I n d . Eng. Chem. Res. 1995,34, 599-606
599
Reverse Micellar Extraction of Amino Acids Using Dioctyldimethylammonium Chloride Wenhua Wang, Martin E. Weber, and Juan H. Vera* Department of Chemical Engineering, McGill University, Montreal, Quebec, Canada H3A 2A7
The effects of pH, ionic strength,and amino acid and surfactant concentrations on the reverse micellar extraction of aspartic acid, glutamic acid, and threonine using dioctyldimethylammonium chloride (DODMAC), were determined. The concentrations of the ionic species in the aqueous phase were measured, and the corresponding concentrations inside the reverse micelles were obtained by material balances and the condition of electrical neutrality. The results show that the amino acids were mainly extracted through an ion exchange mechanism. The net charge of a n amino acid, the competition among anions, the dissociation equilibrium of DODMAC,and the water uptake in the reverse micelles are important factors which dominate the amino acid extraction.
Introduction Progress in biotechnology requires major improvements for large-scale bioproduct purification from fermentation and cell-culture media. The purification steps represent 50-90% of the cost of the final bioproducts; thus it is important to have well-designed separation processes. Reverse micelles are aggregates of surfactant molecules in an apolar solvent surrounding water pools in which biomolecules may be solubilized. The extraction of proteins by solubilization in reverse micelles has been investigated extensively in the last two decades (Goklen and Hatton, 1987; Jolivalt et al., 1990; Krei and Hustedt, 1992; Nishiki et al., 1993). A step toward a better understanding of the mechanism of reverse micellar extraction of proteins is the study of the extraction of amino acids, the building blocks of proteins. Since the charged state of an amino acid in solution is easily altered by changing the pH, amino acids have been used as model biomolecules for the study of reverse micellar extraction (Thien et al., 1988; Furusaki and Kishi, 1990; Leodidis and Hatton, 1990a; Adachi et al., 1991; Hano et al., 1991). In addition, amino acids are valuable bioproducts. There are three methods to produce amino acids (Scheper et al., 1984): (i) by extraction from protein hydrolysates; (ii) by microbiological techniques (fermentation methods and enzymatic methods); (iii) by synthetic techniques. Ion exchange and crystallization are commonly utilized t o separate and concentrate amino acids, usually in batch processes. Thus, amino acid separation by reverse micelles may prove to be a useful step for their largescale production. Two types of surfactants have been commonly employed to form reverse micelles for amino acid extraction from aqueous solution. Aerosol-OT, an anionic surfactant, has been used in the low-pH range for the extraction of positively charged amino acids (Furusaki and Kishi, 1990; Leodidis and Hatton, 1990a,b; Adachi et al., 1991). Trioctylmethylammonium chloride (TOMAC), a cationic surfactant, has been used in the highpH range for the extraction of negatively charged amino acids (Thien et al., 1988; Hano et al., 1991). Amino acids having a net charge are trapped inside the water pools of the reverse micelles due to electrostatic interactions, while uncharged amino acids are located mainly in the interfacial region of the reverse micelles (Leodidis et al., 1990a,b; Adachi et al., 1991). Luisi et al. (1979) studied the transfer of tryptophan into an organic phase 0888-5885/95/2634-0599$09.00l~
composed of TOMAC in cyclohexane. They showed that the effect of pH on the amount transferred was determined by the ionization of the amino group. Thien (1988) reported similar results for Aerosol-OT reverse micelles, obtaining fair agreement between the titration curves for arginine and the amount of amino acid extracted as a function of the pH. Furusaki and Kishi (1990) found that the number of arginine molecules in a reverse micelle is much higher than that in the case of protein extraction where, in general, one single molecule is entrapped. They also found that the concentration of arginine in the reverse micelles is higher than that in the bulk aqueous phase. The complete analysis of all species in the aqueous phase has not been reported in any of these papers. We recently reported the formation of reverse micelles in isooctane with the two tailed cationic surfactant dioctyldimethylammonium chloride (DODMAC),using alcohol as cosurfactant (Wang et al., 1994). The objective of the present work was to determine the effects of pH, ionic strength, and amino acid and surfactant concentrations on the extraction of amino acids using DODMAC reverse micelles. The organic solvent was isooctane, and the cosurfactant was l-decanol. A method to determine the concentrations of all species in the extraction system is described.
