- Email: [email protected]
Studies on emulsion liquid membrane extraction of cephalexin
Journal of Membrane Science 145 (1998) 15±26
Studies on emulsion liquid membrane extraction of cephalexin G.C. Sahoo, N.N. Dutta* Chemical Engineering Division, Regional Research Laboratory, Jorhat 785006, India Received 22 October 1996; received in revised form 5 June 1997; accepted 5 January 1998
Abstract An experimental study on batch extraction of cephalexin using an emulsion liquid membrane system has been reported. The effects of surfactant, carrier and solute concentrations, phase volume ratio, stirring speed, and counterion concentration on the extraction rate were examined. Surfactant, carrier and diluent used were Span-80, Aliquat-336 and n-heptane±kerosene (1:1), respectively. Under the optimised experimental conditions, emulsion swelling was found to be marginal. By maintaining an appropriate pH gradient in the feed and receiving aqueous phase, facilitated transport could be realised. Selective separation of cephalexin from a mixture of 7-aminodeacetoxy cephalosporanic acid (7-ADCA) could be demonstrated in the emulsion liquid membrane system. A mathematical model based on mass transfer across aqueous boundary layer, interfacial chemical reaction and diffusion in the emulsion globule provides a reasonable ®t of the experimental solute concentration versus time pro®les in the emulsion liquid membrane system. # 1998 Elsevier Science B.V. Keywords: Emulsion liquid membranes; Cephalexin; 7-Aminodeacetoxy cephalosporanic acid (7-ADCA); Span-80; Aliquat336; Coupled transport; Ion-exchange extraction
1. Introduction Reactive extraction in liquid membrane or nondispersive extraction in hollow ®bre membrane can provide cost-effective methods for separation and puri®cation of cephalosporin antibiotics [1,2]. We have been studying the liquid membrane technique for separation and puri®cation of cephalosporin antibiotics and the technique was found to be effective in certain speci®c cases [3±6]. In our previous communication, we have demonstrated facilitated uphill transport of cephalexin in a bulk liquid membrane by exploiting the concept of *Corresponding author. Tel.: +91 0376 320353; fax: +91 0376 321158. 0376-7388/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0376-7388(98)00027-1
reactive extraction using Aliquat-336 as the liquid ionexchange carrier [5]. Cephalexin was transported from an aqueous phase of higher pH through the membrane phase (comprising Aliquat-336 in n-butyl acetate as the diluent) to another aqueous phase of lower pH, the transport being facilitated by countertransport of another anion and a strong pH dependence of the distribution coef®cient of cephalexin between the membrane and the aqueous phases. In this paper, we report a comprehensive study to con®rm the applicability of liquid emulsion membrane (LEM) process for separation of cephalexin from aqueous solution such as the one encountered as reaction medium of an enzymatic process. Since the reaction mixture of enzymatically produced cephalexin contains 7-aminodeacetoxy cephalosporanic acid (7-
16
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
ADCA) as the unconverted reactant and selective separation is desirable, complementary studies were made on extraction behaviour of 7-ADCA and its mixture with cephalexin. 2. Theoretical aspects 2.1. Separation mechanism The mechanism of separation by extraction and reextraction in the LEM system considered here correspond to liquid±liquid anion-exchange reaction. The reaction of dissociated cephalexin anion Aÿ a and the carrier (QClo) in organic solvent is k1
ÿ Aÿ a QClo QAo Cla kÿ1
(1)
The equilibrium constant of the reversible complexation cephalexin reaction is given by K
QAo Clÿ a QClo Aÿ a
The 1:1 stoichiometry of the reaction was con®rmed by the result of equilibrium experiment. For the extraction to be effective, the pH of the feed aqueous phase should be maintained above 5.88 (the upper pKa value of cephalexin, the lower pKa being 2.56) to achieve complete dissociation with anionic form only. The driving force for the process may be provided by the difference in chloride ion concentration in the receiving and feed phases, respectively. The mechanism of the countertransport may be represented by the scheme shown in Fig. 1 which indicates transport of cephalexin anion from feed (external) solution to the receiving (internal) solution facilitated by the carrier in a solvent as the membrane phase. At the external±membrane phase interface, the carrier binds with Aÿ a to form a complex, QAo, which is extracted by the membrane. The complex is transported due to concentration gradient to the membrane±internal phase interface, where another interfacial ion-exchange reaction takes place thereby releasing cephalexin into the internal receiving phase. The carrier returns to the external±membrane phase interface to recombine with cephalexin anion, the transport being accompanied by countertransport of Clÿ ion. Alternately, buffer anion may also provide the
Fig. 1. Schematic diagram of the transport mechanism in LEM process for cephalexin separation.
driving force for the transport via another interfacial reaction. In our studies, we use citrate buffer in the internal phase, the other interfacial ion-exchange reaction involving citrate ion (Ctÿ) is Ctÿ QClo QCto Clÿ a
(2)
The extraction equilibrium constants for the reaction given by Eq. (2) is relatively lower than that given by Eq. (1). Using excess Clÿ concentration in the receiving phase, the forward reaction rate of Eq. (2) may be suppressed providing the driving force for ÿ facilitated transport of Aÿ a via countertransport of Cl . ÿ Alternately, using excess Ct and QClo concentration, the Clÿ for countertransport may be maintained. 2.2. Mathematical description of the kinetic model The concentration pro®le for the permeation of cephalexin anion into the emulsion globule is shown in Fig. 2. The following steps are involved in the transport process: diffusion Aÿ a to the feed±membrane phase interface of the emulsion globule from the feed phase (3); reaction of Aÿ a with the carrier, QCl, at the external interface releasing the counterion Clÿ; diffusion of the solute carrier complex, QAo, into the emulsion globule; stripping of the complex at the internal interface between the membrane (2) and internal phase (1); back diffusion of the carrier to the feed (3) and membrane (2) phase interface. The following assumptions are inherent in the above process: the extraction and stripping reactions are located at the interfaces and the amount of carrier dissolved in the aqueous phase is negligible;
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
17
V2 dCQA 1 @ @CQA Deff 2 r2 V1 V2 dt @r r @r S0 ÿ kÿ1 CQA CClÿ V 1 V2
(5)
where S0 is the total interfacial area of the emulsion globule, Deff the effective diffusivity of the solute± carrier complex, and V1 and V2 are the volumes of the internal and membrane phases, respectively. The equilibrium constant KA for reaction given by Eq. (1) can be expressed as KA Fig. 2. Concentration profile of cephalexin in an ELM system.
negligible transport resistance in the internal phase (I) because phase I droplets are very small (R1<5 mm); a quasi-homogeneous state; resistance in the oil layer (surfactant monolayer) at the external interface is negligible. Based on the above, the following equations hold good; the stripping reaction is very fast due to large surface area of phase II. Mass transfer of Aÿ a in the external phase is expressed as dCAÿ 1 ÿ kF A CAÿ ÿ CA ÿ dt V3
(3)
CQA CClÿ k1 CQCl CAÿ kÿ1
(6)
where 0 ÿ CQA CQCl CQCl
(7)
From (6) and (7), we get " # 0 CCl CQCl ÿ1 CAÿ KA CQCl
(8)
Initial and boundary conditions are as follows: CAÿ CA0 ÿ ;
t 0;
CQA 0
0 r R
(9)
Boundary conditions at centre of globules to have no ¯ux are given by @CQA 0 @r
r 0;
t 0
(10)
At the feed±membrane interface, we have
where CAÿ is the solute concentration in the external phase, CA ÿ the concentration at the interface between the external phase and the membrane phase, V3 the volume of the external phase, t the stirring time, kF the mass transfer coef®cient and A is the surface area of emulsion globules. The interfacial reaction rate at 3±2 interface may be expressed as
The following relations are used for solution of the above equations: (i) The number of emulsion globule Nm is given by
dCAÿ A k1 CA ÿ CQCl RF dt V3 extraction reaction rate per unit area of 3ÿ2 interface (4)
where P R is thePmean globule radiusD32/2, and D32 ni D3i = ni D2i (Sauter mean diameter). (ii) Surface area of emulsia globule, A, is
The diffusion of the solute±carrier complex in Phase II is described by Fick's second law and is given by the following equation for phase II. The mass transfer in the emulsion globule is given by
(iii) Total interfacial area of emulsia globule, S0 , is
r R;
Nm
A
Deff
@CQA kF CAÿ ÿ CA ÿ @r
V2 V 1 4=3R3
3 V2 V1 R
S0 ANm
(11)
(12)
(13)
(14)
18
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
Eqs. (3)±(14) may be simultaneously solved numerically by using the methods of line (a combination of ®nite differences method and Gear's algorithm) in the subroutine of IVPAG of IMSL-MATH Library [7,8].
