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A new technique for separation and purification of l -phenylalanine from fermentation liquid: Flotation complexation extraction
Separation and Purification Technology 63 (2008) 487–491
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Short communication
A new technique for separation and purification of l-phenylalanine from fermentation liquid: Flotation complexation extraction P.Y. Bi a , H.R. Dong a,∗ , H.B. Yu a , L. Chang b a b
Faculty of Science, Beijing University of Chemical Technology, Beijing 100029, PR China Qinghai Institute of Salt Lakes, Chinese Academy of Science, Xining Qinghai 810008, PR China
a r t i c l e
i n f o
Article history: Received 29 November 2007 Received in revised form 5 June 2008 Accepted 7 June 2008 Keywords: Flotation complexation extraction Solvent sublation Complexation extraction l-Phenylalanine
a b s t r a c t Combining solvent sublation with complexation extraction, flotation complexation extraction (FCE) is proposed for the first time. This new technique was used to separate and purify l-phenylalanine from fermentation liquid with good results. The effects of extraction solvent, pH of the solution, NaCl concentration, concentration of di-(2-ethylhexyl) phosphoric acid (D2EHPA) in organic phase, nitrogen flow rate and flotation time on the separation efficiency of l-phenylalanine were investigated in detail, and the optimal conditions for FCE were obtained. The flotation product from the fermentation liquid under optimal conditions, after back-extraction and re-crystallization, was characterized by FTIR and HPLC, and its purity was more than 98%. Furthermore, the mechanism of FCE was also primarily discussed. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Amino acids are very important biochemistry products. The key to producing them from fermentation liquid lies in separation and purification techniques and these have attracted much attention [1–4]. The separation of amino acids from fermentation liquid may encounter various problems. For example, amino acids are polar organic compounds, they cannot dissolve in any organic solvent, so the traditional solvent extraction and solvent sublation cannot be used to separate amino acids from fermentation liquid. At present, the ion exchange technique is widely used to separate and purify amino acids in industry, but it has some disadvantages, such as low recovery, low throughput, and is expensive, complicated, time consuming, etc. Recently, because of the application of di-(2-ethylhexyl) phosphoric acid (D2EHPA) in an emulsion liquid membrane [5] and the development of complexation extraction of polar organic compounds [6], amino acids can be separated and purified from the fermentation liquid by complexation extraction containing D2EHPA as a carrier or a catcher [7,8]. In this paper, the mass transfer mode of solvent sublation [9–11] was used in the D2EHPA complexation extraction of amino acids for the first time. In the process, the amino acids are floated on bubble surfaces to the top of the sample solution where they encounter a solvent layer containing D2EHPA; the amino acids can be caught by D2EHPA, and then dissolved in the organic phase. This mass transfer
∗ Corresponding author. Tel.: +86 10 64944151; fax: +86 10 64944151. E-mail address: [email protected] (H.R. Dong). 1383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2008.06.016
mode of bubble adsorption can overcome the emulsification under alkaline conditions in complexation extraction [12], and improve the separation efficiency. Moreover, the mass transfer mode of bubble adsorption is much better than that of mechanical vibration in solvent extraction. The new technique, which combines solvent sublation with complexation extraction, is neither solvent sublation nor complexation extraction, so has been named flotation complexation extraction (FCE) for the first time in this paper. This method has many advantages, such as simultaneous separation and purification, high concentration efficiency, very low wastage of organic solvent, etc. In this work, l-phenylalanine was selected as the model compound, and the distribution ratio of l-phenylalanine between organic phase and aqueous phase was used to optimize the parameters of the FCE. Under the selected optimal conditions, lphenylalanine was separated and purified from fermentation liquid by FCE, and characterized by FTIR and HPLC, and its purity was more than 98%. 2. Experimental 2.1. Reagents The purity of l-phenylalanine standard (Beijing Aoboxing Biotechnology Co. Ltd., China) was more than 99.5%. D2EHPA (Tianjin Jinke Fine Chemical Institute, China), n-octanol, n-hexane, ethanol, sodium acetate and sodium chloride were all of analytical grade (Beijing Chemical Factory, China). The acetonitrile was chro-
