioactive compound is affected by the type of surfactant, bioactive compound and the type of extractor [8,11]. While Dungan et al. [7] have reported that the mass transfer rate of cytochrome c and ␣chymotrypsin is controlled by solubilization resistance at the interface, Nishiki et al. [9] have reported that it is controlled by the diffusion in the aqueous film and solubilization at the same AOT/isooctane reverse micelle system. In the other their study, Nishiki et al. [10] indicated that the extraction of phenyl alanine with AOT/isooctane reversed micelles is controlled by the interfacial rate processes. Lye et al. [8] investigated the mass transfer of lysozyme and ribonuclease in the AOT/isooctane system. They reported that the rate of mass transfer processes is influenced by pH and ionic strength. As can be seen, most of the mass transfer studies are on the AOT/isooctane reversed micelle system. There are
Z. D¨ovyap et al. / Enzyme and Microbial Technology 38 (2006) 557–562
558
Nomenclature a C kaq kd korg ks K0 m N t T
interfacial area (m−1 ) concentration of amino acid or water (kmol m−3 ) mass transfer coefficient in the aqueous phase (m s−1 ) freedom rate coefficient in the interface (m s−1 ) mass transfer coefficient in the organic phase (m s−1 ) solubilization rate coefficient in the interface (m s−1 ) total mass transfer coefficient (m s−1 ) distribution coefficient stirring speed (rpm) time (min) temperature (◦ C)
Sub/superscripts A amino acid aq aqueous phase i interface org organic phase QCl Aliquat-336 w water 0 initial * equilibrium
limited papers published about interfacial transport processes of bioactive compound in the Aliquat-336/isooctane reversed micellar system. Dekker et al. [6] studied the extraction of ␣-amylase with a reversed micelle phase of this cationic surfactant in isooctane. They found that extraction rate is controlled by diffusion in the aqueous film. In this paper, a mass transfer model of amino acid extraction in stirring cell with a flat liquid–liquid interface has been developed. The transport of l-isoleucine, which is a model amino acid, into a reversed micelle solution was examined using a cationic surfactant with pH conditions above its isoelectric point. Transport of water into the surfactant system was also studied.
Fig. 1. Schematic diagram of stirred cell.
2.2. Methods In this study, Aliquat-336 was used as the surfactant, 1-decanol as the co-surfactant and isooctane as the apolar solvent. The reversed micelle phase was prepared by dissolving desired concentrations of surfactant, Aliquat-336 and 20% (v/v) co-surfactant, 1-decanol in isooctane at the orbital shaker (50 ◦ C, 200 rpm). Aqueous phases which were included 5 mM l-Ile as a model amino acid was prepared at pH 12 by NaOH solution. Extraction experiments were performed in the glass cylindrical stirring cell which is 500 ml volume and is equipped with two impellers with four flat plates (Fig. 1). Extraction temperature was kept constant at 30 ◦ C with passing hot water from jacket of the stirring cell. One hundred and fifty milliliters of the aqueous amino acid solution was brought into contact with an equal volume of the reverse micelle phase in the cell and two phases was independently stirred with separate impellers at a speed of 50–100 rpm under conditions of no ripple at the interface. 2.3. Analyses
2. Experimental
In order to determine the time course of amino acid content of the aqueous phase, about 3 ml of samples was drawn with a syringe from aqueous phase and was analyzed by UV Spectrophotometer at 340 nm using OPA Labeling method [12]. The amount of l-isoleucine in the organic phase was obtained by mass balance and the water uptake in the organic phase was measured by Karl Fischer titration.
2.1. Chemicals
2.4. Model
Trioctylmethylammonium chloride (Aliquat-336) was purchased from Aldrich Chemicals. Model amino acid was lisoleucine (molecular weight: 131.17, isoelectric point: 5.94) from Sigma Chemicals. 1-Decanol and isooctane were from Merck Chemicals. All chemicals are purchased from analytical grade.
