以环糊精为基质的扩张床吸附剂的制备及银杏黄酮的分离
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摘要
扩张床吸附(expanded bed adsorption,EBA)技术是一种集成化分离技术,它将固液分离、富集浓缩和初期纯化融合于一个新的单元操作中,能直接从含有固体颗粒的料液中捕获目标产物,缩短分离流程,提高分离效率。目前扩张床吸附技术主要应用于蛋白质的分离纯化。开发新型的吸附剂,并应用于其他生物活性物质的分离,是拓展扩张床吸附技术应用范围的有益尝试。本论文开发出一种新型的以环糊精为基质的扩张床吸附剂,围绕吸附剂的制备、性质和应用等内容展开,并对其在银杏黄酮分离纯化中的应用进行初探。
首先以环氧氯丙烷交联的β-环糊精聚合物为骨架材料,以碳化钨粉为增重剂,通过β-环糊精预聚、反相悬浮成球及交联固化等步骤,制备出具有良好球形度的CroCD-TuC吸附剂,其粒径分布为80μm～320μm。考察了反相悬浮过程的工艺条件,对影响微球粒径分布和形貌的关键参数进行分析,提出了优化的工艺参数：油相为液体石蜡和真空泵油GS-1按质量比5：7混合；油水比4：1(w/w)；分散剂吐温-80占油相质量的1.0%；搅拌转速350rpm～400rpm。通过5个批次重复实验所得微球的物性数据对比,发现工艺具有良好的重现性。
对吸附剂的物性、孔结构、机械强度和化学稳定性等基本性质进行了表征,重点考察碳化钨的用量对吸附剂性质的影响。实验发现,所有吸附剂的粒径均呈对数正态分布,且受碳化钨用量的影响较小,其平均粒径一般在150μm至170μm之间。此外碳化钨粉的用量对吸附剂的孔度和平均孔径的影响亦较小,表明β-环糊精聚合物骨架的结构未发生明显的改变。然而,掺入了碳化钨粉的吸附剂的密度和机械强度有了明显的提高,因此通过控制原料的配比可以制得适应不同需要的吸附剂。
测定了吸附剂在扩张床中的流体力学性质。采用Richardson-Zaki方程拟合实验结果,得到终端沉降速率和扩张指数,并结合Stokes方程和Martin方程的计算、比较对计算值进行了修正。采用停留时间分布法测定扩张床内的流体流动特性,引入Bodenstein准数、床层轴向扩散系数和理论等板高度等参数进行分析讨论。结果发现,流体的黏度和流速是影响吸附剂扩张特性和流体混合性质的主要因素,而吸附剂的密度通过流速一扩张率之间的关系,间接影响了其扩张特性和流体混合性质。论文还比较了几种吸附剂/基质的流体力学性质,发现CroCD-TuC 3的表现较出色,是用于扩张床操作的理想吸附剂。
本文改变以往扩张床多用于蛋白质的做法,把目标物定为生物活性小分子物质,作一个新的尝试。以芦丁为模型化合物,考察CroCD-TuC 3吸附剂对芦丁的吸附性质。研究表明,CroCD-TuC 3对芦丁的吸附等温线遵循Langmuir吸附模型,且吸附量随温度和溶液pH值的升高而减小,随溶剂极性的增强而增大。以结构相近的SephadexTM G-15葡聚糖凝胶为参照物,比较了等量吸附焓和吸附剂表面不均匀度参数,体现出CroCD-TuC 3吸附剂骨架中β-环糊精/芦丁包结复合物的形成对吸附过程的重要贡献。采用孔扩散模型拟合吸附动力学实验数据,得到芦丁在吸附剂内的有效孔扩散系数约为3.8×10-11m2·s-1,远小于芦丁在稀溶液中的分子扩散系数,揭示了吸附质在内孔中的扩散是整个吸附过程的限速步骤。此外,分别在固定床和扩张床中测定了CroCD-TuC 3对芦丁的动态吸附情况,发现其动态吸附效率受流速和料液浓度的影响较大。
在了解吸附剂性质的基础上,本文将CroCD-TuC 3吸附剂进行应用尝试,以扩张床吸附法分离银杏黄酮。采用水提液直接上柱的扩张床操作方式,通过水洗、50%(w/w)乙醇洗脱等一系列步骤,使得产物中黄酮的纯度达到36.5%,纯化倍数为20.8,回收率36.1%,实验证明采用扩张床吸附技术能够实现银杏黄酮的快速、高效纯化,也为扩张床用于活性小分子物质的分离纯化提供了一个有价值的实例。与文献报导的吸附剂相比较,CroCD-TuC 3吸附剂表现出色,具有较好的应用前景。
Expanded bed adsorption (EBA) is a promising technique for bioseparations. It integrates clarification, concentration, and primary purification into a single step, allowing the capture of bio-molecules from unclarified feedstock without prior removal of particulates. EBA simplifies the technical process and shows high efficiency for protein separation. In order to expand the applications of EBA, new adsorbents would be developed and applied to separate more bio-active molecules. The purpose of this work is to develop a novel cyclodextrin-based adsorbent aiming for separating Ginkgo flavonoids with expanded bed mode. This work will focus on the preparation, characterization and application of this novel adsorbent.