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P.Y. Bi et al. / Separation and Purification Technology 63 (2008) 487–491
matographic grade (Fisher, USA). The fermentation liquid was from Dr. Yan Liu and Prof. Tianwei Tan. It was produced by Escherichia coli fermentation method, and was mainly consisted of glucose (few), inorganic salt (few), Vitamin B1 (few), tyrosine (few), E. coli and its metabolites, and a lot of l-phenylalanine (more than 10 g/l). 2.2. Apparatus A Mettler Toledo 320-S pH meter (Mettler, Switzerland) was used to determine the pH of the solution. A U-3010 ultraviolet–visible spectrophotometer (Hitachi, Japan) was used to determine the absorbance of the aqueous phase. An AVATAR 370-FTIR spectrometer (Nicolet, USA) and an Agilent 1100 Series chromatograph (Agilent, USA) with Diamonsil TM C18 column (150 mm × 4.6 mm) were used to characterize the product obtained. A RE-522A rotary evaporator (Yarong, China) and an AB204-N electronic balance (Mettler) were also used. The FCE apparatus was similar as the one mentioned in the earlier reports [10,11]. Fig. 1. Effect of initial pH on FCE.
2.3. Procedure A 300-ml aliquot of the fermentation liquid was transferred into a 400-ml beaker, to which 3.5064 g NaCl was added. This solution was adjusted to pH 11 with NaOH solution, and then transferred to a flotation cell. The l-phenylalanine was floated by bubbling nitrogen gas at a flow rate of 40 ml/min from the bottom of the cell for 60 min, and extracted into 10.00 ml of n-hexane solution containing 80% D2EHPA (v/v) on the surface of the sample solution. The organic phase was transferred into a 100-ml separating funnel, and 10 ml of 0.1 M hydrochloric acid aqueous solution was added for back-extraction. The back-extraction was repeated three times, then the aqueous phase was collected and concentrated, and the product was obtained after re-crystallizing and drying. The product obtained was characterized by FTIR and HPLC. In the HPLC analysis [13], the mobile phase consisted of A and B solutions (92:8, v/v): A, 50 mM of sodium acetate solution (pH 6.5); B, acetonitrile. The flow rate was 1.0 ml/min, detection wavelength was 220 nm, and all chromatographic analysis were performed at room temperature. 3. Results and discussion 3.1. Optimization of the FCE parameters In this work, a standard solution containing 500 g/l of l-phenylalanine, and the distribution ratio of l-phenylalanine between organic phase and aqueous phase were used to optimize the parameters affecting the FCE efficiency of l-phenylalanine. The distribution ratio can be calculated using the following equation. D=
continue to increase the initial pH to 13, a lot of foam will appear on the top of the flotation cell, and the separation process will have to stop. When the initial pH ≤1, the distribution ratio is zero, because the l-phenylalanine cannot complex with D2EHPA. Therefore, the initial pH 11 can be selected as the optimal pH for the efficient FCE. 3.1.2. Effect of NaCl concentration The effect of NaCl concentration on the distribution ratio of l-phenylalanine is shown in Fig. 2. The maximum distribution ratio is observed when the concentration of NaCl is 0.2 M. Lowering the concentration of NaCl can reduce the dissolution of l-phenylalanine, and increase the separation efficiency. When the concentration of NaCl is more than 0.2 M, the distribution ratio decreases with increasing NaCl concentration, however, above 0.75 M, the distribution ratio decreases very slowly. So the optimal concentration of NaCl is 0.2 M. 3.1.3. Effect of concentration of D2EHPA in organic phase The effect of the concentration of D2EHPA in the organic phase on the distribution ratio of l-phenylalanine is shown in Fig. 3. It can be seen that the distribution ratio of l-phenylalanine increases with the increase in concentration of D2EHPA, however, the maximum distribution ratio is obtained when the concentration of D2EHPA is
30 × (A0 − At ) At
where D is the distribution ratio of l-phenylalanine between organic phase and aqueous phase, 30 the volume ratio of aqueous phase and organic phase, A0 the initial absorbance of aqueous phase at 258 nm and At is the absorbance of aqueous phase at time t. 3.1.1. Effect of initial pH D2EHPA was suitable for a non-polar solvent system [12], so n-octanol and n-hexane were selected as extraction solvents. An experiment was performed by changing the initial pH from 0 to 12 by adding suitable hydrochloric acid solution or sodium hydroxide solution. As shown in Fig. 1, the maximum distribution ratio is observed in the range initial pH 10–12, and n-octanol and n-hexane are both suitable for the FCE of l-phenylalanine. Although, we can
Fig. 2. Effect of NaCl concentration on FCE.