Two-film theory has been used to characterize the mass transfer coefficient [9,13]. The mass transfer coefficients of species from bulk aqueous phase to interface and bulk organic phase were determined at given surfactant concentration and stirring speed by measuring the amino acid bulk concentration. The rates were determined from these time-dependent
Z. D¨ovyap et al. / Enzyme and Microbial Technology 38 (2006) 557–562
of the water can be written as: 1 dCw ∗ − Cw ) − = korg (Cw Jw = a dt
559
(6)
Eqs. (5) and (6) are integrated [9,13] as the following, respectively: 1 1 0 (CAorg − CA K0 t ) = −a 1 + ln 1 − 1 + aq m m (7) ln
Fig. 2. Concentrations profiles around the interface in the two-film model.
data. As can be seen in Fig. 2, during the extraction, amino acid molecules diffuse from the bulk of the aqueous phase to the interface; at the interface, amino acid is encapsulated in a layer of the surfactant molecules. The filled reversed micelles diffuse from interface into the bulk organic phase. The relationships for the transfer of amino acid from the aqueous phase to the organic phase can be written as: JA = kaq (CAaq − CAaq,i )
(1)
1 JA = ks CAaq,i − CAorg,i m
(2)
JA = korg (CAorg,i − CAorg )
(3)
where m =
ks kd
=
∗ CA org ∗ CA aq
. In the reverse micelle system, the
interfacial concentrations of the transferable compound are in the equilibrium. In the two-film theory, the overall mass transfer resistance can be described as the sum of three individual resistances [11,14]. The overall mass transfer resistance and rate can be given as follows, respectively: 1 1 1 1 1 = (4) + + K0 kaq ks m korg JA =
dCAaq 1 1 − = K0 CAaq − CAorg a dt m
(5)
where a is the ratio of the interfacial area to the volume of the aqueous phase. In this study, volume of the aqueous phase is equal to the organic phase.In order to define separately the diffusional resistance of aqueous phase and reversed micelle phase, the transport of water to the reversed micelle phase was investigated (Fig. 2). Here, we assumed the diffusion of micelles free from amino acid is the same speed with containing amino acid. Thus, mass transfer coefficients of the free and filled micelles are equal and free micelles only contain water molecules. The relationship for the mass transfer rate
∗ −C Cw w ∗ − C0 Cw w
= −akorg t
(8)
K0 and korg can be obtained from Eqs. (7) and (8) using by the time-dependent concentration of amino acid and water concentration in the reversed micelle phase, respectively.
3. Results and discussion The mass transfer rates of l-isoleucine and water solubilization rate have been determined for the batch extraction in the stirring cell. These rates were measured for various Aliquat-336 concentration and stirring speed. For an amino acid particle, there are three resistances for transport from an aqueous phase to a reversed micelle organic phase. Two of these are diffusional resistances in the aqueous phase and organic phase. By stirring both upper and lower phases, this resistance was reduced to that operating in boundary layer region adjacent to the interface. The effect of increasing the stirring speed was therefore to decrease bulk transport limitations by reducing the thickness of the boundary layer. The total mass transfer coefficient was evaluated by Eq. (7) using l-isoleucine time-dependent concentrations in the organic phase at various stirring speed (Fig. 3a–c). The total mass transfer coefficient was evaluated by Eq. (7) using l-isoleucine time-dependent concentrations in the organic phase at various stirring speed (Fig. 3a–c). As can be seen Fig. 3a–c extraction was reached in equilibrium after 90 and 30 speed, respec minfor 50 and 100 rpm stirring
1 0 tively. ln 1 − 1 + m (CAorg − CAaq ) was plotted against −a 1 + m1 t and values of K0 were obtained from slope of straight lines with Microsoft Excel. The variation of the overall mass transfer coefficient with the stirring speed can be seen in Fig. 4 at 50, 100 and 200 mM Aliquat-336 concentration. As suggestion of Dungan et al. [7] there are linear relation between the bulk resistance and the inverse of the stirring speed. The total mass transfer coefficient increased about three times especially at high Aliquat336 concentration with stirring speed from 50 to 100 rpm because of the reducing the boundary layer of the aqueous and the organic phases. Consequently the transfer of isoleucine is influenced the diffusional resistance of the phases. The effect of solubilization resistance at the interface on this extraction system can be determined by investigating the
560
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Fig. 3. The effect of stirring speed on the time-dependent concentration of l-isoleucine for different Aliquat-336 concentration in the reversed micelle phase. CA0 = 5 mM, pH = 12, T = 30 ◦ C, 1-decanol 20% (v/v). (a) CQCl = 50 mM; (b) CQCl = 100 mM; (c) CQCl = 200 mM.
