Aqueous Two-Phase Electrophoresis for Separation of Amino Acids

中国科技论文在线 http://www.paper.edu.cn

Separation and Puriﬁcation Technology 21 (2001) 197–203 www.elsevier.com/locate/seppur

Aqueous two-phase electrophoresis for separation of amino acids

S.L. Zhai, G.S. Luo *, J.G. Liu

Department of Chemical Engineering, Tsinghua Uni6ersity, Beijing 100084, 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. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Aqueous two-phase system; Electrophoresis; Amino acids

1. Introduction the phase interface is imposed on the system, so oppositely charged particles should move into dif- Two-phase electrophoresis (TPE), coupling tra- ferent phases. With the migration of the charged ditional extraction with electrophoresis, is a novel particles, the original thermodynamics of amino separation method [1]. TPE technique is similar to acids partitioning balance can be changed and electrophoresis and electrodialysis on the one hence the separation is affected [3,4]. With these hand, and to traditional extraction on the other. improvements, TPE provides stability against In this technique, there are two distinct liquid convection mixing and facilitates product to be phases within the separation device to nullify the isolated. harmful effects of convection. One of the phases Aqueous two-phase systems, ﬁrst developed by contains the mixture to be separated and the Albertsson [5], are interesting from both a theo- other acts as a solvent to remove the components retical and a practical point of view. They can be separated [2]. An electric ﬁeld perpendicular to obtained by mixing suitable concentrations of solutions of two different polymers. Unlike the * Corresponding author. Fax: +86-10-62770304. aqueous/organic phase systems of traditional sol- E-mail address: [email protected] (G.S. Luo). vent extraction, both phases of these aqueous

198 S.L. Zhai et al. / Separation/Purification Technology 21 (2001) 197–203 two-phase systems are predominantly water (usu- 2. Materials and methods ally 80–85%), so they are compatible with amino acids, proteins and other solutes of biological Phenylalanine and tryptophan were purchased origin. Since the partition coefficients of amino from Chinese Medicine Company, 6000 average acids are usually not equal to 1 [6–8], i.e. the molecular weight PEG was from Tianjin Xinya solutes are unequally partitioned in the two Industry and Trade, and 185 000 average molecu- phases, the system has great advantage in separat- lar weight dextran was obtained from Sigma. ing mixtures of biomolecules [9,10]. Deionized water was prepared in our laboratory. Aqueous two-phase electrophoresis (ATPE), The concentration of amino acids was measured just like its name, integrates aqueous two-phase by u.v. detection with an HP8452 u.v./vis separation with electrophoresis. Although the two spectrophotometer. phases are electrically conductive, there have been The experiment apparatus is shown in Fig. 1. few applications with electric field imposed on The main equipment, a U-tube, is made of glass these systems. Brooks and Bamberger [11] first and its inner diameter is 10 mm. Using a U-tube, applied an electric field to an aqueous two-phase rather than a straight tube, could avoid the un- system in an attempt to improve the rate of the wanted effects of convection mixing, brought extraction process by speeding the phase settling about by electrolysis gas bubbles. In order to step. Levine and Bier [12] have studied the elec- maintain the experiment temperature at 25°C, a trophoretic mobility of a protein in an aqueous water-bath is used to remove the heat produced two-phase system by using a U-tube electrophore- by the electrode reactions. The two electrodes are sis device, and they noted an impediment to elec- made of platinum wire 0.5 mm in diameter, and tro-separations by applying a field to the system. the distance between the two electrodes is 10 cm. Marando and Clark [13] have used the dextran- The volume ratio (bottom phase/top phase) is 3. polyethylene glycol-water system to separate mix- The electrodes are inserted into different phases. tures of hemoglobin and albumin. The The electric field strength is determined by the experimental result shows a significant improve- output voltage of the electrophoresis instrument ment compared with the separation obtained by DYY-III. partitioning in the same two-phase system with no For each experimental run, the amino acids are applied field under the same conditions. Investiga- first separated by traditional solvent extraction, tions on the separation of the small biomolecules i.e. for the partition of the solutes in the aqueous such as amino acids by aqueous two-phase elec- two-phase system by mixing well and allowing trophoresis have not been reported up to now. enough settling time with no electric field applied Amino acids are amphoteric and have net until an equilibrium state is reached. The concen- charges when pH of the solution is not equal to trations of amino acids in the top phase were 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 dextran- polyethylene glycol-water system. The influence of factors such as separation time, field strength, initial solute concentration and two-phase inter- Fig. 1. Intermittent two-phase electrophoresis apparatus. 1. face are studied in intermittent separation equip- Water-bath; 2. Electrophoresis appartus; 3. U-tube elec- ment. The principle of separating amino acids by trophoresis equipment; 4. Electrode; 5. Voltmeter; 6. Am- ATPE is discussed at the same time. peremeter. 中国科技论文在线 http://www.paper.edu.cn

