一步纯化胰岛素单克隆抗体

本文报道用瑞典Pharmacia公司生产的快速蛋白液相层析仪、强阴离子交换预装柱(MonoQ)一步纯化胰岛素单克隆抗体的方法,结果证明此法快速、简便、重复性和分离效果好。纯化后的单克隆抗体的免疫活性仍在94%左右。     

射干中乳酸脱氢酶抑制剂的筛选分离与质谱分析

本研究采用高效液相色谱-电喷雾质谱联用技术(HPLC-ESI-MS)对射干提取物中异黄酮类化学成分进行分析鉴定,以乳酸脱氢酶作为生物靶分子,运用超滤质谱技术筛选出6种酶抑制剂,分别为鸢尾苷(tectoridin)、鸢尾新苷A(Iristectorin A)、野鸢尾苷(iridin)、鸢尾苷元(tectorigenin)、野鸢尾黄素(irigenin)和次野鸢尾黄素(irisflorentin),并采用半制备型高效液相色谱技术(Semi-prep·HPLC)分离纯化射干提取物中的活性成分。所得单体分离收集液经分析型高效液相色谱法检测,纯度均大于90%。结果证明利用液相色谱-超滤-质谱-半制备型高效液相色谱联用技术可以快速筛选、鉴定、分离射干中的活性物质。此方法对于筛选酶抑制剂有快速和灵敏等优势,具有广泛的应用前景。     

中低压制备色谱制备茜草蒽醌成分的研究

建立新的中低压制备色谱技术,分离纯化茜草蒽醌单体.先采用高压液相色谱技术分析所分离产物的纯度,再用核兹共振谱、红外、质谱分析确证单体的化学结构.结果表明:用中低压色谱分离茜草的乙醇提取物而得到的4个单体,其纯度分别为99.87％、99.56％、99.42％、98.21％,结构鉴定为从石油醚萃取物中分离得到的单体为大叶茜...     

唐菖蒲球茎中抗真菌物质的部分性质研究

本文报道唐菖蒲球茎中抗真菌活性物质的成分及其分子量测定方法和结果。用沸水提取,经过超滤、透析、柱层析,纯化到一种有抗真菌活性的物质。排除了它是蛋白质、核酸、脂类及小分子物质的可能性,确定这是一种多糖,定名为唐菖蒲抗真菌多糖（Gladiolus hntifungal Polysaccharide,简称GAFS）。真空旋转蒸发干燥后,用苯酚-硫酸法测得干燥后的样品糖含量为44．08％,用FPLC（快速蛋白质液相层析仪）测其分子量为15750。     

高效液相色谱法制备Ⅰ型胶原蛋白及其性质研究

从猪皮中分离纯化I型胶原蛋白,并对其部分性质进行研究。采用粗提法,高效液相色谱半制备法进行分离纯化,用分析型高效液相色谱检测纯度,同时对其理化性质进行鉴定,并对所提取的胶原进行全身急性毒性试验及皮肤致敏试验。高效液相色谱测定结果显示所得样品为一单峰,理化性质测定结果符合I型胶原蛋白特征;通过整体水平的安全性评价,表明该方法提取的I型胶原蛋白具有较大的安全性和可靠性。因此,用高效液相色谱可制得高纯度且具有良好生物安全性的I型胶原蛋白。     

大孔吸附树脂结合半制备型高效液相色谱分离纯化栝楼果皮中的水溶性化学成分(英文)北大核心CSCD

本文使用大孔吸附树脂结合半制备型高效液相色谱,对栝楼果皮中的水溶性化学成分进行了分离纯化。栝楼果皮水溶性粗提物经大孔吸附树脂粗分为4个部分,每一部分分别使用半制备型高效液相色谱进一步纯化,最终得到3种核苷酸及6种碱基,其化学结构经紫外光谱及核磁共振鉴定为:胞嘧啶、尿嘧啶、次黄嘌呤、鸟嘌呤、黄嘌呤、腺嘌呤、鸟苷、6-异次黄嘌呤核苷及腺苷。本文对于栝楼果皮中水溶性化学成分进行了较为系统的研究,建立起来的分离纯化方法具有简单、快速、经济和易于放大的优点,适合于天然产物中强极性活性成分的大规模制备。     

