疏水作用色谱介质的合成及色谱特性研究

利用国产大孔硅胶作基质合成了疏水填料。按照高效疏水作用色谱法,采用梯度洗脱方式分离了6种标准蛋白及唾液中α-淀粉酶和基因工程生产的γ-干扰素。柱子不可逆吸附小、被试验的α-淀粉酶和溶菌酶活性几乎定量被回收。应用合成的色谱填料研究了洗脱剂中盐浓度和温度对蛋白质保留行为的影响,论证了合成填料的色谱属性。     

疏水作用色谱填料研究进展

疏水作用色谱法（HIC）是一种有效的蛋白质分离纯化方法。综述了HIC填料的基质和配基的发展过程,举例说明了不同HIC填料对蛋白质的分离纯化效率,指出了制备型HIC填料的现状及其发展趋势。     

浸透限制固定相及其应用

讨论了浸透限制固定相的特性及其在药物分析中的应用。已发展起来的浸透限制固定相主要用于高效液相色谱法对生物体液中药物的直接进样分析。这种固定相填料是在疏水层上覆盖了一层亲水层,而亲水层允许小分子物质如药物自由出入于固定相的疏水部分,从而立体地阻止了大分子物质如蛋白质与固定相的疏水部分相互作用,使小分子物质得到分离。     

一种疏水色谱填料的特性及应用的研究

 以交联壳聚糖为基质 ,正戊醛为配基 ,利用改进的方法制备了疏水作用色谱 (HIC)填料 ,并对该色谱填料的吸附行为和应用作了研究。结果表明 ,此类填料对蛋白质的吸附行为符合疏水相互作用理论 ,对α 淀粉酶的纯化活性回收率大于 80 %。     

用前沿色谱法测定蛋白质吸附等温线时增浓方式的研究

用连续前沿色谱法和断续前沿色谱法测定了七种标准蛋白在疏水色谱填料上的吸附等温线,两种方法之间存在着一定的差别。从流出曲线突跃处斜率测定的不确定性、色谱过程中动力学因素和实验方法本身存在的问题等几个方面探讨了误差的来源,指出了可能解决这一问题的途径。     

亲和色谱法的进展

概述了亲和色谱法分离机理、色谱填料及其应用的新进展,着重介绍了亲和固定相中配基发掘的新途径。全文包括66篇文献。     

新型高效疏水相互作用色谱填料的合成及性能的研究

]本文介绍了在硅胶基质上键合聚乙二醇、聚丙二醇、聚四氢呋喃和改性聚乙二醇等多类疏水色谱填料的合成及其分离蛋白质的色谱特性。其中改性聚乙二醇、聚1,2-丙二醇、聚四氢呋喃型配基的疏水填料为首次合成。文中探讨了硅胶孔径、聚乙二醇链长、改性聚乙二醇配基的端基和不同的聚醚链对蛋白质的保留值、活性回收率、分离度、填料稳定性和键合密度的影响,发现了在聚醚链单元上引入支链改变生物大分子与填料疏水区的接触面积可以改变蛋白质的保留时间和选择性。此外,还介绍了在硅胶上包裹有机胺类化合物再与环氧交联的疏水色谱填料的制备。     

高效疏水作用色谱填料的合成及其在重组人干扰素纯化中的应用

高效疏水作用色谱（HPHIC）是利用不同蛋白质表面疏水区域与填料之间具有不同疏水作用进行分离的．由于HPHIC采用盐水体系作为流动相，配体采用极性的有机基团，使蛋白质可以在十分温和的条件下进行分离，且保持其生物活性基本不变[1，2]．自80年代中期以来，HPHIC在蛋白质的分离纯化上得到了广泛的应用．在90年代初期，随着基因工程技术的发展，HPHIC同时也被应用到基因工程的下游纯化技术上[3，4]．本文中我们合成了一OCH2CH3为端基的填料，检验了该填料的分离效果，并利用该填料对酵母菌表达的人αA-干扰素、大肠杆菌（E．col…     

透明超疏水材料的研究进展

超疏水表面在包括自清洁、防腐、防冰和流体输送过程中的减阻等许多领域都有广泛应用,透明的超疏水表面更是在太阳能光伏电池板和其他光学领域具有自我清洁的潜在应用。本文首先介绍了透明超疏水的相关理论,然后概括了制备超疏水表面常见的方法,重点归纳总结了用于构建粗糙度的不同物质,如SiO2、 TiO2 等,并分析了其优缺点;最后,简单介绍了透明超疏水的应用前景,并对其研究方向进行了展望。     

