两性离子型可回用两水相体系的构建及其分配生物小分子的应用研究
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摘要
制约两水相萃取发展最重要的因素是两水相体系成相聚合物的回收问题,成相聚合物的低回收率使两水相萃取的成本上升且增加了环境的污染。本文合成两个pH响应型聚合物PADB和PADBA,并利用这两个聚合物构建了pH响应型可回用两水相体系,该两水相体系以一个pH条件回收两个成相聚合物,有效的解决了两相体系的回收问题。其中PADB以丙烯酸,甲基丙烯酸二甲氨基乙酯,甲基丙烯酸丁酯为单体无规聚合而成,而PADBA以丙烯酸,甲基丙烯酸二甲氨基乙酯,甲基丙烯酸丁酯,丙烯醇为单体无规聚合而成,通过红外光谱和核磁共振的方法确定了两个聚合物的结构；通过乌氏粘度计方法测定了聚合物的粘度,并计算聚合物的粘均分子量(PADBA为3.37×105,PADB为2.93×106)；通过称重法测定了聚合物的回收率,测得PADBA的回收率为97.18%,而PADB的回收率为98.87%；通过测定不同pH下聚合物的Zeta电位,计算聚合物PADBA的等电点为4.01,而PADB的等电点为2.79。
对于形成的PADB/PADBA两水相体系,通过浊点法测定了该体系的双节线,通过分别测定成相后上下相各个聚合物的浓度来绘制系线。研究了pH对PADB/PADBA可回用两水相体系双节线和系线的影响,结果显示随着pH的增加,双节线往上移动,表示在相同PADBA浓度(或PADB浓度)下,形成两相所需的PADB浓度(或PADBA浓度)增加。而系线随着pH的增加不断变短,表示pH的增加使相同初始浓度的两水相体系分相后上下相差异变小。同时利用NRTL和Flory-Huggins两个模型拟合实验数据,其中Flory-Huggins模型的RMSD在pH为6.05时达到最小值0.8777,而NRTL模型的RMSD在pH为6.05时达到最小值1.0092。相比于NRTL模型,Flory-Huggins模型的RMSD更小,说明Flory-Huggins模型比NRTL模型更适合PADB/PADBA两水相体系的相图拟合。
利用构建的PADB/PADBA可回用两水相体系分配一些抗生素和甜味剂,包括螺旋霉素、红霉素、林可霉素、去甲基金霉素和甜叶菊甙,考察了不同聚合物浓度、体系pH、盐离子、盐离子浓度等因素对这些生物小分子分配的影响。结果表明,聚合物浓度和pH对螺旋霉素、红霉素、林可霉素和甜叶菊甙在PADB/PADBA两水相体系中的分配影响不大,而在去甲基金霉素的分配中,不同pH下的去甲基金霉素的分配系数的范围为0.4到0.8,且当pH为5.28时分配系数最小,为0.467。在加入的多种无机盐中,MgSO4的影响较大,其中螺旋霉素的分配系数随着MgSO4离子浓度增加而增加,在90mmol/l时达到最大值1.99；红霉素的分配系数在低浓度的MgSO4离子中变化不大,而在高浓度MgSO4离子中随着MgSO4浓度的增加而增加,并在1200mmol/l的时候达到最大值5.3；MgSO4使去甲基金霉素的分配系数下降,且当MgSO4浓度达到60mmol/l时,其分配系数达到最小值0.084。同时考察了不同离子加入顺序对甜叶菊甙分配系数的影响,当甜叶菊甙先和MgSO4混合溶解后再与聚合物溶液混合,其分配系数会随着MgSO4浓度的增加而增加,并在MgSO4浓度为200mmol/l时分配系数达到最大值9.0。PADB/PADBA两水相体系更适合于分配带电荷的生物小分子且Mg2+离子对PADB/PADBA两水相体系分配的影响要比其他离子的影响大。利用PADB/PADBA两水相体系来取代有机溶剂萃取分离生物小分子可解决有机溶剂成本高、污染大的问题,具有一定的前景。
采用一个改进的Gibbs系综蒙特卡洛法模拟了聚合物／聚合物两水相体系的液液平衡,其结果和实际的两水相体系中上下相分别富含一种聚合物的现象相同。考察了聚合物分子量(以单体数量来表示不同聚合物分子量)和聚合物浓度(以聚合物链的初始数量表示聚合物浓度)对成相的影响,结果与实验过程中聚合物分子量和浓度对成相的影响相一致。同时,利用上述参数模拟了DEXT40/PEG4000两水相体系在200C的液液平衡数据,结果显示聚合物成相趋势与实验显示的相成趋势一致,其数据值的误差归于成相聚合物参数的估计方法不够完善。
Compared with traditional extraction technology like organic solvent extraction or ion exchange resin, aqueous two-phase systems (ATPS) have been widely used for purification of bioproducts due to the simple manipulation, mild operation condition, and low pollution. However, the key problem was the recovery difficulty of polymers phase-forming ATPS, increasing the cost. In this paper, two pH-response polymers PADB and PADBA were synthesized and used to construct the pH-response aqueous two-phase systems. PADB was synthesized by acrylic acid (AA), dimethylamino-ethyl methacrylate (DMAEMA), butyl methacrylate (BMA) and PADBA was synthesized by AA, DMAEMA, BMA, allyl alcohol (Aal). The structures of these two polymers were confirmed by IR and H NMR. The viscosities of polymers were measured by ubbelohde viscometer and the average molecular weights of two polymers were calculated using Fuoss equation where molecular weight of PADBA is3.37×105and PADB is2.93×106. The recoveries of polymers at different pH were tested and the maximal recovery of PADBA is97.18%and PADB is98.87%. The isoelectric points were tested by measuring Zeta potential of polymers at different pH where pI of PADBA is4.01and PADB is2.79.