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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 be- fore 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 Fig. 2. Influence of time on concentration. other. The electric field is applied as soon as the system is loaded. Under a specified voltage and concentrations of phenylalanine or tryptophan of different times, as much as possible of the top different times were analyzed. The results of re- phase is withdrawn by a syringe and then ana- covery ratio and mass flux can be calculated by lyzed by u.v. detection. The influence of time on the material balance method. The influence of the recovery ratio and the mass transfer flux can time on the separation of amino acids such as be calculated by the concentration of different phenylalanine or tryptophan can be obtained. times. The recovery ratio is defined as the percent To ascertain the influence of the direction of the of the total amino acid in the feed recovered in electric field on the movement of the amino acids, the extract phase. Mass flux is also related to the inverse electric fields were applied in the system of top phase concentration, C: phenylalanine or tryptophan. That is, the anode Q=DC×V×M (1) was inserted in the top phase of phenylalanine system while in the bottom phase of tryptophan D where C is the increment of amino acid concen- system. Since the phenylalanine and tryptophan tration of extract phase caused by electrophoresis, solutions’ pH values were 6.66 and 6.72, respec- V is the volume of the extract phase, and M is the tively, both of them larger than their isoelectric molecular weight. points, phenylalanine and tryptophan were nega- The influences of other factors such as voltage, tively charged in their solutions. Due to the differ- initial solute concentration and different contact ent choice of the external electric field direction, areas of the two phases can be obtained by simi- phenylalanine and tryptophan moved apart. larly analyzing the concentration of the top phase. Therefore, as shown in Fig. 2, the concentration of phenylalanine increases while that of tryp- 3. Results and discussion tophan decreases with increasing time. Figs. 2–4 show that the separation is obviously 3.1. Influence of time improved with increasing time. More time in the applied field allows more of the charged amino To research the effect of ATPE, the aqueous acids to move across the phase interface, and two-phase system of phenylalanine or tryptophan hence for a given velocity on each amino acid was initially separated by conventional extraction, molecule, a longer time would allow more amino and then was subjected to a specified constant acids to move far enough to cross the interface. voltage for different times. The initial top phase In these figures, when time is zero, the concen- concentrations of phenylalanine and tryptophan tration, recovery and mass flux are those obtained were 0.00324 and 0.001318 mol l−1, respectively. by conventional extraction, which has no electric With other conditions unchanged, the top phase field applied. 中国科技论文在线 http://www.paper.edu.cn

