扩张床吸附层析技术介质研发与应用进展

扩张床吸附层析是集固液分离、浓缩和初步纯化于一个操作单元之中的集成化生物分离技术,应用范围较广泛。本文对扩张床吸附层析介质的研究现状及该技术在生物产品分离中的应用进行了综述,以期能为该技术的进一步发展和应用提供理论基础。     

扩张床吸附基质研究进展

针对扩张床吸附这一新型蛋白质分离纯化技术，论述了吸附剂基质对该项技术应用的重要性，并且评述了近十年来扩张床吸附基质的研究状况，在此基础上重点介绍了基质所需的特性、常用的扩张床基质及其制备方法。     

新型高效液相层析方法:连续床层析

连续床层析的研究始于八十年代末,是一种新型的高效液相层析分离方法,可用于从小分子到蛋白质等生物大分子的分离纯化。连续床层析柱具有制备简单、背压低、分辨率高和处理量大等优点。本文综述了近年来连续床层析介质的制备以及连续床层析的应用研究进展。     

气体和蒸气吸附的位势理論及其应用

一、引言吸附技术在近十几年来发展十分迅速,它已经成为一个重要的单元过程。这主要是因为合成材料等工业对原料的纯度和预处理方面提出了一般方法难以完成的要求;移动床、流化床和多段流化床的应用从根本上改善了固体物料的操作技术;原有吸附剂品种的扩充和新型吸附剂的出现。目前,吸附方法日益广泛地被人们用来分离气体混合物;实现气体的精密清净和     

灰熔聚射流流化床适宜操作条件及几何结构

在３００×５０×２６００ｍｍ带有流化分离柱的灰熔聚冷模流化床中，选用具有实际意义的焦粉／灰球体系，通过考察不同操作条件及不同几何结构时灰球的分离效率及分离速率的变化特征，以确定这一射流流床的适宜操作及几何条件。证明了对分离操作而言，射流分离分隔式的流化床具有操作弹性大的优点。基于实验结果及理论分析，指出流出分离柱高／宽比小于３．７时可获得稳定分离操作，并给出分离柱高效分离操作时的气速变化范围。本文     

浅流化床的热响应

众所周知,流化床中气体一颗粒的传递过程和反应过程,主要发生在分布板区。而浅层流化床正具备了分布板区的许多优点,它在涉及热过程的领域中,已经显示了广阔的应用前景。为了考察浅层床中气体一颗粒间的传递特征,本文从气体一颗粒传热入手,采用热响应的实验技术,测定了气-固系浅流化床(L/D_T=0.045～0.17)内空气-海砂的表观传热系数,同时求算了相应的浓相区传热系数,并表述了传热系数随床深的变化趋势。从实用角度,本研究最终将表明,在较浅的颗粒床层中,能给出较大的传热速率但床层压降较小,藉以推广浅流化床的应用。     

一种球形纤维素/钛白粉扩张床吸附剂的研究--组成对性能的影响

采用反相悬浮再生法 ,以超细钛白粉颗粒作增重剂 ,包埋于纤维素骨架之中 ,经环氧氯丙烷活化后与二乙胺连接 ,制得一种球形扩张床吸附剂 .研究表明 ,吸附剂的密度、机械强度和孔结构可以随钛白粉用量的变化而改变 ;钛白粉颗粒的掺入有利于基质的活化 ,活化后环氧基含量可达 2 2 0 μmol mL .吸附剂具有良好的扩张床性能 ,扩张床中的蛋白质吸附行为与填充床中相似 ,吸附容量为 4 8 9mg牛血清白蛋白 mL吸附剂     

气液并流向上三相流化床流体流动性质的研究

本文研究气液并流向上兰相流化床的一般流体流动性质。阐明了不同性质及粒度的固体颗粒、流化床静止床高、气体及液体的表观线速、气体分布板结构等参数对床层的均匀性、气体及液体滞留量、床层膨胀高度及气泡形态及大小等影响的一般规律。提出了在液固两相流化床中通入气体后,床层膨胀比随着气体速度的增大而变化的规律具有五种不同类型。用气泡上升的平均真实速度V_g/ε_g作为V_g及V_L的函数作图,可以表明气体及液体的表观线速对气体滞留量及气泡大小的影响,并用在流化床上部的照片直观地表明了这一点。最后用关联式将本文数据进行关联,误差在±10%以内。     

生物化学中应用的离子交换介质简介

一、前言以间歇操作及色谱操作的离子交换技术是生物化学中重要的分离方法之一。与凝胶渗透法、分配色层法、亲和层析及电泳柱等方法相比,离子交换技术最主要的优点是具有较高的交换吸附容量及分离容量。自八十年代以来离子交换色谱得到高速发展,已工业规模用于分离、纯化产品~([1,2])。     

流化床射流区的颗粒分离

研究了气体垂直射流喷入流化床时待分离物料玻璃珠从喷口排出的稳定分离过程及各参数对初始分离速度U_(IJ)的影响,其经验式为U_(IJ)=1.334U_t.e~(0.1171H)_0C_b~(0.0894)考察了射流区的几何结构对床层流动特性与分离过程的影响,提出用结构流动参数C_5作为选取射流区台理几何结构的重要判据。发现了在粗颗粒射流流亿床中加入一定量的细粒组份将有利于形成射流区的稳定颗粒分离过程。     