The cloud point method was used to calculate the binodal curve of PADB/PADBA aqueous two-phase systems and the tie lines were determined by measurement of each polymer concentration in each phase. The effect of pH on the binodal curve and tie lines of PADB/PADBA aqueous two-phase systems was investigated. The results shows that the two-phase region becomes higher in the phase diagram as pH increases which means that to form ATPS at higher pH, higher concentration of PADB is needed with the same concentration of PADBA.The tie lines becomes shorter as pH increases which means that the difference between two phases becomes smaller after phase separation at the same initial concentrations. The NRTL model and Flory-Huggins model were used to fit the liquid-liquid equilibrium of PADB/PADBA aqueous two-phase systems. The minimal RMSD in Flory-Huggins model is0.8777at pH6.05, and in NRTL model is1.0092at pH6.05. Flory-Huggins model shows better correlation than NRTL model for PADB/PADBA aqueous two-phase systems.
PADB/PADBA aqueous two-phase systems were used to partition some bioproducts, including Spiramycin, Erythromycin, Lincomycin, Stevioside and Demeclocycline. The influence of the polymer concentration, pH, salts, salts concentration on the partition in PADB/PADBA aqueous two-phase systems were investigated. The results shows that the polymer concentration and pH have limited effect on the partition of Spiramycin, Erythromycin and Lincomycin in PADB/PADBA aqueous two-phase systems. The partition coefficient of Demeclocycline in PADB/PADBA aqueous two-phase systems is at the range of0.4to0.8at different pH and reaches minimum (0.467) when pH is5.28. In all the addition of salts, the partition coefficient of Spiramycin in PADB/PADBA aqueous two-phase systems increases with the salt concentration and reaches maximum at90mmol/l (1.99). The partition coefficient of Erythromycin in PADB/PADBA aqueous two-phase systems reaches maximum at high concentration1200mmol/l (5.296).The partition coefficient of Demeclocycline in PADB/PADBA aqueous two-phase systems is at the range of0.1～0.2at the salt range of10mmol/1～90mmol/l and reaches minimum (0.084) at60mmol/l. This result shows extensive application prospect in purification of Demeclocycline. In the investigation of the sequence of adding salts, the partition coefficient of Stevioside in PADB/PADBA aqueous two-phase systems reachs maximum (9.0) when Stevioside is mixed with MgSO4(100mmol/l～200mmol/l) firstly then mixed with polymers solution.