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Fig. 5. Influence of voltage on concentration. Fig. 3. Influence of time on recovery. 3.2. Influence of electric field strength The mass fluxes of phenylalanine and tryptophan are increased with the increasing experi- Similarly, the aqueous two-phase system of mental time. At the beginning, the top phase phenylalanine or tryptophan with the same con- concentration, the recovery ratio and mass flux centration was initially separated by conventional vary rapidly. Gradually the velocity of change extraction, and then was subjected to different slows down. The same trend can be seen from the voltages for the same time. The initial top phase following experimental results. It is estimated the concentrations of phenylalanine and tryptophan main reason for this is that the amino acids were still 0.00324 and 0.001318 mol l−1, respec- having crossed the interface stay mainly near the tively. Inverse electric fields were applied in the interface and hence the concentration of amino system of phenylalanine or tryptophan. The an- acids near the interface increases with time. So an ode was inserted in the top phase of phenylalanine inverse electric field will form with increasing system while in the bottom phase of tryptophan time, which will counteract the migration of amino acids. Furthermore, the higher concentra- system. Since both the amino acids were nega- tion near the interface of the top phase facilitates tively charged in the solutions, phenylalanine and the inverse concentration diffusion, and hence tryptophan moved apart. there are less amino acids across the interface. With other conditions unchanged, the top Therefore, the variance of the top phase concen- phase concentrations under different electric field tration, recovery ratio and total mass flux are strengths were analyzed. The results of recovery decreased. 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 Fig. 4. Influence of time on mass flux. impetus for amino acids to move in each phase is 中国科技论文在线 http://www.paper.edu.cn

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Fig. 6. Influence of voltage on recovery. Fig. 8. Influence of initial concentration on top phase concen- also increased. Consequently, there are more so- tration. lutes transferring across the interface and the recovery ratio is improved. that caused by increasing the initial solute concentration, the top phase concentrations of pheny- 3.3. Influence of initial solute concentration lalanine or tryptophan increase. From Fig. 9, it can be seen that the recovery ratios of the amino With the other experiment conditions un- acids are not linear with the increasing initial changed, the top phase concentrations of different solute concentration. The recovery ratio trend initial solute concentrations were analyzed. The increases in the beginning and decreases with the influence of the initial solute concentration on the increasing initial solute concentration. The expla- separation effects of phenylalanine and tryp- nation for this phenomenon is presumed to be as tophan can be obtained. follows. When the initial concentration is compar- Figs. 8 and 10 show that the top phase concen- atively low, the greater initial solute concentration trations and the mass fluxes of phenylalanine and can cause a larger electric current, which will tryptophan increase with an increase of the initial result in more amino acids participating in the solute concentration. This is because there are far separation process. Therefore, the recovery ratios more solutes to transfer across the interface. Since of the amino acids are improved in the beginning. an increase in the amount of amino acids trans- When the initial concentrations continue to in- ferred from top phase to bottom phase is less than crease, 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. 中国科技论文在线 http://www.paper.edu.cn

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Fig. 12. Influence of contact area on recovery. Fig. 10. Influence of initial concentration on mass flux. 4. Conclusion Consequently, the proportions of amino acids transferring across the interface decrease, and the Our research is a first attempt to study factors recovery ratios are reduced. that influence the transport of small amphoteric biomolecules across the interface of an aqueous two-phase system with an imposed electric field. 3.4. Influence of contact area The main influence factors studied are time, electric field strength, initial concentration and con- With the other experiment conditions un- tact area. The results show that the separation changed, U-tubes with different sectional areas effects of phenylalanine and tryptophan are obvi- were used and the top phase concentrations were ously improved by ATPE, compared with analyzed. The influence of the contact area on the aqueous two-phase partitioning. The conclusion is separation effect of phenylalanine and tryptophan drawn that amphoteric particles can be directed to can be obtained. the phase required by changing the direction of Figs. 11–13 show that the recovery ratio and external electric field. More than 90% of pheny- the mass flux of tryptophan increase with in- lalanine can be obtained by prolonging time and creased contact area. The increase in the contact increasing the electric field strength. The mass area results in a larger mass transfer surface and transfer can be further improved by changing the less resistance of the solution, which results in a operating conditions. It is predicted that the com- larger current. More amino acids can cross the plete separation of amino acids can be achieved interface at the same time, and therefore the mass by adjusting the phase volume ratio and other transfer effect is improved. influencing factors.

Fig. 11. Influence of contact area on concentration. Fig. 13. Influence of contact area on mass flux. 中国科技论文在线 http://www.paper.edu.cn

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