二维毛细管区带电泳/胶束电动毛细管色谱分离尿样中的药物及其对映体

建立了毛细管区带电泳(CZE)/胶束电动毛细管色谱(MEKC)二维毛细管电泳分离平台,CZE毛细管和MEKC毛细管通过一段带微孔的聚四氟乙烯(polytetrafluoroethylene, PTFE)套管固定。样品在CZE毛细管中分离后进入MEKC毛细管进一步分离,在二维转换过程中采用动态pH连接-胶束扫集法避免第一维分离区带在接口处扩散。将该方法成功用于鼠尿样品中4种药物及其对映体的分离,各组分的理论塔板数为(2.8～4.3)×104/m,检出限为0.015～0.052 mg/L,实际样品中峰面积和迁移时间的相对标准偏差(n=7)分别为1.7%～3.8%和1.3%～4.6%。方法重现性好、灵敏度和分离度高、峰容量大,适用于尿样中多种药物组分及其对映体的同时分离检测。     

含双T切换接口的胶束电动色谱-毛细管区带电泳二维电泳芯片上混合氨基酸的分离富集

基于时间顺序设计提出了含双T切换接口的胶束电动色谱(MEKC)和毛细管区带电泳(CZE)的二维芯片,构建了相应的芯片分析测试系统.基于FITC标记的氨基酸样品的一维MEKC和CZE实验结果,对二维进样时间、二维分离启动时间等二维芯片电泳关键操作参数进行了优化.采用所构建的MEKC-CZE二维芯片电泳分析系统对精氨酸、赖氨酸、组氨酸、苯丙氨酸和酪氨酸的混合样品进行了二维芯片电泳分离分析,计算得到两种分离模式的正交性为56.0%.     

二维毛细管电泳电化学检测尿样中的β-阻断剂

设计并制作了在柱电化学(EC)检测池,用于在同一根毛细管中进行中心切割二维毛细管电泳(2D-CE)在线纯化分离检测尿样中的6种β-阻断剂.尿样先在15 mmol/L NaAc缓冲液中进行毛细管区带电泳(CZE)分离,带正电荷的β-阻断剂与中性和带负电荷的干扰物质分成不同区带,然后在检测端施加13.8 kPa压力将干扰成分从毛细管入口端排出,同时将目标组分驱送到毛细管入口端,最后在90 mmol/L NaAc-30 mmol/L SDS缓冲液中进行胶束电动毛细管色谱(MEKC)分离.场放大样品堆积(FASS)/胶束推扫在柱双重富集技术不仅有效抵消压力驱送过程中产生的区带扩散,还可进一步压缩样品区带,提高检测灵敏度.本方法成功用于服药后鼠尿样品中6种β-阻断剂的分离测定,经第一维CZE分离排除干扰后,在未涂层毛细管柱(60 cm ×50 μm i.d.)、90 mmol/L NaAc/HAc-30 mmol/L SDS运行缓冲液、检测电位0.8 V、运行电压10 kV条件下,对6种β-阻断剂进行在线富集分离,峰高、峰面积和迁移时间的相对标准偏差(RSD)分别为2.0%～4.1%, 1.4%～3.7%和0.9%～2.7%(n=6).本研究为毛细管电泳在复杂样品在线纯化分析等方面的应用提供了新方法.     

毛细管电泳法在手性分离中的应用及进展

近年来,毛细管电泳(CE) 手性分离方法的研究主要集中在各种手性添加剂与对映体药物的匹配及实验条件的最优化选择上.目前,较为成熟的CE分离模式有:区带电泳(CZE)、凝胶电泳(CGE)、等速电泳(CITP)、胶束电动色谱(MEKC)和非水电泳(NACE)等,并已成功地用于手性化合物对映体的分离.CE手性分离研究正朝着新型手性选择剂的研制和实现与其他各种定性分析仪器及其他色谱分离模式的联用方向发展.     

微流控芯片二维电泳研究进展

综述了微流控芯片二维电泳技术及其在生命科学中的应用,包括胶束电动力学毛细管色谱(MEKC)与毛细管区带电泳(CZE)、等电聚焦(IEF)与CZE、开管电色谱(OCEC)与CZE耦联等模式的二维微流控芯片。展望了二维微流控芯片的应用前景。     

苯胺及其衍生物的毛细管电泳行为研究

采用紫外吸收检测，毛细管电泳分离，研究了九种苯胺及其衍生物在毛细管区带电泳(CZE)体系和胶束电动毛细管色谱(MECC)体系中的行为特征。讨论了缓冲溶液的浓度与pH、胶束浓度及混合胶束等在不同体系中对分离组分的影响，发现在CZE体系中，控制分离的主要因素是pKb值；在MECC体系中，控制分离的主要因素是溶质分子中碳原子数。建立了一种分离测定九种苯胺及其衍生物的高效毛细管电泳方法。     

中性溶质在MEKC中柱内富集的理论研究

毛细管电动力学色谱(MEKC)中通过“扫”的技术可以使样品组分在线富集。本研究基于柱分离过程弛豫理论的基本方法,对溶质在MEKC中柱内输运过程加以研究,得到了描述进样长度及样品区带和运行区带性质差别对富集效果影响的理论表达式。通过对进样长度及溶质在两区带中的容量因子与富集效果之间关系的进一步探讨,证实溶质在胶束中的溶解度是影响富集的最重要因素;在基本不影响分离效果的情况下,适当加大进样长度一般对富集有利。     