variation of K0 values with Aliquat-336 concentration. K0 increases from 1.30 × 10−5 to 2.17 × 10−5 m s−1 and from 1.77 × 10−5 to 6.02 × 10−5 m s−1 for 50 and 100 rpm stirring rate, respectively, with increasing Aliquat-336 concentration
Fig. 4. Variation of total mass transfer coefficient with stirring speed and Aliquat-336 concentration. CA0 = 5 mM, pH = 12, T = 30 ◦ C, 1-decanol 20% (v/v).
from 50 to 200 mM. It can be said that the number of reverse micelle near the interface increase with increasing initial Aliquat-336 concentration. As these results, the transport of l-isoleucine is strongly affected from the solubilization process at the interface. Nishiki et al. [9] obtained similar result from the protein extraction in the AOT/isooctane reverse micelle system. They reported overall mass transfer coefficient increased with increasing AOT concentration and stirring speed. In order to determine the diffusional resistances separately we can investigate transport of water to the organic phase without amino acid. For this aim, it is assumed that the micelles without l-isoluecine and with l-isoleucine are the same speed in the reverse micellar phase and their trans ∗mass
C −C fer coefficients in the organic film are equal. ln w∗ w0 was Cw −Cw plotted against (−at) using the water content of organic phase data (Fig. 5a–c). korg was obtained from slope of straight lines for various Aliquat-336 concentration and stirring speed with Microsoft Excel. In Fig. 6, the korg values are plotted against the stirring speed as a parameter of the Aliquat-336 concentration. Although the korg values increase a small amount with stirring
Z. D¨ovyap et al. / Enzyme and Microbial Technology 38 (2006) 557–562
561
Fig. 5. The effect of stirring speed on the time-dependent concentration of water in the reversed micelle phase. CA0 = 5 mM, pH = 12, T = 30 ◦ C, 1-decanol 20% (v/v). (a) CQCl = 50 mM; (b) CQCl = 100 mM; (c) CQCl = 200 mM.
rate at low Aliquat-336 concentration, there is almost no difference of that of korg for different stirring speeds at high surfactant concentration. It can be considered that organic phase mass transfer resistance is neglected especially at high surfactant concentration of this system. In the literature, whereas
Adachi et al. [13] reported that the rate limiting step is the fusion process in the liquid–liquid interface for the extraction of trytophan with AOT/n-heptan reverse micelle system, Nishiki et al. [9] found that it is controlled with the diffusion in the aqueous film and solubilization at the interface. Also, in this system, diffusion rate of aqueous phase and solubilization rate at interface are the rate limiting steps in this Aliquat-336/1-decanol/isooctane reverse micelle system.
4. Conclusion
Fig. 6. Variation of organic phase film mass transfer coefficient with stirring speed and Aliquat-336 concentration. CA0 = 5 mM, pH = 12, T = 30 ◦ C, 1decanol 20% (v/v).
The extraction of l-isoleucine from aqueous phase to a reversed micelle phase of the cationic surfactant Aliquat-336 and co-surfactant 1-decanol in isooctane was investigated in the stirring cell. By applying two films model total mass transfer coefficient and organic phase mass transfer coefficient were calculated. The rate limited step was determined at different surfactant concentration and stirring speed. The total mass transfer coefficient is composed of mass transfer coefficient in the aqueous and the organic films and solubilizing rate constant in the interface. The organic film mass transfer coefficient has not been affected with stirring
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Z. D¨ovyap et al. / Enzyme and Microbial Technology 38 (2006) 557–562
rate and Aliquat-336 concentration. The extraction rate of l-isoleucine in the reverse micelle extraction system is controlled by interface solubilization and the diffusion of the amino acid in the aqueous phase boundary layer.
Acknowledgement The authors gratefully acknowledge the financial supports given to this work by Scientific and Technical Research Council of Turkey, TUBITAK (Project No. MISAG-207).
References [1] Alexandridis P, Holzwarth JF, Hatton TA. Kinetics of solute partitioning at surfactant monolayers in water-in-oil microemulsions. J Mol Liq 1997;72:55–68. [2] Dekker M, Riet KV, Bijsterbosch BH, Wolbert RBG, Hilhorst R. Modelling and optimization of the reversed micellar extraction of ␣-amylase. AIChE J 1989;35(2):321–4. [3] Goto M, Ono T, Nakashio F, Hatton TA. Design of surfactants suitable for protein extraction by reversed micelles. Biotechnol Bioeng 1997;54(1):26–32. [4] Shiomori K, Kawano Y, Kuboi R, Komasawa I. Extraction of proteins and water with sodium bis (2-ethylhexyl) sulfosuccinate/long chain alkyl amines mixed micellar system. J Chem Eng Jpn 1999;32(2):177–83.