仿生超疏水性表面的生物应用

自然给科学家和工程师带来仿生的灵感和启发. 近年来, 受自然界中荷叶的启发, 在充分考虑表面形貌和化学组成协同效应的基础上, 人们已经制备出许多仿生超疏水性表面, 这些表面在抗结冰、微流体、生物相容性等领域具有很多潜在的应用价值. 仿生超疏水性表面在生物领域的应用逐渐崭露头角, 研究发现, 超疏水性表面所俘获的空气能够减缓药物释放的速率, 因此利用此类表面作为药物的载体有望实现长期供药. 超疏水特性能在一定程度改善和提高生物体与材料表面之间的相互作用, 例如, 血小板几乎不在超疏水表面上进行粘附和活化避免了造成血栓和血凝, 因此仿生超疏水性表面可用于制备人造血管和与血液相接触的仪器. 细胞和生物分子在不同特殊润湿性表面具有不同的行为和现象, 如粘附、繁殖、吸附等差异, 这有助于进一步探索研究细胞和生物分子的信息功能, 是当前仿生超疏水性表面应用的重要研究方向之一. 本综述简单介绍了经典的润湿模型, 重点总结了仿生超疏水表面在生物领域的应用, 其主要包括控制药物释放、提高血液相容性、蛋白质吸附研究、细胞行为研究、生物分子和细胞微图案化等. 最后, 对仿生超疏水性表面在生物领域研究应用进行了展望.     

分离生物大分子的液相色谱固定相

本文介绍了各类用于生物大分子分离的液相色谱填料,并对它们在生物大分子分离及制备中的性能、基本特征作了评述。     

Preparation of hydrophobic interaction chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application

The monodisperse, poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads with macroporous in the range of 8.0-12.0 microm were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury instrusion method. Based on this media, a hydrophobic interaction chromatographic (HIC) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages for biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The dynamic protein loading capacity of the synthesized HIC packings was 40.0 mg/ml. Six proteins were fast separated in less than 8.0 min using the synthesized HIC stationary phase. The HIC resin was firstly used for the purification and simultaneous renaturation of recombinant human interferon-gamma (rhIFN-gamma) in the extract solution containing 7.0 mol/l guanidine hydrochloride with only one step. The purity and specific bioactivity of the purified of rhIFN-gamma was found more than 95% and 1.3 x 10(8) IU/mg, respectively.     

Anion-exchange chromatography of DNA restriction fragments.

The abilities of several high-performance liquid chromatography (HPLC) anion-exchange packings to separate DNA restriction fragments, ranging in size from 50 to 23,000 base pairs, were studied. The ion exchangers investigated include the porous packings Protein-Pak DEAE-5PW, Nucleogen-DEAE 4000-7, Poros-Q and BakerBond WP-PEI, and the non-porous packings TSK Gel DEAE-NPR, Gen-Pak FAX, and ProPac PA1. The results indicated that the non-porous packings could separate all 18 fragments (less than 600 base pairs) in a pBR322 DNA-HaeIII digest, while of the porous packings, only Nucleogen-DEAE 4000-7 could resolve DNA fragments in this size range. Only Gen-Pak FAX and TSK Gel DEAE-NPR could significantly resolve the very large DNA fragments (125-23,000 base pairs) of a lambda DNA-HindIII digest. The chromatographic parameters governing this separation by Gen-Pak FAX were optimized so that six of eight fragments were resolved. Split-peak phenomena were observed at low flow-rates when employing non-poros packings, but were eliminated by the incorporation of organic modifiers or surfactants, suggesting that, under certain conditions, hydrophobicity may play a significant role in separations on this packing. Gen-Pak FAX also separated 21 of 23 fragments in a 1000-base pair DNA ladder, a performance which, in addition to the quantitative capabilities of HPLC, makes anion-exchange chromatography a powerful method complementary to slab-gel electrophoresis, and perhaps preferable over agarose gel electrophoresis for applications such as the confirmation of plasmid integrity.     