A modified Gibbs Ensemble Monte Carlo method was used to simulate the liquid-liquid equilibrium of aqueous two-phase systems composed of two model polymers. The results show that one polymer is enriched in one phase and the other polymer is enriched in the other phase which is the same with the real system. The effect of molecular weight (using the number of monomers to express the molecular weight) and the polymer concentration (using the number of initial polymer chains to express the polymer concentration) on the phase forming were investigated. Results show that ATPS can't form ATPS at low molecular weight and at low polymer concentration which is also same with real system. And the simulated tie lines are parallel and the tie line length decreases as the concentration decreases, fitting the real system. The program was also used to simulate the experiment data of DEXT40/PEG4000ATPS at20"C, and the results show the same tendency of phase forming with real system.
对于形成的PADB/PADBA两水相体系,通过浊点法测定了该体系的双节线,通过分别测定成相后上下相各个聚合物的浓度来绘制系线。研究了pH对PADB/PADBA可回用两水相体系双节线和系线的影响,结果显示随着pH的增加,双节线往上移动,表示在相同PADBA浓度(或PADB浓度)下,形成两相所需的PADB浓度(或PADBA浓度)增加。而系线随着pH的增加不断变短,表示pH的增加使相同初始浓度的两水相体系分相后上下相差异变小。同时利用NRTL和Flory-Huggins两个模型拟合实验数据,其中Flory-Huggins模型的RMSD在pH为6.05时达到最小值0.8777,而NRTL模型的RMSD在pH为6.05时达到最小值1.0092。相比于NRTL模型,Flory-Huggins模型的RMSD更小,说明Flory-Huggins模型比NRTL模型更适合PADB/PADBA两水相体系的相图拟合。
利用构建的PADB/PADBA可回用两水相体系分配一些抗生素和甜味剂,包括螺旋霉素、红霉素、林可霉素、去甲基金霉素和甜叶菊甙,考察了不同聚合物浓度、体系pH、盐离子、盐离子浓度等因素对这些生物小分子分配的影响。结果表明,聚合物浓度和pH对螺旋霉素、红霉素、林可霉素和甜叶菊甙在PADB/PADBA两水相体系中的分配影响不大,而在去甲基金霉素的分配中,不同pH下的去甲基金霉素的分配系数的范围为0.4到0.8,且当pH为5.28时分配系数最小,为0.467。在加入的多种无机盐中,MgSO4的影响较大,其中螺旋霉素的分配系数随着MgSO4离子浓度增加而增加,在90mmol/l时达到最大值1.99；红霉素的分配系数在低浓度的MgSO4离子中变化不大,而在高浓度MgSO4离子中随着MgSO4浓度的增加而增加,并在1200mmol/l的时候达到最大值5.3；MgSO4使去甲基金霉素的分配系数下降,且当MgSO4浓度达到60mmol/l时,其分配系数达到最小值0.084。同时考察了不同离子加入顺序对甜叶菊甙分配系数的影响,当甜叶菊甙先和MgSO4混合溶解后再与聚合物溶液混合,其分配系数会随着MgSO4浓度的增加而增加,并在MgSO4浓度为200mmol/l时分配系数达到最大值9.0。PADB/PADBA两水相体系更适合于分配带电荷的生物小分子且Mg2+离子对PADB/PADBA两水相体系分配的影响要比其他离子的影响大。利用PADB/PADBA两水相体系来取代有机溶剂萃取分离生物小分子可解决有机溶剂成本高、污染大的问题,具有一定的前景。
采用一个改进的Gibbs系综蒙特卡洛法模拟了聚合物／聚合物两水相体系的液液平衡,其结果和实际的两水相体系中上下相分别富含一种聚合物的现象相同。考察了聚合物分子量(以单体数量来表示不同聚合物分子量)和聚合物浓度(以聚合物链的初始数量表示聚合物浓度)对成相的影响,结果与实验过程中聚合物分子量和浓度对成相的影响相一致。同时,利用上述参数模拟了DEXT40/PEG4000两水相体系在200C的液液平衡数据,结果显示聚合物成相趋势与实验显示的相成趋势一致,其数据值的误差归于成相聚合物参数的估计方法不够完善。
Compared with traditional extraction technology like organic solvent extraction or ion exchange resin, aqueous two-phase systems (ATPS) have been widely used for purification of bioproducts due to the simple manipulation, mild operation condition, and low pollution. However, the key problem was the recovery difficulty of polymers phase-forming ATPS, increasing the cost. In this paper, two pH-response polymers PADB and PADBA were synthesized and used to construct the pH-response aqueous two-phase systems. PADB was synthesized by acrylic acid (AA), dimethylamino-ethyl methacrylate (DMAEMA), butyl methacrylate (BMA) and PADBA was synthesized by AA, DMAEMA, BMA, allyl alcohol (Aal). The structures of these two polymers were confirmed by IR and H NMR. The viscosities of polymers were measured by ubbelohde viscometer and the average molecular weights of two polymers were calculated using Fuoss equation where molecular weight of PADBA is3.37×105and PADB is2.93×106. The recoveries of polymers at different pH were tested and the maximal recovery of PADBA is97.18%and PADB is98.87%. The isoelectric points were tested by measuring Zeta potential of polymers at different pH where pI of PADBA is4.01and PADB is2.79.