[5] Wang W, Weber ME, Vera JH. Effect of the concentration of DODMAC and 1-decanol on the behavior of reverse micelles in the extraction of amino acids. Biotechnol Bioeng 1995;46:343– 50. [6] Dekker M, Riet KV, Fijneman P, Hilhorst R. Mass transfer rate of protein extraction with reversed micelles. Chem Eng Sci 1990;45(9):2949–57. [7] Dungan SR, Bausch T, Hatton TA, Plucinski P, Nitsch W. Interfacial transport processes in the reversed micellar extraction of proteins. J Colloid Interf Sci 1991;145(1):33–50. [8] Lye GJ, Asenjo JA, Pyle DL. Reverse micellar mass-transfer processes: spray column extraction of lysozyme. AIChE J 1996;42(3):713–26. [9] Nishiki T, Sato I, Muto A, Kataok T. Mass transfer characterization in forward and backward extractions of lysozyme by AOT–isooctane reverse micelles across a flat liquid–liquid interface. Biochem Eng J 1998;1:91–7. [10] Nishiki T, Nakamura K, Kato D. Forward and backward extraction rates of amino acid in reversed micellar extraction. Biochem Eng J 2000;4:89–195. [11] Cardoso MM, Viegas RMC, Crespo JPSG. Extraction and reextraction of phenylalanine by cationic reversed micelles in hollow fiber contactors. J Membr Sci 1999;156:303–19. [12] Roth M. Fluorescence reaction for amino acids. Anal Chem 1971;53(7):880–2. [13] Adachi M, Harada M, Nishita R, Shioi A. Extraction kinetics of small charged molecules in water-in-oil microemulsion/brine system. J Phys Chem 1995;99:8722–9. [14] Mc Cabe WL, Smith JC, Harriot P. Unit operation of chemical engineering. New York: McGraw-Hill Book Company; 1985. p. 606–8.
Amino acid extraction and mass transfer rate in the reverse micelle system ¨ u Mehmeto˘glu Zuhal D¨ovyap, Emine Bayraktar ∗ , Ulk¨ Ankara University, Faculty of Engineering, Department of Chemical Engineering, Ankara, Turkey Received 25 November 2004; received in revised form 29 June 2005; accepted 19 July 2005
Abstract The mass transfer of l-isoleucine from NaOH solution of pH 12.0 to a reverse micelle phase includes Aliquat-336 as a cationic surfactant, 1-decanol as a co-surfactant and isooctane as an apolar solvent has been investigated in a stirred cell. Interfacial mass transfer coefficients were determined using two film models. The extraction in this system was found to be controlled by interface solubilization and the diffusion of the amino acid in the aqueous phase boundary layer. The total mass transfer coefficient, K0 , increased from 1.77 × 10−5 to 6.02 × 10−5 m s−1 with the increase of Aliquat-336 concentration from 50 to 200 mM for 100 rpm stirring rate. The organic phase coefficient, korg was evaluated using the data of transport of water to the reversed micelle phase. korg increased from 1.50 × 10−5 to 2.00 × 10−5 m s−1 with the increase of stirring rate from 50 to 100 rpm for 50 mM Aliquat-336 concentration. © 2005 Elsevier Inc. All rights reserved. Keywords: Reverse micelle; Cationic surfactant; Mass transfer coefficient; l-Isoleucine; Aliquat-336
1. Introduction Reverse micelles are the nanometer-sized aggregates of surfactant molecules in apolar organic solvents surrounding an inner core of water. These surfactant aggregation structures are known to be thermodynamically stable and have been shown to be capable of solubilizing the bioactive compound such as amino acids, enzymes or protein molecules [1]. The partitioning of bioactive compound between a reversed micelle phase and an aqueous phase depends on several factors, the most important one being electrostatic interactions between the interface of the reverse micelles and the hydrophobic part of the bioactive compound. It has been demonstrated that under certain condition bioactive compounds can be transferred from the aqueous phase to the reversed micelle phase [2–5]. The coexistence of aqueous and reversed micelle phases thus allows the application of liquid–liquid extraction technology to microbial products extraction and separation. The ∗ Corresponding author. Tel.: +90 3122126720x1373; fax: +90 3122121546. E-mail address: [email protected] (E. Bayraktar).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.07.013