Fine technical process is a warrant for the adsorbent with excellent performance. In this work, a series of adsorbents named CroCD-TuC have been prepared by the methods ofβ-cyclodextrin (β-CD) pre-polymerizing and reversed phase suspension crosslinking. Tungsten carbide (TuC) powder is selected as a densifier embedded inβ-cyclodextrin-epichlorohydrin polymer skeleton. The adsorbents obtained have regular spherical shape and a proper diameter of 80μm～320μm. The reversed phase suspension process is studied in detail and the optimal technical parameters are:the mixture of paraffin liquid and pump oil GS-1 with mass ratio of 5:7 as the oil phase; the mass ratio of oil to polymer solution at 4:1; the content of dispersant tween-80 at 1.0% by weight based on the oil phase; stirring speed of 350 rpm-400 rpm. The results of five batches demonstrate that the technology shows good reproducibility with comparable physical properties of adsorbents prepared.
The basic properties of adsorbents including physical properties, pore properties, mechanical strength and chemical stability are characterized. It is found that the adsorbent beads follow the logarithmic normal size distribution, and the mean diameter slightly changes within the range of 150μm～170μm, regardless of the amount of tungsten carbide used. However, their wet density and mechanical strength significantly increase as the mass ratio TuC/CD increases, providing a potential of preparing adsorbents suitable for different use by controlling the amount of TuC added in the preparation process. The effect of TuC/CD mass ratio on the porosity and pore diameter is insignificant, indicating the structure ofβ-cyclodextrin polymer skeleton is hardly changed.
The hydrodynamic properties of CroCD-TuC adsorbents are determined in an expanded bed. It is found for all CroCD-TuC adsorbents tested, the expansion characteristics can be well correlated with the Richardson-Zaki equation. But the predictions of terminal settling velocity and expansion index with the Stokes' law or Martin model are somewhat deviated from that correlated, needing an improvement with empirical modification. The Bodenstein number (Bo), the axial dispersion coefficient (Dax) and the height equivalent of theoretical plate (HETP) are used to analyze and evaluate the liquid mixing in the expanded bed, based on residence time distribution (RTD) measurement. In summary, fluid velocity and viscosity are the crucial factors influencing the expansion and liquid mixing properties, while adsorbent density directly affects these properties in the form of the expansion factors. A further comparison of hydrodynamic properties of several adsorbents/matrices reveals that CroCD-TuC 3 can be selected as the most promising adsorbent for EBA use.
As a new attempt, the bio-active molecule rutin is adsorbed by CroCD-TuC 3 in aqueous solutions. The isothermal adsorption equilibrium follows the Langmuir adsorption equation, and the adsorption capacity is gradually reduced as the temperature and pH of solution rises, but increases with the increase of solvent polarity. The comparisons of CroCD-TuC 3 with SephadexTM G-15 beads on isosteric enthalpy and adsorbent surface heterogeneity analysis indicate that the formation ofβ-cyclodextrin/rutin inclusion complex in CroCD-TuC 3 skeleton makes significant contribution to the adsorption. The adsorption kinetics data are fitted by pore diffusion model. The effective pore diffusivity of rutin calculated with this model (3.8×10-11 m2·s-1) is much lower than that in diluted solution, indicating the diffusion inside the pores is the rate-restricting step in the whole adsorption process. The dynamic adsorption capacities of rutin with CroCD-TuC 3 are also measured and evaluated with the breakthrough curves in packed and expanded bed. The results reveal that the dynamic adsorption capacity is notably influenced by fluid velocity and feed concentration.
