Synthesis and Application of a Light-sensitive Polymer Forming Aqueous Two-phase Systems
Kong FanQi, Cao Xuejun†, Xia Jinan*, and Byung-Ki Hur**
State Key Laboratory of Bioreactor Engineering, Department of Biochemical Engineering, East China University of Science & Technology, 130 Meilong Rd., Shanghai 200237, China
* Department of Applied Physics, Donghua University, Shanghai 200051, China
** Department of Biological Engineering and Institute of Biotechnological Industry, Inha University, Incheon 402-751, Republic of Korea
Received August 10, 2006; Accepted January 25, 2007
Abstract: A visible light-sensitive polymer that forms aqueous two-phase systems has been synthesized by us- ing N-isopropylacrylamide, n-butyl acrylate, and chlorophyllin sodium copper salt as monomers, and its capa- bility to form aqueous two-phase system was investigated. The polymer formed aqueous two-phase systems with Dextran20000. Over 98 % of the polymer could be recovered by using light radiation at 488 nm. The re- covery experiments were repeated five times. The recoveries were maintained fairly stable. The polymer could be recovered at more than 95 % by using thermo separation. Trypsin constantly maintained a relatively high activity and stability in the aqueous two-phase systems.
Keywords: N-isopropylacrylamide, chlorophyllin sodium copper salt, visible light-sensitive polymer, aqueous two-phase system, recovery
Introduction
1)
Aqueous two-phase systems [1] hold potential as in- dustrial bioseparation techniques. However, the key problem in this field is that polymers forming aqueous two-phase systems often can not be recycled, and result in increasingly expensive bioproducts, purification proc- esses, and environmental pollution. In recent years, re- searchers have focused on this problem and have at- tempted to find new ways of recycling polymers of aque- ous two-phase systems. For example, Asrof and cow- orkers [2] synthesized an anion polymer with N,N dia- llyl-N-carboethoxymethylammonium chloride as a mo- nomer to form aqueous two-phase systems. The polymer was present as a precipitate from a solution of 0.1 M HCl, could form aqueous two-phase systems with PEG-35000, and could be recycled by shifting the pH of the solution. However, the acidic conditions may have caused the inactivation of biomolecules, and one polymer (PEG) could not be recycled. Tjerneld and coworkers [3]
†To whom all correspondence should be addressed.
(e-mail:[email protected])
synthesized polymers with ethylene oxide (EO)/propy- lene oxide (PO) and HM-EOPO to form aqueous two- phase systems. Both polymers were sensitive to heat and were soluble in water. When the temperature was above the critical temperature, the polymers precipitated from the solution. BSA, lysozyme, and pro A-1 were used to test the systems. The recoveries of EO50/PO50 and HM- EOPO were 66 and 45 %, respectively. Polymers could be reused three times. Persson and coworkers [4] applied EO50PO50, EO30PO70, and EO20PO80 to form aqueous two-phase systems with hydroxylpropyl starch. These polymers could be reused four times. At an optimum temperature and separation time, the recovery of EO-POs was over 90 %. Collen and coworkers [5] and Berggren and coworkers [6] investigated the effects of several fac- tors on the partitioning of proteins in new systems. Van Berlo and coworkers [7]developed a new kind of aque- ous two-phase system using NH3 and CO2, which formed NH4NH2CO4, thus generating aqueous two-phase sys- tems with PEG. NH3 and CO2 could be reused if the pres- sure was changed.
In our study, a light-sensitive polymer was synthesized by using N-isopropylacrylamide, n-butyl acrylate, and
Scheme 1. Synthesis of light-sensitive polymer.
chlorophyllin sodium copper salt as monomers. The pol- ymer was applied to form aqueous two-phase systems with Dextran20000; the polymer containing chlor- ophyllin sodium copper salt was sensitive to visible light.
The light-sensitive copolymer would precipitate from solution under light irradiation (488 nm) and could be reused.
