Kinetic study of antibiotic by reverse micelle extraction technique

Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 685–695

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Kinetic study of antibiotic by reverse micelle extraction technique Siti Hamidah Mohd-Setapar *, Hanapi Mat, Siti Norazimah Mohamad-Aziz Centre of Lipids Engineering and Applied Research (CLEAR), Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 September 2011 Received in revised form 12 January 2012 Accepted 19 February 2012 Available online 29 March 2012

The kinetic reverse micelle extraction of penicillin G was studied using the new surfactant; dioleyl phosphoric acid. Studies were conducted for model development on the kinetic partitioning of penicillin G and for investigation of mechanism governing the forward and backward transfers of penicillin G in reverse micelle system. Results were interpreted in terms of a two-film theory for flat interface. The extraction in this system was found to be controlled by interface solubilization and the diffusion of the penicillin G in the aqueous phase boundary layer. The values mass transfer coefficient of forward extraction, KLA increased from 0.2859  107 to 0.6115  107 as the aqueous pH was increased from 5 to 8. While as dioleyl phosphoric acid concentration increased from 5 mM to 10 mM, the value of KLA and forward extraction equilibrium partition coefficient, mf were increased from 0.1285  107 to 0.8971  107 and 0.4954 to 1.40085 respectively. However further increased dioleyl phosphoric acid concentration up to 25 mM the value of KLA and mf were declined because the organic solution becomes saturated with dioleyl phosphoric acid molecules. From the experimental result it was found that the transfer rate and efficiency was reduced dramatically at higher concentration of salt and an increase of penicillin G concentration the driving force between charge polar heads of dioleyl phosphoric acid and opposite charge of penicillin G molecules. ß 2012 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Reverse micelle extraction Dioleyl phosphoric acid Kinetic partitioning

1. Introduction Penicillin G is a b-lactam antibiotics which have been manufacture over 11,000 tons per year [1] and extensively used in the large scale production of 6-aminopenicillanic acid (6-APA) as a raw material for the synthesis of penicillins derivative and cephalosporins such as cephalexin and amoxicillin [2]. This natural antibiotic is also important for bacterial infections treatment caused by gram-positive and gram-negative organisms hence maintain the human and animal health [3]. Penicillin G is produce in industrial by submerged aerobic fermentation using Penicillium chryogenum strains. Conventional solvent extraction at pH 2.0 and 2.5 is currently employed to recover penicillin from the broth, but about 10% losses of penicillin occur because penicillin G is unstable and tends to decompose in the low pH range and ambient temperature. Therefore there is a significant need for efficient method that can separate and concentrate Penicillin G from the broth. In the past decades, reversed micelles have attracted much attention as novel method for separating and purifying many biological product because reversed micelles provide a special

* Corresponding author. Tel.: +60 75535496; fax: +60 75581463. E-mail addresses: [email protected], [email protected], [email protected] (S.H. Mohd-Setapar).

microenvironment in the organic medium [4,5]. Reverse micelles can be referred to as a nanometer-scale droplet of an aqueous solution, stabilized in an a polar environment by the presence of surfactant interface [6,7]. Reverse micelles have already been demonstrated to be capable of hosting large quantities of biomolecules such as protein, hemoglobin and plasmids without causing denaturing by adjusting operational parameters [8–12]. A number of papers have been presented on the interfacial transport processes of proteins between a bulk aqueous and a reverse micelle phase. Dekker et al. [13] investigated the mass transfer rates in the extraction of a-amylase with a reverse micelle phase of the cationic surfactant trioctylmethylammonium chloride (TOMAC) in isooctane. They found that the forward extraction rate is controlled by the diffusion of the enzyme in the aqueous phase boundary layer, and the interfacial process of enzyme released from the reverse micelles controls the back extraction rate. The interfacial transport processes of a-chymotrypsin and cytochrome c between a bulk aqueous and an AOT-isooctane reverse micelle phase were studied by Dungan et al. [14]. They evaluated the interfacial mass transfer coefficients for the transfer of these proteins and indicated that the solubilization resistance at the interface significantly influences the forward extraction rates. The mass transfer processes in lysozyme extraction by AOT-isooctane reverse micelles were also studied by Plucinski and Nitsch [15] and Lye et al. [16] while in their analyses of the transfer kinetics, any resistance arising from the interfacial process was ignored.

1876-1070/$ – see front matter ß 2012 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jtice.2012.02.007

S.H. Mohd-Setapar et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 685–695

686

Symbols A C1 C2 C1 C2 jL kaqA KL kL KLa KLA krmA m mb mf V1 V2 Vr

Total interfacial area between the two phases Concentration of phase 1 at interface Concentration of phase 2 at interface Concentration of phase 1 Concentration of phase 2 Mass transfer rate Individual aqueous phase mass transfer coefficient Overall mass transfer coefficient Solubilization rate constant Volumetric mass transfer coefficient Combined mass transfer coefficient Individual aqueous phase mass transfer coefficients Equilibrium partition coefficient Equilibrium partition coefficient (backward) Equilibrium partition coefficient (forward) Volume of phase 1 Volume of phase 2 Phase volume ratio

The study of kinetic partitioning of antibiotics in reverse micelle system is very important because the information gathered from the study could be used in the design of a continuous liquid–liquid contactor for the continuous extraction of antibiotics from this system [17–19]. Moreover, the mass transfer mechanism governing the extraction of antibiotics in the reverse micelle system could be developed as well. In this study, the effects of pH, surfactant concentration, salt concentration (or ionic strength) and antibiotic concentration on the kinetic partitioning of penicillin G in the reverse micelle system were studied.

