Aqueous two-phase electrophoresis for separation of amino acids

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1 Separation and Purification Technology 21 (2001) Aqueous two-phase electrophoresis for separation of amino acids S.L. Zhai, G.S. Luo *, J.G. Liu Department of Chemical Engineering, Tsinghua Uni ersity, Beijing , PR China Received 10 August 1999; received in revised form 16 May 2000; accepted 18 May 2000 Abstract Two-phase electrophoresis, coupling traditional solvent extraction with electrophoresis, is a novel separation technique. Being bio-compatible, aqueous two-phase electrophoresis provides a successful method for separating mixtures of biomolecules. In this work, the separation of amino acids by aqueous two-phase electrophoresis was examined with dextran-polyethylene glycol-water as a working system. The influences of separation time, field strength, initial solute concentration and two-phase contact area were studied in intermittent separation equipment. The experiment results show that aqueous two-phase electrophoresis is an effective separation technique for amino acids. With time, field strength, and contact area increasing, mass fluxes across the interface are increased quickly Elsevier Science B.V. All rights reserved. Keywords: Aqueous two-phase system; Electrophoresis; Amino acids 1. Introduction Two-phase electrophoresis (TPE), coupling traditional extraction with electrophoresis, is a novel separation method [1]. TPE technique is similar to electrophoresis and electrodialysis on the one hand, and to traditional extraction on the other. In this technique, there are two distinct liquid phases within the separation device to nullify the harmful effects of convection. One of the phases contains the mixture to be separated and the other acts as a solvent to remove the components separated [2]. An electric field perpendicular to * Corresponding author. Fax: address: gsluo@mail.tsinghua.edu.cn (G.S. Luo). the phase interface is imposed on the system, so oppositely charged particles should move into different phases. With the migration of the charged particles, the original thermodynamics of amino acids partitioning balance can be changed and hence the separation is affected [3,4]. With these improvements, TPE provides stability against convection mixing and facilitates product to be isolated. Aqueous two-phase systems, first developed by Albertsson [5], are interesting from both a theoretical and a practical point of view. They can be obtained by mixing suitable concentrations of solutions of two different polymers. Unlike the aqueous/organic phase systems of traditional solvent extraction, both phases of these aqueous /01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S (00) 转载

2 198 S.L. Zhai et al. / Separation/Purification Technology 21 (2001) two-phase systems are predominantly water (usually 80 85%), so they are compatible with amino acids, proteins and other solutes of biological origin. Since the partition coefficients of amino acids are usually not equal to 1 [6 8], i.e. the solutes are unequally partitioned in the two phases, the system has great advantage in separating mixtures of biomolecules [9,10]. Aqueous two-phase electrophoresis (ATPE), just like its name, integrates aqueous two-phase separation with electrophoresis. Although the two phases are electrically conductive, there have been few applications with electric field imposed on these systems. Brooks and Bamberger [11] first applied an electric field to an aqueous two-phase system in an attempt to improve the rate of the extraction process by speeding the phase settling step. Levine and Bier [12] have studied the electrophoretic mobility of a protein in an aqueous two-phase system by using a U-tube electrophoresis device, and they noted an impediment to electro-separations by applying a field to the system. Marando and Clark [13] have used the dextranpolyethylene glycol-water system to separate mixtures of hemoglobin and albumin. The experimental result shows a significant improvement compared with the separation obtained by partitioning in the same two-phase system with no applied field under the same conditions. Investigations on the separation of the small biomolecules such as amino acids by aqueous two-phase electrophoresis have not been reported up to now. Amino acids are amphoteric and have net charges when ph of the solution is not equal to their isoelectric points. Charged amino acids can be directed into the controlled phase by changing the external electric field direction, and the effect of mass transfer can be strengthened greatly. So it is of great importance to study separation of amino acids by ATPE. In the present study, the separation of mixtures by this new technique is investigated in dextranpolyethylene glycol-water system. The influence of factors such as separation time, field strength, initial solute concentration and two-phase interface are studied in intermittent separation equipment. The principle of separating amino acids by ATPE is discussed at the same time. 2. Materials and methods Phenylalanine and tryptophan were purchased from Chinese Medicine Company, 6000 average molecular weight PEG was from Tianjin Xinya Industry and Trade, and average molecular weight dextran was obtained from Sigma. Deionized water was prepared in our laboratory. The concentration of amino acids was measured by u.v. detection with an HP8452 u.v./vis spectrophotometer. The experiment apparatus is shown in Fig. 1. The main equipment, a U-tube, is made of glass and its inner diameter is 10 mm. Using a U-tube, rather than a straight tube, could avoid the unwanted effects of convection mixing, brought about by electrolysis gas bubbles. In order to maintain the experiment temperature at 25 C, a water-bath is used to remove the heat produced by the electrode reactions. The two electrodes are made of platinum wire 0.5 mm in diameter, and the distance between the two electrodes is 10 cm. The volume ratio (bottom phase/top phase) is 3. The electrodes are inserted into different phases. The electric field strength is determined by the output voltage of the electrophoresis instrument DYY-III. For each experimental run, the amino acids are first separated by traditional solvent extraction, i.e. for the partition of the solutes in the aqueous two-phase system by mixing well and allowing enough settling time with no electric field applied until an equilibrium state is reached. The concentrations of amino acids in the top phase were Fig. 1. Intermittent two-phase electrophoresis apparatus. 1. Water-bath; 2. Electrophoresis appartus; 3. U-tube electrophoresis equipment; 4. Electrode; 5. Voltmeter; 6. Amperemeter.