Experimental Method The amino acids used were DL-aspartic acid (Asp),DLglutamic acid (Glu), DL-glUtamine (Gln), and DL-threonine (Thr). They were obtained from Sigma (St. Louis, MO) and used as received. The dioctyldimethylammonium chloride (DODMAC) was purified, as described previously (Wang et al., 19941, from the commercial surfactant Bardac LF-80, obtained from Lonza Inc. (Fair Lawn, NJ). Its final purity was 98%, as determined by sodium lauryl sulfate titration with a Model 93-42 Orion surfactant electrode (Orion Research Inc., Cambridge, MA). o-Phthaldialdehyde (OPA), 2-mercaptoethanol, and sodium tetraborate were purchased from Pfaltz & Bauer Inc. (Waterbury, CT). Absolute ethanol was obtained from Consolidated Alcohols Ltd. (Toronto, ON). All other chemicals were obtained from A&C American Chemicals Ltd. (Montreal, QC). Deionized water, with an electrical conductivity below 0.8 pSlcm, was used for all experiments. The experimental procedure was the following: 30 mL of a solution containing 100 or 200 mM DODMAC and
0 1995 American Chemical Society
600 Ind. Eng. Chem. Res., Vol. 34, No. 2, 1995
250 mM l-decanol in isooctane was shaken for 30 min at 23 "C and 200 rpm with 30 mL of an aqueous solution containing 5, 10, or 20 mM of amino acid. The ionic strength and pH in the aqueous phase were adjusted by adding NaCl and NaOH, respectively. The samples were settled for 1week at 23 "C. The water uptake in the organic phase was measured by Karl Fischer titration using a Model 701 titrator (Metrohm Ltd., Herisau, Switzerland). The pH of the aqueous phase was measured by a Model 691 pH meter (Metrohm Ltd.). The sodium concentration in the aqueous phase was measured by atomic absorption on a Model Smith-Hieftje 11 (Therm0 Jarrell Ash, Franklin, MA) spectrophotometer at 330.2 nm wavelength. The standard samples contained from 20 to 140 ppm sodium; thus it was necessary to dilute all phase samples at least 20 times. The concentration of DODMAC in the aqueous phase was measured by gas chromatography on a Model 5890A chromatograph (Hewlett-Packard, Palo Alto, CAI with a flame ionization detector using 2-butoxyethanol as an internal standard. The DODMAC tailing was reduced by using a 10% Carb. 20M 2% KOH 80/100 column of 0.125 in. diameter and 8 ft length, supplied by Chromatographic Specialties Inc. (Brockville, ON). The DODMAC standard samples ranged in concentration from 0.5 to 6.0 mM; hence all phase samples were diluted approximately four times. The concentration of chloride ions in the aqueous phase was measured by ion chromatography on a Model 4500i Dionex chromatograph (Dionex, Sunnyvale, CA) using an Ionpac AG4ASC guard column and an Ionpac AS4A-SC analytical column. The chloride standards ranged from 2 to 10 ppm; thus the phase samples were diluted at least 100 times. The concentration of amino acid in the aqueous phase was measured with a Cary 1/3 W spectrophotometer (Varian Techtron Pty Ltd., Victoria, Australia) using the OPA-labeling method (Roth, 1971; Church et al., 19831, which is based on the fact that the a-amino groups of the amino acid released by hydrolysis react with o-phthaldialdehyde and 2-mercaptoethanol to form an adduct that absorbs strongly at 340 nm. After preliminary studies, we adopted the following OPAlabeling procedure. The OPA reagent was prepared mixing 0.8 g of o-phthaldialdehyde with 20 mL of ethanol and then adding 10 g of sodium tetraborate, 2 mL of 2-mercaptoethanol and, finally, water to 1000 mL. This reagent was used immediately after preparation. A 20 mL sample of OPA reagent was mixed with 0.150.80 mL of the sample solution from the aqueous phase or the standard amino acid solution, and then the W absorbance at 340 nm was measured 5 min after mixing. The concentrations of the amino acid in the mixture of OPA reagent with the experimental samples or standard amino acid solution were between 0.04 and 0.16 mM.