2. Experimental 2.1. Chemicals The aqueous phases were made up of reagent salt, potassium chloride (KCl), which was obtained from E. Merck and used as received. The buffer solutions used were, phosphate buffers series (H3PO4: pH = 1.83; KH2PO4: pH = 5.40; K2HPO4: pH = 9.02) and carbonate buffers (KHCO3: pH = 8.73; K2CO3: pH = 11.7) to adjust the aqueous solution pHs, which were also obtained from E. Merck and used as received. The organic solvent employed in this study was isooctane of analytical grade procured from Sigma Chemical Co. Acetone (70%) and de-ionized water were used in the cleaning of all equipment to remove any microorganism or residues of biological materials that may be attached to the equipment. De-ionized water, obtained from Millipore filtration unit with an electrical conductivity below

Fig. 1. Chemical structure of Penicillin G.

0.8 mS/cm was used for aqueous solution preparation. In all the experiments reported here, the anionic surfactant used was dioleylphosphoric acid (DOLPA). The antibiotic used in this study was penicillin G which was obtained from Sigma Chemical Co. 2.2. Extraction procedures Forward transfer was carried out by contacting equal amounts of 40 ml of the aqueous phase and the reverse micelle phase in a 100 ml Biejour bottle containing a magnetic stirring bar at a speed of 400 rpm. The bottle was covered to prevent loss of solution by splashing or evaporation. Mixing was carried out for 30 min. While mixing, 5 ml of the sample, equal for both phases, was taken out at different time intervals. The samples were then centrifuged at 4000 rpm for 15 min to facilitate phase separation. For the backward transfer process, again the equal amounts of the reverse micelle phase containing penicillin G from forward transfer and the fresh aqueous phase were brought into contact in a Biejour bottle containing a magnetic stirring bar at a speed of 400 rpm and the bottle was covered. The two phases were mixed for 30 min. While mixing, 5 ml of the sample was taken out at different time intervals. The mixture was then centrifuged for 15 min at 4000 rpm to facilitate phase separation. The time-based experiments were carried out for all the parameters affecting the kinetic partitioning of penicillin G. The concentrations of penicillin G in both phases were assayed by absorbance at 257 nm using an UVSpectrophotometer model obtained from Shimadzu Co (Fig. 1). 3. Model development In this research two-film theory for flat interface applies to characterize the mass transfer coefficient [20] and it is known that this can be successfully applied in the reverse micelle extraction system [16,21,22]. The partitioning of a penicillin G is considered to occur between two well-mixed liquid phases. The mass transfer coefficients of species from bulk aqueous phase to interface and bulk organic phase were determined at given pH, surfactant concentration, salt concentration, penicillin G concentration by measuring the penicillin G bulk concentration. The determination of rates is based on these time-dependent data. The two-film theory for reverse micelles extraction is described in Fig. 2(a) and (b). According to the previous researches, reverse micelle system follows the two-film theory [20,22]. This can be justifying by the mechanism of partitioning of penicillin G from aqueous phase into organic phase. During the extraction process, penicillin G molecules are diffuse from the bulk of the aqueous phase to the interface at the interface between aqueous and organic phase (C1 ). Then, penicillin G is encapsulated in a layer of the surfactant molecules. The reversed micelles aggregates containing penicillin G are further diffuse from interface into the bulk organic phase and vice versa for the backward extraction. The reverse micelle extraction is governed by two important processes, which are the forward extraction where the solute is transferred from an aqueous phase to a reverse micelle phase and the backward extraction process, which is the opposite of forward extraction. There are two phases involved in this study; phase 1 (with constant volume V1 and penicillin G concentration at time t of C1(t) for phase 1) and phase 2 (with constant volume V2 and penicillin G concentration C2(t) for phase 2). For forward extraction, phase 1 represents the aqueous phase and phase 2 represents the reverse micelle phase. At the start of the forward extraction experiments, all the penicillin G reside in phase 1, and thus the concentration of phase 1 is C1(t) = C1(0) and C2(t) = 0. So the mass balance of penicillin G in the system at any given time is: V 1 C 1 ð0Þ ¼ V 1 C 1 ðtÞ þ V 2 C 2 ðtÞ

(1)

S.H. Mohd-Setapar et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 685–695

687

Fig. 2. Concentrations profiles around the interface in for two-film theory in the reverse micelle system (a) forward extraction (b) backward extraction.

At the equilibrium, the relative penicillin G concentrations would be given by C1 ¼ m C2 , where m is the equilibrium partition coefficient (assumed to be constant under given conditions). C1 and C2 are the equilibrium concentrations of penicillin G in phases 1 and 2, respectively. Therefore the value of equilibrium partition coefficient is given by: mf ¼

C 1 ð1Þ C 2 ð1Þ

(2)

The definition of m is reversed here (compared to the common definition for equilibrium partition coefficient) as mf = Caq/Crm for the forward extraction whilst for the backward extraction m is defined as mb = Crm/Caq where ‘‘aq’’ and ‘‘rm’’ stand for aqueous and reverse micelle phases respectively. However this definition is applied only for the purpose of model calculation. As a two-film model is used to describe solute transfer, the mass transfer rate is given by: jL ¼ K L AðC 1 ðtÞ  C1 Þ

(3)

where KL is the overall mass transfer coefficient and A is the total interfacial area between the two phases and C1 ¼ m f C 2 ðtÞ. Thus: V1

dC 1 ¼ K L AðC 1  m f C 2 ðtÞ dt

(4)

In this study, the actual interfacial area between the two phases is unknown, and thus we can only obtain a combined mass transfer coefficient, KLA, having units of m3/s from which the more familiar volumetric mass transfer coefficient, KLa (s1) can be calculated. Rearrange Eq. (1) in terms of C2(t). Then substituting C2(t) in Eq. (4) and rearranging gives:     m f Vr dC 1 KLA ¼ ð1 þ m f V r Þ C 1 ðtÞ  C 1 ð0Þ V1 dt 1 þ m f Vr

(5)

where Vr is the phase volume ratio (=V1/V2). Integrating Eq. (5) between the limits C1(0) and C1(t) and rearranging gives: C 1 ðtÞ  b ¼ ð1  bÞexp  ðatÞ C 1 ð0Þ

