Ind. Eng. Chem. Res. 1996, 35, 3665-3672
3665
Extraction of Zwitterionic Amino Acids with Reverse Micelles in the Presence of Different Ions Hamid R. Rabie and Juan H. Vera* Department of Chemical Engineering, McGill University, Montreal, Quebec, Canada H3A 2A7
The effects of salt type and concentration, of amino acid and surfactant concentrations, of volume ratio, and of solvent on the reverse micellar extraction of tryptophan and phenylalanine at their zwitterionic condition using Aerosol-OT were determined. The effect of solvent on the ion distribution was also examined. The extraction was mainly controlled by the chemical and electrostatic interactions between the amino acids and the surfactant headgroups at the active reverse micellar interface. A simple model which accounts for the effect of different variables and accurately predicts the amino acid solubilization in the reverse micellar phase is proposed. 1. Introduction The amino acid production methods fall into three classes (Barrett, 1985): (i) extraction from protein hydrolyzate; (ii) microbiological methods (fermentation processes and enzymatic processes); and (iii) chemical synthesis. Ion exchange and crystallization are commonly used to separate and concentrate amino acids, usually in batch processes. However, there is still a need for efficient methods to separate and concentrate amino acids continuously. While liquid-liquid extraction is used extensively in the antibiotics industry (Krei and Hustedt, 1992), it has found only limited application for amino acids. Organic solvents are not suitable here, since amino acids have a very low solubility in organic media. An alternative approach is the extraction of amino acids using reverse micelles of ionic surfactants in organic solvents. Reverse micelles are aggregates of surfactant molecules containing microscopic polar cores of solubilized water, called water pools, in an apolar solvent. Reverse micellar extraction of amino acids has recently received a great deal of attention. The first reported experiments on the reverse micellar extraction of amino acids were performed by Fendler et al. (1975). They measured the solubility of 12 amino acids in hexane in the presence of decylammonium propionate using radio-isotope labeling. They concluded that electrostatic interactions played the major role in the solubility of amino acids in the reverse micelles. Once amino acids are extracted to the reverse micellar phase, they can be back extracted by contacting the organic phase, containing the amino acids, with a fresh aqueous phase at a proper pH or at high ionic strength. Rodgers and Lee (1984) studied the fluorescence quenching of tryptophan by singlet molecular oxygen in reverse micelles of Aerosol-OT (bis(2-ethylhexyl) sodium sulfosuccinate). They concluded that tryptophan was largely located in the reverse micellar interface. Luisi’s group (Dossena et al., 1976; Luisi et al., 1979) reported experimental results of the solubilization of tryptophan and some dipeptides in reverse micelles formed by trioctylmethylammonium chloride. They also found that extraction was mainly influenced by the electrostatic interaction between the surfactant and the solute. A more relevant work to this study was done by Leodidis and Hatton (1990). They defined an adsorp* To whom all correspondence should be addressed.
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tion-based partition coefficient in order to model extraction. However, they found that this partition coefficient was a function of different system variables such as salt type and concentration and surfactant concentration. From the data reported in that work (Leodidis and Hatton, 1990), it is difficult to separate the effect of the independent variables of the system on the extraction of amino acids. In most of the previous studies, the pH was adjusted to provide the suitable condition for extraction. In this work, we study the extraction of amino acids at neutral pH instead of adding acids or bases for pH adjustment. In a recent work (Rabie and Vera, 1995), we developed a chemical theory for extraction of ions in reverse micellar systems, discussed the mechanism of ion distribution, and identified the main driving forces for extraction. We found that ion-exchange reactions had major contributions for the solubilization of ions. While for charged amino acids at the proper pH such a treatment is directly applicable, for amino acids at their zwitterionic condition, the mechanism of extraction is far from clear. The objective of the present study is to report experimental results for extraction of different amino acids at their zwitterionic condition and clearly show the effect of different independent variables on the extraction. In addition, we develop a model which can accurately predict the extraction under different conditions. In this study, we have chosen tryptophan and phenylalanine as model amino acids. Some available data from previous work (Leodidis and Hatton, 1990) are also used for comparison. 2. Experimental Methods AOT of 99% purity and reagent-grade octane, nonane, and decane were obtained from Sigma (St. Louis, MO). Reagent-grade isooctane was purchased from Fisher Scientific (Montreal, Quebec, Canada). Monobasic and dibasic phosphate salts of sodium and potassium were received from A & C (Montreal). Tryptophan and phenylalanine were obtained from Sigma and used as received. Deionized water, with an electrical conductivity below 0.8 µS/cm, was used for all experiments. A solution containing AOT in one of the solvents was contacted with an aqueous solution of an amino acid and salt. The volume ratio of two phases was set at a fixed value. The volume ratio was set to unity for most experiments, and it was varied from 0.25 to 4.5 in the experiments designed to study the effect of this variable. © 1996 American Chemical Society
3666 Ind. Eng. Chem. Res., Vol. 35, No. 10, 1996
Figure 1. Diagram of the experimental procedure and the solubilization sites according to the active interface model. (solid dot with double legs) AOT surfactant, (cross-hatched oval) solute.