新型离子交换硅胶键合相的制备及评价

二甲基氯硅烷与硅胶表面反应,形成牢固的ＳｉＨ键之后,连接上活泼的中间体——烯丙基缩甘油醚作为柔软的分子臂,最后接上二乙基氨基,由此制得了新型的离子交换硅胶键合相。经漫反射红外光谱、元素分析和高效液相色谱法对键合相进行了鉴定和评价。结果表明：键合反应按预定路线进行,键合相具有较好的色谱性能。此种方法可有效地运用于无孔硅胶填料的制备。     

Superficially porous silica microspheres for fast high-performance liquid chromatography of macromolecules

Very fast reversed-phase separations of biomacromolecules are performed using columns made with superficially porous silica microsphere column packings ("Poroshell"). These column packings consist of ultra-pure "biofriendly' silica microspheres composed of solid cores and thin outer shells with uniform pores. The excellent kinetic properties of these new column packings allow stable, high-resolution gradient chromatography of polypeptides, proteins, nucleic acids, DNA fragments, etc. in a fraction of the time required for conventional separations. Contrasted with <2-microm non-porous particles, Poroshell packings can be used optimally with existing equipment and greater sample loading capacities, while retaining kinetic (and separation speed) advantages over conventional totally porous particles.     

Hydrophobic Interaction Chromatography of t-RNA's and Proteins

Abstract

Various microparticulate siliceous bonded stationary phases having weakly hydrophobic ligates were developed for HPLC of proteins and t-RNA's by hydrophobic interaction chromatography (HIC). It was confirmed that optimal separation of different types of biopolymers can be obtained by using a set of stationary phases having appropriate hydrophobic properties. Thus, the separation of t-RNA's is best carried out on stationary phases which are more hydrophobic than those optimal for HIC of proteins. Plots of log k' of both proteins and t-RNA's against the salt molality in the eluent yielded straight lines at sufficiently high salt concentrations in the eluent. The limiting slopes represent the hydrophobic interaction parameter for the particular chromatographic system and can serve as measures of the hydrophobic character of either the biopolymer or the stationary phase. Stationary phases with covalently bound polyether chains at the surface were found to be most suitable for HIC of proteins and t-RNA's.     

Packings and stationary phases in preparative column liquid chromatography

Although the theoretical treatment of chromatographic processes on a preparative scale provides guidelines to the extent to which packing and stationary phase properties affect the target quantities such as sample input, throughput and resolution times sample input, a series of additional criteria were established to judge the quality of a packing in preparative column liquid chromatography. These include bed stability and flow resistance, chemical resistance and purity, solute accessibility, mass and biological recovery, fouling, regeneration and cost. Applying these criteria, the relative importance of physical and chemical structure parameters of packings and stationary phases was assessed. Commercial packings with mean particle diameters dp greater than 20 micron were listed for adsorption, size exclusion, ion-exchange and affinity chromatography. An analysis of the characteristic features of phase systems showed that adsorption media offer a high selectivity combined with adequate loadability, whereas ion exchangers and affinity media were best suited for biospecific solutes, particularly biopolymers, which can be attributed to their high selectivity and loadability.     

Options for rapid analysis of peptides and proteins, using wide-pore, superficially porous, high-performance liquid chromatography particles with unique bonded-phase ligands

The large size and complexity of many proteins constrains the reversed-phase high-performance liquid chromatography packings that are useful for their separation. Wide-pore, superficially porous, silica-based packings with solid 4.5-microm cores and a 0.25-microm porous outer layer (Poroshell) demonstrate a variety of characteristics that are beneficial for the separation of proteins. A shorter diffusion distance allows separations of large molecules at high linear velocities. This benefit over totally porous particles is clearly shown using separations of a peptide-protein standard. The structure and reduced surface area (4.5 m2/g) of these superficially porous particles simplifies interactions with its surface, resulting in improved peak shapes and resolution. Specialized bonding chemistries for low- and high-pH operation may be used to change band-spacing and achieve atypical separations. These rapid analysis options are demonstrated using protein standards and very high molecular weight glycosylated proteins including intact monoclonal antibodies, IgM, alpha2-macroglobulin, and glycophorin. In liquid chromatography-mass spectrometry analysis of a myoglobin peptide digest, bidentate-C18-bonded superficially porous packings achieve complete runs in 4 min and demonstrate an elution pattern that is unique from that of material bonded with sterically protected C18 ligands.     

Chemically modified resins for solid-phase extraction

The packings most widely used for solid-phase extraction are hydrophobic and make poor surface contact with aqueous samples unless the resins are first treated with an activating organic solvent such as methanol. Insertion of an acetyl- or hydroxymethyl group into a porous polystyrene-divinylbenzene resin provides a more hydrophilic surface that is easily wetted by water alone. Small columns of the chemically modified resins were found to be very efficient for the solid-phase extraction of many types of organic solutes from aqueous samples. Comparative recovery studies showed that the modified resins are superior to both silica packings and unmodified organic resins for the solid-phase extraction of organic compounds, and especially for polar organics such as phenols.