Table 1 Experimental parameters for LEM extraction of cephalexin
3. Experimental
External feed phase
3.1. Materials Cephalexin and Aliquat-336 were procured from Aldrich, USA. Span-80 was obtained from Sigma, USA. n-Heptane, sodium citrate, sodium carbonate/ phosphate, citric acid, NaCl, etc. were procured from E. Merck (India) and were of analytical grade. Superior grade kerosene was procured from local supplier. 3.2. Method Emulsion liquid membrane experiments were conducted in a mixer±settler device used in our earlier work [9]. The membrane phase consisted of a 1:1 v/v mixture of n-heptane and kerosene, Span-80 (8±15% v/v) and Aliquat-336 (1±16% v/v). High surfactant to carrier concentration ratio was necessary as a criterion to maintain a stable w/o emulsion [11]. Citrate buffer was taken as the internal aqueous receiving phase maintaining a pH of 5 at which cephalexin is relatively stable [10]. A water in oil emulsion was prepared by slow addition of the internal aqueous phase (50 ml) to the organic membrane phase (50 ml) using a high speed homogeniser (Remi, India) at 10 000 rpm for 20 min. The external feed solution of cephalexin (20 mM) was prepared in carbonate and phosphate buffers. It may be noted that extraction from carbonate rather than phosphate buffer solution is preferred because of relatively high coextraction of the latter [12]. By taking 50 ml of feed phase in the mixer± settler the w/o emulsion (10±50 ml) was dispersed at a constant stirring speed of the mixer±settler. All experiments were conducted at 328C with the parameters shown in Table 1. The effect of emulsion to feed phase volume ratio, surfactant and carrier concentration, feed phase pH, stirring speed and Clÿ concentration in the internal phase were investigated. The stirring speed was measured by a digital non-contact type tachometer. During
Internal receiving phase Membrane phase
VR:VM1:1 in w/o emulsion, Vem:VF1:1 to 1:5
Citrate buffer, pH 5, volume (VR): 50 ml Volume (VM): 50 ml, Aliquat-336: 1±6% v/v, Span-80: 8±15% v/v, solvent: 1:1 v/v mixture of n-heptane and kerosene Volume (VF): 50 ml, cephalexin: 20 mM, pH: up to 9 by carbonate and phosphate buffer
experiments, samples were drawn from the vessel at regular intervals and concentration of cephalexin in the aqueous solution were analysed in a UV±Vis spectrophotometer (Schimadzu 160A). Changes in the volume of internal phase by water transport (swelling) were estimated by volume balance after breaking the emulsion at the end of the experiments. The emulsion was broken by thermal demulsi®cation as well as by passing the emulsion through a hydrophilic membrane (Millipore). Complementary studies of extraction equilibrium of cephalexin and 7-ADCA were carried out in an agitated 100 ml glass round bottom ¯ask with respect to the effect of aqueous phase pH value on the cephalexin and 7-ADCA distribution coef®cient (Fig. 3). Experiments on the pH
Fig. 3. Distribution coefficient of cephalexin and 7-ADCA versus pH of aqueous phase.
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
19
effect (pH varied between 5 and 10) were conducted for 2 h with 20 ml each of the aqueous and organic phases by taking 10 mM solute and 10 mM Aliquat336 in the respective phases. The value of the distribution coef®cient, ``m'', was calculated using the following equation: m
QAo eq Vo Aÿ a eq Va
(15)
[QAo]eq was estimated from ÿ Vo QAo eq Va fAÿ a t0 ÿ Aa eq g
(16)
4. Results and discussion 4.1. Effect of pH on distribution coefficient In order to establish the optimal pH values for both the feed and receiving aqueous phases for an optimal LEM system, the results of equilibrium studies were used as the guideline. Fig. 3 shows the variation of distribution coef®cient, ``m'', with pH of cephalexin and 7-ADCA solutions. As expected, increase of pH increases the ``m'' value up to pH of 9.5, beyond which ``m'' value appears to decrease marginally. This decrease in ``m'' value may be attributed to the hydrolytic decomposition of the solutes at higher pH. Indeed, the decomposition rate of cephalexin increases sharply when the pH is raised from 8 to 10 as reported elsewhere [10]. Lower values of ``m'' at low pH may be realised by incomplete dissociation of the solutes. Since the maximum value of distribution coef®cient is achieved in the pH range 9±9.5 and cephalexin is stable up to pH 9, our experimental protocol for LEM primarily involves extraction from an aqueous solution of high pH and stripping to another aqueous phase of relatively low pH, which will be optimised as discussed in Section 4.2. 4.2. Effect of surfactant concentration The effect of surfactant concentration was studied at a Aliquat-336 concentration of 5% v/v in the membrane phase with feed and receiving phase pH values of 9 and 5, respectively. The ratio of emulsion phase to feed phase volume was maintained at 1:1 and Span-80 concentration was varied from 10 to 15% v/v.
Fig. 4. Effect of Span-80 concentration (% v/v) on the batch permeation of cephalexin. Aÿ a 20 mM; rpm 450: * (5), ~ (10), & (12.5), 5 (15).
It may be noted that the dilute mixture used for preparation of the stable emulsion was the optimised one. Further, the volume ratio of Span-80 and Aliquat336 affects the emulsion stability, being greater for high Span-80 concentration. A ratio of 2:1 for surfactant to carrier generates the most stable emulsion. Fig. 4 shows the effects of Span-80 concentration on the overall dimensionless concentration of cephalexin in the external phase versus time and initial permeation rate calculated from the initial slope of the concentration pro®le curves. It is evident that increase of surfactant concentration increases both the initial extraction rate and degree of removal. The initial extraction rate increases almost linearly with Span-80 concentration in the range 5±15%. It may be expected that this surfactant effect may be realised through enhanced emulsion stability alone. Similar effect of surfactant was also observed in case of extraction of lactic acid [13,14] and penicillin-G [15±18]. The decreased rate can be attributed to solute leakages from the internal phase thus indicating emulsion breakage at low surfactant concentration. It is believed that Span-80 affects solute transport because of its in¯uence on water transport and swelling via
20
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
osmotic effects [19,20]. However, in our investigation, swelling was found to be very marginal. The change in internal volume with increase of Span-80 concentration was also marginal. From an initial emulsion volume of 50 ml, the ®nal volume increased up to 60 ml only at the highest Span-80 concentration used in our work. 4.3. Effect of volume ratio of the emulsion Vem and feed phase VF The condition maintained during the study of the effect of phase volume ratio is essentially the same as mentioned in Section 4.2. However, the Span-80 concentration was maintained at 15% v/v. Fig. 5 shows the effect of Vem/VF on the batch permeation rate of cephalexin. As expected, an increase of this ratio tends to increase the extraction rate due to the increase of the amount of the carrier in the membrane phase and the improvement in the dispersibility of the w/o/w emulsion system. Furthermore, the increase of this ratio provides increased capacity of the membrane and internal phase for enhanced permeation and stripping
Fig. 5. Effect of the volume ratio of emulsion (Vem) to feed (VF) phase on batch permeation of cephalexin: [Span-80]15%, [QCl]2%, Vem/VF, 1/5 ( ), 2/5 (~), 3/5 (&), 1/1 ( ).
of cephalexin. However, further increase in the Vem/VF ratio is unlikely to provide increased extraction rate because of the increase in viscosity which strongly in¯uences the emulsion swelling. From the experimental data, it appears that the separation ef®ciency reaches a limiting value of 70± 80% at optimal condition of the membrane phase composition, phase volume ratio, etc. Our experiments on bulk liquid membrane [5] extraction revealed that the feed phase pH affects the extraction ef®ciency, a high pH being favourable for the system due to the presence of the solute predominantly in an anionic form. However, at pH>9.5, hydrolytic decomposition of cephalexin occurs. During the course of experiments, the feed phase pH was found to marginally decrease probably due to co-extraction of buffer anion by Aliquat-336. It was observed that the pH of the feed phase dropped from 9.5 to 8.5 after 50 min of extraction time. Thus, the limiting value of extraction ef®ciency obtained in our study may be explained from the feed phase pH dependence of the extraction rate. 4.4. Effect of Aliquat-336 concentration The effect of Aliquat-336 (carrier) concentration was studied at Vem/VF of 1/5 and the highest level of Span-80 concentration. As shown in Fig. 6, increase in extraction rate with increase in Aliquat-336 concentration corresponds to increased diffusion of solute± carrier complex across the membrane. The concentration of carrier in the membrane phase should phenomenologically increase the interfacial cephalexin concentration and hence the driving force for extraction providing an increased extraction rate. However, it appears that there is a limit to the carrier concentration that will give signi®cant increase in the extraction rate, probably as a result of the equilibrium of the stripping reaction. On the other hand, Aliquat-336 having surfactant property tends to act antagonistically to the emulsion stabilising surfactant such that the carrier can reduce the membrane stability. A limiting carrier concentration of 5% v/v reported for lactic acid permeation [12] seems to be in agreement with the results obtained in the present work. However, our results indicate monotonic changes of feed phase cephalexin concentration with time at all concentrations studied here. Had there been an increase of feed phase cephalexin concentration at
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
21
Fig. 6. Effect of Aliquat-336 (QCl) concentration on the batch permeation of cephalexin. Vem/VF1/5, [Span-80]15% v/v, [QCl336] (% v/v); 1 (~), 2 (&), 4 ( ), 6 (*).
