拟分维高效液相色谱法分离纯化双峰驼精清中的活性蛋白

利用一种新的高效液相色谱洗脱技术，建立了ＰＨ缓冲液／离子对二元洗脱模式，对双它精清的两种提取物的反相柱上进行了分离，得到了较纯的活性蛋白组分。该法操作简单，快速，比常规ＨＰＬＣ有更高的选择及及峰容量，并可减少活性蛋白的失活。     

31ku激发子蛋白的液相色谱分离纯化

利用阴离子交换色谱和疏水相互作用色谱从烟草疫霉菌培养液中分离出一种新的31ku激发子蛋白，选择了阴离子交换色谱流动相的的最佳pH值6．0，建立了疏水相互作用色谱硫酸铵Tris缓冲液－水洗脱模式，简化了纯化步骤和减少了活性损失的危险。     

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。     

转Bt基因植物表达产物Cry1Ab蛋白的制备纯化方法研究

以转Bt基因水稻为试材,研究其表达产物Cry1Ab蛋白的提取、分离及纯化的方法。实验结果表明,DEAE-纤维素填料对Bt蛋白有较好捕获效果。根据生物信息学方法预测了目标蛋白和主要共存蛋白的等电点和疏水性差异。合理地选择了阴离子交换色谱与疏水作用色谱组合方法。提取液经DEAE-Sephadex A-50柱层析及Phenyl-Sepharose Fast Flow疏水层析分离后,目标蛋白得到了显著的纯化。考察了疏水层析中用不同洗脱液洗脱Cry1Ab蛋白对活性回收率和纯度的影响,结果表明：以0．25mol／L KSCN作洗脱液对活性影响最小,HIC一步纯化倍数可达8倍,总纯化倍数达100倍。     

动态细胞骨架网络的自组织与力学性能

细胞骨架是由微管、肌动蛋白丝和中间纤维三种蛋白丝为主要成分组成的复合动态网络结构，在结合蛋白、辅助调节蛋白和马达蛋白的参与下帮助细胞实现运动、分裂和生长等基本生命过程。研究体外纯化的细胞骨架蛋白和马达蛋白网络，可以深入了解控制自组织亚细胞结构动力学行为的基本原理，为设计类似生命的活性物质和机器提供方向。本文综述了近年来基于纯化蛋白在体外简化环境中实现的细胞骨架蛋白-马达蛋白网络，重点介绍其非平衡本质、活性应力和动态网络的产生，以及这种动态网络对亚细胞结构和宏观尺度活性材料自组织过程的影响。此外，还简要介绍了细胞骨架蛋白-马达蛋白网络在构建体外仿生系统中的应用。     

液相色谱法分离纯化烟草疫霉菌激发子

利用阴离子交换色谱和疏水相互作用色谱从烟草疫霉菌培养液中分离了一种新的９０ＫＤ激发子蛋白,他阴离子交换色谱流动相的最佳ｐＨ值,建立了疏水相互作用色谱,硫酸铵与三羟甲基氨基甲烷（Ｔｒｉｓ）缓冲液／水洗脱模式,简化了纯化步骤和活性损失的危险。     

强阴离子交换色谱结合分子排阻色谱纯化血清中免疫球蛋白和白蛋白

动物血清中免疫球蛋白和白蛋白的等电点分别约为7.8和4.8,根据它们等电点的较大差别,利用Q SepharoseTM-XL强阴离子交换色谱结合分子排阻色谱同时分离纯化这2种蛋白。以0.02 mol/L pH 8.0的Tris-HCl缓冲液平衡离子交换色谱柱并将已稀释10倍的高免疫的兔血清上样,采用pH分段洗脱。在pH 6.0时以0.3 mL/min低流速洗脱得到高纯度的免疫球蛋白,继续在pH 4.0时洗脱,再辅以Sephadex G-75分子排阻色谱可获得纯度大于95%的白蛋白。对纯化后的蛋白进行活性检测,证明所纯化的免疫球蛋白和白蛋白都保持正常的生物活性。蛋白质含量测定说明免疫球蛋白的纯化回收率达到95%以上,而白蛋白的纯化回收率大于90%。该法简便快速,可同时从动物血清中纯化出保持生物活性的免疫球蛋白和白蛋白,纯化效率高。     

新型免疫磁性微球的制备及其在α-2b干扰素分离纯化中的应用

采用琼脂糖、纤维素和聚乙烯醇3种材料制备表面带有活性羟基的新型磁性微球,对其性能进行了表征并探讨其致敏条件。通过性能的比较,选择纤维素磁性微球作为分离纯化载体,并建立α-2b干扰素的免疫磁性分离方法。通过对α-2b干扰素发酵菌体裂解液的预处理、上样量和稀释倍数以及洗脱条件进行考察,确立免疫磁性分离条件,用于α-2b干扰素的一步分离纯化,其活性回收率达到88%,平均比活性为1.5×108IU/mg,纯度大于99%。实验结果表明,纤维素磁性微球作为性能优良的新型磁性载体,更适于目的蛋白的免疫磁性分离。     