(6)

where,

a¼

KLA ð1 þ m f V r Þ V1

(7)

m f Vr 1 þ m f Vr

(8)

  C 1 ðtÞ  bC 1 ð0Þ ¼ at Ln ð1  bÞC 1 ð0Þ

(9)

The b value can be calculated directly from the equilibrium partition coefficient and the phase volume ratio. While this was subsequently used to calculate values of a, i.e. KLA, from the experimental results using a computer based iterative procedure (MATLAB). The model was fitted to C1(t) versus t data and recalculation was forced until the value of a fitted the data points allowing a maximum error of about 10%. The values of a and b were then inserted into Eq. (6) to calculate the variation of penicillin G concentration in phase 1, C1(t), during the course of extraction. Values for the corresponding uptake of protein into phase 2, C2(t), were calculated by mass balance. The interfacial area between the two phases will be very large and can be assumed to be similar in all cases (the mixing speed employed produces complete dispersion of the two phases; the size of the dispersed phase droplets being below the level that could be discerned by the human eye). The overall resistance to antibiotics transfer, as represented by KLA, can be described as the sum of the resistances of both the aqueous and organic boundary films. Assuming that any resistance arising from the interfacial solubilization step is negligible [3,4,7], then the overall, combined mass transfer coefficient can be represented as: 1 1 m ¼ þ K L A k1 A k2 A

(10)

It should then be probable to estimate the individual aqueous phase and reverse micelle phase mass transfer coefficients since a plot of 1/KLA against m should yield a straight line of gradient 1/k2A and intercept 1/k1A. Again due to the nature of the experimental system employed, the value of the interfacial area, A is unknown and so we can only estimate the combined film mass transfer coefficients (k1A = kaqA and k2A = krmA), having units m3/s. For the backward extraction process, at the start of the experiment, all the penicillin G is in phase 2, with the concentration of phase 2 is C2(t) = C2(0). So the mass balance of penicillin G in the system at any given time is: V 2 C 2 ð0Þ ¼ V 2 C 2 ðtÞ þ V 1 C 1 ðtÞ

(11)

The partition coefficient of backward extraction process is given by:

and,

b¼

Eqs. (6), (7) and (8) described the penicillin G transfer between phase 1 to phase 2. Furthermore,

mb ¼

C 2 ð1Þ C 1 ð1Þ

(12)

S.H. Mohd-Setapar et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 685–695

As a two-film model is used for transfer, the mass transfer rate is given by: K L AðC2

jL ¼

 C 2 ðtÞÞ

Substituting C2(t) from Eq. (11) and rearranging gives:     dC 2 K LA mb V r ¼ ð1 þ mb V r Þ C 2 ðtÞ  C 2 ð0Þ V2 dt 1 þ mb V r

(14)

(15)

pH 5 Calc pH 5 exp pH 7 Calc pH 7 exp pH 8 Calc pH 8 exp

4 3.5 3 2.5 2

Vr is the phase volume ratio. Integrating Eq. (15) between the limits C2(0) and C2(t) and rearranging gives: C 2 ðtÞ  b ¼ ð1  bÞexp  ðatÞ C 2 ð0Þ

5

4.5

(13)

where KL is the overall mass transfer coefficient, A is the total interfacial area between the two phases. Thus: dC 2 ¼ K L AðC 2  mb C 1 ðtÞÞ V2 dt

(a)

Concentration (mM)

688

1.5 1

(16)

0

5

10

15

20

25

30

Time (minute)

where

a¼

KLA ð1 þ mb V r Þ V2

(17)

(b) 4.5 4

And, 3.5

(18)

Eqs. (16), (17) and (18) described the penicillin G transfer between phase 1 and 2. The suitability of applying this model to reverse micellar system can be checked by rearranging Eq. (16) to give:   C 2 ðtÞ  bC 2 ð0Þ (19) ¼ at Ln ð1  bÞC 2 ð0Þ

Concentration (mM)

mb V r b¼ 1 þ mb V r

3 2.5 2 1.5 pH 5 Calc pH 5 exp pH 7 Calc pH 7 exp pH 8 Calc pH 8 exp

1

4. Results and discussion 4.1. Forward transfer 4.1.1. Effect of aqueous phase pH The pH is used to control the charge distribution over the solute surface [23]. The charge distribution is depending on the initial chemistry of the solute but remains fixed in the whole range of pH of the solution therefore by tuning the pH of the solution, the total charge distribution in the aqueous phase can be control throughout the process although in the present of salt in the solution. In ionic surfactant micelle systems, extraction is strongly affected by change in the solution pH owing to the altered electrostatic interaction between the polar head of the surfactant and solute surface [24]. The effect of aqueous phase pH on the kinetic partitioning of penicillin G is shown in Fig. 3(a) and (b). The results show that small amount of penicillin G are transferred to the reverse micelle phase at high pH (pH 8) whereas as the pH is decreased to pH 7, the rate of extraction is considerably enhanced and practically reaches the highest degree of extraction rate. By using the kinetic model that was developed in Section 3, the combined mass transfer coefficient of the forward extraction (KLA) and the forward extraction equilibrium partition coefficient (mf) were calculated as shown in Table 1. The values of mf and KLA increase as the aqueous phase pH increases, but decrease when pH is further increased. This subsequently indicates that at medium range pH, the solubilization of penicillin G into the reverse micelle system was increased. When less penicillin G was taken up into reverse micelle phase, there was a decrease in the transfer efficiency. The main reason for this is due to the decrease in the electrostatic interaction between the penicillin G and the charged surfactant head groups.

