薄层色谱在高速逆流色谱溶剂系统选择过程中的应用

本文对薄层色谱在高降流色谱的溶剂系统选择过程中的应用进行了研究，发现根据薄层色谱的斑点色度可以观察样品中各组分的含量差别及其在两相溶剂中分配系数间的差异，由此来判断溶剂系统的适用性，并确定哪一层适宜作流动相。     

高速逆流色谱分离同分异构体

应用高速逆流色谱法对同分异构体的分离纯化进行研究。实验结果表明,以正己烷-乙酸乙酯-水(体积比为1∶10∶10)为两相溶剂系统,上相为固定相,下相为流动相,进行二次高速逆流色谱分离,可从茶多酚中分离出g级的儿茶素同分异构体——(-)-表没食子儿茶素没食子酸酯(EGCG)和(-)-没食子儿茶素没食子酸酯(GCG),其高效液相色谱纯度均在98%以上。选择四氯化碳-氯仿-甲醇-水(体积比为7∶3∶7∶3)为溶剂系统,下相为固定相,上相为流动相,经一次高速逆流色谱即可将药物中间体溴代苯胺同分异构体进行有效的分离。     

高速逆流色谱法分离制备中药诃子中的没食子酸

利用高速逆流色谱法从100 mg诃子醇提物中一次性分离制备得到8.6 mg没食子酸。通过分析型高速逆流色谱对5种溶剂系统进行筛选,确定以正己烷-乙酸乙酯-甲醇-水(体积比为1:5:1:5)为两相溶剂体系并放大到制备型上,以上相为固定相,下相为流动相,在主机转速850 r/min、流动相流速2 mL/min、检测波长254 nm的条件下进行分离制备,获得4个分离峰(组分Ⅰ、Ⅱ、Ⅲ、Ⅳ)。经高效液相色谱检测,按照面积归一法计算,其中组分Ⅲ的纯度达96.40%。经电喷雾电离质谱分析,并结合与没食子酸标准品的高效液相色谱测定结果的对比,确定组分Ⅲ为没食子酸。该方法简便、快速、重复性好,适合于诃子中没食子酸的分离制备。     

高速逆流色谱法分离当归挥发油中有效成分藁本内酯及其气相色谱-质谱法的分析和鉴别北大核心CSCD

提出了应用高速逆流色谱从当归挥发油中分离有效成分藁本内酯,探索这一分离条件的关键是两相溶剂体系的选择。根据分离目标物的极性以及选择两相溶剂应满足的3个条件((1)分层时间不超过30s;(2)对样品不引起分解;(3)目标成分的分配系数在0.5～2.0之间),经薄层色谱法及高效液相色谱法预选,最终从几种溶剂体系中筛选得到两相溶剂体系为正己烷-乙酸乙酯-乙醇-水(1+1+1+1)体系。采用这一溶剂体系,一次进样500mg当归挥发油,可达到目标组分与其他杂质完全分离,目标组分回收后得到48mg藁本内酯。对此分离所得组分经气相色谱-质谱法(GC-MS)分析,其纯度达到98.5%以上,GC-MS还对此组分进行结构鉴定,证明其确为藁本内酯。     

石英毛细管气相色谱中的双峰现象——种可能的溶剂效应

 〕本文较系统地观察了毛细管气相色谱中出现的“双峰现象”即单一纯组分在某些气相色谱条件下，会出现两个类似独立组分的双峰。本工作研究了柱温、柱子固定相极性、样品溶剂种类、仪器结构以及操作等因素对双峰出现的影响。实验结果表明，在柱温较低、溶剂蒸发潜热较大的情况下，容易引起双峰的事实可能说明，毛细管气相色谱中的双峰现象是一种溶剂效应。事实上，通过更换合适的溶剂，可以消除这种现象。本文对有关实验结果进行了讨论。     

石英毛细管气相色谱中的双峰现象——种可能的溶剂效应

〕本文较系统地观察了毛细管气相色谱中出现的“双峰现象”即单一纯组分在某些气相色谱条件下，会出现两个类似独立组分的双峰。本工作研究了柱温、柱子固定相极性、样品溶剂种类、仪器结构以及操作等因素对双峰出现的影响。实验结果表明，在柱温较低、溶剂蒸发潜热较大的情况下，容易引起双峰的事实可能说明，毛细管气相色谱中的双峰现象是一种溶剂效应。事实上，通过更换合适的溶剂，可以消除这种现象。本文对有关实验结果进行了讨论。     