P.Y. Bi et al. / Separation and Purification Technology 63 (2008) 487–491
Fig. 3. Effect of concentration of D2EHPA in organic phase on FCE.
489
Fig. 5. Effect of back-extraction time on the extraction efficiency of back solvent extraction.
80% (v/v), and when the concentration is more than 80%, the distribution ratio decreases. Therefore, the concentration of D2EHPA in organic phase can be fixed at 80% in this work. 3.1.4. Effects of nitrogen flow rate and flotation time Fig. 4 shows the effects of nitrogen flow rate and flotation time on the distribution ratio of l-phenylalanine. The change in nitrogen flow rate can influence the velocity of mass transfer, but cannot influence the thermodynamic equilibrium. The higher flow rate can reduce the flotation time. But the large number of bubbles cannot rapidly burst at a higher flow rate, and they accumulate on the top of the flotation cell. As shown in Fig. 4, a nitrogen flow rate of 40 ml/min and a flotation time of 60 min can be selected. According to the above optimization experiments, the optimal conditions for l-phenylalanine FCE can be obtained, that is pH 11, 0.2 M NaCl concentration, n-hexane solution containing 80% D2EHPA (v/v) as organic phase, nitrogen flow rate of 40 ml/min and flotation time of 60 min. Under the optimization conditions, the distribution ratio between organic phase and aqueous phase reached 24–25, and it was better than the one in previous reports [2–8].
Fig. 4. Effects of nitrogen flow rate and flotation time on FCE.
Fig. 6. Microscope images of the final product and l-phenylalanine standard.
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P.Y. Bi et al. / Separation and Purification Technology 63 (2008) 487–491
Fig. 7. Infrared spectra of the final product and l-phenylalanine standard.
3.2. Back-extraction of l-phenylalanine It follows from Fig. 1 that l-phenylalanine can be released from organic phase to aqueous phase at pH ≤1. When 0.1 M hydrochloric acid aqueous solution was used for the back-extraction of l-phenylalanine, the curve of the back-extraction time vs. efficiency is shown in Fig. 5. It is clear that the back-extraction efficiency is more than 90% after back-extraction was repeated three times.
Fig. 8. Chromatograms of fermentation liquid, final product and l-phenylalanine standard.