Fig. 7. Effect of feed phase cephalexin concentration (mM) on the batch permeation of cephalexin. Vem/VF1/1, [Span-80]15% v/v, [QCl]2% v/v; * (10), ~ (15), & (20).
long times of permeation particularly for higher carrier concentration, the inference of membrane breakage could have been drawn. Thus, the membrane breakage could be considered negligible in the present LEM system. In this regard, the chemical nature of the counterion in the receiving phase may be important in determining the membrane swell and the extent of separation. Thien et al. [21], while discussing the effect of the chemical nature of counterion from the so-called ``lyotrop number'' concept, observed that acetate ions with lowest ``lyotrop number'' provide higher degree of separation of lactic acid as compared to that achieved with Clÿ, Brÿ and Iÿ ions and that the membrane swelling was also the lowest. Thus, the observation in our LEM system containing acetate and/or citrate ion in the receiving phase may be considered reasonable.
increase with a decrease of the cephalexin concentration. However, at a concentration of 5 mM, the rate tends to reach an asymptote after 20 min of extraction. As the solute concentration is increased, the solute transport rate may decrease largely due to the reduced capacity of the internal phase to strip the transported solute anion. When the solute concentration is high, the internal droplets in the peripheral region are more rapidly saturated with the solute, and the complex must diffuse through the membrane phase to the more inner region of the drop to release the solute in the receiving phase such that the internal mass transfer resistance is signi®cant. When the solute concentration is low, the external mass transfer is controlling and the extraction rate is higher than that at high solute concentration. It may be noted that the interfacial area for stripping is much larger than that for extraction. The observation of high solute transport rate and recovery at low solute concentration is important from process design point of view. It is possible to achieve the required degree of separation with small solute concentration in the feed using a multi-stage train of mixer±settler devices in a practical recovery process.
4.5. Effect of feed phase cephalexin concentration The effect of feed phase cephalexin concentration was studied at 5, 10 and 12.5 mM under otherwise identical conditions. As shown in Fig. 7, the extraction rate and the degree of extraction appear to
22
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
Fig. 8. Effect of stirring speed (rpm) on the batch permeation of cephalexin. Vem/VF1/1, [QCl]2% v/v; [Span-80]15% v/v, rpm: * (250), ~ (350), & (450).
4.6. Effect of stirring speed As shown in Fig. 8, increase in stirring speed increases the extraction rate, obviously due to reduction of the emulsion globule size thereby providing high interfacial contact area between the external and membrane phases and increase of the external phase mass transfer coef®cient. However, due to very viscous nature of the dispersion, high stirring speed may be prohibitive from the process point of view. As shown in Fig. 8, increase of stirring speed from 250 to 450 rpm does not result in appreciable increase of extraction rate and recovery. This may be attributed to the fact that during extraction the system becomes viscous and at long contact time it became hard to disperse the emulsion because of high surfactant concentration used in the membrane phase. 4.7. Effect of counterion in the receiving phase From mechanistic consideration, it may be expected that the Clÿ ion concentration gradient provides the driving force for separation. Thus Clÿ effect was studied by using NaCl in the receiving phase at a
Fig. 9. Effect of chloride ion concentration in receiving phase on the batch permeation of cephalexin. Vem/VF1/1, [QCl]2% v/v; [Span-80]15% v/v, rpm250; [Clÿ] mM: (0), ~ (30).
concentration of 30 mM. As shown in Fig. 9, increase of Clÿ concentration does not markedly increase the extraction rate and recovery of cephalexin. In our previous studies also [5] on bulk liquid membrane transport of cephalexin under the condition of appropriate pH gradient in the aqueous phases, similar observation was made and the extraction rate was only marginally increased for an increase of Clÿ concentration from 0 to 10 mM and the rate was essentially the same at 10 and 30 mM. It may be attributed to the fact that Clÿ ion added in the receiving phase will be cotransported to the external phase where Clÿ ion accumulation occurs, thereby reducing the forward rate constant of the complexation reaction. However, this observation is contrary to that of the results in bulk liquid membrane transport of 7-aminocephalosporanic acid [4] and of citric acid [22] where the facilitated transport was associated with an optimum Clÿ concentration. Although it is dif®cult to explain, it is perceivable that the facilitated transport is the result of pH gradient in the aqueous phases (cephalexin distribution coef®cient) and the probable reaction of the buffer anion with the excess carrier at the receiving phase membrane phase interface. In fact, citrate buffer was used in the receiving phase and the
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
buffer anion can well take part in another ionexchange reaction providing countertransport for release of cephalexin anion in the receiving phase at an appropriate stripping rate. As reported elsewhere [21], it may be possible that increase of Clÿ concentration may well increase the osmotic gradient across the membrane thereby increasing membrane swelling which results in unfavourable condition for solute separation. 4.8. Relative transport of cephalexin and 7-aminodeacetoxy cephalosporanic acid (7-ADCA) The reaction mixture of biosynthetically produced cephalexin contains unconverted 7-ADCA and thus selective separation of cephalexin is desirable. In order to examine this aspect, batch extraction experiments were carried out with 7-ADCA solution under identical experimental conditions. In Fig. 10, the results of batch experiments conducted independently with the individual solutes are shown, whereas Fig. 11 shows the results of experiments conducted with a 1:1 mixture of the solutes. It is clear that the extraction rate and recovery of cephalexin are markedly higher
Fig. 10. Comparison of cephalexin and 7-ADCA permeation. Conditions are the same as in Table 1, rpm450.
23
than those achieved with 7-ADCA alone implying high selectivity for cephalexin. This selectivity is further enhanced when the extraction is carried out with a mixed solute feed. The selectivity may be solely attributed to the differences in pH-dependent equilibrium constants of cephalexin and 7-ADCA. As evident from Fig. 3, the equilibrium constant for cephalexin at a pH of 9.0 of the feed phase is around ®ve times greater than that of 7-ADCA whereas, at the pH of 5.0 of the receiving phase the equilibrium constant is higher for 7-ADCA. 4.9. Comparison of model and experimental data For comparison of model and experimental data batch extraction result of Fig. 6 at 6% Aliquat-336 was considered. The model parameter values were estimated as brie¯y outlined below. The external mass transfer coef®cient, kF, was estimated from the correlation of Calderbank and Moo-Young [22] given by kF
1:3 10ÿ3 Re0:25 m=s Sc0:67
(17)
Fig. 11. Comparison of cephalexin and 7-ADCA permeation in experiments with a 1:1 solute mixture.