transport of the bioactive compounds by the reversed micelles is very complicated. Determination of mass transfer rates are important not only for designing an extraction process using reverse micelle phases, but also gaining a more fundamental understanding of the physical processes occurring the interfacial solubilization of amino acids. In the literature, there are many different studies based on the mass transfer characterization [1,6–10]. Mass transfer of bioactive compound is affected by the type of surfactant, bioactive compound and the type of extractor [8,11]. While Dungan et al. [7] have reported that the mass transfer rate of cytochrome c and ␣chymotrypsin is controlled by solubilization resistance at the interface, Nishiki et al. [9] have reported that it is controlled by the diffusion in the aqueous film and solubilization at the same AOT/isooctane reverse micelle system. In the other their study, Nishiki et al. [10] indicated that the extraction of phenyl alanine with AOT/isooctane reversed micelles is controlled by the interfacial rate processes. Lye et al. [8] investigated the mass transfer of lysozyme and ribonuclease in the AOT/isooctane system. They reported that the rate of mass transfer processes is influenced by pH and ionic strength. As can be seen, most of the mass transfer studies are on the AOT/isooctane reversed micelle system. There are
Z. D¨ovyap et al. / Enzyme and Microbial Technology 38 (2006) 557–562
558
Nomenclature a C kaq kd korg ks K0 m N t T
interfacial area (m−1 ) concentration of amino acid or water (kmol m−3 ) mass transfer coefficient in the aqueous phase (m s−1 ) freedom rate coefficient in the interface (m s−1 ) mass transfer coefficient in the organic phase (m s−1 ) solubilization rate coefficient in the interface (m s−1 ) total mass transfer coefficient (m s−1 ) distribution coefficient stirring speed (rpm) time (min) temperature (◦ C)
Sub/superscripts A amino acid aq aqueous phase i interface org organic phase QCl Aliquat-336 w water 0 initial * equilibrium
limited papers published about interfacial transport processes of bioactive compound in the Aliquat-336/isooctane reversed micellar system. Dekker et al. [6] studied the extraction of ␣-amylase with a reversed micelle phase of this cationic surfactant in isooctane. They found that extraction rate is controlled by diffusion in the aqueous film. In this paper, a mass transfer model of amino acid extraction in stirring cell with a flat liquid–liquid interface has been developed. The transport of l-isoleucine, which is a model amino acid, into a reversed micelle solution was examined using a cationic surfactant with pH conditions above its isoelectric point. Transport of water into the surfactant system was also studied.
Fig. 1. Schematic diagram of stirred cell.
2.2. Methods In this study, Aliquat-336 was used as the surfactant, 1-decanol as the co-surfactant and isooctane as the apolar solvent. The reversed micelle phase was prepared by dissolving desired concentrations of surfactant, Aliquat-336 and 20% (v/v) co-surfactant, 1-decanol in isooctane at the orbital shaker (50 ◦ C, 200 rpm). Aqueous phases which were included 5 mM l-Ile as a model amino acid was prepared at pH 12 by NaOH solution. Extraction experiments were performed in the glass cylindrical stirring cell which is 500 ml volume and is equipped with two impellers with four flat plates (Fig. 1). Extraction temperature was kept constant at 30 ◦ C with passing hot water from jacket of the stirring cell. One hundred and fifty milliliters of the aqueous amino acid solution was brought into contact with an equal volume of the reverse micelle phase in the cell and two phases was independently stirred with separate impellers at a speed of 50–100 rpm under conditions of no ripple at the interface. 2.3. Analyses
2. Experimental
In order to determine the time course of amino acid content of the aqueous phase, about 3 ml of samples was drawn with a syringe from aqueous phase and was analyzed by UV Spectrophotometer at 340 nm using OPA Labeling method [12]. The amount of l-isoleucine in the organic phase was obtained by mass balance and the water uptake in the organic phase was measured by Karl Fischer titration.
2.1. Chemicals
2.4. Model
Trioctylmethylammonium chloride (Aliquat-336) was purchased from Aldrich Chemicals. Model amino acid was lisoleucine (molecular weight: 131.17, isoelectric point: 5.94) from Sigma Chemicals. 1-Decanol and isooctane were from Merck Chemicals. All chemicals are purchased from analytical grade.