over the range 14.69–235 g/ml. The regression equation was y = 1.34x − 0.32 (R2 = 0.9999), where y is the peak area and x is the concentration (g/ml), and this was used to determine the purities of l-phenylalanine obtained by the proposed method. The purity of l-phenylalanine obtained was 98.67%, and the R.S.D. was 2.24% (n = 5). 3.4. Mechanism of flotation complexation extraction
3.3. Separation and purification of l-phenylalanine from fermentation liquid The optimized experimental conditions were applied to a real fermentation sample to evaluate the efficiency of the separation and purification of l-phenylalanine by FCE. Under the optimal conditions, the target compound in the fermentation liquid was floated and extracted into n-hexane phase containing 80% D2EHPA, then the n-hexane phase was back-extracted with 0.1 M hydrochloric acid aqueous solution. The back-extraction was repeated three times, the aqueous phase was collected and concentrated, and the
product was obtained after re-crystallizing and drying. The product was of white acicular crystals, and similar to the l-phenylalanine standard (as shown in Fig. 6). Structural identification of the product was carried out by FTIR and HPLC (Figs. 7 and 8). Comparing the infrared spectra and chromatogram of l-phenylalanine standard with those of the product, it can be seen that they are both identical in peak positions, shape and number, so it can be concluded that the product was l-phenylalanine. The purity of the product was determined by HPLC with the external standard method. Under the selected chromatographic conditions [13], the retention time of l-phenylalanine was 3.52 min. The calibration curve for l-phenylalanine was obtained
Based on the technique described in this paper, it can be divided into three processes: (1) bubble mass transfer, (2) reaction between l-phenylalanine and D2EHPA, and (3) the complex compound dissolve in organic phase. In the process of the bubble mass transfer, the l-phenylalanine is adsorbed on the bubble surface, and carried to the top of flotation column with a layer of D2EHPA/n-hexane solution. After bubble rupturing, the aqueous layer around the bubble transforms to a small water droplet. It is well known that the l-phenylalanine cannot directly dissolve in the organic phase until react with D2EHPA [7,8] as follows:
In the equations, HP is D2EHPA. Then the complex compound can enter into the organic phase from the interface between aqueous phase and organic phase. The reaction process is very similar to the traditional complexation extraction. But, compared with the traditional complexation extraction under the same conditions, the concentration of lphenylalanine on the bubble surface is higher than the O/W interface. Furthermore, after bubble rupturing, the bubble surface transforms into the O/W interface, and the total area of the interface is rapidly reduced. However, the amount of l-phenylalanine on the interface is not reduced. In other words, the concentration of l-phenylalanine on the O/W interface is greatly increased, so
P.Y. Bi et al. / Separation and Purification Technology 63 (2008) 487–491
the reaction balance greatly turns to right. That is the basic reason why the distribution ratio of the FCE is higher than the traditional complexation extraction. 4. Conclusion l-Phenylalanine was successfully separated and purified by FCE from fermentation liquid, and the conception (FCE) was proposed for the first time. The experimental results prove that FCE is a good technique with high selectivity for separating and purifying amino acid from fermentation liquid. The new technique, with advantages of simultaneous separation and purification, excellent concentration efficiency, and very low wastage of organic solvent, has a considerable number of potential applications. Acknowledgements The authors are very much obliged to express their acknowledgement to Dr. Yan Liu and Prof. Tianwei Tan (Faculty of Life Science and Technology, Beijing University of Chemical Technology) for supplying l-phenylalanine fermentation liquid and analyzing the main components. References [1] M.P. Thien, T.A. Hatton, Liquid emulsion membrane and their application in biochemical processing, Sep. Sci. Technol. 23 (1988) 819–853.
491