Expanded Bed Adsorption Chromatography for Recovery of Phycocyanins from the Microalga Spirulina Platensis

We carried out the purification of C-phycocyanin and allophycocyanin from Spirulina platensis taking advantage of the adsorption properties of the expanded beds. Initially, phycobiliproteins were released from the microalga cells by osmotic shock. Next, phycocyanins were recovered by applying the centrifuged cell suspension directly to the anion exchanger Streamline-DEAE using expanded bed columns, equilibrated with 50 mM sodium phosphate buffer, pH 7.0. After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and phycocyanins rich solution was eluted with a downward flow of 500 mM sodium phosphate buffer, pH 7.0. Finally, we utilized conventional gel filtration and ion exchange chromatography methods for separation and purification of C-phycocyanin and allophycocyanin. The purification steps were monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the purity of recovered phycocyanins was confirmed by absorption and emission spectroscopy. The main advantage of this new method is the high yield achieved in the steps of product extraction and adsorption by expanded bed adsorption, so reducing both processing times and costs.     

Combination of integrated expanded bed adsorption chromatography and countercurrent chromatography for the direct extraction and purification of pseudohypericin and hypericin from St. John's wort (Hypericum perforatum L.)

St. John's wort has attracted particular attention because of its beneficial effects as an antidepressant, antiviral, and anticancer agent. A method for the combination of integrated expanded bed adsorption chromatography and countercurrent chromatography for the simultaneous extraction and purification of pseudohypericin and hypericin from the herb is presented in this paper. Firstly, the constituents were extracted and directly adsorbed by expanded bed adsorption chromatography under optimal conditions. The stepwise elution was then performed by expanded bed adsorption chromatography that enriched the targets with higher purities and recoveries compared to other methods. Secondly, the eluent fractions from expanded bed adsorption chromatography were further separated by two‐step high‐speed countercurrent chromatography. A two‐step high‐speed countercurrent chromatography method with a biphasic solvent system composed of n‐hexane/ethyl acetate/methanol/water with a volume ratio of 1:2:1:2 was performed by stepwise changing the flow rate of the mobile phase. Consequently, 5.6 mg of pseudohypericin and 2.2 mg of hypericin with purities of 95.5 and 95.0%, respectively, were successfully obtained from 40 mg of crude sample.     

Affinity purification of proteins using expanded beds.

The use of expanded beds of affinity adsorbents for the purification of proteins from feedstocks containing whole or broken cells is described. It is demonstrated that such feedstocks can be applied to the bed without prior removal of particulate material by centrifugation or filtration thus showing considerable potential for this approach in simplifying downstream processing flow-sheets. A stable, expanded bed can be obtained using simple equipment adapted from that used for conventional packed bed adsorption and chromatography processes. Circulation and mixing of the adsorbent particles is minimal and liquid flow through the expanded bed shows characteristics similar to those of plug flow. Frontal analysis performed with the highly selective affinity system involving the adsorption of human polyclonal immunoglobulin G onto Protein A Sepharose Fast Flow indicate that the adsorption performance of the expanded bed is similar to that achieved when the same amount of adsorbent is used in a packed configuration at the same volumetric flow-rate. The adsorption performance of the expanded bed was not diminished when adsorption was carried out in the presence of intact yeast cells. Batch adsorption experiments also indicated that the adsorption characteristics of the affinity system were not greatly altered in the presence of cells in contrast to results from a less selective ion-exchange system. An expanded bed of Cibacron Blue Sepharose Fast Flow was used to purify phosphofructokinase from feedstock of disrupted yeast prepared by high pressure homogenisation without the need for prior removal of particulate material. The potential for the use of expanded beds in large scale purification systems is discussed.     

Use of expanded bed adsorption to purify flavonoids from Ginkgo biloba L.