24
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
The correlation is based on the rigid sphere assumption applicable for the present case also since the size of the emulsion is around 1 mm. The value of kF so estimated was found to be 1.5010ÿ6 m/s under the set of experimental conditions. For comparison, the membrane phase mass transfer coef®cient, kM, was determined through Lewis-cell experiment by a procedure suggested by Gu et al. [23] and the value was found to be 1.6510ÿ5 m/s which is about 10 times higher than kF. The forward rate constant, k1, for the complexation reaction was determined from separate experiments in Lewis-cell following a procedure reported by Reisinger and Marr [24]. The k1 value so determined was found to be 1.3910ÿ4 m4/mol s. The value of the equilibrium constant experimentally determined at the speci®c pH value was found to be 0.90. The value of the backward reaction rate constant, kÿ1, is 1.510ÿ5 m4/mol s. The effective diffusivity value, Deff, was calculated following a procedure ®rst proposed by Jefferson et al. [25] and further extended by Reisinger and Marr [24] for analysis of amino acid transport in an LEM system. For this purpose, the interval drop size of the emulsion was determined in a Leitz microscope. The calculated Sauter mean diameter was 3 mm. The emulsion globule sizes also measured photographically and expressed in terms of the Sauter mean diameter ranged from 1 to 1.5 mm in the range of stirring speed used in the work. In the calculation of Deff, the value of the molecular diffusivities of the solute in the aqueous phase and of the solute±carrier complex was calculated by Wilke±Chang [26] correlation. The value of Deff was found to be 4.410ÿ11 m2/s. The calculated concentration pro®le under a set of experimental conditions is shown by the dotted line in Fig. 6. It is evident that there is a marginal deviation of the theoretical pro®le from the experimentally determined values which may be attributed to the assumption of negligible diffusion resistance in the surfactant monolayer. In order to assess the rate determining step, a semiquantitative analysis was also made by considering the following dimensionless numbers to express the mass transfer resistances: DaF
k1 CQCl ; kF
DaM
k1 CQCl ; kM
Table 2 Dimensionless numbers for mass transfer in the LEM system DaF
DaM
2
NES
0.033
0.365
2.52
0.045
2
k1 CQCl R ; Deff
NES
k1 CQCl r kÿ1 RCCl
Table 2 lists the values of the dimensionless numbers for the system. It is apparent that the important dimensionless number is q2, its value being indicative that under optimised conditions, the diffusional resistance for the transfer of solute from feed phase to the interface, transport of the solute±carrier complex into the surfactant monolayer and re-extraction to the stripping phase are relatively unimportant as compared to the diffusional resistance through the membrane phase and the resistance due to extraction reaction. However, the diffusional resistance may be considered as the major rate controlling step. 5. Conclusion Liquid emulsion membrane can be effective in separation of cephalexin from dilute solution. Selective separation of cephalexin from a practically relevant mixture of cephalexin and 7-ADCA can be achieved in the emulsion liquid membrane system. Mass transfer analysis made on the basis of single component batch permeation experiments reveals that the permeation of cephalexin is controlled by the reaction and diffusional resistance in the emulsion globule. 6. List of symbols A C Deff Da k1 kÿ1
surface area of emulsion globule (m2) concentration (mol/m3) effective diffusivity second Damkohler number (dimensionless) rate constant for forward reaction (m4/mol s) rate constant for the backward reaction (m4/mol s)
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
kF kM KA m NES Nm r R Re Sc S0 t V 2
external (feed phase) mass transfer coefficient (m/s) internal (membrane phase) mass transfer coefficient (m/s) equilibrium constant (dimensionless) distribution coefficient of cephalexin (dimensionless) dimensionless number comparing extraction and stripping reaction number of emulsion globule radius of internal droplet (m) radius of emulsion globule (m) Reynolds number (dimensionless) Schmidt number (dimensionless) total interfacial area of the emulsion globule (m2) time (s) volume modified Thiele modulus (dimensionless)
Subscripts a aqueous eq equilibrium F feed phase M membrane phase o oil R stripping phase Superscripts 0 initial
[3] [4] [5] [6] [7] [8] [9] [10] [11]
[12]
[13]
[14]
Acknowledgements [15]
Financial support from DST-New Delhi vide sanction no. III-4 (15) 94-ET is gratefully acknowledged. The authors are grateful to Dr. T.R. Krishna Mohan of C-MMACS, NAL-Bangalore, for guidance in the computer simulation work.
[17]
References
[18]
[1] A.C. Ghosh, M.M. Borah, N.N. Dutta, Developments in liquid membrane separation of b-lactam antibiotics, Bioseparation 6 (1996) 91±105. [2] A.C. Ghosh, R.K. Mathur, N.N. Dutta, Extraction and purification of cephalosporin antibiotics, in: Th. Scheper
[16]
[19]
25
(Ed.), Adv. Biochem. Eng./Biotechnol., vol. 56, Springer, Berlin, 1997. A.C. Ghosh, S. Borthakur, M.K. Roy, N.N. Dutta, Extraction of cephalosporin-C in a supported liquid membrane, Sep. Technol. 5 (1995) 121. G.C. Sahoo, A.C. Ghosh, N.N. Dutta, R.K. Mathur, Facilitated transport of 7-aminocephalosporanic acid in a bulk liquid membrane, J. Membr. Sci. 112 (1996) 147. G.C. Sahoo, A.C. Ghosh, N.N. Dutta, Recovery of cephalexin from dilute solution in a bulk liquid membrane, Proc. Biochem. 32(4) (1997) 265±272. G.C. Sahoo, N.N. Dutta, R.L. Bezbaruah, Liquid membrane extraction of cephalosporin-C from fermentation broth, J. Chem. Tech. Biotech. (1997), submitted. Users Manual ± IMSL Math/Library, Version 2.0, MALBUSM-Perfect-EN-9109-20, September 1991. J.B. Riggs, An Introduction to Numerical Methods for Chemical Engineers, Texas Technical University Press, Austin, TX, 1988, pp. 209±219. A.C. Ghosh, G.S. Patil, N.N. Dutta, Liquid membrane permeation of aromatic hydrocarbons in LPG condensate, Fuel Proc. Technol. 38 (1994) 17. T. Yamana, A. Tsuji, Comparative stability of cephalosporins in aqueous solution: kinetics and mechanism of degradation, J. Pharma. Sci. 65 (1976) 1563±1574. G. Trouve, E. Malher, P. Colinart, H. Reuon, A moving film apparatus for measurements of mass transfer in emulsion, and its application to active transport of phosphate ion, Chem. Eng. Sci. 37 (1982) 1225±1233. T. Hano, T. Ohtake, M. Matsumoto, D. Kitayama, F. Hori, F. Nakashio, Extraction equilibrium of amino acids with quaternary ammonium salt, J. Chem. Eng. Japan 24 (1991) 20. J.B. Chaudhury, D.L. Pyle, Emulsion liquid membrane extraction of organic acids I: A theoretical model for lactic acid extraction with emulsion swelling, Chem. Eng. Sci. 471 (1992) 41. J.B. Chaudhury, D.L. Pyle, Emulsion liquid membrane extraction of organic acids II: Experimental, Chem. Eng. Sci. 47 (1992) 49. T. Hano, T. Ohtake, M. Matsumoto, S. Ogawa, F. Hari, Extraction of penicillins with liquid surfactant membrane, J. Chem. Eng. Japan 23 (1990) 771. S.C. Lee, W.K. Lee, Extraction of penicillin-G from simulated media by an emulsion liquid membrane process, J. Chem. Tech. Biotechnol. 53 (1992) 251. K.H. Lee, S.C. Lee, W.K. Lee, Penicillin-G extraction from model media using an emulsion liquid membrane: A theoretical model of product decomposition, J. Chem. Tech. Biotechnol. 59 (1994) 356. K.H. Lee, S.C. Lee, W.K. Lee, Penicillin-G extraction from model media using an emulsion liquid membrane: Determination of optimum extraction condition, J. Chem. Tech. Biotechnol. 59 (1994) 371. K.J. Marr, J. Draxler, in: W.S.W. Ho, K.K. Sirkar (Eds.), Membrane Handbook, Van Nostrand-Reinhold, New York, 1992, pp. 701±717.
26
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
[20] S.A. Hong, H.J. Choi, S.W. Nam, Concentration of amino acids by a liquid emulsion membrane with a cationic extractant, J. Membr. Sci. 70 (1992) 225±235. [21] M.P. Thien, T.A. Hulton, D.I.C. Wang, Separation and concentration of amino acids using liquid emulsion membranes, Biotech. Bioeng. 32 (1998) 604. [22] P.H. Calderbank, M.B. Moo-Young, The continuous phase heat and mass transfer properties of dispersions, Chem. Eng. Sci. 16 (1961) 39. [23] Z.M. Gu, D.T. Wasan, N.N. Li, Interfacial mass transfer in
ligand accelerated metal extraction by liquid surfactant membranes, Sep. Sci. Technol. 20 (1985) 599. [24] H. Reisinger, R. Marr, Comparison of the separation of lactic acid and L-leucin by liquid emulsion membranes, J. Membr. Sci. 80 (1993) 85. [25] T.B. Jefferson, O.W. Witzell, W.L. Sibbit, Thermal conductivity of graphite±silicon oil and graphite±water suspensions, Ind. Eng. Chem. 50 (1958) 1589. [26] R.C. Reid, J.M. Prausnitz, B.E. Polling, The Properties of Gases and Liquids, McGraw-Hill, New York, 1987.