Experimental
Materials
N-Isopropylacrylamide (synthesized in our laboratory), n-butyl acrylate, and isopropylamine were obtained from Ling Feng Co. Ltd. (Shanghai, China), and chlorophyllin sodium copper salt was purchased from Sigma. All other reagents were of analytical grade.
Synthesis of N-Isopropylacrylamide
Isopropylamine and triethylamine were added to a flask with benzene as solvent, and hydroquinone (anti- poly- merization) was dropped into the mixture at 0 oC. The re- action was carried out for 3 h with stirring, and the re- sultant mixture was then filtered. The filtrate was evapo- rated under a pressure of 2000 Pa at 110 oC, and N-iso- propylacrylamide was obtained. The product was re- crystallized from n-hexane and dried under vacuum.
Synthesis of a Visible Light-Sensitive Polymer PNBC
N-Isopropylacrylamide, n-butyl acrylate, and chloro- phyllin sodium copper salt were added pro rata into a conical flask containing benzene to synthesized a poly- mer described as PNBC in this work. The reaction was ini- tialized by 2,2'-azobisisobutyronitrile (AIBN) in a N2 at-
mosphere, with shaking for 24 h in a water bath at 60 oC.
After the reaction was completed, the solvent was evapo- rated and the product was purified with n-hexane and dried under vacuum.
Molecular Weight Measurement of PNBC
A sample (0.10 g) of the polymer PNBC was dissolved in water at a 10 g/L concentration. An Ubbelohde dilution viscometer was applied to measure the viscosity of the polymer at room temperature (20 oC). Firstly, 10 mL of the solution was added into the Ubbelohde viscometer and the viscosity was measured, the solution was then di- luted with 5 mL of H2O for four times in turn. The vis- cosity of each solution was measured three times to cal- culate average data.
Probability of Polymers Forming Aqueous Two-Phase Systems
We investigated whether light-sensitive polymers could form aqueous two-phase systems. We used some tradi- tional polymers and salts to test the polymer. Here, EOPO (40 % w/w), PEG6000 (20 % w/w), PEG20000 (20 % w/w), polypropylene glycol (PPG, 40 % w/w), Dextran20000 (10 % w/w), SDS (20 % w/w), Tween80, (NH4)2SO4 (40 % w/w), and isopropyl alcohol (IPA) were chosen to investigate whether aqueous two-phase systems could be formed with PNBC (5 % w/w).
Phase diagrams of aqueous two-phase systems, stock solutions of PNBC (Mw 3.5 × 105) and dextran (Mw 2.0 × 104), were prepared at concentrations of 5 and 20 % (w/w), respectively. The phase diagram of the PNBC/ Dextran20000 was determined using the following meth- od: a series of top-bottom phase mixtures in different volume ratios (such as 2:1 and 2:3) at different tie-line lengths were prepared in test tubes; the test tubes were shak acutely; the test tubes were leff to form two phases;
the concentrations of the two polymers in the top and bottom phases were measured and calculated, and these points were connected by a binodal curve.
Polymer solutions containing light-sensitive polymer were placed into a water bath and treated with light for several minutes at 488 nm or at 30 oC, after which time two phases appeared. The solutions were centrifuged, and the absorbance of the supernatant was detected at a wavelength of 488 nm. The recovery of the polymer was then calculated.
Recovery of Single-Phase (Light-Sensitive Phase) Polymer
AIBN and chlorophyllin sodium copper salt as parame- ters (one factor) were used to analyze the polymer recov- ery using thermal treatment and light. NaCl, Na2SO4, Na3PO4, and NaClO4 were added to the solution, and the salts were sufficiently resolved with stirring. The salt
Figure 1. H-NMR spectrum of the polymer (PNBC).
concentration was 50 mM. After precipitation with tem- perature and light, the remaining solution was detected using a spectrophotometer operated at a wavelength of 488 nm.