高效液相色谱法制备Ⅰ型胶原蛋白及其性质研究

从猪皮中分离纯化Ⅰ型胶原蛋白,并对其部分性质进行研究。采用粗提法,高效液相色谱半制备法进行分离纯化,用分析型高效液相色谱检测纯度,同时对其理化性质进行鉴定,并对所提取的胶原进行全身急性毒性试验及皮肤致敏试验。高效液相色谱测定结果显示所得样品为一单峰,理化性质测定结果符合Ⅰ型胶原蛋白特征;通过整体水平的安全性评价,表明该方法提取的Ⅰ型胶原蛋白具有较大的安全性和可靠性。因此,用高效液相色谱可制得高纯度且具有良好生物安全性的Ⅰ型胶原蛋白。     

大孔吸附树脂结合半制备型高效液相色谱分离纯化栝楼果皮中的水溶性化学成分（英文）

本文使用大孔吸附树脂结合半制备型高效液相色谱,对栝楼果皮中的水溶性化学成分进行了分离纯化。栝楼果皮水溶性粗提物经大孔吸附树脂粗分为4个部分,每一部分分别使用半制备型高效液相色谱进一步纯化,最终得到3种核苷酸及6种碱基,其化学结构经紫外光谱及核磁共振鉴定为:胞嘧啶、尿嘧啶、次黄嘌呤、鸟嘌呤、黄嘌呤、腺嘌呤、鸟苷、6-异次黄嘌呤核苷及腺苷。本文对于栝楼果皮中水溶性化学成分进行了较为系统的研究,建立起来的分离纯化方法具有简单、快速、经济和易于放大的优点,适合于天然产物中强极性活性成分的大规模制备。     

二维液相色谱法在羊草地上部总蛋白分离中的应用

取8周龄羊草的地上部分,用三氯乙酸-丙酮法沉淀总蛋白,沉淀裂解后将缓冲液置换为起始缓冲液,进行第一维色谱聚焦分离。将第一维分离收集的pH值为8.5至4.0之间的组分分别进行第二维无孔硅胶反相高效液相色谱分离,利用ProteoVue软件获得羊草植株总蛋白pI/UV图谱,即羊草植株总蛋白质表达谱。文中对二维液相色谱法分离羊草蛋白质进行了方法学的研究,在第二维分离中尝试用3种不同的洗脱梯度条件进行分离,优化二维液相色谱分离条件并与传统凝胶双向电泳进行了比较,另外还对二维液相色谱的重现性和准确性进行了检验。实验建立了利用二维液相色谱分离羊草总蛋白的技术方法。     

UPLC/Q-TOFMS对三七中皂苷类成分的快速化学表征北大核心CSCD

本文主要运用超高效液相串联四级杆飞行时间质谱联用技术(UPLC/Q-TOF MS)对三七中皂苷类成分进行快速分离和鉴定。以超高效液相为分离手段,飞行时间质谱仪为鉴定方法,水饱和正丁醇为提取溶剂,以0.05%甲酸水(A)-0.05%甲酸乙腈(B)为流动相进行梯度洗脱,采用ESI负离子模式进行数据采集。根据实验结果,并结合相关参考文献,二级质谱裂解数据以及元素组成,分离、鉴定出17种皂苷类成分,并发现三七中含有姜糖酯B成分。实验结果表明经过超高效液相的分离,以及Q-TOF MS的二级负离子信息的鉴定,为分析三七中皂苷类成分提供了一种快速、简便、可靠的方法。     

金标法与免疫发光法检测CEA在健康体检中的应用

目的：探讨金标法和免疫发光法检测CEA在健康体检中的应用价值。方法：对728例健康体检的个人同时应用金标法和免疫发光法测定CEA,对结果进行分析。结果：金标法测定的阳性率为0．69％,免疫发光法测定的阳性率为0．41％,两者具有高度一致性。结论：对健康体检人群应先用金标法进行定性,对阳性结果再用免疫发光法进行定量。     