3 S.L. Zhai et al. / Separation/Purification Technology 21 (2001) analyzed with the time of mixing and settling, and it was found that the concentrations were not changed after 12-h mixing and 2-day settling. So, in the traditional extraction experiment in the absence of an electric field, the time of mixing is 12 h and the setting time is 2 days. The clarified top phase and bottom phase are isolated by an extraction funnel. The recovery of the amino acids by conventional extraction can be obtained by analyzing the concentration of the top phase before electrophoresis. Then some (12 ml) of the bottom phase is introduced into one leg of the U-tube, and 4-ml top phase is loaded into the other. The electric field is applied as soon as the system is loaded. Under a specified voltage and different times, as much as possible of the top phase is withdrawn by a syringe and then analyzed by u.v. detection. The influence of time on the recovery ratio and the mass transfer flux can be calculated by the concentration of different times. The recovery ratio is defined as the percent of the total amino acid in the feed recovered in the extract phase. Mass flux is also related to the top phase concentration, C: Q= C V M (1) where C is the increment of amino acid concentration of extract phase caused by electrophoresis, V is the volume of the extract phase, and M is the molecular weight. The influences of other factors such as voltage, initial solute concentration and different contact areas of the two phases can be obtained by similarly analyzing the concentration of the top phase. 3. Results and discussion 3.1. Influence of time To research the effect of ATPE, the aqueous two-phase system of phenylalanine or tryptophan was initially separated by conventional extraction, and then was subjected to a specified constant voltage for different times. The initial top phase concentrations of phenylalanine and tryptophan were and mol l 1, respectively. With other conditions unchanged, the top phase Fig. 2. Influence of time on concentration. concentrations of phenylalanine or tryptophan of different times were analyzed. The results of recovery ratio and mass flux can be calculated by the material balance method. The influence of time on the separation of amino acids such as phenylalanine or tryptophan can be obtained. To ascertain the influence of the direction of the electric field on the movement of the amino acids, inverse electric fields were applied in the system of phenylalanine or tryptophan. That is, the anode was inserted in the top phase of phenylalanine system while in the bottom phase of tryptophan system. Since the phenylalanine and tryptophan solutions ph values were 6.66 and 6.72, respectively, both of them larger than their isoelectric points, phenylalanine and tryptophan were negatively charged in their solutions. Due to the different choice of the external electric field direction, phenylalanine and tryptophan moved apart. Therefore, as shown in Fig. 2, the concentration of phenylalanine increases while that of tryptophan decreases with increasing time. Figs. 2 4 show that the separation is obviously improved with increasing time. More time in the applied field allows more of the charged amino acids to move across the phase interface, and hence for a given velocity on each amino acid molecule, a longer time would allow more amino acids to move far enough to cross the interface. In these figures, when time is zero, the concentration, recovery and mass flux are those obtained by conventional extraction, which has no electric field applied.