The cloud point method was used to calculate the binodal curve of PADB/PADBA aqueous two-phase systems and the tie lines were determined by measurement of each polymer concentration in each phase. The effect of pH on the binodal curve and tie lines of PADB/PADBA aqueous two-phase systems was investigated. The results shows that the two-phase region becomes higher in the phase diagram as pH increases which means that to form ATPS at higher pH, higher concentration of PADB is needed with the same concentration of PADBA.The tie lines becomes shorter as pH increases which means that the difference between two phases becomes smaller after phase separation at the same initial concentrations. The NRTL model and Flory-Huggins model were used to fit the liquid-liquid equilibrium of PADB/PADBA aqueous two-phase systems. The minimal RMSD in Flory-Huggins model is0.8777at pH6.05, and in NRTL model is1.0092at pH6.05. Flory-Huggins model shows better correlation than NRTL model for PADB/PADBA aqueous two-phase systems.
PADB/PADBA aqueous two-phase systems were used to partition some bioproducts, including Spiramycin, Erythromycin, Lincomycin, Stevioside and Demeclocycline. The influence of the polymer concentration, pH, salts, salts concentration on the partition in PADB/PADBA aqueous two-phase systems were investigated. The results shows that the polymer concentration and pH have limited effect on the partition of Spiramycin, Erythromycin and Lincomycin in PADB/PADBA aqueous two-phase systems. The partition coefficient of Demeclocycline in PADB/PADBA aqueous two-phase systems is at the range of0.4to0.8at different pH and reaches minimum (0.467) when pH is5.28. In all the addition of salts, the partition coefficient of Spiramycin in PADB/PADBA aqueous two-phase systems increases with the salt concentration and reaches maximum at90mmol/l (1.99). The partition coefficient of Erythromycin in PADB/PADBA aqueous two-phase systems reaches maximum at high concentration1200mmol/l (5.296).The partition coefficient of Demeclocycline in PADB/PADBA aqueous two-phase systems is at the range of0.1～0.2at the salt range of10mmol/1～90mmol/l and reaches minimum (0.084) at60mmol/l. This result shows extensive application prospect in purification of Demeclocycline. In the investigation of the sequence of adding salts, the partition coefficient of Stevioside in PADB/PADBA aqueous two-phase systems reachs maximum (9.0) when Stevioside is mixed with MgSO4(100mmol/l～200mmol/l) firstly then mixed with polymers solution.
A modified Gibbs Ensemble Monte Carlo method was used to simulate the liquid-liquid equilibrium of aqueous two-phase systems composed of two model polymers. The results show that one polymer is enriched in one phase and the other polymer is enriched in the other phase which is the same with the real system. The effect of molecular weight (using the number of monomers to express the molecular weight) and the polymer concentration (using the number of initial polymer chains to express the polymer concentration) on the phase forming were investigated. Results show that ATPS can't form ATPS at low molecular weight and at low polymer concentration which is also same with real system. And the simulated tie lines are parallel and the tie line length decreases as the concentration decreases, fitting the real system. The program was also used to simulate the experiment data of DEXT40/PEG4000ATPS at20"C, and the results show the same tendency of phase forming with real system.
引文
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