Finally, the practical application of CroCD-TuC 3 adsorbent is carried out in an expanded bed for separating the flavonoids from natural resources. The feedstock extracted from Ginkgo biloba L. leaves with water is directly loaded onto CroCD-TuC 3 in an expanded bed. After washed with water, the flavonoids are eluted with 50%(w/w) ethanol from the adsorbent. The recovery is 36.1% with the flavonoids purity of 36.5% and the purification factor of 20.8. The results demonstrate that CroCD-TuC 3 is suitable for flavonoids separation and purification. It is expected that the novel adsorbent CroCD-TuC 3 would be a promising candidate for flavonoids separation and purification.
首先以环氧氯丙烷交联的β-环糊精聚合物为骨架材料,以碳化钨粉为增重剂,通过β-环糊精预聚、反相悬浮成球及交联固化等步骤,制备出具有良好球形度的CroCD-TuC吸附剂,其粒径分布为80μm～320μm。考察了反相悬浮过程的工艺条件,对影响微球粒径分布和形貌的关键参数进行分析,提出了优化的工艺参数：油相为液体石蜡和真空泵油GS-1按质量比5：7混合；油水比4：1(w/w)；分散剂吐温-80占油相质量的1.0%；搅拌转速350rpm～400rpm。通过5个批次重复实验所得微球的物性数据对比,发现工艺具有良好的重现性。
对吸附剂的物性、孔结构、机械强度和化学稳定性等基本性质进行了表征,重点考察碳化钨的用量对吸附剂性质的影响。实验发现,所有吸附剂的粒径均呈对数正态分布,且受碳化钨用量的影响较小,其平均粒径一般在150μm至170μm之间。此外碳化钨粉的用量对吸附剂的孔度和平均孔径的影响亦较小,表明β-环糊精聚合物骨架的结构未发生明显的改变。然而,掺入了碳化钨粉的吸附剂的密度和机械强度有了明显的提高,因此通过控制原料的配比可以制得适应不同需要的吸附剂。
测定了吸附剂在扩张床中的流体力学性质。采用Richardson-Zaki方程拟合实验结果,得到终端沉降速率和扩张指数,并结合Stokes方程和Martin方程的计算、比较对计算值进行了修正。采用停留时间分布法测定扩张床内的流体流动特性,引入Bodenstein准数、床层轴向扩散系数和理论等板高度等参数进行分析讨论。结果发现,流体的黏度和流速是影响吸附剂扩张特性和流体混合性质的主要因素,而吸附剂的密度通过流速一扩张率之间的关系,间接影响了其扩张特性和流体混合性质。论文还比较了几种吸附剂/基质的流体力学性质,发现CroCD-TuC 3的表现较出色,是用于扩张床操作的理想吸附剂。
本文改变以往扩张床多用于蛋白质的做法,把目标物定为生物活性小分子物质,作一个新的尝试。以芦丁为模型化合物,考察CroCD-TuC 3吸附剂对芦丁的吸附性质。研究表明,CroCD-TuC 3对芦丁的吸附等温线遵循Langmuir吸附模型,且吸附量随温度和溶液pH值的升高而减小,随溶剂极性的增强而增大。以结构相近的SephadexTM G-15葡聚糖凝胶为参照物,比较了等量吸附焓和吸附剂表面不均匀度参数,体现出CroCD-TuC 3吸附剂骨架中β-环糊精/芦丁包结复合物的形成对吸附过程的重要贡献。采用孔扩散模型拟合吸附动力学实验数据,得到芦丁在吸附剂内的有效孔扩散系数约为3.8×10-11m2·s-1,远小于芦丁在稀溶液中的分子扩散系数,揭示了吸附质在内孔中的扩散是整个吸附过程的限速步骤。此外,分别在固定床和扩张床中测定了CroCD-TuC 3对芦丁的动态吸附情况,发现其动态吸附效率受流速和料液浓度的影响较大。
在了解吸附剂性质的基础上,本文将CroCD-TuC 3吸附剂进行应用尝试,以扩张床吸附法分离银杏黄酮。采用水提液直接上柱的扩张床操作方式,通过水洗、50%(w/w)乙醇洗脱等一系列步骤,使得产物中黄酮的纯度达到36.5%,纯化倍数为20.8,回收率36.1%,实验证明采用扩张床吸附技术能够实现银杏黄酮的快速、高效纯化,也为扩张床用于活性小分子物质的分离纯化提供了一个有价值的实例。与文献报导的吸附剂相比较,CroCD-TuC 3吸附剂表现出色,具有较好的应用前景。
Expanded bed adsorption (EBA) is a promising technique for bioseparations. It integrates clarification, concentration, and primary purification into a single step, allowing the capture of bio-molecules from unclarified feedstock without prior removal of particulates. EBA simplifies the technical process and shows high efficiency for protein separation. In order to expand the applications of EBA, new adsorbents would be developed and applied to separate more bio-active molecules. The purpose of this work is to develop a novel cyclodextrin-based adsorbent aiming for separating Ginkgo flavonoids with expanded bed mode. This work will focus on the preparation, characterization and application of this novel adsorbent.