4 200 S.L. Zhai et al. / Separation/Purification Technology 21 (2001) Fig. 3. Influence of time on recovery. The mass fluxes of phenylalanine and tryptophan are increased with the increasing experimental time. At the beginning, the top phase concentration, the recovery ratio and mass flux vary rapidly. Gradually the velocity of change slows down. The same trend can be seen from the following experimental results. It is estimated the main reason for this is that the amino acids having crossed the interface stay mainly near the interface and hence the concentration of amino acids near the interface increases with time. So an inverse electric field will form with increasing time, which will counteract the migration of amino acids. Furthermore, the higher concentration near the interface of the top phase facilitates the inverse concentration diffusion, and hence there are less amino acids across the interface. Therefore, the variance of the top phase concentration, recovery ratio and total mass flux are decreased. Fig. 4. Influence of time on mass flux. Fig. 5. Influence of voltage on concentration Influence of electric field strength Similarly, the aqueous two-phase system of phenylalanine or tryptophan with the same concentration was initially separated by conventional extraction, and then was subjected to different voltages for the same time. The initial top phase concentrations of phenylalanine and tryptophan were still and mol l 1, respectively. Inverse electric fields were applied in the system of phenylalanine or tryptophan. The anode was inserted in the top phase of phenylalanine system while in the bottom phase of tryptophan system. Since both the amino acids were negatively charged in the solutions, phenylalanine and tryptophan moved apart. With other conditions unchanged, the top phase concentrations under different electric field strengths were analyzed. The results of recovery ratio and mass flux were also calculated by the material balance method. The influence of the electric field strength on the separation effects of phenylalanine and tryptophan can be obtained. Figs. 5 7 show that the top phase concentration of phenylalanine is increased and that of tryptophan is obviously decreased, and the recovery ratio and the whole mass flux of both amino acids are increased with increased voltage. The reason for this phenomenon is that the larger voltage leads to the stronger force of the charged particles in electric field, and hence the solutes more easily overcome the impedance of the interface to enter into another phase. Meanwhile the impetus for amino acids to move in each phase is

5 S.L. Zhai et al. / Separation/Purification Technology 21 (2001) Fig. 6. Influence of voltage on recovery. also increased. Consequently, there are more solutes transferring across the interface and the recovery ratio is improved Influence of initial solute concentration With the other experiment conditions unchanged, the top phase concentrations of different initial solute concentrations were analyzed. The influence of the initial solute concentration on the separation effects of phenylalanine and tryptophan can be obtained. Figs. 8 and 10 show that the top phase concentrations and the mass fluxes of phenylalanine and tryptophan increase with an increase of the initial solute concentration. This is because there are far more solutes to transfer across the interface. Since an increase in the amount of amino acids transferred from top phase to bottom phase is less than Fig. 8. Influence of initial concentration on top phase concentration. that caused by increasing the initial solute concentration, the top phase concentrations of phenylalanine or tryptophan increase. From Fig. 9, it can be seen that the recovery ratios of the amino acids are not linear with the increasing initial solute concentration. The recovery ratio trend increases in the beginning and decreases with the increasing initial solute concentration. The explanation for this phenomenon is presumed to be as follows. When the initial concentration is comparatively low, the greater initial solute concentration can cause a larger electric current, which will result in more amino acids participating in the separation process. Therefore, the recovery ratios of the amino acids are improved in the beginning. When the initial concentrations continue to increase, the inverse diffusions of amino acids increase with the increasing initial concentration. Fig. 7. Influence of voltage on mass flux. Fig. 9. Influence of initial concentration on recovery.