0.5 0

0

5

10

15

20

25

30

Time (minute) Fig. 3. (a) Effect of pH for the forward extraction of penicillin G at Vr = 1 (aqueous phase). Experiment conditions: [Penicillin G] = 5 mM; [DOLPA] = 10 mM; [KCl] = 0.1 M. (b). Effect of pH for the forward extraction of penicillin G at Vr = 1 (reverse micelle phase). Experiment conditions: [Penicillin G] = 5 mM; [DOLPA] = 10 mM; [KCl] = 0.1 M.

In the reverse micelle system, pH of an aqueous phase solution could be manipulated so that the penicillin G would exhibit a net charge opposite to that of the surfactant thus allowing electrostatic interaction between penicillin G and surfactant in two different phases. The overall penicillin G charge is determined by the pH of the aqueous phase and ionization constant (pKa) of the biomolecule. If the pH of aqueous phase is higher than the ionization constant, penicillin G will be negative but if the is lower than ionization constant the charge is positive. The ionization constant of penicillin G is 2.74. It is expected to have strong attraction with the surfactant head group at pH < 2.74 in which the negative head group of DOLPA will attract the positive charges of penicillin G hence increase the solubility of penicillin G. However the results show different, it was found that the DOLPA system does not form reverse micelle at pH lower than 5 since there is no extraction of penicillin G was detected into the organic phase. At lower pH, DOLPA exists as a monomer of undissociated form and does not adsorb at interface, lowering the interfacial tension and forming reverse micelles due to high concentration of ion in the aqueous phase. While at medium pH DOLPA molecules

S.H. Mohd-Setapar et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 685–695

Parameter pHaqueous

Surfactant concentration (mM)

Salt concentration (M)

Penicillin G concentration (mM)

5 7 8 5 10 25 0.1 0.5 1.0 0.5 5 25

7

3

mf (C1/C2)*

KLA  10 (m /s)

0.8634 1.9793 1.1577 0.4954 1.9793 1.4085 1.9793 1.5202 1.2338 0.9306 1.9793 0.7698

0.2859 1.6465 0.6115 0.1285 1.6451 0.8971 1.6451 1.3509 0.6937 0.7269 1.6451 0.3382

(a)

5

[dolpa]=5 mM Calc [dolpa]=5 mM exp [dolpa]=10 mM Calc [dolpa]=10 mM exp [dolpa]=25 mM Calc [dolpa]=25 mM exp

4.5 4

Concentration (mM)

Table 1 Values of combined mass transfer coefficient (KLA) and equilibrium partition coefficient (mf) for the effects of pH, surfactant concentration, salt concentration and penicillin G concentration on the forward extraction of penicillin G (* sign indicate as equilibrium).

689

3.5 3 2.5 2 1.5 1

0

5

10

15

20

25

30

Time (minute)

4.1.2. Effect of surfactant concentration Fig. 4(a) and (b) shows the results of the effect of surfactant (DOLPA) concentration on the kinetic partitioning of penicillin G in the reverse micelle system. The surfactant molecules formed by DOLPA become a barrier to mass transfer in a reverse micelle process when the interface is saturated with surfactant molecules. However, the initial extraction rate based on normalized concentration of penicillin G increased with the concentration of DOLPA, because DOLPA functions as surfactants. The results show that increasing DOLPA concentration would increase the transfer rate of penicillin G. This indicates that higher surfactant concentrations form more reverse micelles at the interface, leading to higher extraction rate of penicillin G in the reverse micelles phase. On the other hand, increasing DOLPA concentration above 10 mM exhibited a less significant effect to the size of reverse micelle. High amount of surfactant produce a stable reverse micelle structure, and thus the reverse micelle phase becomes saturated to solubilize more penicillin G molecules. This suggests that the surfactant concentration controls the number of reverse micelles formed and hence the capacity to entrap penicillin G.

Table 2 Calculated individual mass transfer coefficients for the effects of pH, surfactant concentration, salt concentration and penicillin G concentration at optimum value in forward extraction. Parameter pH Surfactant concentration (mM) Salt concentration (M) Penicillin G concentration (Mm)

7 10 0.1 25

kaqA  107

krmA  107

1.2664 2.4331 1.4493 3.0159

0.8143 0.9947 0.8218 0.9932

(b)

3.5 3

Concentration (mM)

dissociate to form anionic ions, adsorb at the interface and lowering the interfacial tension. However the interfacial tension is not low enough which is a thermodynamic requirement for forming a clear and thermodynamically stable reverse micelle due to the lack of DOLPA ions adsorbed at the interface. At higher pH, the water solubilization reaches saturation that indicates all molecules dissociates and exist in the ionic form and forming reverse micelles. This can be proved as a result of hydrophobic interaction between non-polar group surfactant and penicillin G through the formation of hydrogen bonding. When appropriate pH condition is applied, more reverse micelle molecules dissociate to form anionic ions, diffuse at the interface and lower the interfacial tension to make higher diffusion of penicillin G at the interface. The smaller value of krmA compared to kaqA as shown in Table 2 indicates that diffusion in the reverse micelle phase is the rate limiting step for the effect of pH.

4

2.5 2

1.5 [dolpa]=5 mM Calc [dolpa]=5 mM exp [dolpa]=10 mM Calc [dolpa]=10 mM exp [dolpa]=25 mM Calc [dolpa]=25 mM exp

1

0.5 0

0

5

10

15

20

25

30

Time (minute) Fig. 4. (a) Effect of surfactant concentration for the forward extraction of penicillin G at Vr = 1 (aqueous phase). Experiment conditions: [Penicillin G] = 5 mM; pH = 7; [KCl] = 0.1 M. (b) Effect of surfactant concentration for the forward extraction of penicillin G at Vr = 1 (reverse micelle phase). Experiment conditions: [Penicillin G] = 5 mM; pH = 7; [KCl] = 0.1 M.