Three techniques (liquid–liquid extraction, packed bed adsorption and expanded bed adsorption) have been compared for the purification of flavonoids from the leaves of Ginkgo biloba L. A crude Ginkgo extract was obtained by refluxing with ethanol for 3 h. The yield of flavonoids achieved by this crude extraction was about 19% (w/w) and the purity of flavonoids in the concentrated extract was between 1.9 and 2.3% (w/w). The crude extract was then dissolved in deionized water and centrifuged where necessary to prepare clarified feedstock for further purification. For the method using liquid–liquid extraction with ethyl acetate, the purity, concentration ratio and yield of flavonoids were 25.4–31.0%, 16–18 and >98%, respectively. For the method using packed bed adsorption, Amberlite XAD7HP was selected as the adsorbent and clarified extract was used as the feedstock. The dynamic adsorption breakthrough curves and elution profiles were measured. For a feedstock containing flavonoids at a concentration of 0.25 mg/mL, the appropriate loading volume to reach a 5% breakthrough point during the adsorption stage was estimated to be 550–600 mL for a packed bed of volume 53 mL and a flow rate of 183 cm/h. The results from the elution stage indicated that the majority of impurities were eluted by ethanol concentrations of 40% (v/v) or below and efficient separation of flavonoids from the impurities could be achieved by elution of the flavonoids with 50–80% ethanol reaching an average purity of ∼25%. The recovery yield of flavonoids using the packed bed purification method was about 60% of the flavonoids present in the clarified feedstock (corresponding to around 30% for the total flavonoids in the unclarified crude extract). For the method using expanded bed adsorption also conducted with Amberlite XAD7HP as the adsorbent, the optimal operation conditions scouted during the packed bed experiments were used but unclarified crude extract could be loaded directly into the column. For an expanded bed with a settled bed height of 30 cm, the loss of flavonoids in the column flow-through was about 30%. The two-step elution protocol again proved to be effective in separating the adsorbed impurities and flavonoids. More than 96% of the bound impurities were completely removed by 40% ethanol in the first elution stage and less than 4% remained in the final product eluted by 90% ethanol in the second elution stage. Also, ∼74% of the adsorbed flavonoids on column (corresponding to 51% of the total flavonoids in the unclarified feedstock) were recovered in the product. In addition to higher recovery yield, the average process time to obtain the same amount of product was decreased in the expanded bed adsorption (EBA) process. The results suggest that the adoption of EBA procedures can greatly simplify the process flow sheet and in addition reduce the cost and time to purify flavonoids from Ginkgo biloba. These results clearly demonstrate the potential for the use of EBA to purify pharmaceuticals from plant sources.     

Adsorption Strategy of Plasmid DNA Nanoparticulate: Preparative Purification by a Simple Custom Expanded Bed Column

This paper summarizes the critical examination of the hydrodynamic performance of the NBG expanded bed contactor operated with streamline-DEAE adsorbent under various operating conditions for expanded bed adsorption of plasmid DNA nanoparticles from alkaline lysate. The purification process is not RNase-free. In this study, a rapid and efficient scaleable purification protocol obtaining, plasmid DNA nanoparticles (average size of 40 nm) with a high purity level for use as therapeutic agent in customized NBG expanded bed columns was developed. This technique allows efficient levels of binding to the column media and vector purification without centrifugation or filtration steps. Residence time distribution (RTD) studies were exploited to achieve the optimal condition of plasmid DNA nanoparticle (pDNA) recovery upon anion exchange adsorbent in this contactor. In addition, the purification experiments were carried out in the expanded bed columns with settle bed height of 6.0 ± 0.2 cm. NaCl gradient elution enabled the isolation of supercoiled plasmid from low-Mr RNA, cDNA and plasmid variants. Subsequently dynamic binding capacity of the adsorbent was calculated while these values decreased with increase in flow velocity. Moreover, the effect of pH upon the performance of this recovery process and the feedstock volume upon the expanded bed anion exchange purification was investigated. The results demonstrated that separation of low-Mr RNA from plasmid DNA isoforms in the range of pH between 5.5 and 7.5 is achievable in this column. The yield of recovery of pDNA in optimal condition was higher than 88.51% which was a superior result in one-pass frontal chromatography. The generic application of simple customized NBG expanded bed column and its potential for the purification and recovery of plasmid DNA as a nanoparticulate bioproduct is strongly discussed.     

Mass spectrometric strategies for the analysis of polar industrial chemicals and their by-products in wastewater and surface water

The purification of a 6x-histidine tagged viral coat protein (L1) in expanded mode directly following chemical extraction from the cytoplasm of Escherichia coli HMS174(DE3) is investigated. Chelating adsorbents based on the ligands iminodiacetic acid (IDA) and nitrilotriacetic acid, using chelated metal ions Ni2+ and Cu2+, were compared. The use of Ni2+-IDA resulted in a high purification factor (9.7) and moderate recovery yield (58%). However, the eluted fractions had an overall L1 purity less than 50% and were therefore significantly contaminated with other host proteins. In batch tests, Cu2+-IDA was found to be superior to all other combinations as it was characterised by higher binding capacities and faster adsorption kinetics. A subsequent immobilised metal affinity chromatography-expanded bed adsorption experiment using Cu2+-IDA resulted in a higher L1 purification factor (20), recovery yield (71%) and purity (89%). The process presented here combines direct chemical extraction with expanded bed recovery. It is simpler than traditional methods, and should find more widespread application in the recovery of inclusion body proteins. Robust pseudo-affinity ligands such as metal chelates show potential for selective primary recovery of unfolded proteins, and could be used for further processing such as on-column refolding.     

纳豆激酶分离纯化和酶活性测定的研究进展

该文对纳豆激酶的分离纯化和酶活性测定进行了综述。重点讨论了溶剂沉淀法、柱层析法、磁性微球吸附法、膨胀床法、反相胶束法、三相分割法等分离方法。对酶活性的不同测定方法进行了讨论和比较。提出了将核酸适配体识别技术用于纳豆激酶分离纯化和酶活性测定的可行性。