Two-film theory has been used to characterize the mass transfer coefficient [9,13]. The mass transfer coefficients of species from bulk aqueous phase to interface and bulk organic phase were determined at given surfactant concentration and stirring speed by measuring the amino acid bulk concentration. The rates were determined from these time-dependent
Z. D¨ovyap et al. / Enzyme and Microbial Technology 38 (2006) 557–562
of the water can be written as: 1 dCw ∗ − Cw ) − = korg (Cw Jw = a dt
559
(6)
Eqs. (5) and (6) are integrated [9,13] as the following, respectively: 1 1 0 (CAorg − CA K0 t ) = −a 1 + ln 1 − 1 + aq m m (7) ln
Fig. 2. Concentrations profiles around the interface in the two-film model.
data. As can be seen in Fig. 2, during the extraction, amino acid molecules diffuse from the bulk of the aqueous phase to the interface; at the interface, amino acid is encapsulated in a layer of the surfactant molecules. The filled reversed micelles diffuse from interface into the bulk organic phase. The relationships for the transfer of amino acid from the aqueous phase to the organic phase can be written as: JA = kaq (CAaq − CAaq,i )
(1)
1 JA = ks CAaq,i − CAorg,i m
(2)
JA = korg (CAorg,i − CAorg )
(3)
where m =
ks kd
=
∗ CA org ∗ CA aq
. In the reverse micelle system, the
interfacial concentrations of the transferable compound are in the equilibrium. In the two-film theory, the overall mass transfer resistance can be described as the sum of three individual resistances [11,14]. The overall mass transfer resistance and rate can be given as follows, respectively: 1 1 1 1 1 = (4) + + K0 kaq ks m korg JA =
dCAaq 1 1 − = K0 CAaq − CAorg a dt m
(5)
where a is the ratio of the interfacial area to the volume of the aqueous phase. In this study, volume of the aqueous phase is equal to the organic phase.In order to define separately the diffusional resistance of aqueous phase and reversed micelle phase, the transport of water to the reversed micelle phase was investigated (Fig. 2). Here, we assumed the diffusion of micelles free from amino acid is the same speed with containing amino acid. Thus, mass transfer coefficients of the free and filled micelles are equal and free micelles only contain water molecules. The relationship for the mass transfer rate
∗ −C Cw w ∗ − C0 Cw w
= −akorg t
(8)
K0 and korg can be obtained from Eqs. (7) and (8) using by the time-dependent concentration of amino acid and water concentration in the reversed micelle phase, respectively.
3. Results and discussion The mass transfer rates of l-isoleucine and water solubilization rate have been determined for the batch extraction in the stirring cell. These rates were measured for various Aliquat-336 concentration and stirring speed. For an amino acid particle, there are three resistances for transport from an aqueous phase to a reversed micelle organic phase. Two of these are diffusional resistances in the aqueous phase and organic phase. By stirring both upper and lower phases, this resistance was reduced to that operating in boundary layer region adjacent to the interface. The effect of increasing the stirring speed was therefore to decrease bulk transport limitations by reducing the thickness of the boundary layer. The total mass transfer coefficient was evaluated by Eq. (7) using l-isoleucine time-dependent concentrations in the organic phase at various stirring speed (Fig. 3a–c). The total mass transfer coefficient was evaluated by Eq. (7) using l-isoleucine time-dependent concentrations in the organic phase at various stirring speed (Fig. 3a–c). As can be seen Fig. 3a–c extraction was reached in equilibrium after 90 and 30 speed, respec minfor 50 and 100 rpm stirring
1 0 tively. ln 1 − 1 + m (CAorg − CAaq ) was plotted against −a 1 + m1 t and values of K0 were obtained from slope of straight lines with Microsoft Excel. The variation of the overall mass transfer coefficient with the stirring speed can be seen in Fig. 4 at 50, 100 and 200 mM Aliquat-336 concentration. As suggestion of Dungan et al. [7] there are linear relation between the bulk resistance and the inverse of the stirring speed. The total mass transfer coefficient increased about three times especially at high Aliquat336 concentration with stirring speed from 50 to 100 rpm because of the reducing the boundary layer of the aqueous and the organic phases. Consequently the transfer of isoleucine is influenced the diffusional resistance of the phases. The effect of solubilization resistance at the interface on this extraction system can be determined by investigating the
560
Z. D¨ovyap et al. / Enzyme and Microbial Technology 38 (2006) 557–562
Fig. 3. The effect of stirring speed on the time-dependent concentration of l-isoleucine for different Aliquat-336 concentration in the reversed micelle phase. CA0 = 5 mM, pH = 12, T = 30 ◦ C, 1-decanol 20% (v/v). (a) CQCl = 50 mM; (b) CQCl = 100 mM; (c) CQCl = 200 mM.