高速逆流色谱法对当归中石油醚萃取部位化学成分的分离纯化

采用高速逆流色谱法对当归石油醚萃取部位中4个主要化学成分进行分离纯化。选择Arizona溶剂体系作为高速逆流色谱分离的溶剂系统,经过实验条件优化,确定最佳溶剂为:正己烷-乙酸乙酯-甲醇-水(体积比6∶0.6∶3∶1)。高速逆流色谱分离时,采用有机相(上相)为固定相,水相(下相)为流动相,正相洗脱,流速为2mL/min,转速为900r/min,检测波长为254nm。当归石油醚萃取部位中的4种化学成分获得较好分离,经高效液相色谱检测,其纯度分别为78.5%、93.2%、98.9%、88.7%。通过1HNMR、IR和质谱分析,鉴别出其中1种化合物为藁本内酯。     

高速逆流色谱溶剂体系及洗脱模式研究进展

高速逆流色谱所具有的诸多优点,使其在过去的几十年间从理论研究到实际应用均获得了长足的发展。溶剂体系的筛选和洗脱模式的设置对于高速逆流色谱的分离是最为关键和重要的环节。本文根据高速逆流色谱溶剂体系的要求、筛选方法及梯度洗脱、双向洗脱、循环洗脱、推挤洗脱四种洗脱模式的原理、应用范围等进行了综述,以期为高速逆流色谱研究者提供相关参考。     

硅胶柱色谱结合高速逆流色谱法分离纯化荷花中黄酮类化合物

采用硅胶柱色谱结合高速逆流色谱法分离纯化了荷花中3种黄酮类化合物。荷花粗提物先经过硅胶柱色谱初步分离,得到黄酮含量高的组分,再经过高速逆流色谱分离,以乙酸乙酯-乙醇-水-乙酸(4:1:5:0.025, v/v/v/v)为两相溶剂系统,上相为固定相,下相为流动相,在主机转速800 r/min、流速2.0 mL/min、检测波长254 nm条件下,从150 mg样品中一次性分离制备得到6.1 mg槲皮素-3-O-β-D-葡萄糖醛酸苷(I), 14.8 mg杨梅素-3-O-β-D-葡萄糖苷(II)和20.2 mg紫云英苷(III),经高效液相色谱检测其纯度分别为97.0%、95.4%、96.3%,并通过质谱和核磁共振氢谱、碳谱鉴定各化合物的结构。该方法简便、快速、节省溶剂,可以对荷花中的黄酮类化合物进行快速有效的分离纯化,具有较好的实用价值,为荷花资源的进一步开发应用提供了参考依据。     

高速逆流色谱分离独活中水合氧化前胡素

采用高速逆流色谱从独活的乙醇粗提物中分离纯化水合氧化前胡素。用氯仿-甲醇-水(4+4+2)为两相溶剂系统,上相为固定相,下相为流动相。所得下相溶剂经高效液相色谱-紫外检测器测定及高分辨电子轰击质谱和核磁共振氢谱鉴定,为水合氧化前胡素,其纯度为98.3%。     

Advances in screening enzyme inhibitors by capillary electrophoresis

Capillary electrophoresis (CE) has attracted lots of attention due to its simplicity, low sample consumption, low solvent volume, high resolution, and high speed. Based on these advantages, it has been widely used in enzyme inhibitor screening. There are two main operation modes on enzyme inhibitor screening: off‐line (precapillary enzyme assays) in which process CE was used as an analytical tool; online (in‐capillary enzyme assays) which combined the sample injection, mix, reaction, separation, and detection within a single run. Additionally, diverse of new materials were introduced to immobilize enzyme, which has been coupled with CE for the study of enzyme activity and its inhibitor screening. This review gives an overview of the developments and applications for the CE‐based enzyme inhibitor screening.     