+
Results and Discussion We planned originally to extract four amino acids: glutamine, aspartic acid, glutamic acid, and threonine. In the development of the OPA-labeling procedure, we found that the glutamine calibration curve at the highpH values, which were of interest for extraction, was a strong function of settling time (i.e., the time before mixing with the OPA reagent). Figure 1 shows that the W absorbance at 340 nm, measured 5 min after mixing the standard glutamine solution with the OPA reagent, decreased as the settling time of the standard glutamine solution increased. This indicates a decrease in the
- -1 .o I
1*
1 hr I Week4 Weeks
0.8
m
/
0.4
n 0
0.05
I
I
0.10
0.15
0.20
Gln Concentration ( mM ) Figure 1. Effect of settling time of the standard glutamine solution on the UV calibration curve of glutamine by the OPAlabeling method: pH 12.5, readings taken 5 min after mixing the OPA reagent with the standard glutamine solution.
number of a-amino groups and a change in structure. The calibrations for aspartic acid, glutamic acid, and threonine were stable over the settling time of the standard amino acid solutions; hence extraction experiments were conducted only with these three amino acids. Aspartic acid and glutamic acid have similar charged states. The three pK values for aspartic acid are 2.09, 3.86, and 9.82 and for glutamic acid are 2.19, 4.25, and 9.67 (Lehninger, 1975). However, their structures are different; glutamic acid has one more CH2 group in the hydrocarbon chain than aspartic acid. On the other hand, aspartic acid and threonine have the same number of carbon atoms but different charged states. Threonine has the two pK values: 2.63 and 10.43 (Lehninger, 1975). On the basis of our previous results on the water uptake by DODMAC reverse micelles (Wang et al., 19941, the initial organic phase was 100 or 200 mM DODMAC and 250 mM l-decanol in isooctane in all the extraction experiments. In previous work on extraction of proteins (Goklen and Hatton, 1987; Jolivalt et al., 1990; Krei and Hustedt, 1992) and amino acids (Leodidis and Hatton, 1990a,b; Adachi et al., 1991; Hano et al., 19911, the pH was adjusted by adding a buffer solution. The buffer interferes with the extraction of an amino acid because both the buffer and the amino acid are weak salts and their degree of dissociation is a function of pH. The buffer anions also compete with the amino acid in the extraction process; hence we adjusted the pH with NaOH and the ionic strength of the aqueous phase with NaC1. In order to have aspartic acid and glutamic acid with two negative charges and threonine with one negative charge, the pH of the initial aqueous phase was -12.5, except when the effect of pH was investigated. For studying the effect of pH, the concentration of NaCl in the initial aqueous phase was fured at 51.2 mM. This value represented a compromise between two opposite tendencies. If there is too little salt, no reverse micelles are formed (at medium pH); if there is too much salt, the effect of pH is obscured. The aqueous phase in our extraction system contained three anions, amino acid, C1- and OH-, and three cations, H+, Na+, and DODMAC. We measured the concentrations of all of these species in the aqueous phase. For most aqueous samples, the difference between the total anions and cations measured was less than 10%of the total ions in solution (for a few samples, the difference was between 10 and 15%)(Wang, 1994). Because the dielectric constant of the water pools inside the reverse micelles is lower than in the bulk aqueous
Ind. Eng. Chem. Res., Vol. 34, No. 2, 1995 601 e .-
30
140
Y
20
-
*
100
.-E
$u
10
60
C
.-
Y
0
tZiFz? 6
8
IO
12
Equilibrium pH
Figure 2. Effect of equilibrium pH on the partition coefficients of aspartic acid, glutamic acid and threonine: initial organic, 200 mM DODMAC, 250 mM 1-decanol; initial aqueous, 5 mM amino acid, 51.2 mM NaCl.