The phases were vigorously shaken at 23 °C for 2 h. The electrolytes used in the initial aqueous phase were monobasic and dibasic sodium or potassium in variable concentrations. The ratio of the monobasic to the dibasic form was adjusted so that the pH of the amino acid solution was between 6 and 6.5. The samples were left to stand for 2 weeks at the same temperature to reach equilibrium. The phases were then carefully separated for analysis. The pH of the aqueous phase was measured by a Model 691 pH meter (Metrohm Ltd.). The concentrations of tryptophan and phenylalanine in the aqueous phase were measured by a Cary 1/3 UV spectrophotometer (Varian Techtron Pty. Ltd., Victoria, Australia) at wavelengths above 240 nm. The concentration of amino acid solubilized in the reverse micelles was determined from analysis of the bulk aqueous phase. The final pH in the aqueous phase was also measured to ensure that no significant pH change occurred after contacting the two phases. The maximum pH change was less than 0.3 unit, which was considered acceptable for the purposes of this work. 3. Theory There are essentially four distinct regions in reverse micellar systems where a solute can be solubilized. They are (i) the organic continuum, (ii) the interface of the surfactant hydrophillic groups and the water pool in the reverse micelles, (iii) the water pool inside the reverse micelles, and (iv) the excess aqueous phase. These regions are shown in Figure 1. In the following equations, a notation with a bar is used for the organic phase even though the solute may be localized in any of the first three regions, while a notation without a bar is for excess aqueous phase. The superscript “0” indicates the value of the properties at the initial conditions. All symbols without this superscript denote the values of the properties at equilibrium. Amino acids are essentially insoluble in organic solvents, even in mixtures containing ethanol up to 33%
by volume (Fendler et al., 1975). Thus, in this study, we only consider the latter three regions. Here, we assume that all the surfactant molecules are aggregated. Such an assumption presupposes that (i) the cmc of the surfactant in the organic phase is low and (ii) the solubility of surfactant in water is negligible. Considering the results of Aveyard et al. (1986), the first assumption is reasonable for AOT. The second assumption is indeed the case for most of the surfactants in a Windsor II system. For example, under Windsor II conditions, less than 1% of AOT resides in excess aqueous phase at equilibrium (Rabie and Vera, 1995). Reverse Micellar Interface. The reverse micellar interface is where the surfactant molecules are placed shielding the water pool from contact with organic solvent. The interface is considered to be a uniform solubilization environment. Currently, there are three ways of visualizing a uniform interface. (i) Pseudophase Model. This model has been used in studies of biological membranes (Diamond and Wright, 1969; Katz and Diamond, 1974; Rogers and Davis, 1980), micelles (Birdi and Ben-Naim, 1981; Moroi, 1980; Treiner, 1982), and microemulsions (Fletcher, 1986). In this model, the surfactant tails are assumed to be an independent organic pseudophase where lipophilic solutes are solubilized. This model was found to be unrealistic for many solute-membrane systems (Leodidis and Hatton, 1990). (ii) Surface-Monolayer Model. In this model, the solute is assumed to be adsorbed at the reverse micellar interface. A mole-fraction-based partition coefficient is used to quantify extraction. However, it was found that such a partition coefficient depends strongly on type and concentration of the external electrolyte and it also depends weakly on the surfactant concentration (Leodidis and Hatton, 1990). Obviously, such a partition coefficient is of limited use for the prediction of solubilization. (iii) Active Interface Model. In this work, we adopt a very different way to visualize the reverse micellar interface. Due to the strong electrostatic effect of the surfactant headgroups, the interface is chemically active. Different solutes are able to react with surfactant headgroups and form different complexes. Thus, the nature of the solute at the interface is different from that in the water pool where the solute is more likely to be in a similar state as in the excess water. In this model, the key to solving the solubilization problem is to determine the specific reactions happening between different solutes and this active interface and also to measure the equilibrium constants of these reactions. This way of quantifying extraction by reverse micelles has been successful in our previous work (Rabie and Vera, 1995). A simple ion-exchange reaction between any cations present in the aqueous phase and AOT counterion, sodium, provided excellent predictions for ion distribution (Rabie and Vera, 1995). A dimensionless form of the model based on dimensionless groups reduced the number of independent variables. The accuracy of the results obtained with the dimensionless form suggests that the model is physically meaningful. For extraction of amino acids at their zwitterionic condition, the following reactions are proposed in this work:
SNa + Ai,f( a SAiNa
(1)
where the free zwitterionic amino acid, Ai,f(, in the
Ind. Eng. Chem. Res., Vol. 35, No. 10, 1996 3667
water pool forms a complex with the surfactant SNa. In this equation, S stands for an anionic surfactant such as AOT whose counterion is sodium, Na. Subscript f refers to the free species inside the water pool and serves to distinguish them from those at the reverse micellar interface. The equilibrium constant of this reaction in terms of concentrations is
Ki-Na ) c
C h i-Na,c
(2)
C h Na,bC h i,f
where C h i-Na,c is the molar concentration of the ith amino acid in the form of surfactant-sodium complex at the reverse micellar interface, C h i,f is the molar concentration of free amino acid in the water pool, and C h Na,b is the molar concentration of sodium bound to the surfactant headgroup at the reverse micellar interface. Equation 2 presupposes that Kγ, the product of activity coefficients of the bound sodium and the free amino acid over the activity coefficient of the complex, is constant. Furthermore, we assume that this complex can dissociate to release sodium into the water pool and leave a surfactant-amino acid anion with one negative charge:
acids with reactions similar to (1) and (3). Similar to reaction 1, we can write for the jth cation
S(zj)Mj + zjAi,f( a S(zj)Ai(zj)Mj
(7)
with the equilibrium constant written similar to the eq 2 as
Ki-j c )
C h i-j,c
(8)
C h j,bC h i,f
where Ci-j,c is the molar concentration of the ith amino acid in the surfactant complex of the jth cation. However, the dissociation reaction for this complex is not independent and can be obtained from eqs 1, 3, 5, and 7. The equilibrium constant of the dissociation reaction for this complex, Ki-j d , can be then found from the following equation:
Ki-j d )
Ki-Na )zj (Ki-Na c d j-Na Ki-j c K
(9)
It should be noted that
SAiNa a SAi + Naf -
(3)
+
The equilibrium constant of this reaction is written as
) Ki-Na d
h Na,f C h i,dC
(4)
C h i-Na,c
where C h i,d is the molar concentration of the dissociated amino acid-surfactant complex at the reverse micellar interface and C h Na,f is the molar concentration of free sodium in the water pool. Similar to eq 2, the Kγ term in eq 4 is also assumed to be constant. These equations are written for a monovalent anionic surfactant with sodium as the counterion; however, they can be easily applied for any other kinds of surfactants. Any cation in the water pool undergoes a reaction of ion exchange with the cations bound to the surfactant headgroups, counterions. Following Rabie and Vera (1995), the ion exchange for an anionic surfactant with sodium as the counterion is represented by a reversible reaction of the form zj+
zjSNa + Mj,f
a S(zj)Mj + zjNaf
+
(5)
where the free cation Mj,f in the water pool replaces the surfactant-bound counterion, sodium, at the reverse micellar interface. In this equation, zj is the charge number of cation Mj. The equilibrium constant of this reaction, Kj-Na, is
Kj-Na )
( )( ) C h j,b C h Na,f
C h j,f C h Na,b
zj
(6)
where C h j,b and C h j,f are the molar concentrations of cation j bound to the surfactant headgroup at the reverse micellar interface and free cation j in the water pool, respectively. Reaction 5 produces new forms of surfactants, different from the original one, SNa. These new forms of surfactants are also able to react with amino
C h i-j,c ) zjC h j-i,c
(10)
Water Pool of Reverse Micelles. The water pool is assumed to be a well-mixed solubilization environment. Despite the fact that a solute concentration gradient exists in the water pool, an average value can conveniently represent this environment. The partitioning of any solute from an excess aqueous phase to the water pool of reverse micelles is characterized by a partition coefficient, Λ:
Λ)C h f /C
(11)
where C is the molar concentration of a solute in the aqueous phase and C h f is the molar concentration of that solute in the water pool. Equation 11 results from phase equilibrium considerations. At equilibrium, the chemical potential of a solute molecule must be the same throughout the two-phase system. Thus, the chemical potential of a solute in the excess aqueous phase must be equal to that in the water pool
(12)
µ)µ jf which leaves the partition coefficient as
Λ)
[
µΘ - µj Θ γ f exp γ jf RT
]
(13)