强阳离子交换毛细管液相色谱-反相加压毛细管电色谱二维系统的构建及其在中药黄柏提取物分离中的应用

加压毛细管电色谱(pCEC)具有电泳和液相色谱的双重分离机理,其柱效高、选择性强、分辨率高和分离速度快并可进行梯度洗脱。我们在此基础上加入离子交换色谱模式,构建了强阳离子交换-反相加压毛细管液相色谱(micro strong cation exchange liquid chromatography/reversed phase pressurized capillary electrochromatography, μ-SCXLC/RP-pCEC)二维系统,并对中药黄柏的提取物进行了优化分离。第一维μ-SCXLC采用线性盐梯度分离,样品被切割成11个馏分洗脱收集后进入第二维,第二维脱盐后,采用RP-pCEC进行分离分析,梯度洗脱。以中药黄柏提取物为样品,此二维系统的分辨率和峰容量都较一维系统有很大提高,理论峰容量可达900左右,证明构建的二维体系非常适合复杂样品的分离分析。     

Study of the selectivity of inorganic anions in hydro-organic solvents using indirect capillary electrophoresis

In capillary electrophoresis (CE) analysis of small inorganic anions, the ability to control the electroosmotic flow (EOF) and the ability to alter the electrophoretic mobility of the ions are essential to improve resolution and separation speed. In this work, a CE method for separation of small inorganic anions using indirect detection in mixed methanol/water buffers is presented. The suitability of different UV absorbing probes commonly used for indirect detection including chromate, iodide, phthalate, benzoate, trimellitate, and pyromellitate, in mixed methanol/water buffers is examined. The effect of the electrolyte buffer system, including the pH, buffer concentration and the organic solvent on the electrophoretic mobility of the probes and analytes are also investigated. The EOF was reversed using cationic surfactant, cetyltrimethylammonium bromide (CTAB) so ions were separated under co-EOF mode. The organic solvent alters the electrophoretic mobility of the probes and the analytes differently and hence choice of the appropriate probe is essential to achieve high degree of detection sensitivity. Separations of six anions in less than 2.5 min were accomplished in buffers containing up to 30% MeOH. Adjustment of the methanol content helps to improve the selectivity and resolution of inorganic anions. Limit of detection, reproducibility and application of the method for quantification of anions in water samples will also be discussed.     

Determination of pharmaceuticals and related impurities by capillary electrophoresis

In the first part of this work, the use of capillary electrophoresis (CE) for the separation of two groups of pharmaceuticals, namely a metabolite of tamoxifen and a basic drug substance, DS1, was investigated. The effects of pH and types of modifiers, e.g. surfactant, bile salt, γ-cyclodextrin and hydroxypropyl-β-cyclodextrin on selectivity, separation and peak shape were studied. Besides achieving complete separation of the compounds, the CE system was capable of providing separation with significant improvements in overall peak shape of the compounds compared with HPLC. In the case of the basic drug substance DS1, validation of the CE system developed in terms of linearity, selectivity, sensitivity and reproducibility was satisfactorily performed. At the same time, a study of the sample solvent matrix effects on the separation of this group of compounds was examined. The system was successfully applied to the analysis of laboratory-synthesized samples. Good correlation was observed between CE and HPLC, although higher efficiency and faster speed of separation were obtained using the CE system developed. For the tamoxifen metabolite, special emphasis was placed on the use of CE for the separation of the pair of isomers. This was readily achieved through the introduction of γ-cyclodextrin in the electrolyte. Resolution of at least 1.5 was obtained for the isomers using the CE method.     

High-speed separation of proteins by sodium dodecyl sulfate-capillary gel electrophoresis with partial translational spontaneous sample injection

In this study, we developed a picoliter-scale partial translational spontaneous injection approach which is suitable for high-speed protein separation under sodium dodecyl sulfate-capillary gel electrophoresis mode. On the basis of this approach, we built a high-speed CE system for protein separation based on a short capillary and slotted-vial array. The system has the advantages of simple structure, ease of building without the requirement of microfabricated devices, convenient operation, and low cost. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ～65?μm plug length) were obtained, which ensured both the high speed and the high efficiency in protein separation. Five fluorescein isothiocyanate labeled proteins including myoglobin, egg albumin, bovine serum albumin, phosphorylase b, and myosin were separated within 60?s with an effective separation length of 1.5?cm. Theoretical plates per meter ranging from 2.58×10? to 1.28×10? (corresponding to 0.78-3.88?μm plate height) were obtained. The separation speed and separation efficiency of the present system are comparable to those of most microchip-based capillary electrophoresis systems for protein separation. The relative standard deviations of the migration times were in the range of 0.9-1.3% (n=5). Good linear relationships between log relative molecular mass and migration time were obtained in the molecular weigh range of 17,200-500,000, which demonstrate the present system can be applied in protein relative molecular mass determination.     