phase (Leodidis and Hatton, 1989; Karpe and Ruckenstein, 19911, the ionic species inside reverse micelles are expected to be only partly dissociated and the ion product of water inside reverse micelles is different from that in the bulk aqueous phase. The concentrations of amino acid, C1, Na, and DODMAC in the reverse micelles were calculated from the difference of mole number between the initial and the equilibrium aqueous phases divided by the volume of water in the reverse micelles. For the Aerosol-OT reverse micelles, the water pool is slightly more acidic than the bulk aqueous phase (Smith and Luisi, 1980; El Seoud and Chinelatto, 1983; Karpe and Ruckenstein, 1990). Contrary to the case of the anionic reverse micelles, the water pool inside the cationic reverse micelles should be slightly more basic than the bulk aqueous phase. Hence, based on the assumptions that the pH inside the reverse micelles is close to the pH in the bulk aqueous phase and the charge number of the amino acids is determined by the aqueous phase pH, a t the pH of the aqueous phase of -12.5 the concentration of the bound and free H+ ions inside reverse micelles can be neglected and the total concentration of the bound and free OH- ions in the reverse micelles can be calculated by the condition of electric neutrality. The small effect caused by the concentration of the ionic species a t the interface between aqueous and organic phases was neglected. 1. Partition Coefficient of Amino Acids. It was shown experimentally that the three amino acids did not dissolve in the organic phase without DODMAC (Wang, 1994). Since ionized amino acids were extracted into the organic phase only when reverse micelles were present, we believe that the amino acid is located inside the reverse micelles. Hence, the partition coefficient of an amino acid, KA, is defined as the ratio of the concentration of amino acid in the water pool inside the reverse micelles, CA~P, to the concentration of amino acid in the equilibrium aqueous phase, C A ~ ~ :
Figure 2 shows the partition coefficients of the amino acids as a function of the pH of the equilibrium aqueous phase. The partition coefficients of aspartic acid and glutamic acid at pH values between 6 and 8 are independent of pH due to the charge since both amino acids carry a charge of -1. The partition coefficients of these amino acids increase significantly at pH values between 8 and 11,where both amino acids change from
a
20
0
0
50
100
150
200
Initial Aqueous NaCl Concentration (mM) Figure 3. Effect of initial aqueous NaCl concentration on the partition coefficient of glutamic acid (initial organic, 100 mM DODMAC, 250 mM 1-decanol; initial aqueous, 5, 10, or 20 mM Glu; pH 12.5.
one negative charge to two negative charges. The amino acids with a charge of -2 have strong potential to replace the C1- counterions of DODMAC in the reverse micelles. At pH higher than 12, the partition coefficients of aspartic acid and glutamic acid decrease rapidly with increasing pH as OH- ions compete with the amino acid ions as the counterions for DODMAC. It is evident that the competition of the ions to participate as anions of DODMAC depends not only on their charge but also on their size. The partition coefficient of threonine, at pH values between 8 and 10.7,increases due to the change in charge of threonine from 0 to -1, and decreases at pH larger than 11 due to the strong competition from OH- ions. The partition coefficients of all three amino acids are almost the same at the same charged state of -1, which occurs at pH -7 for aspartic and glutamic-acids and a t pH -10.7 for threonine. In general, the partition coefficients of aspartic and glutamic acids, as a function of pH, fall on a common curve and they are much higher than the partition coefficient of threonine, at equal pH. These results show that the electrostatic force is the main driving force for the extraction of amino acids by reverse micelles. Using small-angle neutron scattering (SANS) for the study of protein extraction, Sheu et al. (1986) also found that the driving force for protein extraction was reduced with the decrease of the attractive electrostatic force between the protein and the charged wall of the reverse micelles. Based on the assumption that the pK values of amino acids inside the reverse micelles are same as those in the bulk water (Smith and Luisi, 1980; El Seoud and Chinelatto, 1983; Karpe and Ruckenstein, 19901, the partition coefficients of the amino acids, as shown in Figure 2, indicate that the pH inside the reverse micelles is close t o the pH in the bulk aqueous phase. Figure 3 shows the partition coefficient of glutamic acid as a function of the initial NaCl concentration in the aqueous phase for three initial glutamic acid concentrations. The partition coefficient decreases rapidly with initial NaCl concentration. Glutamic acid, which has two negative charges in the aqueous phase, replaces the C1- counterions of DODMAC in the reverse micelles. The addition of NaCl may have the following effects: (i) The increase of the aqueous C1- concentration reduces the driving force for the transfer of the C1counterions of DODMAC from the reverse micelles to the aqueous phase. (ii) The decrease of the water uptake reduces the dielectric constant (Leodidis and Hatton, 1989; Karpe and Ruckenstein, 1991) and increases the C1- concentration inside the reverse mi-