where γ is the activity coefficient and superscript Θ refers to the standard state. As in eqs 2 and 4, if the ratio of the activity coefficients is assumed to be constant, the partition coefficient is only a function of temperature. This consideration of partitioning for the solute between the water pool and excess aqueous phase is more general than needed. For droplets with radius larger than 30-40 Å, water in the water pool is very similar to that in the excess aqueous phase, with respect to its solubilization capacity (Leodidis and Hatton, 1990). For most of the experiments reported in this paper, the conditions were similar to those used by
3668 Ind. Eng. Chem. Res., Vol. 35, No. 10, 1996
Leodidis and Hatton (1990); thus, the values of the partition coefficients have been assumed to be unity in all cases. For the mass balance of amino acid, surfactant, and cations, the experimental conditions are described as follows. Initially, an aqueous phase of volume V0 containing water, n different amino acids at their zwitterionic condition with concentrations C0i , and m different cations, including sodium, with concentrations C0j , is in contact with an organic phase of volume V h0 containing isooctane and AOT with concentration of C h 0s. At equilibrium, the aqueous phase has a volume of V which contains surfactant, amino acids, and cations at the concentrations of Cs, Ci, and Cj, respectively. The organic phase of volume V h contains isooctane, AOT (C h s), free amino acids (C h i,f), surfactant-amino acid complex with the jth cation (C h i-j,c), dissociated surfactant-amino acid complexes (C h i,d), free cations (C h j,f), bound cations (C h j,b), and the ith amino acid complex (C h j-i,c), and water. The concentrations of free solutes in the water pool of the organic phase are defined as the moles of solute per unit volume of water pool, while the concentrations of the surfactant, the bound cations, and the surfactant-amino acid complexes are defined as the moles of solute per unit volume of water-free organic phase. This rather arbitrary definition of concentration in the organic phase has been adopted as a way of making these concentrations independent of the water uptake. The mass balance of the ith amino acid present in the initial aqueous phase is m
(C h i-j,c) + rC h i,d ) C0i ∑ j)1
Ci + r
(14)
where r is the initial volume ratio of organic phase to aqueous phase defined by the following equation:
r)V h 0/V0
(15)
The mass balance of sodium is then formulated as n
0 (C h Na-i,c) ) CNa + rC h 0s ∑ i)1
CNa + rC h Na,b + r
(16)
Equation 16 presupposes that the surfactant solubilized in the aqueous phase is completely dissociated. For each of the other cations present in the initial aqueous phase, the mass balance is n
(C h j-i,c) ) ∑ i)1
Cj + rC h j,b + r
C0j
(17)
In eqs 14, 16, and 17, we have assumed that the values of Λ are all unity. From eq 10, we can write the following relation for the complexes of surfactant and the ith amino acid: m
(zjC h j-i,c) ) C h i,c ∑ j)1
(18)
where C h i,c is the total molar concentration of the ith amino acid in the form of a complex at the reverse micellar interface. Assuming that in the reverse micelles all the surfactant sites are filled with either
sodium or other cations or amino acids, then the mass balance of surfactant is m
∑ j)1
n m
(zjC h j,b) +
∑ ∑ i)1 j)1
n
(C h i-j,c) +
(C h i,d) ) C h 0s ∑ i)1
(19)
Equation 19 assumes that at equilibrium, all the surfactant molecules in the organic phase are at the reverse micellar interface and that the concentration of the AOT surfactant in the aqueous phase is negligible. These assumptions have been discussed earlier. In order to find the equilibrium distribution of all cations and all amino acids in different regions, the above equations have to be solved simultaneously. The results then provide information of the ion distribution including the overall extraction of each amino acid from the aqueous phase to the organic phase. For a system initially consisting of n different amino acids at their zwitterionic condition and m different cations, including sodium, there are several species in different regions at equilibrium. These are mn different complexes, n different dissociated complexes, m different cations bound to the surfactant headgroups, and m different cations and n different amino acids in the excess aqueous phase, assuming that the water pool is similar to the aqueous phase. Thus, there are 2n + 2m + mn different concentrations to be determined. The number of equations are mn equations of the type of eqs 2 and 7, n equations of the type of eq 4, m - 1 equations of the type of eq 5, n equations of the type of eq 14, m 1 equations of the type of eq 17, and one equation of each of the equations (16) and (19), which comes to a total of 2n + 2m + mn independent equations. For some systems at specified conditions, the solution to the above equations is analytical. These specific cases and the evaluation of equilibrium constants are discussed below. In the Results and Discussion section, when we refer to the predictions of the model, it means the complete solution of the above equations. However, for some specific systems in which there are analytical solutions, we have used the relevant equations which are detailed below and mentioned specifically by the equation number. 3.1. Single Counterion, Single Amino Acid System. The solution for a system consisting of a single counterion which has to be sodium for AOT and one amino acid is