Studies on emulsion liquid membrane extraction of cephalexin G.C. Sahoo, N.N. Dutta* Chemical Engineering Division, Regional Research Laboratory, Jorhat 785006, India Received 22 October 1996; received in revised form 5 June 1997; accepted 5 January 1998
Abstract An experimental study on batch extraction of cephalexin using an emulsion liquid membrane system has been reported. The effects of surfactant, carrier and solute concentrations, phase volume ratio, stirring speed, and counterion concentration on the extraction rate were examined. Surfactant, carrier and diluent used were Span-80, Aliquat-336 and n-heptane±kerosene (1:1), respectively. Under the optimised experimental conditions, emulsion swelling was found to be marginal. By maintaining an appropriate pH gradient in the feed and receiving aqueous phase, facilitated transport could be realised. Selective separation of cephalexin from a mixture of 7-aminodeacetoxy cephalosporanic acid (7-ADCA) could be demonstrated in the emulsion liquid membrane system. A mathematical model based on mass transfer across aqueous boundary layer, interfacial chemical reaction and diffusion in the emulsion globule provides a reasonable ®t of the experimental solute concentration versus time pro®les in the emulsion liquid membrane system. # 1998 Elsevier Science B.V. Keywords: Emulsion liquid membranes; Cephalexin; 7-Aminodeacetoxy cephalosporanic acid (7-ADCA); Span-80; Aliquat336; Coupled transport; Ion-exchange extraction
1. Introduction Reactive extraction in liquid membrane or nondispersive extraction in hollow ®bre membrane can provide cost-effective methods for separation and puri®cation of cephalosporin antibiotics [1,2]. We have been studying the liquid membrane technique for separation and puri®cation of cephalosporin antibiotics and the technique was found to be effective in certain speci®c cases [3±6]. In our previous communication, we have demonstrated facilitated uphill transport of cephalexin in a bulk liquid membrane by exploiting the concept of *Corresponding author. Tel.: +91 0376 320353; fax: +91 0376 321158. 0376-7388/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0376-7388(98)00027-1
reactive extraction using Aliquat-336 as the liquid ionexchange carrier [5]. Cephalexin was transported from an aqueous phase of higher pH through the membrane phase (comprising Aliquat-336 in n-butyl acetate as the diluent) to another aqueous phase of lower pH, the transport being facilitated by countertransport of another anion and a strong pH dependence of the distribution coef®cient of cephalexin between the membrane and the aqueous phases. In this paper, we report a comprehensive study to con®rm the applicability of liquid emulsion membrane (LEM) process for separation of cephalexin from aqueous solution such as the one encountered as reaction medium of an enzymatic process. Since the reaction mixture of enzymatically produced cephalexin contains 7-aminodeacetoxy cephalosporanic acid (7-
16
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
ADCA) as the unconverted reactant and selective separation is desirable, complementary studies were made on extraction behaviour of 7-ADCA and its mixture with cephalexin. 2. Theoretical aspects 2.1. Separation mechanism The mechanism of separation by extraction and reextraction in the LEM system considered here correspond to liquid±liquid anion-exchange reaction. The reaction of dissociated cephalexin anion Aÿ a and the carrier (QClo) in organic solvent is k1
ÿ Aÿ a QClo QAo Cla kÿ1
(1)
The equilibrium constant of the reversible complexation cephalexin reaction is given by K
QAo Clÿ a QClo Aÿ a
The 1:1 stoichiometry of the reaction was con®rmed by the result of equilibrium experiment. For the extraction to be effective, the pH of the feed aqueous phase should be maintained above 5.88 (the upper pKa value of cephalexin, the lower pKa being 2.56) to achieve complete dissociation with anionic form only. The driving force for the process may be provided by the difference in chloride ion concentration in the receiving and feed phases, respectively. The mechanism of the countertransport may be represented by the scheme shown in Fig. 1 which indicates transport of cephalexin anion from feed (external) solution to the receiving (internal) solution facilitated by the carrier in a solvent as the membrane phase. At the external±membrane phase interface, the carrier binds with Aÿ a to form a complex, QAo, which is extracted by the membrane. The complex is transported due to concentration gradient to the membrane±internal phase interface, where another interfacial ion-exchange reaction takes place thereby releasing cephalexin into the internal receiving phase. The carrier returns to the external±membrane phase interface to recombine with cephalexin anion, the transport being accompanied by countertransport of Clÿ ion. Alternately, buffer anion may also provide the
Fig. 1. Schematic diagram of the transport mechanism in LEM process for cephalexin separation.
driving force for the transport via another interfacial reaction. In our studies, we use citrate buffer in the internal phase, the other interfacial ion-exchange reaction involving citrate ion (Ctÿ) is Ctÿ QClo QCto Clÿ a
(2)
The extraction equilibrium constants for the reaction given by Eq. (2) is relatively lower than that given by Eq. (1). Using excess Clÿ concentration in the receiving phase, the forward reaction rate of Eq. (2) may be suppressed providing the driving force for ÿ facilitated transport of Aÿ a via countertransport of Cl . ÿ Alternately, using excess Ct and QClo concentration, the Clÿ for countertransport may be maintained. 2.2. Mathematical description of the kinetic model The concentration pro®le for the permeation of cephalexin anion into the emulsion globule is shown in Fig. 2. The following steps are involved in the transport process: diffusion Aÿ a to the feed±membrane phase interface of the emulsion globule from the feed phase (3); reaction of Aÿ a with the carrier, QCl, at the external interface releasing the counterion Clÿ; diffusion of the solute carrier complex, QAo, into the emulsion globule; stripping of the complex at the internal interface between the membrane (2) and internal phase (1); back diffusion of the carrier to the feed (3) and membrane (2) phase interface. The following assumptions are inherent in the above process: the extraction and stripping reactions are located at the interfaces and the amount of carrier dissolved in the aqueous phase is negligible;
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
17
V2 dCQA 1 @ @CQA Deff 2 r2 V1 V2 dt @r r @r S0 ÿ kÿ1 CQA CClÿ V 1 V2
(5)
where S0 is the total interfacial area of the emulsion globule, Deff the effective diffusivity of the solute± carrier complex, and V1 and V2 are the volumes of the internal and membrane phases, respectively. The equilibrium constant KA for reaction given by Eq. (1) can be expressed as KA Fig. 2. Concentration profile of cephalexin in an ELM system.
negligible transport resistance in the internal phase (I) because phase I droplets are very small (R1<5 mm); a quasi-homogeneous state; resistance in the oil layer (surfactant monolayer) at the external interface is negligible. Based on the above, the following equations hold good; the stripping reaction is very fast due to large surface area of phase II. Mass transfer of Aÿ a in the external phase is expressed as dCAÿ 1 ÿ kF A CAÿ ÿ CA ÿ dt V3
(3)
CQA CClÿ k1 CQCl CAÿ kÿ1
(6)
where 0 ÿ CQA CQCl CQCl
(7)
From (6) and (7), we get " # 0 CCl CQCl ÿ1 CAÿ KA CQCl
(8)
Initial and boundary conditions are as follows: CAÿ CA0 ÿ ;
t 0;
CQA 0
0 r R
(9)
Boundary conditions at centre of globules to have no ¯ux are given by @CQA 0 @r
r 0;
t 0
(10)
At the feed±membrane interface, we have
where CAÿ is the solute concentration in the external phase, CA ÿ the concentration at the interface between the external phase and the membrane phase, V3 the volume of the external phase, t the stirring time, kF the mass transfer coef®cient and A is the surface area of emulsion globules. The interfacial reaction rate at 3±2 interface may be expressed as
The following relations are used for solution of the above equations: (i) The number of emulsion globule Nm is given by
dCAÿ A k1 CA ÿ CQCl RF dt V3 extraction reaction rate per unit area of 3ÿ2 interface (4)
where P R is thePmean globule radiusD32/2, and D32 ni D3i = ni D2i (Sauter mean diameter). (ii) Surface area of emulsia globule, A, is
The diffusion of the solute±carrier complex in Phase II is described by Fick's second law and is given by the following equation for phase II. The mass transfer in the emulsion globule is given by
(iii) Total interfacial area of emulsia globule, S0 , is
r R;
Nm
A
Deff
@CQA kF CAÿ ÿ CA ÿ @r
V2 V 1 4=3R3
3 V2 V1 R
S0 ANm
(11)
(12)
(13)
(14)
18
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
Eqs. (3)±(14) may be simultaneously solved numerically by using the methods of line (a combination of ®nite differences method and Gear's algorithm) in the subroutine of IVPAG of IMSL-MATH Library [7,8].