Light-Sensitive Polymer Recovery of Two Phases All polymer concentrations were calculated as % (w/w).
A solution of PNBC and Dextran20000 formed two phases;
NaCl, Na2SO4, Na3PO4, and NaClO4 were added to de- termine the recovery of light-sensitive polymer. A certain volume was taken from the top and bottom phase, moved into a test tube, and mixed. The systems were separated at room temperature, and the phase separation was en- hanced by centrifugation for 5 min at 2000 g. The phases were separated and the top phase PNBC was placed in a water bath or was irradiated with visible light for phase separation.
Results and Discussion
Synthesis and Characterization of Polymer
N-Isopropylacrylamide, n-butyl acrylate, and chloro- phyllin sodium copper [8] salt were dissolved into ben- zene at a specified ratio in the presence of AIBN. Oxy- gen was removed by nitrogen (N2), and the mixture was shaken for 24 h in a water bath maintained at 60 oC. The solvent was evaporated, the product was purified using n-hexane, and polymer was obtained after drying under vacuum.
In the polymerization reaction, the amount of AIBN in- fluenced the molecular weight and the viscosity of the polymer. A polymer with a high molecular weight will result in increased viscosity. The amount of AIBN was within the range 0.3∼2.0 % (mol %). The recovery of the polymer in aqueous solution ranged from 97.3 to 99.5
% with light-sensitive radiation of 488 nm. All polymers
synthesized using various amounts of AIBN resulted in relatively high recoveries after irradian.
Chlorophyllin sodium copper salt contains a light-sensi- tive group, and can absorb visible light. A polymer is sensitive to visible light when it is copolymerized with chlorophyllin as a monomer. As a result, a polymer will precipitate under light irradiation. Here, chlorophyllin so- dium copper salt and N-isopropylacrylamide were copo- lymerized to the PNBC polymer. The polymer was re- cycled after light irradiation and reused. Different mole ratios of chlorophyllin sodium copper salt in the polymer synthesis step influenced the recovery of polymers. For example, when the amount of chlorophyllin sodium cop- per salt increased from 1.38 × 10-5 to 6.9 × 10-5 mol, the recovery decreased dramatically, from 99.4 to 88.3 %.
No precipitation appeared when the amount of chloro- phyllin sodium copper salt was further increased. chlor- ophyllin sodium copper salt contains a hydrophilic group. As its content in the polymer increased, the pro- portion of hydrophilic groups also increased and it was difficult to stimulate the polymer to precipitate from the solution. The appropriate amount of chlorophyllin sodi- um copper salt was 0.08∼0.24 % (mol).
The polymer was characterized using 1H-NMR, spectro- scopy with DMSO as solvent. According to the 1H- NMR spectrum, the monomers ratio in the polymer was 408：8：1, while the added monomers ratio was 427 : 9.：1 (N : B : C) during the synthesis of polymer PNBC.
The ratio of N-isopropylacrylamide and n-butyl acrylate in polymer PNBC was slightly higher than the ratio of the two added monomers. We conclude that the three mono- mers were reacted into the polymer (PNBC). The 1H-NMR spectrum is shown as Figure 1. An Ubbelohde visc- ometer was used to measure the PNBC viscosity and the molecular weight was determined according to Mark- Houwink equation; utilizing the equation [η] = 14.5 × 10-2n0.5 (H2O, 20 oC), to get a molecular weight of ca.
n = 3.5 × 105 [9].
Capacity to Form Aqueous Two-Phase Systems PNBC is a novel polymer used in aqueous two-phase sys- tems; some polymers, inorganic salts, and surfactants were chosen to test the probability of forming two phases. The results of this experiment are shown in Table 1. Different phase compositions were tested for each group system. We found that PNBC polymer could form aqueous two-phase systems with Dextran20000. A phase diagram of the aqueous two-phase systems was prepared (Figure 2). The region above the binodal curve is the two-phase region. The tie-line connecting two points on the binodal curve describes the concentration of polymer in the top and bottom phases and the total composition of the two polymers in the aqueous two-phase systems.