[2] M.P. Thien, T.A. Hatton, D.I.C. Wang, Separation and concentration of amino acids using liquid emulsion membranes, Biotechnol. Bioeng. 32 (1988) 604– 615. [3] H. Itoh, M.P. Thien, T.A. Hatton, D.I.C. Wang, A liquid emulsion membrane process for the separation of amino acids, Biotechnol. Bioeng. 35 (1990) 853– 860. [4] M. Teramoto, T. Yamashiro, A. Inoue, Extraction of amino acids by emulsion liquid membranes containing di(2-ethylhexyl) phosphoric acid as a carrier biotechnology; coupled, facilitated transport; diffusion, J. Membr. Sci. 58 (1991) 11–32. [5] S.A. Hong, J.W. Yang, Process development of amino acid concentration by a liquid emulsion membrane technique, J. Membr. Sci. 86 (1994) 181–192. [6] Q.H. Shi, Y. Sun, L. Liu, S. Bai, Distribution behavior of amino acid extraction with di(2-ethylhexyl) phosphoric acid, Sep. Sci. Technol. 32 (1997) 2051– 2067. [7] Y.S. Liu, Y.Y. Dai, J.D. Wang, Distribution behavior of l-phenylalanine by extraction with di(2-ethylhexyl) phosphoric acid, Sep. Sci. Technol. 34 (1999) 2165–2176. [8] Y.S. Liu, Y.Y. Dai, J.D. Wang, Distribution behavior of l-phenylalanine by extraction with di(2-ethylhexyl) phosphoric acid, Sep. Sci. Technol. 35 (2000) 1439–1454. [9] Y.J. Lu, X.H. Zhu, A mathematical model of solvent sublation of some surfactants, Talanta 57 (2002) 891–898. [10] Q. Cheng, H.R. Dong, Solvent sublation using dithizone as a ligand for determination of trace elements in water samples, Microchim. Acta 150 (2005) 59– 65. [11] H.R. Dong, P.Y. Bi, S.H. Wang, Separation and enrichment of Baicalin in SBG by solvent sublation and its determination by HPLC and spectroscopy, Anal. Lett. 38 (2005) 257–270. [12] Y.Y. Dai, W. Qin, J. Zhang, Complexation Extraction Technique of Organic Compound, Chemical Industry Press, Beijing, 2003, p. 165, 180. [13] M.R. Amin, Y. Tomita, R. Onodera, Rapid determination of phenylalanine and its related compounds in rumen fluid by high-performance liquid chromatography, J. Chromatogr. B 663 (1995) 201–207.
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Short communication
A new technique for separation and purification of l-phenylalanine from fermentation liquid: Flotation complexation extraction P.Y. Bi a , H.R. Dong a,∗ , H.B. Yu a , L. Chang b a b
Faculty of Science, Beijing University of Chemical Technology, Beijing 100029, PR China Qinghai Institute of Salt Lakes, Chinese Academy of Science, Xining Qinghai 810008, PR China
a r t i c l e
i n f o
Article history: Received 29 November 2007 Received in revised form 5 June 2008 Accepted 7 June 2008 Keywords: Flotation complexation extraction Solvent sublation Complexation extraction l-Phenylalanine
a b s t r a c t Combining solvent sublation with complexation extraction, flotation complexation extraction (FCE) is proposed for the first time. This new technique was used to separate and purify l-phenylalanine from fermentation liquid with good results. The effects of extraction solvent, pH of the solution, NaCl concentration, concentration of di-(2-ethylhexyl) phosphoric acid (D2EHPA) in organic phase, nitrogen flow rate and flotation time on the separation efficiency of l-phenylalanine were investigated in detail, and the optimal conditions for FCE were obtained. The flotation product from the fermentation liquid under optimal conditions, after back-extraction and re-crystallization, was characterized by FTIR and HPLC, and its purity was more than 98%. Furthermore, the mechanism of FCE was also primarily discussed. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Amino acids are very important biochemistry products. The key to producing them from fermentation liquid lies in separation and purification techniques and these have attracted much attention [1–4]. The separation of amino acids from fermentation liquid may encounter various problems. For example, amino acids are polar organic compounds, they cannot dissolve in any organic solvent, so the traditional solvent extraction and solvent sublation cannot be used to separate amino acids from fermentation liquid. At present, the ion exchange technique is widely used to separate and purify amino acids in industry, but it has some disadvantages, such as low recovery, low throughput, and is expensive, complicated, time consuming, etc. Recently, because of the application of di-(2-ethylhexyl) phosphoric acid (D2EHPA) in an emulsion liquid membrane [5] and the development of complexation extraction of polar organic compounds [6], amino acids can be separated and purified from the fermentation liquid by complexation extraction containing D2EHPA as a carrier or a catcher [7,8]. In this paper, the mass transfer mode of solvent sublation [9–11] was used in the D2EHPA complexation extraction of amino acids for the first time. In the process, the amino acids are floated on bubble surfaces to the top of the sample solution where they encounter a solvent layer containing D2EHPA; the amino acids can be caught by D2EHPA, and then dissolved in the organic phase. This mass transfer