variation of K0 values with Aliquat-336 concentration. K0 increases from 1.30 × 10−5 to 2.17 × 10−5 m s−1 and from 1.77 × 10−5 to 6.02 × 10−5 m s−1 for 50 and 100 rpm stirring rate, respectively, with increasing Aliquat-336 concentration
Fig. 4. Variation of total mass transfer coefficient with stirring speed and Aliquat-336 concentration. CA0 = 5 mM, pH = 12, T = 30 ◦ C, 1-decanol 20% (v/v).
from 50 to 200 mM. It can be said that the number of reverse micelle near the interface increase with increasing initial Aliquat-336 concentration. As these results, the transport of l-isoleucine is strongly affected from the solubilization process at the interface. Nishiki et al. [9] obtained similar result from the protein extraction in the AOT/isooctane reverse micelle system. They reported overall mass transfer coefficient increased with increasing AOT concentration and stirring speed. In order to determine the diffusional resistances separately we can investigate transport of water to the organic phase without amino acid. For this aim, it is assumed that the micelles without l-isoluecine and with l-isoleucine are the same speed in the reverse micellar phase and their trans ∗mass
C −C fer coefficients in the organic film are equal. ln w∗ w0 was Cw −Cw plotted against (−at) using the water content of organic phase data (Fig. 5a–c). korg was obtained from slope of straight lines for various Aliquat-336 concentration and stirring speed with Microsoft Excel. In Fig. 6, the korg values are plotted against the stirring speed as a parameter of the Aliquat-336 concentration. Although the korg values increase a small amount with stirring
Z. D¨ovyap et al. / Enzyme and Microbial Technology 38 (2006) 557–562
561
Fig. 5. The effect of stirring speed on the time-dependent concentration of water in the reversed micelle phase. CA0 = 5 mM, pH = 12, T = 30 ◦ C, 1-decanol 20% (v/v). (a) CQCl = 50 mM; (b) CQCl = 100 mM; (c) CQCl = 200 mM.
rate at low Aliquat-336 concentration, there is almost no difference of that of korg for different stirring speeds at high surfactant concentration. It can be considered that organic phase mass transfer resistance is neglected especially at high surfactant concentration of this system. In the literature, whereas
Adachi et al. [13] reported that the rate limiting step is the fusion process in the liquid–liquid interface for the extraction of trytophan with AOT/n-heptan reverse micelle system, Nishiki et al. [9] found that it is controlled with the diffusion in the aqueous film and solubilization at the interface. Also, in this system, diffusion rate of aqueous phase and solubilization rate at interface are the rate limiting steps in this Aliquat-336/1-decanol/isooctane reverse micelle system.
4. Conclusion
Fig. 6. Variation of organic phase film mass transfer coefficient with stirring speed and Aliquat-336 concentration. CA0 = 5 mM, pH = 12, T = 30 ◦ C, 1decanol 20% (v/v).
The extraction of l-isoleucine from aqueous phase to a reversed micelle phase of the cationic surfactant Aliquat-336 and co-surfactant 1-decanol in isooctane was investigated in the stirring cell. By applying two films model total mass transfer coefficient and organic phase mass transfer coefficient were calculated. The rate limited step was determined at different surfactant concentration and stirring speed. The total mass transfer coefficient is composed of mass transfer coefficient in the aqueous and the organic films and solubilizing rate constant in the interface. The organic film mass transfer coefficient has not been affected with stirring
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rate and Aliquat-336 concentration. The extraction rate of l-isoleucine in the reverse micelle extraction system is controlled by interface solubilization and the diffusion of the amino acid in the aqueous phase boundary layer.
Acknowledgement The authors gratefully acknowledge the financial supports given to this work by Scientific and Technical Research Council of Turkey, TUBITAK (Project No. MISAG-207).
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