C0A - CA KcCA(rC h 0s - C0A + CA)
)1+ Kd
0 CNa
+
C0A
- CA - KcCA(rC h 0s - C0A + CA)
(20)
where the subscript i for the ith amino acid has been replaced with subscript A for the only amino acid in the system. Equation 20 suggests that the effect of the initial volumes of the aqueous and organic phases and the initial surfactant concentration can be considered through only one independent variable as rC h 0s . Solution to eq 20 provides the amino acid concentration at equilibrium in the excess aqueous phase for various conditions of the initial surfactant, amino acid and salt concentrations, and the initial volume ratios of organic to aqueous phase. The equilibrium concentration of
Ind. Eng. Chem. Res., Vol. 35, No. 10, 1996 3669
sodium in the excess aqueous phase can be obtained from the following equation: 0 CNa ) CNa + rC h 0s - (1 + KcCA)(rC h 0s - C0A + CA)
(21)
and the concentration of the bound sodium to the surfactant at the reverse micellar interface is
h 0s - (C0A - CA)/r C h Na,b ) C
(22)
If any other monovalent anionic surfactant with a monovalent counterion, such as SM, is used instead of AOT, the above equations are still valid. However, all the notations referring to sodium in these equations should be replaced by the cation M. 3.2. Low Concentration of Amino Acid. If the presence of amino acid in the system has negligible effect on the distribution of the other ions, a simple solution can be found. This assumption depends on the concentration of the amino acid in comparison with the concentrations of other species and the relative values of the equilibrium constants of extraction. For a system consisting of one amino acid and m different cations including sodium, we can write Na C0A ) CA + rKNa c Kd
m C h Na,b CA + r (zjKjcC h j,b(CA)zj) (23) CNa j)1
∑
If the presence of amino acid has no significant effect on the concentrations of bound cations at the reverse micellar interface and on the concentration of sodium in the excess aqueous phase, the extraction of the amino acid can be calculated from eq 23. It is interesting to note that eq 23 can still be used for a system of mixed amino acids and mixed cations. In this case, there is one equation similar to eq 23 for each amino acid. The concentrations of cations at equilibrium, in this case, can be found from solving the equations of ionexchange reactions, eq 5, for each cation and the mass balances of surfactant and all cations neglecting the presence of amino acids. Such analytical solutions are similar to those discussed by Rabie and Vera (1995). Appendix A provides the analytical solutions for a few systems which are of interest in this work. 3.3. Evaluation of Equilibrium Constants. To evaluate the equilibrium constants of sodium-amino acid, a system consisting of AOT-sodium-amino acid needs to be studied. At least two data points are necessary to calculate the values of the equilibrium constants. The use of a least-squares fit for a set of data is preferable. Knowing the values of the equilibrium constants, it is possible to predict the equilibrium state of the system for various conditions of initial concentrations of AOT, sodium salt, and amino acid and initial phase volume ratios. If one more cation is added, cation M, to the above system, there will be two more equilibrium constants to be evaluated. However, one of them can be calculated from eq 9 if the equilibrium constant of the ion-exchange reaction between cation M and sodium of the surfactant, KM-Na, is known. The equilibrium constants of ionexchange reactions for various monovalent and divalent cations in the AOT system have been measured by Rabie and Vera (1995). Thus, there is only one equilibrium constant to be evaluated in the presence of an additional cation M. At least, one data point from the
Figure 2. Effect of salt concentration on the extraction of tryptophan and phenylalanine. Initial organic phase, 0.2 M AOT in isooctane; initial aqueous phase, 5 mM amino acid, sodium buffer; and an initial volume ratio of unity. Solid lines represent the predictions of the active interface model.