弱阳离子交换色谱中疏水作用对蛋白保留的影响

选取了四种常用的弱阳离子交换(WCX)商品柱以研究标准蛋白在其上的色谱保留行为。发现在疏水色谱(HIC)模式下,蛋白在这四种WCX商品柱上也有不同程度的保留特征,且洗脱曲线呈现出保留值随盐浓度变化的"U"型。从分子力学角度定性解释了因疏水相互作用力的存在影响了蛋白在WCX色谱柱上洗脱顺序的改变。运用计量置换理论(SDT)中的两组线性方程进一步证实了WCX和HIC中蛋白与固定相间相互作用力的性质,在HIC中为非选择性作用力,而在离子交换色谱(IEC)中为选择性作用力。这四种色谱柱中的两种事实上可在WCX和HIC两种模式下,对标准蛋白进行分离且有较好的分离效果,有可能作为二维色谱柱来使用。     

阴阳离子交换色谱串联分离纯化苦瓜籽核糖体失活蛋白

将阴阳离子交换色谱柱在灌注色谱工作站上串联,使苦瓜籽粗提液中未被阴离子色谱柱吸附的蛋白质直接在阳离子色谱柱上吸附,用盐线性梯度洗脱从阳离子色谱柱上分离出两个具有抗真菌活性的蛋白。两个抗真菌蛋白经鉴定都达到了电泳纯,相对分子质量均为30000左右,N-端氨基酸序列分别为DVSFRLSGADPRSYGMFI与DVNFDLSTATAK,表明所得到的两种蛋白为苦瓜籽中的2个Ⅰ型核糖体失活蛋白α-苦瓜素(α-MMC) 和β-苦瓜素(β-MMC)。抗菌实验表明,α-MMC对香蕉枯萎菌和果腐霉菌均具有较强的抑制作用,β     

Separation of tricyclic psychosedative drugs by high-speed ion-pair partition and liquid-solid adsorption chromatography.

The application of high-speed ion-pair partition and liquid-solid adsorption chromatography to the separation of twenty common tricyclic tranquilizers and antidepressant drugs is described. In the ion-pair system, amine-perchlorate ion-pairs were extracted from an aqueous stationary phase supported on 10-mum silica gel by organic eluents containing a chloromethane and a higher aliphatic alcohol, and chromatographic parameters for elution by eight eluent mixtures are presented. Using 5 mm times 120 mm columns good separations, according to chemical class, were achieved. For adsorption chromatography, the components were eluted from 20-mum spherical alumina using eluents containing methylene chloride, n-hexane or n-pentane, and acetic acid. Chromatographic parameters are given for eight eluent compositions. Components differing little in structure are well separated by liquid-solid adsorption chromatography. Compared with ion-pair partition chromatography, adsorption chromatography is much more selective for compounds of the same chemical type. The two methods are therefore complementary. Both methods gave plate heights in the range of 0.1 to 0.3 mm.     

Determination of the urinary metabolites of caffeine and theophylline by high-performance liquid chromatography. A comparative study of a direct injection and an ion-pair extraction procedure

Chromatographic separation of methylxanthine metabolites was achieved using a Hypersil octadecylsilane column with a simple concave gradient elution programme of 0-12.75% acetonitrile in 1% tetrahydrofuran, pH 4.8, and the eluted components were detected by monitoring their absorption at 280 nm. An extraction procedure involving the formation of an ion-pair complex was developed which gave significant improvements over previously described methods including a shorter chromatographic run of 20 min. A thorough comparison of this procedure with a more convenient alternative involving direct injection of diluted urine specimens showed that the latter analysis was adequate for the quantitation of the major urinary metabolites of caffeine and theophylline.     

Optimisation of solvent desorption conditions for chemical warfare agent and simulant compounds from Porapak Q using experimental design. Part 2: Extraction of sulphur mustard from steel and glass Porapak tubes

The ion-pair solid-phase extraction (SPE) of 4-alkylphenols followed by derivatization with pentafluoropyridine is demonstrated. Under alkaline conditions, the 4-alkylphenols could be efficiently adsorbed on a C18 SPE cartridge conditioned with an ion-pair reagent, tetra-n-hexylammonium bromide. The ion pairs, ammonium phenolates, formed on the C18 solid phase, were eluted with a solvent containing the derivatizing reagent, pentafluoropyridine, and completely derivatized during the elution. After optimization of the adsorption and derivatization, we established a method for the determination of the 4-alkylphenols in water samples. The method showed good linearity between 20 and 1000 ng (200-10,000 ng for nonylphenol). By processing 20-ml samples, the method detection limits (MDL) were in the range of 5.2-8.9 ng/l for the 4-alkylphenols (76 ng/l for nonylphenol). To evaluate its applicability to a real aqueous matrix, several river water samples were analyzed.     