Fine technical process is a warrant for the adsorbent with excellent performance. In this work, a series of adsorbents named CroCD-TuC have been prepared by the methods ofβ-cyclodextrin (β-CD) pre-polymerizing and reversed phase suspension crosslinking. Tungsten carbide (TuC) powder is selected as a densifier embedded inβ-cyclodextrin-epichlorohydrin polymer skeleton. The adsorbents obtained have regular spherical shape and a proper diameter of 80μm～320μm. The reversed phase suspension process is studied in detail and the optimal technical parameters are:the mixture of paraffin liquid and pump oil GS-1 with mass ratio of 5:7 as the oil phase; the mass ratio of oil to polymer solution at 4:1; the content of dispersant tween-80 at 1.0% by weight based on the oil phase; stirring speed of 350 rpm-400 rpm. The results of five batches demonstrate that the technology shows good reproducibility with comparable physical properties of adsorbents prepared.
The basic properties of adsorbents including physical properties, pore properties, mechanical strength and chemical stability are characterized. It is found that the adsorbent beads follow the logarithmic normal size distribution, and the mean diameter slightly changes within the range of 150μm～170μm, regardless of the amount of tungsten carbide used. However, their wet density and mechanical strength significantly increase as the mass ratio TuC/CD increases, providing a potential of preparing adsorbents suitable for different use by controlling the amount of TuC added in the preparation process. The effect of TuC/CD mass ratio on the porosity and pore diameter is insignificant, indicating the structure ofβ-cyclodextrin polymer skeleton is hardly changed.
The hydrodynamic properties of CroCD-TuC adsorbents are determined in an expanded bed. It is found for all CroCD-TuC adsorbents tested, the expansion characteristics can be well correlated with the Richardson-Zaki equation. But the predictions of terminal settling velocity and expansion index with the Stokes' law or Martin model are somewhat deviated from that correlated, needing an improvement with empirical modification. The Bodenstein number (Bo), the axial dispersion coefficient (Dax) and the height equivalent of theoretical plate (HETP) are used to analyze and evaluate the liquid mixing in the expanded bed, based on residence time distribution (RTD) measurement. In summary, fluid velocity and viscosity are the crucial factors influencing the expansion and liquid mixing properties, while adsorbent density directly affects these properties in the form of the expansion factors. A further comparison of hydrodynamic properties of several adsorbents/matrices reveals that CroCD-TuC 3 can be selected as the most promising adsorbent for EBA use.
As a new attempt, the bio-active molecule rutin is adsorbed by CroCD-TuC 3 in aqueous solutions. The isothermal adsorption equilibrium follows the Langmuir adsorption equation, and the adsorption capacity is gradually reduced as the temperature and pH of solution rises, but increases with the increase of solvent polarity. The comparisons of CroCD-TuC 3 with SephadexTM G-15 beads on isosteric enthalpy and adsorbent surface heterogeneity analysis indicate that the formation ofβ-cyclodextrin/rutin inclusion complex in CroCD-TuC 3 skeleton makes significant contribution to the adsorption. The adsorption kinetics data are fitted by pore diffusion model. The effective pore diffusivity of rutin calculated with this model (3.8×10-11 m2·s-1) is much lower than that in diluted solution, indicating the diffusion inside the pores is the rate-restricting step in the whole adsorption process. The dynamic adsorption capacities of rutin with CroCD-TuC 3 are also measured and evaluated with the breakthrough curves in packed and expanded bed. The results reveal that the dynamic adsorption capacity is notably influenced by fluid velocity and feed concentration.
Finally, the practical application of CroCD-TuC 3 adsorbent is carried out in an expanded bed for separating the flavonoids from natural resources. The feedstock extracted from Ginkgo biloba L. leaves with water is directly loaded onto CroCD-TuC 3 in an expanded bed. After washed with water, the flavonoids are eluted with 50%(w/w) ethanol from the adsorbent. The recovery is 36.1% with the flavonoids purity of 36.5% and the purification factor of 20.8. The results demonstrate that CroCD-TuC 3 is suitable for flavonoids separation and purification. It is expected that the novel adsorbent CroCD-TuC 3 would be a promising candidate for flavonoids separation and purification.
引文
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