system of AOT-sodium-cation M-amino acid is needed to calculate this equilibrium constant. 4. Results and Discussion The predictions of the model proposed here are compared with experimental data for the extraction of amino acids in the presence of different salts. All the data reported here were obtained in Windsor type II systems. The data are reported as the equilibrium concentrations of solutes in different regions and as the overall fraction of amino acid extracted to the organic phase. The overall fraction extracted is calculated from the following equation:
f ) (C0A - CA)/C0A
(24)
4.1. Effects of Salt Type and Concentration. The effect of salt concentration on extraction of tryptophan and phenylalanine is presented in Figure 2. The extraction of both amino acids decreases with an increase of the salt concentrations at low salt concentration and remains almost constant at higher salt concentrations. The values of the equilibrium constants, Na KNa c and Kd , were obtained from a least-squares fit of eq 20 to the data of Figure 2 for each amino acid. They are KT-Na ) 3.57 M-1 and KT-Na ) 0.038 M for trypc d -1 and KP-Na ) 0.026 M for tophan and KP-Na ) 1.18 M c d phenylalanine. At high sodium concentrations, most of the amino acid molecules are in the form of a complex. Thus, no effect of sodium is observed at high salt concentrations, as suggested by eqs 1 and 3. The results calculated with eq 20 using the two equilibrium constants, shown as solid lines in Figure 2, reproduce the experimental results within 0.5 to 2%. The same values of equilibrium constants were used throughout this work. Figure 3 shows the equilibrium concentration of tryptophan in the final aqueous phase vs initial cation, sodium or potassium, concentration. The data of sodium from Figure 2 are included for comparison. There is a strong dependence on the cation type and concentration. The aqueous-phase amino acid concentrations for potassium are significantly larger than those obtained with sodium, which means there is less extrac-
3670 Ind. Eng. Chem. Res., Vol. 35, No. 10, 1996
Figure 3. Cation effect on the final tryptophan concentration in the excess aqueous phase. Initial organic phase, 0.2 M AOT in isooctane; initial aqueous phase, 5 mM tryptophan, sodium or potassium buffer; and an initial volume ratio of unity. Solid lines represent the predictions of the active interface model, while dotted lines represent the predictions of the surface-monolayer model (Leodidis and Hatton, 1990).
tion with potassium. This is due to the fact that the potassium equilibrium constant for the formation of the complex is smaller than that for sodium. This equilibrium constant for potassium and tryptophan, KT-K , c found with a least-squares fit of eq 23 to the data of potassium in Figure 3, was 1.15 M-1. The second equilibrium constant was calculated using eq 9, where the value of KK-Na ) 1.96 was taken from the literature (Rabie and Vera, 1995). The second equilibrium constant for potassium was then calculated as KT-K ) c 0.0597 M. It is very interesting to note that at lower concentrations of cation, the data of potassium approach those of sodium. At this condition, a small fraction of sodium bound to the surfactant has been exchanged by potassium, as indicated by eq 5. Thus, the system behaves like one in which only the sodium salt of surfactant is present. The predictions of the surfacemonolayer model (Leodidis and Hatton, 1990) is also shown in this figure. No effect of salt type and concentration is predicted by this model. The solid line for potassium in Figure 3 is the predictions of eq 23 using a single adjustable parameter. For comparison, the predictions of the complete solution of equations for potassium were also calculated and no difference was obtained between the approximate results of eq 23 and the predictions of the complete solution. This is due to the fact that in the range of concentrations studied, the presence of amino acid had no significant effect on the distribution of the other species. 4.2. Effects of Amino Acid Type and Concentration. Figure 4 shows the effect of initial amino acid concentration on the final concentration of amino acid in the aqueous phase for various amino acids. The initial sodium concentration was kept constant at 0.2 M, and the initial AOT concentration in the organic phase was 0.3 M with an initial phase volume ratio of unity. Similar experimental results have been reported by Leodidis and Hatton (1990). For comparison, their data for tryptophan are shown in this figure. For any amino acid, an almost linear variation was observed with a slight increase in the slope of the curve at higher concentrations of amino acid. An excellent agreement
Figure 4. Effect of initial amino acid concentration on the residual amino acid concentration in the excess aqueous phase. Initial organic phase, 0.3 M AOT in isooctane; initial aqueous phase, 0.2 M Na+; and an initial volume ratio of unity. Solid lines represent the predictions of the active interface model. The closed symbols are the data from the present study, while the open symbols are the data from the literature (Leodidis and Hatton, 1990). Table 1. Effect of Tryptophan Concentration on the Distribution of Different Species in the Reverse Micellar Systema C0A
CA (E)
CA (M)
C h A,c (M)
C h A,d (M)
C h Na,b (M)
2.0 5.0 7.5 12.5 20.0 25.0 32.5
0.88 2.20 3.30 5.64 8.82 11.10 14.55
0.88 2.21 3.33 5.57 8.99 11.30 14.80
0.94 2.34 3.52 5.83 9.28 11.55 14.92
0.18 0.44 0.66 1.09 1.73 2.15 2.78
298.88 297.21 295.83 293.07 289.00 286.30 282.30
a All concentrations are given in mM. In this table (E) refers to experimental results and (M) refers to the results obtained from the model.