Table 1 Experimental parameters for LEM extraction of cephalexin
3. Experimental
External feed phase
3.1. Materials Cephalexin and Aliquat-336 were procured from Aldrich, USA. Span-80 was obtained from Sigma, USA. n-Heptane, sodium citrate, sodium carbonate/ phosphate, citric acid, NaCl, etc. were procured from E. Merck (India) and were of analytical grade. Superior grade kerosene was procured from local supplier. 3.2. Method Emulsion liquid membrane experiments were conducted in a mixer±settler device used in our earlier work [9]. The membrane phase consisted of a 1:1 v/v mixture of n-heptane and kerosene, Span-80 (8±15% v/v) and Aliquat-336 (1±16% v/v). High surfactant to carrier concentration ratio was necessary as a criterion to maintain a stable w/o emulsion [11]. Citrate buffer was taken as the internal aqueous receiving phase maintaining a pH of 5 at which cephalexin is relatively stable [10]. A water in oil emulsion was prepared by slow addition of the internal aqueous phase (50 ml) to the organic membrane phase (50 ml) using a high speed homogeniser (Remi, India) at 10 000 rpm for 20 min. The external feed solution of cephalexin (20 mM) was prepared in carbonate and phosphate buffers. It may be noted that extraction from carbonate rather than phosphate buffer solution is preferred because of relatively high coextraction of the latter [12]. By taking 50 ml of feed phase in the mixer± settler the w/o emulsion (10±50 ml) was dispersed at a constant stirring speed of the mixer±settler. All experiments were conducted at 328C with the parameters shown in Table 1. The effect of emulsion to feed phase volume ratio, surfactant and carrier concentration, feed phase pH, stirring speed and Clÿ concentration in the internal phase were investigated. The stirring speed was measured by a digital non-contact type tachometer. During
Internal receiving phase Membrane phase
VR:VM1:1 in w/o emulsion, Vem:VF1:1 to 1:5
Citrate buffer, pH 5, volume (VR): 50 ml Volume (VM): 50 ml, Aliquat-336: 1±6% v/v, Span-80: 8±15% v/v, solvent: 1:1 v/v mixture of n-heptane and kerosene Volume (VF): 50 ml, cephalexin: 20 mM, pH: up to 9 by carbonate and phosphate buffer
experiments, samples were drawn from the vessel at regular intervals and concentration of cephalexin in the aqueous solution were analysed in a UV±Vis spectrophotometer (Schimadzu 160A). Changes in the volume of internal phase by water transport (swelling) were estimated by volume balance after breaking the emulsion at the end of the experiments. The emulsion was broken by thermal demulsi®cation as well as by passing the emulsion through a hydrophilic membrane (Millipore). Complementary studies of extraction equilibrium of cephalexin and 7-ADCA were carried out in an agitated 100 ml glass round bottom ¯ask with respect to the effect of aqueous phase pH value on the cephalexin and 7-ADCA distribution coef®cient (Fig. 3). Experiments on the pH
Fig. 3. Distribution coefficient of cephalexin and 7-ADCA versus pH of aqueous phase.
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
19
effect (pH varied between 5 and 10) were conducted for 2 h with 20 ml each of the aqueous and organic phases by taking 10 mM solute and 10 mM Aliquat336 in the respective phases. The value of the distribution coef®cient, ``m'', was calculated using the following equation: m
QAo eq Vo Aÿ a eq Va
(15)
[QAo]eq was estimated from ÿ Vo QAo eq Va fAÿ a t0 ÿ Aa eq g
(16)
4. Results and discussion 4.1. Effect of pH on distribution coefficient In order to establish the optimal pH values for both the feed and receiving aqueous phases for an optimal LEM system, the results of equilibrium studies were used as the guideline. Fig. 3 shows the variation of distribution coef®cient, ``m'', with pH of cephalexin and 7-ADCA solutions. As expected, increase of pH increases the ``m'' value up to pH of 9.5, beyond which ``m'' value appears to decrease marginally. This decrease in ``m'' value may be attributed to the hydrolytic decomposition of the solutes at higher pH. Indeed, the decomposition rate of cephalexin increases sharply when the pH is raised from 8 to 10 as reported elsewhere [10]. Lower values of ``m'' at low pH may be realised by incomplete dissociation of the solutes. Since the maximum value of distribution coef®cient is achieved in the pH range 9±9.5 and cephalexin is stable up to pH 9, our experimental protocol for LEM primarily involves extraction from an aqueous solution of high pH and stripping to another aqueous phase of relatively low pH, which will be optimised as discussed in Section 4.2. 4.2. Effect of surfactant concentration The effect of surfactant concentration was studied at a Aliquat-336 concentration of 5% v/v in the membrane phase with feed and receiving phase pH values of 9 and 5, respectively. The ratio of emulsion phase to feed phase volume was maintained at 1:1 and Span-80 concentration was varied from 10 to 15% v/v.
Fig. 4. Effect of Span-80 concentration (% v/v) on the batch permeation of cephalexin. Aÿ a 20 mM; rpm 450: * (5), ~ (10), & (12.5), 5 (15).
It may be noted that the dilute mixture used for preparation of the stable emulsion was the optimised one. Further, the volume ratio of Span-80 and Aliquat336 affects the emulsion stability, being greater for high Span-80 concentration. A ratio of 2:1 for surfactant to carrier generates the most stable emulsion. Fig. 4 shows the effects of Span-80 concentration on the overall dimensionless concentration of cephalexin in the external phase versus time and initial permeation rate calculated from the initial slope of the concentration pro®le curves. It is evident that increase of surfactant concentration increases both the initial extraction rate and degree of removal. The initial extraction rate increases almost linearly with Span-80 concentration in the range 5±15%. It may be expected that this surfactant effect may be realised through enhanced emulsion stability alone. Similar effect of surfactant was also observed in case of extraction of lactic acid [13,14] and penicillin-G [15±18]. The decreased rate can be attributed to solute leakages from the internal phase thus indicating emulsion breakage at low surfactant concentration. It is believed that Span-80 affects solute transport because of its in¯uence on water transport and swelling via
20
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
osmotic effects [19,20]. However, in our investigation, swelling was found to be very marginal. The change in internal volume with increase of Span-80 concentration was also marginal. From an initial emulsion volume of 50 ml, the ®nal volume increased up to 60 ml only at the highest Span-80 concentration used in our work. 4.3. Effect of volume ratio of the emulsion Vem and feed phase VF The condition maintained during the study of the effect of phase volume ratio is essentially the same as mentioned in Section 4.2. However, the Span-80 concentration was maintained at 15% v/v. Fig. 5 shows the effect of Vem/VF on the batch permeation rate of cephalexin. As expected, an increase of this ratio tends to increase the extraction rate due to the increase of the amount of the carrier in the membrane phase and the improvement in the dispersibility of the w/o/w emulsion system. Furthermore, the increase of this ratio provides increased capacity of the membrane and internal phase for enhanced permeation and stripping
Fig. 5. Effect of the volume ratio of emulsion (Vem) to feed (VF) phase on batch permeation of cephalexin: [Span-80]15%, [QCl]2%, Vem/VF, 1/5 ( ), 2/5 (~), 3/5 (&), 1/1 ( ).
of cephalexin. However, further increase in the Vem/VF ratio is unlikely to provide increased extraction rate because of the increase in viscosity which strongly in¯uences the emulsion swelling. From the experimental data, it appears that the separation ef®ciency reaches a limiting value of 70± 80% at optimal condition of the membrane phase composition, phase volume ratio, etc. Our experiments on bulk liquid membrane [5] extraction revealed that the feed phase pH affects the extraction ef®ciency, a high pH being favourable for the system due to the presence of the solute predominantly in an anionic form. However, at pH>9.5, hydrolytic decomposition of cephalexin occurs. During the course of experiments, the feed phase pH was found to marginally decrease probably due to co-extraction of buffer anion by Aliquat-336. It was observed that the pH of the feed phase dropped from 9.5 to 8.5 after 50 min of extraction time. Thus, the limiting value of extraction ef®ciency obtained in our study may be explained from the feed phase pH dependence of the extraction rate. 4.4. Effect of Aliquat-336 concentration The effect of Aliquat-336 (carrier) concentration was studied at Vem/VF of 1/5 and the highest level of Span-80 concentration. As shown in Fig. 6, increase in extraction rate with increase in Aliquat-336 concentration corresponds to increased diffusion of solute± carrier complex across the membrane. The concentration of carrier in the membrane phase should phenomenologically increase the interfacial cephalexin concentration and hence the driving force for extraction providing an increased extraction rate. However, it appears that there is a limit to the carrier concentration that will give signi®cant increase in the extraction rate, probably as a result of the equilibrium of the stripping reaction. On the other hand, Aliquat-336 having surfactant property tends to act antagonistically to the emulsion stabilising surfactant such that the carrier can reduce the membrane stability. A limiting carrier concentration of 5% v/v reported for lactic acid permeation [12] seems to be in agreement with the results obtained in the present work. However, our results indicate monotonic changes of feed phase cephalexin concentration with time at all concentrations studied here. Had there been an increase of feed phase cephalexin concentration at
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
21
Fig. 6. Effect of Aliquat-336 (QCl) concentration on the batch permeation of cephalexin. Vem/VF1/5, [Span-80]15% v/v, [QCl336] (% v/v); 1 (~), 2 (&), 4 ( ), 6 (*).