6 202 S.L. Zhai et al. / Separation/Purification Technology 21 (2001) Fig. 10. Influence of initial concentration on mass flux. Consequently, the proportions of amino acids transferring across the interface decrease, and the recovery ratios are reduced Influence of contact area With the other experiment conditions unchanged, U-tubes with different sectional areas were used and the top phase concentrations were analyzed. The influence of the contact area on the separation effect of phenylalanine and tryptophan can be obtained. Figs show that the recovery ratio and the mass flux of tryptophan increase with increased contact area. The increase in the contact area results in a larger mass transfer surface and less resistance of the solution, which results in a larger current. More amino acids can cross the interface at the same time, and therefore the mass transfer effect is improved. Fig. 12. Influence of contact area on recovery. 4. Conclusion Our research is a first attempt to study factors that influence the transport of small amphoteric biomolecules across the interface of an aqueous two-phase system with an imposed electric field. The main influence factors studied are time, electric field strength, initial concentration and contact area. The results show that the separation effects of phenylalanine and tryptophan are obviously improved by ATPE, compared with aqueous two-phase partitioning. The conclusion is drawn that amphoteric particles can be directed to the phase required by changing the direction of external electric field. More than 90% of phenylalanine can be obtained by prolonging time and increasing the electric field strength. The mass transfer can be further improved by changing the operating conditions. It is predicted that the complete separation of amino acids can be achieved by adjusting the phase volume ratio and other influencing factors. Fig. 11. Influence of contact area on concentration. Fig. 13. Influence of contact area on mass flux.

7 S.L. Zhai et al. / Separation/Purification Technology 21 (2001) Acknowledgements We acknowledge the support of the National Science Foundation of China and Tsinghua University Science Foundation for this work. References [1] G.S. Luo, M.J. Yu, W.B. Jiang, S.L. Zhu, Y.Y. Dai, Electro-extraction separation of acid chrome blue K, in: Y. Qian, H.Q. Chen (Eds.), Advances in Environmental Engineering and Chemical Engineering, Guangzhou, PR China, 1997, pp [2] K.J. Ptasinski, P.J.A.M. Kerkhof, Electric field driven separations: phenomena and applications, Sep. Sci. Technol. 27 (8 9) (1992) [3] W.M. Clark, Electrophoresis-enhanced extractive separation, Chemtech July (1992) [4] J. Stichlmair, J. Schmidt, R. Proplesch, Electro-extraction: novel separation technique, Chem. Eng. Sci. 47 (12) (1992) [5] P.A. Albertsson, Partition of Cell Particles and Macromolecules, Wiley, New York, 1986, p [6] T. Furuya, Y. Iwai, Y. Tanaka, H. Uchida, S. Yamada, Y. Arai, Measurement and correlation of liquid-liquid equilibria for dextran-poly (ethylene glycol)-water aqueous two-phase systems at 20.0 C, Fluid Phase Equilibria 103 (1995) [7] F. Zhou, S. Yamada, K. Kakisaka, T. Furuya, Y. Iwai, Y. Arai, Measurement of partition coefficients of amino acids in dextran+poly(ethylene glycol)+water aqueous two-phase systems at 20.0 C, Solvent Extraction Res. Dev. Jpn. 3 (1996) [8] F. Zhou, K. Kakisaka, T. Ishidao, Y. Iwai, Y. Arai, Measurement of partition coefficients of amino acids in dextran+poly(ethylene glycol)+water aqueous twophase systems, J. Chem. Eng. Jpn. 30 (2) (1997) [9] Z.Q. Zhu, Y.X. Guan, M. Li, Progress in extracting small molecular biomolecules by aqueous two-phase partitioning, Chem. Eng. Dev. 4 (1996) [10] C.W. Theos, W.M. Clark, Electroextraction two-phase electrophoresis, Appl. Biochem. Biotechnol. 154 (1995) [11] D.E. Brooks, S. Bamberger, in: G.E. Rindone (Ed.), Materials Processing in the Reduced Gravity Environment of Space, Elsevier, New York, 1982, pp [12] M.L. Levine, M. Bier, Electrophoretic transport of solutes in aqueous two-phase systems, Electrophoresis 11 (1990) [13] M.A. Marando, W.M. Clark, Two-phase electrophoresis of proteins, Sep. Sci. Technol. 28 (8) (1993)