Chiral counter-current chromatography of gemifloxacin guided by capillary electrophoresis using (+)-(18-crown-6)-tetracarboxylic acid as a chiral selector

(+)-(18-crown-6)-tetracarboxylic acid (18C6H4) has been known as a highly efficient chiral selector for resolving primary amine enantiomers in capillary electrophoresis (CE). We investigated the chiral separation of gemifloxacin using 18C6H4 in analytical counter-current chromatography (CCC). The separation conditions for CE, including the binding constant, pH, and run buffer constituents, provided a helpful guideline for chiral CCC. A successful separation of gemifloxacin enantiomers could be achieved using a two-phase solvent system composed of 1-butanol-ethyl-acetate-bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane acetate buffer with a small amount of 18C6H4. The hydrophobicity of the solvent system and the 18C6H4 concentration were varied to optimize the chiral separation.     

Review of recent developments of on-line sample stacking techniques and their application in capillary electrophoresis

Capillary electrophoresis (CE) has become one of the most useful tools in separation science because of its high separation efficiency, low cost, versatility, ease of sample preparation and automation. However, some limitations of CE, such as poor concentration sensitivity due to its lower sample loading and shorter optical path length, limits its further applications in separation science. In order to solve this problem, various on-line sample preconcentration techniques such as transient isotachophoresis preconcentration, field-enhanced sample stacking, micelle to solvent stacking, micelle collapse, dynamic pH junction, sweeping, solid phase extraction, single drop microextraction and liquid phase microextraction have been combined with CE. Recent developments, applications and some variants together with different combinations of these techniques integrating in CE are reviewed here and our discussions will be confined to the past three years (2008–2011).      

Nonaqueous capillary electrophoresis in coated capillaries: an interesting alternative for proteomic applications

This work brings together some contributions for the use of nonaqueous media for proteomic analysis, for both capillary electrophoresis (CE) separation and the preparation of tryptic digests. First, a ternary nonaqueous buffer consisting of 60/30/10 v/v methanol/acetonitrile/acetic acid with 12.5 mmol/L ammonium acetate was optimized for CE separation of the tryptic digest of lysozyme. Lysozyme was chosen as a model system for the protein digestion, which has also been prepared in an organic-rich medium with methanol/50 mmol/L NH(4)HCO(3), pH 8.0 (60/40 v/v). The separation results were compared to in silico (PeptideCutter program) digestion conditions, and high-efficiency peak separation (18 peaks) was obtained in 20 min with an electric field of 350 V/cm. In addition, we have evaluated the stability of a coated capillary with poly-N,N-dimethylacrylamide (60/30 cm total/effective length and 75 microm ID) for over 100 runs of tryptic digest with the nonaqueous background electrolyte solvent system. The migration times for ten selected peptide peaks presented 3-7% relative standard deviation.     

High-performance liquid chromatographic,capillary electrophoretic and capillary electrophoretic-electrospray ionisation mass spectrometric analysis of selected alkaloid groups

Systems for efficient separation of selected alkaloid groups by high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and capillary electrophoresis coupled with electrospray ionisation mass spectrometry (CE-ESI-MS) are described. The optimized HPLC system was applied for the separation of 23 standard indole alkaloids as well as for qualitative and quantitative analyses of crude alkaloid extracts of Rauvolfia serpentina X Rhazya stricta hybrid cell cultures. The developed conditions for CE analysis proved to be efficient for separation of mixtures of standard indole and beta-carboline alkaloids. The described buffer system is also applicable in the combination of CE with electrospray ionisation mass spectrometry. This analytical technique allowed the separation and identification of components of standard indole alkaloid mixture as well as crude extracts of R. serpentina roots, R. serpentina cell suspension cultures and cortex of Aspidosperma quebracho-blanco. The influence of buffer composition and analyte structures on separation is discussed.     

Spherical molecularly imprinted polymer particles: a promising tool for molecular recognition in capillary electrokinetic separations

Spherical molecularly imprinted polymer particles obtained via precipitation polymerization, were introduced as a pseudostationary phase in capillary electrophoresis (CE) to study molecular recognition. Analyses were performed via a partial filling technique using (+)-ephedrine-imprinted microspheres (100-200 nm) which were polymerized from methacrylic acid and 1,1,1-Tris(hydroxymethyl)propanetrimethacrylate using acetonitrile as the solvent. The influence of pH and the modifier content on the separation was investigated. A 0.1% w/v suspension in an aqueous 10 mM phosphate buffer (pH 2.5 with 40% acetonitrile) was hydrodynamically injected into the CE system (80% of the effective capillary length) and led to full baseline separation of racemic ephedrine within 10 min.