Table 1. Probability of Forming Aqueous Two-Phase Systems with PNBC with Different Components of Varied Composition
System 1 (% w/w) 2 (% w/w) 3 (% w/w) Two-phases
PNBC：PEG6000 3.3:6.7 2.5:10 3.0:8.0 no
PNBC：PEG20000 3.3:6.7 2.5:10 3.0:8.0 no
PNBC：PPG 3.3:13.3 2.5:20 3.0:16 turbid
PNBC：Dex20000 3.3:3.3 2.5:6.7 3.0:4.0 yes
PNBC：SDS 3.3:6.7 2.5:10 3.0:8.0 turbid
PNBC：Tween80 3.3:33.3 2.5:50 3.0:40 no
PNBC：EO30PO70 3.3:6.7 2.5:10 3.0:8.0 no
PNBC：IPA 3.3:33.3 2.5:50 3.0:40 no
PNBC：(NH4)2SO4 3.3:6.7 2.5:10 3.0:8.0 precipitation
*The numbers 1, 2, and 3 denote initial volume ratios of 1:1, 2:1, and 3:2, respectively. All systems were rested for 1 day at 20 oC.
Figure 2. Phase diagram for PNBC-Dextran20000 in a water sol- ution at 20 oC.
Figure 3. Repeated recovery of polymer in aqueous solution.
It is well known that some polymer-polymer and poly- mer-salt mixtures can form aqueous two-phase systems.
The former mixtures result in aqueous two-phase sys- tems because of polymer-polymer incompatibility; the latter form aqueous two-phase systems by salting-out of the polymer. For the PNBC-PEG6000 and PNBC-PEG 20000 systems, aqueous two-phase systems could not be formed at any of the three ratios of phase composition;
the components dissolved each other to form a single phase. In PNBC/Tween, PNBC/EO30PO70, and PNBC/IPA, the same phenomena were observed as that for PNBC/PPG:
the solution of PNBC became turbid immediately after the
addition of PPG. For the PNBC/Dextran20000 systems, all systems at each of the three phase compositions were ca- pable of forming aqueous two-phase systems.
Repeated Recovery of Polymer
Since the end of the 20th century, researchers have fo- cused on the recycling of polymers. Thus far, the prob- lem has not been resolved very well. In our study, the re- covery of polymer was investigated through five re- peated cycles (Figure 3).
The polymer was precipitated from solution by light ir- radiation at 488 nm, dried to a constant weight, dissolved in water again, placed into a water bath at room temper- ature, and precipitated under light irradiation. These steps were repeated for five cycles.
The polymer recoveries could be maintained at 98.5∼
99.6 % during the five cycles. The values were much higher than the recoveries of EO50/PO50 (73 %) and HM- EOPO (97.5 %). Theoretically, the polymer could be re- cycled more than 60 times. It is very interesting that light-sensitive irradiation (488 nm), unlike the case with the thermo-sensitive polymer, does not influence bio- molecule stability. The polymer would, therefore, likely show broader application potential in aqueous two-phase systems.
As noted above, Dextran20000 was able to form aque- ous two-phase systems with PNBC. Further, recovery of PNBC in PNBC/Dextran20000 aqueous two-phase systems was investigated by comparison of light-sensitive irradi- ation and thermo-sensitive separation. Different phase systems with diverse phase compositions resulting from different tie-lengths were prepared. In general, some salts must be added during extraction of aqueous two-phase systems to improve partitioning of biomolecules. There- fore, we chose some inorganic salts and investigated their influence on the recovery of PNBC polymer. Here, NaCl, Na2SO4, Na3PO4, and NaClO4 were used to inves- tigate the effect of inorganic salts on the recovery of PNBC
polymer in aqueous two-phase systems.