Fig. 7. Effect of feed phase cephalexin concentration (mM) on the batch permeation of cephalexin. Vem/VF1/1, [Span-80]15% v/v, [QCl]2% v/v; * (10), ~ (15), & (20).
long times of permeation particularly for higher carrier concentration, the inference of membrane breakage could have been drawn. Thus, the membrane breakage could be considered negligible in the present LEM system. In this regard, the chemical nature of the counterion in the receiving phase may be important in determining the membrane swell and the extent of separation. Thien et al. [21], while discussing the effect of the chemical nature of counterion from the so-called ``lyotrop number'' concept, observed that acetate ions with lowest ``lyotrop number'' provide higher degree of separation of lactic acid as compared to that achieved with Clÿ, Brÿ and Iÿ ions and that the membrane swelling was also the lowest. Thus, the observation in our LEM system containing acetate and/or citrate ion in the receiving phase may be considered reasonable.
increase with a decrease of the cephalexin concentration. However, at a concentration of 5 mM, the rate tends to reach an asymptote after 20 min of extraction. As the solute concentration is increased, the solute transport rate may decrease largely due to the reduced capacity of the internal phase to strip the transported solute anion. When the solute concentration is high, the internal droplets in the peripheral region are more rapidly saturated with the solute, and the complex must diffuse through the membrane phase to the more inner region of the drop to release the solute in the receiving phase such that the internal mass transfer resistance is signi®cant. When the solute concentration is low, the external mass transfer is controlling and the extraction rate is higher than that at high solute concentration. It may be noted that the interfacial area for stripping is much larger than that for extraction. The observation of high solute transport rate and recovery at low solute concentration is important from process design point of view. It is possible to achieve the required degree of separation with small solute concentration in the feed using a multi-stage train of mixer±settler devices in a practical recovery process.
4.5. Effect of feed phase cephalexin concentration The effect of feed phase cephalexin concentration was studied at 5, 10 and 12.5 mM under otherwise identical conditions. As shown in Fig. 7, the extraction rate and the degree of extraction appear to
22
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
Fig. 8. Effect of stirring speed (rpm) on the batch permeation of cephalexin. Vem/VF1/1, [QCl]2% v/v; [Span-80]15% v/v, rpm: * (250), ~ (350), & (450).
4.6. Effect of stirring speed As shown in Fig. 8, increase in stirring speed increases the extraction rate, obviously due to reduction of the emulsion globule size thereby providing high interfacial contact area between the external and membrane phases and increase of the external phase mass transfer coef®cient. However, due to very viscous nature of the dispersion, high stirring speed may be prohibitive from the process point of view. As shown in Fig. 8, increase of stirring speed from 250 to 450 rpm does not result in appreciable increase of extraction rate and recovery. This may be attributed to the fact that during extraction the system becomes viscous and at long contact time it became hard to disperse the emulsion because of high surfactant concentration used in the membrane phase. 4.7. Effect of counterion in the receiving phase From mechanistic consideration, it may be expected that the Clÿ ion concentration gradient provides the driving force for separation. Thus Clÿ effect was studied by using NaCl in the receiving phase at a
Fig. 9. Effect of chloride ion concentration in receiving phase on the batch permeation of cephalexin. Vem/VF1/1, [QCl]2% v/v; [Span-80]15% v/v, rpm250; [Clÿ] mM: (0), ~ (30).
concentration of 30 mM. As shown in Fig. 9, increase of Clÿ concentration does not markedly increase the extraction rate and recovery of cephalexin. In our previous studies also [5] on bulk liquid membrane transport of cephalexin under the condition of appropriate pH gradient in the aqueous phases, similar observation was made and the extraction rate was only marginally increased for an increase of Clÿ concentration from 0 to 10 mM and the rate was essentially the same at 10 and 30 mM. It may be attributed to the fact that Clÿ ion added in the receiving phase will be cotransported to the external phase where Clÿ ion accumulation occurs, thereby reducing the forward rate constant of the complexation reaction. However, this observation is contrary to that of the results in bulk liquid membrane transport of 7-aminocephalosporanic acid [4] and of citric acid [22] where the facilitated transport was associated with an optimum Clÿ concentration. Although it is dif®cult to explain, it is perceivable that the facilitated transport is the result of pH gradient in the aqueous phases (cephalexin distribution coef®cient) and the probable reaction of the buffer anion with the excess carrier at the receiving phase membrane phase interface. In fact, citrate buffer was used in the receiving phase and the
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
buffer anion can well take part in another ionexchange reaction providing countertransport for release of cephalexin anion in the receiving phase at an appropriate stripping rate. As reported elsewhere [21], it may be possible that increase of Clÿ concentration may well increase the osmotic gradient across the membrane thereby increasing membrane swelling which results in unfavourable condition for solute separation. 4.8. Relative transport of cephalexin and 7-aminodeacetoxy cephalosporanic acid (7-ADCA) The reaction mixture of biosynthetically produced cephalexin contains unconverted 7-ADCA and thus selective separation of cephalexin is desirable. In order to examine this aspect, batch extraction experiments were carried out with 7-ADCA solution under identical experimental conditions. In Fig. 10, the results of batch experiments conducted independently with the individual solutes are shown, whereas Fig. 11 shows the results of experiments conducted with a 1:1 mixture of the solutes. It is clear that the extraction rate and recovery of cephalexin are markedly higher
Fig. 10. Comparison of cephalexin and 7-ADCA permeation. Conditions are the same as in Table 1, rpm450.
23
than those achieved with 7-ADCA alone implying high selectivity for cephalexin. This selectivity is further enhanced when the extraction is carried out with a mixed solute feed. The selectivity may be solely attributed to the differences in pH-dependent equilibrium constants of cephalexin and 7-ADCA. As evident from Fig. 3, the equilibrium constant for cephalexin at a pH of 9.0 of the feed phase is around ®ve times greater than that of 7-ADCA whereas, at the pH of 5.0 of the receiving phase the equilibrium constant is higher for 7-ADCA. 4.9. Comparison of model and experimental data For comparison of model and experimental data batch extraction result of Fig. 6 at 6% Aliquat-336 was considered. The model parameter values were estimated as brie¯y outlined below. The external mass transfer coef®cient, kF, was estimated from the correlation of Calderbank and Moo-Young [22] given by kF
1:3 10ÿ3 Re0:25 m=s Sc0:67
(17)
Fig. 11. Comparison of cephalexin and 7-ADCA permeation in experiments with a 1:1 solute mixture.