is found between the predictions of the model, without any adjustment of the parameters, shown as solid lines, and the experimental results. The data of amino acids which do not react with the reverse micellar interface should fall on the 45° line. It means that the value of the equilibrium constant of the formation of the complex is close to zero, so no amino acid can be extracted. Under this category are zwitterionic threonine, glycine, and alanine; the data are not shown for alanine and glycine, and the data of threonine are taken from the literature (Leodidis and Hatton, 1990). The larger the deviation from the 45° line, the more is the extraction for an amino acid. From Figure 4, we immediately conclude that the equilibrium constants of different amino acids follow the order tryptophan > phenylalanine > threonine. Figure 2 also shows that tryptophan exhibits higher extraction than phenylalanine over a wide range of sodium concentration. Table 1 shows the concentrations of bound sodium to the surfactant headgroups and the concentration of amino acid in complex and dissociated forms, in mM, for the data of tryptophan reported in Figure 4. These data show that the presence of tryptophan at low concentrations has no significant effect on the concentration of bound sodium. A less than 5% decrease in the amount of bound sodium to the surfactant is
Ind. Eng. Chem. Res., Vol. 35, No. 10, 1996 3671
Figure 5. Effect of the initial tryptophan concentration on the final tryptophan concentration in the excess aqueous phase for various surfactant concentrations. Initial organic phase, AOT in isooctane; initial aqueous phase, 0.2 M Na+; and an initial volume ratio of unity. Solid lines represent the predictions of the active interface model.
observed for up to 30 mM concentration of tryptophan. It is also interesting to note that less than 16% of tryptophan at the reverse micellar interface is in the dissociated form with a very slight decrease at higher amino acid concentrations. The numerical results of Table 1 show less than 2% deviation from the experimental values. Figure 5 shows the effect of the initial tryptophan concentration on the equilibrium concentration of tryptophan in the aqueous phase for different initial AOT concentrations. An almost linear variation with a slight increase in the slope at higher amino acid concentrations was observed for any initial surfactant concentrations. Figure 5 also shows that at higher AOT concentrations, more amino acid is extracted to the organic phase. Similarly to previous cases, the predictions of the model are very encouraging. 4.3. Effect of Surfactant Concentration. Figure 6 presents the results of the final tryptophan concentrations in the aqueous phase vs the initial AOT concentration in the organic phase for various initial sodium concentrations. The equilibrium concentration of tryptophan in the excess aqueous phase decreases with surfactant concentration for any initial sodium concentrations. This type of surfactant effect, also found in Figure 5, confirms the specific interaction between surfactant molecules and amino acids. A very good agreement is found between the predictions of the present model and experimental data. Figure 7 shows the fraction of tryptophan extracted to the organic phase as a function of the term rC h 0s . The variation in this term originates from the variation in the initial volume ratio of organic to aqueous phase and of the initial surfactant concentration. The interesting experimental finding is that these data collapse onto one curve which is exactly what the model, eq 20, predicts. The solid line shows the predictions of the model. 4.4. Effect of the Nature of the Solvent. Table 2 contains the experimental results of the equilibrium concentrations of tryptophan in the excess aqueous phase for various solvents. These solvents are octane, isooctane, nonane, and decane. The initial concentration of tryptophan was 5 mM in the aqueous phase. In this table, the initial potassium concentration varies
Figure 6. Effect of surfactant concentration on the final tryptophan concentration in the excess aqueous phase. Initial organic phase, AOT in isooctane; initial aqueous phase, 5 mM tryptophan, sodium buffer; and an initial volume ratio of unity. Solid lines represent the predictions of the active interface model. The closed symbols are the data from the present study, while the open symbols are the data from the literature (Leodidis and Hatton, 1990).