Again Table 1 shows the values of KLA and mf for the effect of surfactant concentration. KLA value was increased when the surfactant concentration used increased from 5 mM to 10 mM whilst the value of mf also increased at the same time. In the previous paper [5] it was reported that the critical micelle concentration of DOLPA in isooctane was found to be around 0.1 mM. This confirms that penicillin G is being solubilized into reverse micelle phase at surfactant concentration more than critical micelle concentration of DOLPA. However, when the surfactant concentration was increased to 25 mM, the values of KLA and mfmf declined showing that less penicillin G were taken up into reverse micelle phase when the system became saturated with DOLPA. More surfactant molecules attached to the interface as higher surfactant concentration was used. As a result, the solubilization in the reverse micelle phase is the rate limiting step for the process on the surfactant concentration effect as smaller value of krmA was obtained. 4.1.3. Effect of salt concentration Fig. 5(a) and (b) shows the results of the effect of salt concentration (or ionic strength) on the kinetic partitioning of penicillin G in the reverse micelle system. The addition of salt to the aqueous phase containing penicillin G is very significant to avoid the formation of a stable emulsion hence decrease the

S.H. Mohd-Setapar et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 685–695

690

(a)

(a) 25

5

Concentration (mM)

4.5 4

Concentration (mM)

[KCl]=0.1 M Calc [KCl]=0.1 M exp [KCl]=0.5 M Calc [KCl]=0.5 M exp [KCl]=1 M Calc [KCl]=1 M exp

3.5 3 2.5 2

[Antibiotic]=0.5 mM Calc [Antibiotic]=0.5 mM exp [Antibiotic]=5 mM Calc [Antibiotic]=5 mM exp [Antibiotic]=25 mM Calc [Antibiotic]=25 mM exp

20

15

10

5

1.5 1

0 0

5

10

15

Time (minute)

20

25

30

10

15

3.5 3 2.5 2

20

25

30

Time (minute)

Concentration (mM)

4

Concentration (mM)

5

(b) 25

(b) 4.5

[Antibiotic]=0.5 mM Calc [Antibiotic]=0.5 mM exp [Antibiotic]=5 mM Calc [Antibiotic]=5 mM exp [Antibiotic]=25 mM Calc [Antibiotic]=25 mM exp

20

15

10

5 [KCl]=0.1 M Calc [KCl]=0.1 M exp [KCl]=0.5 M Calc [KCl]=0.5 M exp [KCl]=1 M Calc [KCl]=1 M exp

1.5 1 0.5 0

0

0

5

10

15

20

25

0

0

5

10

15

20

25

30

Time (minute)

30

Time (minute) Fig. 5. (a) Effect of salt concentration for the forward extraction of penicillin G at Vr = 1 (aqueous phase). Experiment conditions: [Penicillin G] = 5 mM; pH = 7; [DOLPA] = 10 mM. (b) Effect of salt concentration for the forward extraction of penicillin G at Vr = 1 (reverse micelle phase). Experiment conditions: [Penicillin G] = 5 mM; pH = 7; [DOLPA] = 10 mM.

extraction efficiency. Moreover, a minimum amount of salt is necessary to salt out the surfactant from the excess aqueous phase into the organic phase and form reverse micelle [25]. Reverse micelle molecule will not form if there is too little salt in the aqueous phase however if there is too much salt the efficiency of the surfactant molecules to solubilize penicillin G reduced. These findings show that increasing salt concentration in the aqueous phase causes a significant reduction in transfer rate and efficiency. It is attributed to the primary effect of salt by shielding electrostatic attraction between micellar wall of surfactant and penicillin G. The extraction efficiency were varied with salt concentration indicated the existence of electrostatic attraction between the penicillin G and surfactant [26]. At higher ionic strength, the interactions between molecules of antibiotics and DOLPA polar head group were reduced, resulting in a decrease in the amount of water contained in the reverse micelles. The increase in ionic strength compresses the range of electrostatic attraction therefore it can overcome the thermal motion of penicillin G molecules. This effect is identifying as Debye electrostatic screening. Many previous researchers refer the low solubilization capacity of reverse micelles extraction is having relationship with high ionic strength aqueous solutions lead to size exclusion phenomenon [27]. Size exclusion occurs when the reversed micelles size is reduced and consequently the larger size of protein molecules also is excluded.

Fig. 6. (a) Effect of antibiotic concentration for the forward extraction of penicillin G at Vr = 1 (aqueous phase). Experiment conditions: [KCl] = 0.1 M; pH = 7; [DOLPA] = 10 mM. (b) Effect of penicillin G concentration for the forward extraction of penicillin G at Vr = 1 (reverse micelle phase). Experiment conditions: [KCl] = 0.1 M; pH = 7; [DOLPA] = 10 mM.

Consequently, this leads to a decrease in the amount of penicillin G being transferred into reverse micelles. From Table 1, it can be observed that as the ionic strength is increased, the calculated value of KLA decreases, together with a decrease in mf value, indicating that less penicillin G are being taken up into the reverse micelle phase. The rate limiting step on the effect of salt concentration is the diffusion in the reverse micelle phase (krmA < kaqA). However, when the theoretical and experimental results of effect of salt concentration on the extraction of penicillin G are compared in Fig. 5(b), several general observations is obvious illustrated and large deviations were obtained. Therefore, the theoretically curves do not fit the experimental curves accurately due to the natural significance of the great rigidity and roughness of the model. In general, it cannot be pretend to represent all the factors are in correct between theoretical and experimental result. This is phenomenon occurs because the fact that the simplifying assumption resistance arising from the interfacial solubilization step is negligible of this model gives a poor approximation to reality, especially when the concentration of salt changes. 4.1.4. Effect of penicillin G concentration The effect of initial penicillin G concentration on the kinetic partitioning of penicillin G in reverse micelle system is demonstrated in Fig. 6(a) and (b). It was found that the transfer rate was the highest at the initial penicillin G concentration of 25 mM. However, cautious concern should be made that this hypothesis is only valid if the extraction of penicillin G is referred to as the amount of penicillin G transferred into the reverse micelle phase.