∗ Corresponding author. Tel.: +86 10 64944151; fax: +86 10 64944151. E-mail address: [email protected] (H.R. Dong). 1383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2008.06.016
mode of bubble adsorption can overcome the emulsification under alkaline conditions in complexation extraction [12], and improve the separation efficiency. Moreover, the mass transfer mode of bubble adsorption is much better than that of mechanical vibration in solvent extraction. The new technique, which combines solvent sublation with complexation extraction, is neither solvent sublation nor complexation extraction, so has been named flotation complexation extraction (FCE) for the first time in this paper. This method has many advantages, such as simultaneous separation and purification, high concentration efficiency, very low wastage of organic solvent, etc. In this work, l-phenylalanine was selected as the model compound, and the distribution ratio of l-phenylalanine between organic phase and aqueous phase was used to optimize the parameters of the FCE. Under the selected optimal conditions, lphenylalanine was separated and purified from fermentation liquid by FCE, and characterized by FTIR and HPLC, and its purity was more than 98%. 2. Experimental 2.1. Reagents The purity of l-phenylalanine standard (Beijing Aoboxing Biotechnology Co. Ltd., China) was more than 99.5%. D2EHPA (Tianjin Jinke Fine Chemical Institute, China), n-octanol, n-hexane, ethanol, sodium acetate and sodium chloride were all of analytical grade (Beijing Chemical Factory, China). The acetonitrile was chro-
488
P.Y. Bi et al. / Separation and Purification Technology 63 (2008) 487–491
matographic grade (Fisher, USA). The fermentation liquid was from Dr. Yan Liu and Prof. Tianwei Tan. It was produced by Escherichia coli fermentation method, and was mainly consisted of glucose (few), inorganic salt (few), Vitamin B1 (few), tyrosine (few), E. coli and its metabolites, and a lot of l-phenylalanine (more than 10 g/l). 2.2. Apparatus A Mettler Toledo 320-S pH meter (Mettler, Switzerland) was used to determine the pH of the solution. A U-3010 ultraviolet–visible spectrophotometer (Hitachi, Japan) was used to determine the absorbance of the aqueous phase. An AVATAR 370-FTIR spectrometer (Nicolet, USA) and an Agilent 1100 Series chromatograph (Agilent, USA) with Diamonsil TM C18 column (150 mm × 4.6 mm) were used to characterize the product obtained. A RE-522A rotary evaporator (Yarong, China) and an AB204-N electronic balance (Mettler) were also used. The FCE apparatus was similar as the one mentioned in the earlier reports [10,11]. Fig. 1. Effect of initial pH on FCE.
2.3. Procedure A 300-ml aliquot of the fermentation liquid was transferred into a 400-ml beaker, to which 3.5064 g NaCl was added. This solution was adjusted to pH 11 with NaOH solution, and then transferred to a flotation cell. The l-phenylalanine was floated by bubbling nitrogen gas at a flow rate of 40 ml/min from the bottom of the cell for 60 min, and extracted into 10.00 ml of n-hexane solution containing 80% D2EHPA (v/v) on the surface of the sample solution. The organic phase was transferred into a 100-ml separating funnel, and 10 ml of 0.1 M hydrochloric acid aqueous solution was added for back-extraction. The back-extraction was repeated three times, then the aqueous phase was collected and concentrated, and the product was obtained after re-crystallizing and drying. The product obtained was characterized by FTIR and HPLC. In the HPLC analysis [13], the mobile phase consisted of A and B solutions (92:8, v/v): A, 50 mM of sodium acetate solution (pH 6.5); B, acetonitrile. The flow rate was 1.0 ml/min, detection wavelength was 220 nm, and all chromatographic analysis were performed at room temperature. 3. Results and discussion 3.1. Optimization of the FCE parameters In this work, a standard solution containing 500 g/l of l-phenylalanine, and the distribution ratio of l-phenylalanine between organic phase and aqueous phase were used to optimize the parameters affecting the FCE efficiency of l-phenylalanine. The distribution ratio can be calculated using the following equation. D=