24
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
The correlation is based on the rigid sphere assumption applicable for the present case also since the size of the emulsion is around 1 mm. The value of kF so estimated was found to be 1.5010ÿ6 m/s under the set of experimental conditions. For comparison, the membrane phase mass transfer coef®cient, kM, was determined through Lewis-cell experiment by a procedure suggested by Gu et al. [23] and the value was found to be 1.6510ÿ5 m/s which is about 10 times higher than kF. The forward rate constant, k1, for the complexation reaction was determined from separate experiments in Lewis-cell following a procedure reported by Reisinger and Marr [24]. The k1 value so determined was found to be 1.3910ÿ4 m4/mol s. The value of the equilibrium constant experimentally determined at the speci®c pH value was found to be 0.90. The value of the backward reaction rate constant, kÿ1, is 1.510ÿ5 m4/mol s. The effective diffusivity value, Deff, was calculated following a procedure ®rst proposed by Jefferson et al. [25] and further extended by Reisinger and Marr [24] for analysis of amino acid transport in an LEM system. For this purpose, the interval drop size of the emulsion was determined in a Leitz microscope. The calculated Sauter mean diameter was 3 mm. The emulsion globule sizes also measured photographically and expressed in terms of the Sauter mean diameter ranged from 1 to 1.5 mm in the range of stirring speed used in the work. In the calculation of Deff, the value of the molecular diffusivities of the solute in the aqueous phase and of the solute±carrier complex was calculated by Wilke±Chang [26] correlation. The value of Deff was found to be 4.410ÿ11 m2/s. The calculated concentration pro®le under a set of experimental conditions is shown by the dotted line in Fig. 6. It is evident that there is a marginal deviation of the theoretical pro®le from the experimentally determined values which may be attributed to the assumption of negligible diffusion resistance in the surfactant monolayer. In order to assess the rate determining step, a semiquantitative analysis was also made by considering the following dimensionless numbers to express the mass transfer resistances: DaF
k1 CQCl ; kF
DaM
k1 CQCl ; kM
Table 2 Dimensionless numbers for mass transfer in the LEM system DaF
DaM
2
NES
0.033
0.365
2.52
0.045
2
k1 CQCl R ; Deff
NES
k1 CQCl r kÿ1 RCCl
Table 2 lists the values of the dimensionless numbers for the system. It is apparent that the important dimensionless number is q2, its value being indicative that under optimised conditions, the diffusional resistance for the transfer of solute from feed phase to the interface, transport of the solute±carrier complex into the surfactant monolayer and re-extraction to the stripping phase are relatively unimportant as compared to the diffusional resistance through the membrane phase and the resistance due to extraction reaction. However, the diffusional resistance may be considered as the major rate controlling step. 5. Conclusion Liquid emulsion membrane can be effective in separation of cephalexin from dilute solution. Selective separation of cephalexin from a practically relevant mixture of cephalexin and 7-ADCA can be achieved in the emulsion liquid membrane system. Mass transfer analysis made on the basis of single component batch permeation experiments reveals that the permeation of cephalexin is controlled by the reaction and diffusional resistance in the emulsion globule. 6. List of symbols A C Deff Da k1 kÿ1
surface area of emulsion globule (m2) concentration (mol/m3) effective diffusivity second Damkohler number (dimensionless) rate constant for forward reaction (m4/mol s) rate constant for the backward reaction (m4/mol s)
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
kF kM KA m NES Nm r R Re Sc S0 t V 2
external (feed phase) mass transfer coefficient (m/s) internal (membrane phase) mass transfer coefficient (m/s) equilibrium constant (dimensionless) distribution coefficient of cephalexin (dimensionless) dimensionless number comparing extraction and stripping reaction number of emulsion globule radius of internal droplet (m) radius of emulsion globule (m) Reynolds number (dimensionless) Schmidt number (dimensionless) total interfacial area of the emulsion globule (m2) time (s) volume modified Thiele modulus (dimensionless)
Subscripts a aqueous eq equilibrium F feed phase M membrane phase o oil R stripping phase Superscripts 0 initial
[3] [4] [5] [6] [7] [8] [9] [10] [11]
[12]
[13]
[14]
Acknowledgements [15]
Financial support from DST-New Delhi vide sanction no. III-4 (15) 94-ET is gratefully acknowledged. The authors are grateful to Dr. T.R. Krishna Mohan of C-MMACS, NAL-Bangalore, for guidance in the computer simulation work.
[17]
References
[18]
[1] A.C. Ghosh, M.M. Borah, N.N. Dutta, Developments in liquid membrane separation of b-lactam antibiotics, Bioseparation 6 (1996) 91±105. [2] A.C. Ghosh, R.K. Mathur, N.N. Dutta, Extraction and purification of cephalosporin antibiotics, in: Th. Scheper
[16]
[19]
25
(Ed.), Adv. Biochem. Eng./Biotechnol., vol. 56, Springer, Berlin, 1997. A.C. Ghosh, S. Borthakur, M.K. Roy, N.N. Dutta, Extraction of cephalosporin-C in a supported liquid membrane, Sep. Technol. 5 (1995) 121. G.C. Sahoo, A.C. Ghosh, N.N. Dutta, R.K. Mathur, Facilitated transport of 7-aminocephalosporanic acid in a bulk liquid membrane, J. Membr. Sci. 112 (1996) 147. G.C. Sahoo, A.C. Ghosh, N.N. Dutta, Recovery of cephalexin from dilute solution in a bulk liquid membrane, Proc. Biochem. 32(4) (1997) 265±272. G.C. Sahoo, N.N. Dutta, R.L. Bezbaruah, Liquid membrane extraction of cephalosporin-C from fermentation broth, J. Chem. Tech. Biotech. (1997), submitted. Users Manual ± IMSL Math/Library, Version 2.0, MALBUSM-Perfect-EN-9109-20, September 1991. J.B. Riggs, An Introduction to Numerical Methods for Chemical Engineers, Texas Technical University Press, Austin, TX, 1988, pp. 209±219. A.C. Ghosh, G.S. Patil, N.N. Dutta, Liquid membrane permeation of aromatic hydrocarbons in LPG condensate, Fuel Proc. Technol. 38 (1994) 17. T. Yamana, A. Tsuji, Comparative stability of cephalosporins in aqueous solution: kinetics and mechanism of degradation, J. Pharma. Sci. 65 (1976) 1563±1574. G. Trouve, E. Malher, P. Colinart, H. Reuon, A moving film apparatus for measurements of mass transfer in emulsion, and its application to active transport of phosphate ion, Chem. Eng. Sci. 37 (1982) 1225±1233. T. Hano, T. Ohtake, M. Matsumoto, D. Kitayama, F. Hori, F. Nakashio, Extraction equilibrium of amino acids with quaternary ammonium salt, J. Chem. Eng. Japan 24 (1991) 20. J.B. Chaudhury, D.L. Pyle, Emulsion liquid membrane extraction of organic acids I: A theoretical model for lactic acid extraction with emulsion swelling, Chem. Eng. Sci. 471 (1992) 41. J.B. Chaudhury, D.L. Pyle, Emulsion liquid membrane extraction of organic acids II: Experimental, Chem. Eng. Sci. 47 (1992) 49. T. Hano, T. Ohtake, M. Matsumoto, S. Ogawa, F. Hari, Extraction of penicillins with liquid surfactant membrane, J. Chem. Eng. Japan 23 (1990) 771. S.C. Lee, W.K. Lee, Extraction of penicillin-G from simulated media by an emulsion liquid membrane process, J. Chem. Tech. Biotechnol. 53 (1992) 251. K.H. Lee, S.C. Lee, W.K. Lee, Penicillin-G extraction from model media using an emulsion liquid membrane: A theoretical model of product decomposition, J. Chem. Tech. Biotechnol. 59 (1994) 356. K.H. Lee, S.C. Lee, W.K. Lee, Penicillin-G extraction from model media using an emulsion liquid membrane: Determination of optimum extraction condition, J. Chem. Tech. Biotechnol. 59 (1994) 371. K.J. Marr, J. Draxler, in: W.S.W. Ho, K.K. Sirkar (Eds.), Membrane Handbook, Van Nostrand-Reinhold, New York, 1992, pp. 701±717.
26
G.C. Sahoo, N.N. Dutta et al. / Journal of Membrane Science 145 (1998) 15±26
[20] S.A. Hong, H.J. Choi, S.W. Nam, Concentration of amino acids by a liquid emulsion membrane with a cationic extractant, J. Membr. Sci. 70 (1992) 225±235. [21] M.P. Thien, T.A. Hulton, D.I.C. Wang, Separation and concentration of amino acids using liquid emulsion membranes, Biotech. Bioeng. 32 (1998) 604. [22] P.H. Calderbank, M.B. Moo-Young, The continuous phase heat and mass transfer properties of dispersions, Chem. Eng. Sci. 16 (1961) 39. [23] Z.M. Gu, D.T. Wasan, N.N. Li, Interfacial mass transfer in
ligand accelerated metal extraction by liquid surfactant membranes, Sep. Sci. Technol. 20 (1985) 599. [24] H. Reisinger, R. Marr, Comparison of the separation of lactic acid and L-leucin by liquid emulsion membranes, J. Membr. Sci. 80 (1993) 85. [25] T.B. Jefferson, O.W. Witzell, W.L. Sibbit, Thermal conductivity of graphite±silicon oil and graphite±water suspensions, Ind. Eng. Chem. 50 (1958) 1589. [26] R.C. Reid, J.M. Prausnitz, B.E. Polling, The Properties of Gases and Liquids, McGraw-Hill, New York, 1987.