离子对色谱法同时分离测定吡啶及咪唑离子液体阳离子

建立了用紫外检测的反相离子对色谱梯度淋洗同时分离测定4种吡啶离子液体阳离子和5种咪唑离子液体阳离子的方法。实验采用ZORBAX Eclipse XDB-C18反相色谱柱,以离子对试剂水溶液(用柠檬酸调节pH值)+乙腈为流动相,考察了离子对试剂种类和浓度、乙腈浓度和色谱柱温度对保留的影响,探讨了相关保留规律,确定最佳色谱条件为:流速1.0 mL/min、柱温30℃,以1.0 mmol/L庚烷磺酸钠水溶液(pH 4.0)-乙腈为淋洗液进行梯度洗脱。在此条件下,4种吡啶阳离子和5种咪唑阳离子在15 min内达到基线分离。检出限(S/N=3)为0.31～0.54 mg/L,峰面积的相对标准偏差为0.10%～0.75%。将该方法用于实验室合成的离子液体样品分析,加标回收率为94%～98%。该方法准确、可靠,具有较好的实用性。     

Retention behaviour of an homologous series of oligodeoxythymidilic acids using reversed-phase ion-pair chromatography

Summary The retention behaviour of an homologous series of phosphorylated oligodeoxythymidylic acid (pd(T)_5–18) oligonucleotides was studied using reversed-phase ion-pair chromatography with isocratic elution conditions. The effects of temperature, pH, eluent ionic strength, percentage organic modifier, concentration and alkyl chain length of the ion-pairing reagent were investigated. The retention behaviour was generally explicable by current theoretical models of ion-pair chromatography. However, the marked effect of mobile phase pH on the retention of the oligonucleotides was unexpected, and this was ascribed to the presence of ionisable residual silanols on the surface of the reversed-phase packing material.     

Simultaneous Determination of Succinic Acid and Water-Soluble Vitamins by Ion-Pair High-Performance Liquid Chromatography

A procedure is proposed for the determination of succinic acid, riboxin, nicotinamide, and riboflavin by ion-pair HPLC with UV detection. Owing to the addition of an ion-pair modifier to the mobile phase and the selection of gradient elution conditions, the optimal retention and resolution of peaks of the components to be determined are achieved. Specificity, linearity, accuracy, and precision of the developed procedure are proved on an example of the determination of active substances in the Cerebronorm® preparation.     

Solid Phase Extraction and Subsequent Identification by Gas-Chromatography-Mass Spectrometry of a Germination Cue Present in Smoky Water

ABSTRACT

Aqueous extracts of plant-derived smoke (smoky water) are a complex mixture of thousands of components. Solid phase extraction (SPE) cartridges of varying polarity were trialed for their effectiveness in cleaning up smoky water, prior to subsequent analysis. The most effective SPE cartridge used was normal phase silica bonded with aminopropyl (NH₂) with hexane/dichloromethane/methanol mixtures as eluting solvents. This system retained all the active components and permitted elution of two active fractions, separated by an inactive fraction. Other cartridges tested either did not retain all active components or eluted active fractions successively, with more overlap between compositions. Active fractions, as identified by germination tests, were examined by GC-MS and were found to be sufficiently simplified to allow the identification of a new germination cue, 1, 8-cineole.     

染料配体固相萃取剂的制备及萃取性能

脲醛树脂-zro2复合微球;染料配体;固相萃取剂     

Factors affecting selectivity in ion chromatography

Methods for separation of ions by ion-exchange, ion-pair, and zwitterion ion chromatography share at least one common thread--the induced formation of a cation-anion pair in the stationary phase. Selectivity can be defined as the relative ability of sample ions to form such a pair. Examples are given in anion-exchange chromatography to show the effect of variations in the geometry, bulkiness and polarity of the resin cation on selectivity. The type of resin matrix, the hydrophobic nature of the resin surface and the degree of solvation also affect chromatographic behavior. The selectivity series observed in ion chromatography seems to be best explained by the interplay of two components: electrostatic attraction (ES) and the enforced-pairing (EP) that is brought about by hydrophobic attraction and by water-enforced ion pairing. Selectivity in ion-pair chromatography (IPC) and in zwitterion ion chromatography (ZIC) is affected by both the mobile phase cation and anion. This leads to elution orders for anions that are different from conventional ion-exchange chromatography (IC) of anions where cations are excluded from the stationary phase and have little effect on a separation. The elution order of anions in ZIC is similar to that in IC except for small anions of 2-charge, which are retained more weakly in ZIC. A unique advantage of ZIC is that sample ions can be eluted as ion pairs with pure water as the eluent and a conductivity detector. The mechanism for separation of anions on a zwitterionic stationary phase has been a subject for considerable debate. The available facts point strongly to a partitioning mechanism or a mixed mechanism in which partitioning is dominant with a weaker ion-exchange component.