continue to increase the initial pH to 13, a lot of foam will appear on the top of the flotation cell, and the separation process will have to stop. When the initial pH ≤1, the distribution ratio is zero, because the l-phenylalanine cannot complex with D2EHPA. Therefore, the initial pH 11 can be selected as the optimal pH for the efficient FCE. 3.1.2. Effect of NaCl concentration The effect of NaCl concentration on the distribution ratio of l-phenylalanine is shown in Fig. 2. The maximum distribution ratio is observed when the concentration of NaCl is 0.2 M. Lowering the concentration of NaCl can reduce the dissolution of l-phenylalanine, and increase the separation efficiency. When the concentration of NaCl is more than 0.2 M, the distribution ratio decreases with increasing NaCl concentration, however, above 0.75 M, the distribution ratio decreases very slowly. So the optimal concentration of NaCl is 0.2 M. 3.1.3. Effect of concentration of D2EHPA in organic phase The effect of the concentration of D2EHPA in the organic phase on the distribution ratio of l-phenylalanine is shown in Fig. 3. It can be seen that the distribution ratio of l-phenylalanine increases with the increase in concentration of D2EHPA, however, the maximum distribution ratio is obtained when the concentration of D2EHPA is
30 × (A0 − At ) At
where D is the distribution ratio of l-phenylalanine between organic phase and aqueous phase, 30 the volume ratio of aqueous phase and organic phase, A0 the initial absorbance of aqueous phase at 258 nm and At is the absorbance of aqueous phase at time t. 3.1.1. Effect of initial pH D2EHPA was suitable for a non-polar solvent system [12], so n-octanol and n-hexane were selected as extraction solvents. An experiment was performed by changing the initial pH from 0 to 12 by adding suitable hydrochloric acid solution or sodium hydroxide solution. As shown in Fig. 1, the maximum distribution ratio is observed in the range initial pH 10–12, and n-octanol and n-hexane are both suitable for the FCE of l-phenylalanine. Although, we can
Fig. 2. Effect of NaCl concentration on FCE.
P.Y. Bi et al. / Separation and Purification Technology 63 (2008) 487–491
Fig. 3. Effect of concentration of D2EHPA in organic phase on FCE.
489
Fig. 5. Effect of back-extraction time on the extraction efficiency of back solvent extraction.
80% (v/v), and when the concentration is more than 80%, the distribution ratio decreases. Therefore, the concentration of D2EHPA in organic phase can be fixed at 80% in this work. 3.1.4. Effects of nitrogen flow rate and flotation time Fig. 4 shows the effects of nitrogen flow rate and flotation time on the distribution ratio of l-phenylalanine. The change in nitrogen flow rate can influence the velocity of mass transfer, but cannot influence the thermodynamic equilibrium. The higher flow rate can reduce the flotation time. But the large number of bubbles cannot rapidly burst at a higher flow rate, and they accumulate on the top of the flotation cell. As shown in Fig. 4, a nitrogen flow rate of 40 ml/min and a flotation time of 60 min can be selected. According to the above optimization experiments, the optimal conditions for l-phenylalanine FCE can be obtained, that is pH 11, 0.2 M NaCl concentration, n-hexane solution containing 80% D2EHPA (v/v) as organic phase, nitrogen flow rate of 40 ml/min and flotation time of 60 min. Under the optimization conditions, the distribution ratio between organic phase and aqueous phase reached 24–25, and it was better than the one in previous reports [2–8].
Fig. 4. Effects of nitrogen flow rate and flotation time on FCE.
Fig. 6. Microscope images of the final product and l-phenylalanine standard.
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P.Y. Bi et al. / Separation and Purification Technology 63 (2008) 487–491
Fig. 7. Infrared spectra of the final product and l-phenylalanine standard.
3.2. Back-extraction of l-phenylalanine It follows from Fig. 1 that l-phenylalanine can be released from organic phase to aqueous phase at pH ≤1. When 0.1 M hydrochloric acid aqueous solution was used for the back-extraction of l-phenylalanine, the curve of the back-extraction time vs. efficiency is shown in Fig. 5. It is clear that the back-extraction efficiency is more than 90% after back-extraction was repeated three times.
