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Aqueous Two-Phase Electrophoresis for Separation of Amino Acids

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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 so- lutions 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 1383-5866/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S1383-5866(00)00193-3 转载 中国科技论文在线 http://www.paper.edu.cn 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 S.L. Zhai et al. / Separation/Purification Technology 21 (2001) 197–203 199 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.
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