Use of cyclodextrins as chiral selectors for direct resolution of the enantiomers of fluoxetine and its metabolite norfluoxetine by HPLC

The goal of this work is to investigate the direct chromatographic separation of the enantiomers of fluoxetine and its active metabolite norfluoxetine. The liquid chromatographic retention behavior of these enantiomers on a β-cyclodextrin bonded-phase column was investigated with respect to mobile phase composition, pH, ionic strength, and solvent selectivity. Relationships were established between these factors and the three most important chromatographic parameters: retention time, resolution, and selectivity. Most of the evidence suggests that the unique selectivity of this column isdue to inclusion complex formation, which provides the physical basis for enantiomeric resolution. After these studies a set of optimum chromatographic conditions was chosen for the simultaneous separation/determination of a mixture of the four enantiomers using fluorescence detector. © 1993 Wiley-Liss, Inc.     

Quantifying process tradeoffs in the operation of chromatographic sequences

A method for the rapid representation of key process tradeoffs that need to be made during the analysis of chromatographic sequences has been proposed. It involves the construction of fractionation and maximum purification factor versus yield diagrams, which can be completed easily on the basis of chromatographic data. The output of the framework developed reflects the degree of tradeoff between levels of yield and purity and provides a fast and precise prediction of the sample fraction collection strategy needed to meet a desired process specification. The usefulness of this approach for the purposes of product purification and contaminant removal in a single chromatographic step has been successfully demonstrated in an earlier paper and it is now extended by application to a chromatographic sequence: the separation of a hypothetical three-component protein system by hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC). The HIC operation has a strong impact upon the subsequent SEC step. The studies show how the analysis of performance in such a chromatographic sequence can be carried out easily and in a straightforward fashion using the fractionation diagram approach. The methodology proposed serves as a useful tool for identifying the process tradeoffs that must be made during operation of a sequence of chromatographic steps and indicates the impact on further processing of the cut-point decisions that are made.     

Identification and quantitative analysis of alkyl isocyanates as their urea derivatives

A gas chromatographic procedure is described for the quantitative analysis of dilute solutions of short-chain alkyl isocyanates. The procedure is based upon measurement of urea derivatives formed from reaction of the isocyanates with amines. Gas chromatographic and thin-layer chromatographic separations of a number of urea derivatives are described. Application of the method is demonstrated in studies of the decomposition of and urea formation from isocyanates in aqueous solutions.     

Resolution of saturated and unsaturated 5 beta-cholanoic acids by gas-liquid and thin-layer chromatography.

The thin-layer and gas-liquid chromatographic properties of the methyl ester acetates were determined for a series of 20 monounsaturated 5 beta-cholanoic acids representing the simple chemical and enzymic dehydration products of the common bile acids. The unsaturated acids were generally indistinguishable from their saturated analogues by thin-layer chromatography on plain silica gel, but resolution was achieved on silica gel impregnated with silver nitrate for compounds having sterically exposed double bonds. The gas-liquid chromatographic behaviour of the unsaturated bile acids on the OV-225, SE-30, and Poly-S-179 liquid phases was closely similar to that observed for the saturated bile acids. The 5 beta-cholenoic acids obeyed the general rules of chromatographic mobility based on the overall shape of the molecule and the number and configuration of functional groups, with a constant retention factor attributable to the olefinic bond. The structural information provided by the chromatographic behaviour of the standard unsaturated bile acids allows a distinction to be made among most of the isomeric 5 beta-cholenoates. A complete identification of all isomeric olefins is possible when chromatographic and mass spectrometric data are combined.     