S.H. Mohd-Setapar et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 685–695

If the result is interpreted into the percentage of transfer efficiency (%E) therefore the highest %E should be at the penicillin G concentration of 5 mM. The transfer efficiency could be determined by: Transfer efficiency ð%EÞ ¼

Concentration of penicillin G extracted Total concentration of penicillin G  100% (20)

Basically, an increase in the initial penicillin G concentration should increase the driving force between charge polar heads of surfactant and opposite charge of penicillin G molecules. The observation in this study indicates that, besides the driving force from the electrostatic interaction, the ratio of surfactant to the amount of transferred solute may also be the factor affecting the extraction of biomolecules into the reverse micelles [28]. This is generally appropriate for the initial quantity of penicillin G used. Table 1 shows that KLA increases when initial penicillin G concentration used increases from 0.5 mM to 5 mM; the value of mf increases simultaneously. This shows that more penicillin G are being transferred and solubilized into the reverse micelle phase. In contrast, when initial penicillin G concentration is increased to 25 mM, the values of KLA and mf decrease. This confirms that less penicillin G are taken up into the reverse micelle phase in terms of

(a)

3

Concentration (mM)

2.5 2

1.5 pH 3 Calc pH 3 exp pH 5 Calc pH 5 exp pH 8 Calc pH 8 exp

1

0.5 0

0

5

10

15

20

25

30

Time (minute)

(b)

3

pH 3 Calc pH 3 exp pH 5 Calc pH 5 exp pH 8 Calc pH 8 exp

Concentration (mM)

2.5 2

transfer efficiency. The solubilization in the reverse micelle phase is the rate limiting step for the effect of penicillin G concentration (krmA < kaqA). 4.1.5. Effect of aqueous phase pH Fig. 7(a) and (b) shows the effect of the aqueous phase pH for backward extraction process. The buffered aqueous phase was made up of 1.0 M KCl and was maintained at pH 7 while the ionic strength was estimated 0.0537. The low ionic strength is maintained throughout the backward extraction to prevent reextraction of penicillin G back into the reverse micelles following the backward extraction process. Modeling was carried out using the MATLAB program to investigate the combined mass transfer coefficients, KLA and the equilibrium partition coefficients, mb (Table 3) for backward extraction. It was found that the optimum transfer rate is at pH 3, and it decreases as pH is further increased. Even though the backward extraction of penicillin G is influenced significantly by the initial amount of penicillin G extracted to the reverse micelle phase, the pH also imposes a significant effect to the backward extraction rate. Table 3 shows that the highest values of partition coefficient (mb) and mass transfer coefficient (KLA) were obtained at pH 3. A decrease in KLA and mb at higher pH showed that the diffusion in the reverse micelle phase and the diffusion at the interface were slower. Interaction between the penicillin G and reverse micelle is difficult to occur when unfavorable pH is adopted, because there are only few reverse micelles formed. The diffusion in the reverse micelle phase is faster than the one in the aqueous phase. The rate limiting step for pH effect on the backward extraction is the diffusion at the aqueous phase (kaqA < krmA as shown in Table 4). 4.1.6. Effect of surfactant concentration Fig. 8(a) and (b) shows the effect of initial surfactant concentration on the backward extraction kinetics of penicillin G. It could be observed that the maximal concentration of penicillin G that could be extracted in the backward extraction is obtained by using an initial surfactant concentration of 10 mM DOLPA. At higher surfactant concentration, more penicillin G molecules can be solubilized at the interface leading to an increase in the release of penicillin G to a fresh aqueous phase. The calculated values of KLA and mb are shown in Table 3. It was found that when the initial surfactant concentration was 10 mM, both these values satisfy the conditions wherein the backward extraction rate and the recovery of penicillin G into the aqueous phase are the highest. Lower kaqA designates that the solubilization in the aqueous phase is the rate limiting step for the surfactant concentration effect. In Fig. 8(a) and (b) the solid lines represent the results of the two film model the points with labels are the experimental results for Table 3 Values of combined mass transfer coefficient (KLA) and equilibrium partition coefficient (mb) for the effects of pH, surfactant concentration, salt concentration and penicillin G concentration on the backward extraction of penicillin G (* sign indicate as equilibrium).

1.5 1

Parameter pHaqueous

0.5 0

691

0

5

10

15

20

25

30

Surfactant concentration (mM)

Time (minute) Fig. 7. (a) Effect of pH for the backward extraction of penicillin G at Vr = 1 (reverse micelle phase). Forward extraction conditions: pH = 7; [Penicillin G] = 5 mM; [DOLPA] = 10 mM. Backward extraction conditions: [KCl] = 1.0 M. (b) Effect of pH for the backward extraction of penicillin G at Vr = 1 (fresh aqueous phase). Forward extraction conditions: pH = 7; [Penicillin G] = 5 mM; [DOLPA] = 10 mM. Backward extraction conditions: [KCl] = 1.0 M.

Salt concentration (M)

Penicillin G concentration (mM)

3 5 8 5 10 25 0.1 0.5 1.0 0.5 5 25

mb (C2/C1)*

KLA  108 (m3/s)

0.9945 0.6561 0.5237 0.6406 0.9945 0.5035 0.4534 0.6739 0.9945 0.4368 0.9945 0.5385

0.3997 0.1892 0.1162 0.1879 0.3998 0.0339 0.1115 0.1871 0.4027 0.1226 0.4027 0.2014

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692

Table 4 Calculated individual mass transfer coefficients for the effects of pH, surfactant concentration, salt concentration and penicillin G concentration at optimum value in backward extraction. Parameter pH Surfactant concentration (mM) Salt concentration (M) Penicillin G concentration (mM)