Figure 7. Effect of the number of moles of surfactant on the extraction of tryptophan. Initial organic phase, AOT in isooctane; initial aqueous phase, 5 mM tryptophan, 0.2 M Na+. Solid lines represent the predictions of the active interface model. Table 2. Effect of the Solvent on the Equilibrium Tryptophan Concentration in the Excess Aqueous Phasea C0K, mM
octane
isooctane
nonane
decane
80 100 140 200
2.72 2.90 3.05 3.21
2.75 2.90 3.03 3.25
2.77 2.85 3.01 3.23
2.76 2.92 2.98 3.26
a Initial concentration of tryptophan, 5 mM; initial concentration of AOT, 200 mM; volume ratio, unity.
between 80 and 200 mM. The initial AOT concentration was fixed at 200 mM with an initial volume ratio of unity. The results of this table clearly indicate that no effect of solvent is observed on the extraction of tryptophan. Similar results were obtained for phenylalanine. Although a solvent has a definite effect on the water uptake and size of the reverse micelles, it does not have any significant effect on the reactions happening inside
3672 Ind. Eng. Chem. Res., Vol. 35, No. 10, 1996 Table 3. Influence of the Solvent on the Ion Distribution. Molar Ratio of Potassium to Sodium in the Excess Aqueous Phase at Equilibrium for Various Solvents C0K, mM
octane
isooctane
nonane
decane
80 100 140 200
4.08 3.23 2.15 1.41
4.12 3.20 2.16 1.39
4.14 3.16 2.15 1.36
4.13 3.19 2.18 1.43
the reverse micelles between different solutes and the surfactant headgroups. This is indeed in accord with the predictions of the present model. Thus, the values of equilibrium constants found for any pair of amino acid-cation can be used for any solvent. It is also interesting to study the effect of the solvent on the distribution of other species. The experimental results of the equilibrium molar ratio of potassium to sodium in the excess aqueous phase for various solvents and different initial potassium concentrations are reported in Table 3. The conditions are similar to those in Table 2 except that no amino acid was present in the system. Similarly, the effect of solvent was not found to be significant. Notice that no sodium salt was initially added to the aqueous phase. However, there was some sodium in the final aqueous phase due to the ion-exchange reaction, eq 5. For more experimental results and discussion on the ion distribution in reverse micellar systems, refer to the work of Rabie and Vera, 1995.
Appendix A The solution for the cation distribution at equilibrium in a binary mixture of sodium and another cation, Mjzj+, is
Kj-Na ) rzj-1
(Rj - R0j )(1 + R0j )zj-1Rzj j (δ(1 + Rj) - Rj + R0j )zj
(A-1)
where
Rj )
R0j
CNa zjCj 0 CNa
)
zjC0j
V h 0C h 0s
δ)
n-1
V0
(A-2)
(A-3)
(A-4)
(zjC0j ) ∑ i)1
In eq A-4, the summation is over all counterions excluding sodium, which is the original surfactant counterion. For a binary system, the denominator in eq A-4 is simply the initial number of moles of cation Mjzj+ added to the system.
5. Conclusions We have investigated in detail the solubilization of zwitterionic amino acids in AOT reverse micelles. The effects of the amino acid type and concentration, surfactant concentrations, salt type and concentration, volume ratio, and nature of solvent have been examined. The final amino acid concentration in the excess aqueous phase increases linearly with the initial amino acid concentration, decreases with the initial surfactant concentration and volume ratio, and increases with the initial salt concentration. However, the nature of the solvent had no significant effect on the distribution of amino acid and on the distribution of ions. The data of amino acid extraction for different surfactant concentrations and different volume ratios collapse onto one single curve when the data are plotted as a function of the term rC h 0s . A simple solubilization theory has been developed to predict the equilibrium distribution of zwitterionic amino acids from information of the initial conditions of the system. This theory is based on the chemical and electrostatic interactions between the amino acids and the active reverse micellar interface. The predictions of the model are in excellent agreement with the experimental results. Acknowledgment We are grateful to the Natural Sciences and Engineering Research Council of Canada for financial support.
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Received for review January 26, 1996 Revised manuscript received June 27, 1996 Accepted July 2, 1996X IE960053I
X Abstract published in Advance ACS Abstracts, September 1, 1996.