Purification of human B cell growth factor

Human B cell growth factor (BCGF, 12,000 to 14,000 daltons) has been purified from lectin-stimulated, peripheral blood mononuclear cell-conditioned medium. The purification procedure involves a series of column chromatographic steps incorporating ion exchange, affinity binding, and gel filtration. This procedure is centered around a relatively high yield single chromatographic step, for the removal of co-eluting cytokines from BCGF, that is based on differential binding characteristics to the weak ion-exchange matrix, hydroxylapatite. Reverse-phase high-pressure liquid chromatographic separation on a C18-Bondapak column effectively separates the BCGF and TCGF moieties, yet is characterized by poor yields. High-pressure liquid chromatographic procedures on anion exchange and size exclusion provided the final purification step for BCGF, at an analytical level, resulting in a single band with a m.w. of 12,000 on a SDS-polyacrylamide gel.     

High-throughput process development for recombinant protein purification

Methods development in chromatographic purification processes is a complex operation and has traditionally relied on trial and error approaches. The availability of a large number of commercial media, choice of different modes of chromatography, and diverse operating conditions contribute to the challenging task of accelerating methods development. In this paper, we describe a novel microtiter-plate based screening method to identify the appropriate sequence of chromatographic steps that result in high purities of bioproducts from their respective culture broths. Protein mixtures containing the bioproduct were loaded on aliquots of different chromatographic media in microtiter plates. Serial step elution of the proteins, in concert with bioproduct-specific assays, resulted in the identification of "active fractions" containing the bioproduct. The identification of a successful chromatographic step was based on the purity of the active fractions, which were then pooled and used as starting material for screening the next chromatographic dimension. This procedure was repeated across subsequent dimensions until single band purities of the protein were obtained. The sequence of chromatographic steps and the corresponding operating conditions identified from the screen were validated under scaled-up conditions. Various modes of chromatography including hydrophobic interaction, ion exchange (cation and anion exchange) and hydrophobic charge-induction chromatography (HCIC), and different operating conditions (pH, salt concentration and type, etc.) were employed in the screen. This approach was employed to determine the sequence of chromatographic steps for the purification of recombinant alpha-amylase from its cell-free culture broth. Recommendations from the screen resulted in single-band purity of the protein under scaled-up conditions. Similar results were observed for an scFv-beta-lactamase fusion protein. The use of a miniaturized screen enables the parallel screening of a wide variety of actual bioprocess media and conditions and represents a novel paradigm approach for the high-throughput process development of recombinant proteins.     

Direct chiral separation of unnatural amino acids by high-performance liquid chromatography on a ristocetin a-bonded stationary phase.

Direct high-performance liquid chromatographic chiral separation of numerous underivatized unnatural amino acids on a ristocetin A-bonded chiral stationary phase used in the reversed-phase and in the polar organic chromatographic modes is reported. The effects of different parameters such as mobile phase composition, temperature, and the structure of the analytes on the selectivity in both chromatographic modes are discussed. By variation of the parameters, the separation of the stereoisomers was optimized and, as a result, baseline resolution was achieved in most cases.     

A new method for normalization of the peptide retention times in chromatographic/mass-spectrometric experiments

Proteomic studies with the use of mass spectrometry required comparison of different experimental data (for example, control with a pathology or labeled and unlabeled samples). Identification of chromatographic peaks of the same substance in different chromatograms (time normalization of chromatograms) is complicated if peptides are identified in different experiments according only to their exactly evaluated masses and retention times on a chromatographic column. Retention times of the same peptides would vary from one experiment to another due to inevitable differences in experimental chromatographic conditions (replacement of chromatographic columns, slight changes in flow rates of a mobile phase or in a solvent concentration, or in other conditions). We proposed a reliable method for selection of peaks that corresponded to the same peptides from chromatography/mass spectra for the subsequent alignment of retention times (both a linear and some other monotone function).     

金标法与免疫发光法检测CEA 在健康体检中的应用

目的:探讨金标法和免疫发光法检测CEA在健康体检中的应用价值。方法:对728例健康体检的个人同时应用金标法和免疫发光法测定CEA,对结果进行分析。结果:金标法测定的阳性率为0.69%,免疫发光法测定的阳性率为0.41%,两者具有高度一致性。结论:对健康体检人群应先用金标法进行定性,对阳性结果再用免疫发光法进行定量。