Fig. 8. Chromatograms of fermentation liquid, final product and l-phenylalanine standard.
over the range 14.69–235 g/ml. The regression equation was y = 1.34x − 0.32 (R2 = 0.9999), where y is the peak area and x is the concentration (g/ml), and this was used to determine the purities of l-phenylalanine obtained by the proposed method. The purity of l-phenylalanine obtained was 98.67%, and the R.S.D. was 2.24% (n = 5). 3.4. Mechanism of flotation complexation extraction
3.3. Separation and purification of l-phenylalanine from fermentation liquid The optimized experimental conditions were applied to a real fermentation sample to evaluate the efficiency of the separation and purification of l-phenylalanine by FCE. Under the optimal conditions, the target compound in the fermentation liquid was floated and extracted into n-hexane phase containing 80% D2EHPA, then the n-hexane phase was back-extracted with 0.1 M hydrochloric acid aqueous solution. The back-extraction was repeated three times, the aqueous phase was collected and concentrated, and the
product was obtained after re-crystallizing and drying. The product was of white acicular crystals, and similar to the l-phenylalanine standard (as shown in Fig. 6). Structural identification of the product was carried out by FTIR and HPLC (Figs. 7 and 8). Comparing the infrared spectra and chromatogram of l-phenylalanine standard with those of the product, it can be seen that they are both identical in peak positions, shape and number, so it can be concluded that the product was l-phenylalanine. The purity of the product was determined by HPLC with the external standard method. Under the selected chromatographic conditions [13], the retention time of l-phenylalanine was 3.52 min. The calibration curve for l-phenylalanine was obtained
Based on the technique described in this paper, it can be divided into three processes: (1) bubble mass transfer, (2) reaction between l-phenylalanine and D2EHPA, and (3) the complex compound dissolve in organic phase. In the process of the bubble mass transfer, the l-phenylalanine is adsorbed on the bubble surface, and carried to the top of flotation column with a layer of D2EHPA/n-hexane solution. After bubble rupturing, the aqueous layer around the bubble transforms to a small water droplet. It is well known that the l-phenylalanine cannot directly dissolve in the organic phase until react with D2EHPA [7,8] as follows:
In the equations, HP is D2EHPA. Then the complex compound can enter into the organic phase from the interface between aqueous phase and organic phase. The reaction process is very similar to the traditional complexation extraction. But, compared with the traditional complexation extraction under the same conditions, the concentration of lphenylalanine on the bubble surface is higher than the O/W interface. Furthermore, after bubble rupturing, the bubble surface transforms into the O/W interface, and the total area of the interface is rapidly reduced. However, the amount of l-phenylalanine on the interface is not reduced. In other words, the concentration of l-phenylalanine on the O/W interface is greatly increased, so
P.Y. Bi et al. / Separation and Purification Technology 63 (2008) 487–491
the reaction balance greatly turns to right. That is the basic reason why the distribution ratio of the FCE is higher than the traditional complexation extraction. 4. Conclusion l-Phenylalanine was successfully separated and purified by FCE from fermentation liquid, and the conception (FCE) was proposed for the first time. The experimental results prove that FCE is a good technique with high selectivity for separating and purifying amino acid from fermentation liquid. The new technique, with advantages of simultaneous separation and purification, excellent concentration efficiency, and very low wastage of organic solvent, has a considerable number of potential applications. Acknowledgements The authors are very much obliged to express their acknowledgement to Dr. Yan Liu and Prof. Tianwei Tan (Faculty of Life Science and Technology, Beijing University of Chemical Technology) for supplying l-phenylalanine fermentation liquid and analyzing the main components. References [1] M.P. Thien, T.A. Hatton, Liquid emulsion membrane and their application in biochemical processing, Sep. Sci. Technol. 23 (1988) 819–853.
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