3 10 1 25

kaqA  108

krmA  108

1.6499 1.3966 1.8252 2.0674

4.8972 3.2982 6.5062 13.2626

effect of surfactant concentration for the backward extraction of penicillin G are not fit very well. It is because in backward extraction, the structural change of proteins and micelle due to their strong interaction as the surfactant concentration is increased. Hence the backward transfer of protein from a reverse micelle to an aqueous phase is relatively slow and the result of experimental shows large deviations from the model. 4.1.7. Effect of salt concentration In reverse micelle systems, penicillin G backward extraction from the reverse micelle organic phase is conventionally

(a)

4.1.8. Effect of Penicillin G concentration Fig. 10(a) and (b) shows the effect of initial penicillin G concentration on the backward extraction kinetics of penicillin G. It was found that the highest mass transfer rate of backward extraction was achieved at penicillin G concentration of 25 mM, similar to what has been observed for the forward extraction. The results point out that the backward kinetic partitioning of

3

(a)

2.5 2 1.5 [dolpa]=5 mM Calc [dolpa]=5 mM exp [dolpa]=10 mM Calc [dolpa]=10 mM exp [dolpa]=25 mM Calc [dolpa]=25 mM exp

1 0.5 0

3 2.5

Concentration (mM)

Concentration (mM)

conducted by adjusting the backward extraction phase so that it has high ionic strength. The effect of salt concentration of the aqueous phase in the backward extraction process is shown in Fig. 9(a) and (b). It was found that the highest mass transfer rate of backward extraction was achieved at 1.0 M KCl. It can be seen that the backward extraction of penicillin G to the aqueous phase increased significantly in rate and extent of transfer by increasing KCl concentration. At higher ionic strength, the size of the water pool approached the size of solute leading to its release from the reverse micelle phase by size exclusion [29]. Table 3 shows the KLA and mb values of the transport of penicillin G in the backward extraction process. The results show that both KLA and mb increased for the backward extraction of penicillin G as the initial ionic strength increased. The rate limiting step for the effect of salt concentration is diffusion in aqueous phase (kaqA < krmA).

0

5

10

15

20

25

30

2 1.5 [KCl]=0.1 M Calc [KCl]=0.1 M exp [KCl]=0.5 M Calc [KCl]=0.5 M exp [KCl]=1 M Calc [KCl]=1 M exp

1 0.5

Time (minute) 0

(b)

5

10

15

20

25

30

Time (minute)

3

[dolpa]=5 mM Calc [dolpa]=5 mM exp [dolpa]=10 mM Calc [dolpa]=10 mM exp [dolpa]=25 mM Calc [dolpa]=25 mM exp

2.5 2

(b)

[KCl]=0.1 M Calc [KCl]=0.1 M exp [KCl]=0.5 M Calc [KCl]=0.5 M exp [KCl]=1 M Calc [KCl]=1 M exp

3 2.5

Concentration (mM)

Concentration (mM)

0

1.5 1

2 1.5 1

0.5 0.5

0

0

5

10

15

20

25

30

Time (minute)

0

0

5

10

15

20

25

30

Time (minute) Fig. 8. (a) Effect of surfactant concentration for the backward extraction of penicillin G at Vr = 1 (reverse micelle phase). Forward extraction conditions: pH = 7; [Penicillin G] = 5 mM; [KCl] = 0.1 M. Backward extraction conditions: [KCl] = 1.0 M. (b) Effect of surfactant concentration for the backward extraction of penicillin G at Vr = 1 (fresh aqueous phase). Forward extraction conditions: pH = 7; [Penicillin G] = 5 mM; [KCl] = 0.1 M. Backward extraction conditions: [KCl] = 1.0 M.

Fig. 9. (a) Effect of salt concentration for the backward extraction of penicillin G at Vr = 1 (reverse micelle phase). Forward extraction conditions: pH = 7; [Penicillin G] = 5 mM; [DOLPA] = 10 mM; [KCl] = 0.1 M. (b) Effect of salt concentration for the backward extraction of penicillin G at Vr = 1 (fresh aqueous phase). Forward extraction conditions: pH = 7; [Penicillin G] = 5 mM; [DOLPA] = 10 mM; [KCl] = 0.1 M.

S.H. Mohd-Setapar et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 685–695

4.2. Mass transfer mechanism

10 [Antibiotic]=0.5 mM Calc [Antibiotic]=0.5 mM exp [Antibiotic]=5 mM Calc [Antibiotic]=5 mM exp [Antibiotic]=25 mM Calc [Antibiotic]=25 mM exp

9

Concentration (mM)

8 7 6 5 4 3 2 1 0

0

5

10

15

20

25

30

Time (minute)

(b)

5 [Antibiotic]=0.5 mM Calc [Antibiotic]=0.5 mM exp [Antibiotic]=5 mM Calc [Antibiotic]=5 mM exp [Antibiotic]=25 mM Calc [Antibiotic]=25 mM exp

4.5

Concentration (mM)

4 3.5 3 2.5 2 1.5 1 0.5 0

0

5

10

15

20

25

30

Time (minute) Fig. 10. (a) Effect of penicillin G concentration for the backward extraction of penicillin G at Vr = 1 (reverse micelle phase). Forward extraction conditions: pH = 7; [DOLPA] = 10 mM; [KCl] = 0.1 M. Backward extraction conditions: [KCl] = 1.0 M. (b) Effect of penicillin G concentration for the backward extraction of penicillin G at Vr = 1 (fresh aqueous phase). Forward extraction conditions: pH = 7; [DOLPA] = 10 mM; [KCl] = 0.1 M. Backward extraction conditions: [KCl] = 1.0 M.

penicillin G is dependent of the initial amount of penicillin G that could be extracted into the reverse micelle phase. However, the values of KLA and mb as shown in Table 3 give a different phenomenon as the highest value of KLA and mb were at 5 mM. This suggests that even though the highest transfer rate was achieved at the highest initial concentration of penicillin G, there is an optimal concentration limit that offers the highest solubilization and diffusion rate of the solutes for the backward extraction of penicillin G. The solubilization in the aqueous phase is the rate limiting step for the penicillin G concentration effect since kaqA < krmA. From the graph, it shows that the curve fitting between model and experimental for penicillin G concentration of 25 mM are poor. As reported by Mohd-Setapar et al. [7], in the backward extraction the large mass of penicillin G present in the reverse micelle phase after forward extraction stayed in the reverse micelle and did not transfer into a fresh aqueous phase. Many researchers found that the protein is difficult to solubilize from reverse micelle phase after forward extraction and difficult to accomplish due to the large interfacial resistance is mass transfer [7,30,31] therefore the experimental result does not fit well the model. However the trend of the curve fitting is well enough to show the behavior of the penicillin G extraction in backward extraction.