602 Ind. Eng. Chem. Res., Vol. 34, No. 2, 1995 140
20mM IOmM 5mM .f
Q
100
Glu 0
100
60
20
n 100
200
0
300
100
celles, thus shifting the dissociation equilibrium of DODMAC toward the undissociated form (Wang et al., 1994). The increase of undissociated DODMAC reduces the number of the exchangeable C1-ions inside the reverse micelles. (iii) In an effect opposite to (ii), a decrease in the water uptake increases the concentration of amino acid inside the reverse micelles. (iv) The salting-out of DODMAC from the aqueous phase (Wang et al., 1994; Wang, 1994) increases the concentration of DODMAC in the reverse micelles. The decrease of the partition coefficient of glutamic acid shown in Figure 3 suggests that effects i and ii are dominant. At 200 mM NaC1, the effects of NaCl dominate the amino acid extraction, and the partition coefficients are independent of the initial concentrations of the amino acid. When no NaCl is added to the initial aqueous phase, the partition coefficients decrease, almost proportionally, with an increase in the initial glutamic acid concentration, thus indicating that the number of the exchangeable C1- counterions of DODMAC in the reverse micelles is almost constant. Figure 4 shows that the partition coefficients of an amino acid are a function only of the equilibrium ionic strength and are independent of the initial concentration of the amino acid in the aqueous phase. The ionic strength is defined in molarity units (Krei and Hustedt, 1992):
where zi is the ionic valence and Ci is the molarity of the ionic species i measured in the aqueous phase. The partition coefficients of aspartic and glutamic acids are much higher than that of threonine because amino acids with two negative charges have stronger potential than amino acids with one negative charge to replace the C1counterions of DODMAC inside the reverse micelles. Although both aspartic and glutamic acids have the same charged state, the partition coefficient of aspartic acid is slightly higher because it has one CH2 group less and thus its volume is smaller than the volume of glutamic acid. Figure 5 shows that, at the same equilibrium ionic strength, the partition coefficient of aspartic acid decreases with an increase of the DODMAC concentration in the organic phase. This is due to the predominance of effect iii over effect ii. In this case, the water uptake increases -4 times while the DODMAC concentration increases only 2 times (Wang, 1994).
300
Equilibrium Ionic Strength (mM)
Equilibrium Ionic Strength (mM)
Figure 4. Effect of equilibrium ionic strength on the partition coefficients of aspartic acid, glutamic acid, and threonine: initial organic, 100 mM DODMAC, 250 mM 1-decanol; initial aqueous, 5, 10, or 20 mM amino acid; pH 12.5 for Asp and Glu, pH 12.6 for Thr.
200
Figure 5. Effect of equilibrium ionic strength on the partition coefficient of aspartic acid initial organic, 100 or 200 mM DODMAC, 250 mM 1-decanol; initial aqueous, 5 or 20 mM amino acid: pH 12.5).
-
0.6
(II
6
8
10
12
Equilibrium pH Figure 6. Effect of equilibrium aqueous pH on the selectivities of amino acids relative to C1: initial organic, 200 mM DODMAC, 250 mM 1-decanol; initial aqueous, 5 mM amino acid, 51.2 mM NaC1.
2. Selectivityof Amino Acid Relative to C1. The ionic species in the reverse micelles are only partly dissociated as confirmed by Wong et al. (Wong et al., 19771, who reported that the degree of dissociation of Aerosol-OT was 28%. Since our organic phase containing 250 mM decanol as cosurfactant does not favor the migration of the undissociated DODMAC to the bulk organic phase, it is assumed that all the DODMAC in the organic phase resides in the interfacial region of the reverse micelles. Hence, the selectivity of amino acid relative to C1, a, can be defined as the ratio of the partition coefficient of the amino acid, KA, to the partition coefficient of the C1, Kcl:
a = KA/Kcl
(3)
Kcl= CClwP/Cclaq
(4)
where
The C1 in the water pool may be present as ions (Cl-) or associated with the head groups of DODMAC in its undissociated form. Figure 6 shows the selectivity of the three amino acids relative to C1. For pH