Figure 4. Comparison of the recoveries of light-sensitive irradi- ation and thermo-sensitive precipitation of PNBC polymer in two- phase systems. The concentration of salts in the total phases was 50 mM. The initial volume ratio was 3:2 (mL/mL).
As shown in Figure 4, the recovery increased with the ratio of the top phase to the bottom phase (without add- ing salts). The recovery of polymer with Na3PO4 was re- markably low (only 78.24 %) compared with that of oth- er systems. The recoveries of polymers in other aqueous two-phase systems were over 90 %. The recovery of pol- ymer in systems containing salts was greater than that in systems without salts. When comparing the polymer re- coveries under light-sensitive irradiation and thermo-sen- sitive phase separation, the former were greater than those of the latter. The recoveries in the systems with NaCl increased from 95.3 to 98.57 %, while those for systems with Na2SO4 or NaClO4, rose by roughly 1.0 %.
The recovery was lower than that in a single phase be- cause the light-sensitive polymer existed in the bottom phase. In this case, it was difficult to stimulate the poly- mer to precipitate from the solution.
It is advantageous to maintain the viability of bio- molecule activity in aqueous two-phase systems. Here, the viability of trypsin in the aqueous two-phase systems was examined. Its activity remained at the basal level (106.7 U/mL) after 14 h. Irradiation with light would not cause a rise in temperature, which would destroy the ac- tivity of biomolecules (e.g., proteins, nuclear acids).
After precipitation, the polymer could be reused. The polymer has potential for application in aqueous two- phase systems. Of course, the polymer could probably al- so be applied in other areas, such as affinity precip- itation, or as a biocatalyst, and possibly in other arenas in the future.
Conclusion
A light-sensitive polymer was synthesized and its appli- cation to aqueous two-phase systems was tested. Dextran could form aqueous two-phase systems with the polymer.
The polymer could be recycled after irradiation with visi- ble light at 488 nm or by increasing the temperature to levels above 28 oC; the polymer could be recovered at levels above 95 %. It is advantageous to maintain the vi- ability of biomaterials in the system through light ir- radiation. Irradiation with light did not cause heat, which could destroy the activity of biomaterials (e.g., proteins, nuclear acids). After precipitation, the polymer could be reused. The polymer has potential for application in aqueous two-phase systems, e.g., for affinity precip- itation or as a biocatalyst. At present, we are synthesizing another light-sensitive polymer that forms aqueous two- phase systems together with PNBC. We will attempt to re- cover the two polymer components using a single meth- od, or using a combination of light, heat, and pH.
Acknowledgment
This project was supported by the Natural Scientific Foundation of China (20474016) and sponsored by the Scientific Research Foundation for Returned Overseas Chinese Scholars.
References
1. R. Hatti-Kaul, Aqueous Two-Phase Systems Totowa, pp. 1-10, Humana Press, New Jersey (2000).
2. A. Hassan, A. Muallem, I. M. Mohamed, and A. S.
K. Asrof, Polymer, 43, 1041 (2002).
3. J. Persson, H.-O. Johansson, and F. Tjernekd, J.
Chromatogr. A, 864, 31 (1999).
4. J. Persson, A. Kaul, and F. Tjerneld, J. Chromatogr.
B, 743, 115 (2000).
5. A. Collen, M. Ward, F. Tjerneld, and H. Stalbrand, J. Chromatogr. A, 910, 275 (2001).
6. K. Berggren, H.-O. Johansson, and F. Tjerneld, J.
Biotechnol., 87, 179 (2001).
7. M. van Berlo, K. Luyben, and L. Wielen, J.
Chromatogr. B, 711, 61 (1998).
8. A. Suzuki and T. Tanaka, Nature, 346, 345 (1990).
9. S. T. Kim, J. Y. Lim, H. J. Choi, and H. Hyun, J.
Ind. Eng. Chem., 12, 161 (2006).