To date, several papers have been presented on the interfacial transport process of biomolecules such as proteins between a bulk aqueous phase and a reverse micelle phase [32]. Dekker et al. [19] investigated the mass transfer rates in the extraction of a-amylase with a reverse micelle phase of the cationic surfactant (TOMAC) in isooctane. They found that the forward extraction rate is controlled by the diffusion of the enzyme in the aqueous phase boundary layer and the interfacial process of enzyme released from the reverse micelles controls the back extraction rate. In another study on the mass transfer of reverse micelle system, Adachi and Harada [33] suggested that ionic surfactant interacts strongly with the opposite charge residues on the solute surface by electrostatic interactions. They proposed that many kinds of solutes such as protein can be extracted to the reverse micelle phase under usual operating conditions. The following sections will discuss the forward and backward extraction mechanisms of antibiotics using the reverse micellar solubilization process. 4.2.1. Forward extraction mechanism The driving force has a very significant effect to mass transfer process. In most cases it is accepted that the driving force for this process of transfer is the electrostatic interaction between the solutes and the charged surfactant head groups [34]. In this study, the forward extraction of penicillin G into the reverse micelle phase was favored at pH 7 and low salt concentration where attractive electrostatic interactions existed between the penicillin G molecules and the reverse micelles. From experimental observations, the rate of penicillin G transfer increased with decreasing salt concentration and increasing surfactant as well as penicillin G concentrations. The proposed kinetic model suggests that by manipulating KLA and mf values, the mass transfer rate and recovery in the reverse micelle system can be enhanced. Both KLA and mf indicate the diffusion and solubilization rates of the solutes. Fig. 11 shows the plot of combined mass transfer coefficients of aqueous and organic reverse micelle films for the forward extraction of penicillin G at Vr = 1 for the effect of pH. These results proved that the forward extraction rates are controlled by the diffusion in the reverse micelle film and the solubilization at the interface. 4.2.2. Backward extraction mechanism The backward extraction of penicillin G into the fresh aqueous phase was favored at pH 3 and high salt concentration. From experimental findings, the rate of penicillin G transfer is found

1.8 1.6 1.4

1/KLA x 10 7

(a)

693

1.2 1 0.8 0.6 0.4 0.2 0 0.7

0.9

1.1

1.3

1.5

1.7

1.9

2.1

mf Fig. 11. Plot of combined mass transfer coefficient (1/KLA) against the equilibrium partition coefficient (mf) for the forward extraction process for the effect of pH.

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694

0.45

H.B. Mat, and Universiti Teknologi Malaysia for the research scholarship (UTM-PTP) awarded to S.H. Mohd-Setapar.

0.4 0.35

References

1/KLA 10 8

0.3 0.25 0.2 0.15 0.1 0.05 0 0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

mb Fig. 12. Plot of combined mass transfer coefficient (1/KLA) against the equilibrium partition coefficient (mb) for the backward extraction process for the effect of pH.

significantly influenced by the amount of penicillin G loaded in the reverse micelle phase from the forward extraction. Fig. 12 shows the plot of combined mass transfer coefficients of aqueous and organic films for the backward extraction of penicillin G at Vr = 1 for the effect of pH. The model suggested that the backward extraction rates are controlled by the diffusion in the aqueous film and the solubilization at the interface. 5. Conclusions The effects of several important parameters on the kinetic transfer of penicillin G between an aqueous phase and reverse micelles were studied. Based on the results obtained from the kinetic study, the kinetic transfer process was successfully modeled using the general two-film theory of mass transfer to flat interface. This study further demonstrated the suitability of reverse micelle extraction as an initial operation in bioproduct purification. Antibiotic solubilization in reverse micelles can be explained as the electrostatic interactions between the penicillin G and the surfactant head groups. A mass transfer model, based on the two-film theory, is found successfully describe the solubilization of penicillin G between the aqueous and reverse micelle phases in a well-mixed system. From the proposed kinetic model, the overall mass transfer coefficient (KLA) and the equilibrium partition coefficient (m) were calculated. The forward extraction rates are controlled by the diffusion in the reverse micelle film and the solubilization at the interface, while the backward extraction rates are controlled by the diffusion in the aqueous film and the solubilization at the interface. In forward extraction when the aqueous pH is increased from 5 to 8 the values mass transfer coefficient, KLA increased from 0.2859  107 to 0.6115  107. In addition, as DOLPA concentration increase from 5 mM to 10 mM, the value of KLA and forward extraction equilibrium partition coefficient, mf are also increased from 0.1285  107 to 0.8971  107 and 0.4954 to 1.40085 respectively. On the other hand further increased DOLPA concentration up to 25 mM the value of KLA and mf were declined because the organic solution becomes saturated with DOLPA molecules. The result shows that the transfer rate and efficiency was reduced dramatically at higher concentration of salt and an increase of penicillin G concentration the driving force between charge polar heads of DOLPA and opposite charge of penicillin G molecules. Acknowledgments Authors gratefully acknowledge the support of the Hitachi Scholarship Foundation for the research fellowship awarded to

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