准静态扫集胶束电动毛细管色谱法测定扇贝样品中的贝类毒素

采用准静态扫集胶束电动毛细管色谱(MEKC)法测定了扇贝样品中的2种贝类毒素。毛细管内首先充满含十二烷基硫酸钠(SDS)的缓冲溶液,调节缓冲溶液的pH值,使电渗流等于SDS胶束的电泳流速,电动进样时,带正电荷的贝类毒素离子被SDS扫集吸附,由于SDS在毛细管内处于准静止状态,可使进样时间延长至320 s。与常规电动进样MEKC相比,石房蛤毒素和软骨藻酸的检测灵敏度分别提高950和810倍。该方法对石房蛤毒素和软骨藻酸的检出限分别为0.05,0.12 ng/m L。方法可实现对扇贝样品中2种贝类毒素的快速、灵敏检测。     

肌肽类生物活性肽的毛细管电泳在线富集技术

建立了毛细管区带电泳(CZE)在线富集3种肌肽类活性肽(肌肽、鹅肌肽和高肌肽)的两种简便有效的方法。一种是大体积进样反向压力排除基体富集（LVSRP）技术,即通过流体动力学进样,在不改变电源极性的条件下,利用反向压力排除样品基体,电堆积富集后进行CZE分离;另一种是大体积进样电渗流排除基体富集（LVSEP）技术,即通过流体动力学进样,于运行缓冲液中加入溴化十六烷基三甲基铵(CTAB)动态修饰毛细管表面,通过电渗流排除样品基体,改变电源极性后进行CZE分离。与常规CZE相比,LVSRP技术和LVSEP技术使检测灵敏度提高了40~60倍。对影响两种富集过程的一些因素进行了研究,在最优富集条件下考察本方法的线性范围为0.080~5.0 μmol/L。对3种生物活性肽的检测限(S/N=3)分别为LVSRP 41~58 nmol/L,LVSEP 35~43 nmol/L。     

胶束毛细管电动色谱法在线富集联用技术灵敏检测乳制品和饲料样品中的三聚氰胺

建立了胶束毛细管电泳(MEKC)在线富集技术灵敏检测三聚氰胺的方法,采用场放大进样(FASS)联用胶束扫集(Sweep)测定多种样品中的三聚氰胺.试样用乙腈反复提取3次,在优化实验条件下,三聚氰胺的检测灵敏度提高了约1000倍,检出限由原来的2 mg/L降到1.8 μg/L(S/N=3).本方法用于配方奶粉和动物饲料中...     

The multi-concentration and two-dimensional capillary electrophoresis method for the analysis of drugs in urine samples

A novel method has been developed by integration of multi-concentration and two-dimensional(2D) capillary electrophoresis(CE) for simultaneous enhancement of detection sensitivity and separation power in complex samples.Capillary zone electrophoresis(CZE) was used as the first dimension separation according to mobilities,from which the effluent fractions were further analyzed by micellar electrokinetic capillary chromatography(MEKC) acting as the second dimension.Cation-selective exhaustive injection(CSEI) ...     

Capillary sieving electrophoresis-micellar electrokinetic chromatography fully automated two-dimensional capillary electrophoresis analysis of Deinococcus radiodurans protein homogenate

We report the one- and two-dimensional (1-D and 2-D) capillary electrophoresis separation of Deinococcus radiodurans protein homogenate. Proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), which reacts with lysine residues and creates a highly fluorescent product. Detection is by laser-induced fluorescence. 1-D capillary sieving electrophoresis (CSE) produces over 150,000 plates and micellar electrokinetic capillary chromatography (MEKC) produces over 900,000 plates for components in a D. radiodurans protein homogenate. In a 2-D separation, proteins are first separated by CSE. Fractions are repetitively transferred to a second capillary for further separation based on MEKC. The 2-D separation has a approximately 550 spot capacity. Over 150 components are partially resolved from the homogenate. Resolution is limited in the first dimension by diffusion of proteins during the long separation period and in the second dimension by the combination of a long fraction-transfer time and short separation period.     

Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Mass spectrometry (MS)-based proteomics is currently dominated by the analysis of peptides originating either from digestion of proteins separated by two-dimensional gel electrophoresis (2-DE) or from global digestion; the simple peptide mixtures obtained from digestion of gel-separated proteins do not usually require further separation, while the complex peptide mixtures obtained by global digestion are most frequently separated by chromatographic techniques. Capillary electrophoresis (CE) provides alternatives to 2-DE for protein separation and alternatives to chromatography for peptide separation. This review attempts to elucidate how the most promising CE modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), might best be applied to MS-based proteomics. CE-MS interfacing, mass analyzer performance, column coating to minimize analyte adsorption, and sample stacking for CZE are considered prior to examining numerous applications. Finally, multidimensional systems that incorporate CE techniques are examined; CZE often finds use as a fast, final dimension before ionization for MS, while CIEF, being an equilibrium technique, is well-suited to being the first dimension in automated fractionation systems.     

Two-dimensional separation system of coupling capillary liquid chromatography to capillary electrophoresis for analysis of Escherichia coli metabolites

A two-dimensional (2-D) separation system of coupling chromatography to electrophoresis was developed for profiling Escherichia coli metabolites. Capillary liquid chromatography (LC) with a monolithic silica-octadecyl silica column (500 x 0.2 mm ID) was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension. Field-enhanced stacking was selectively employed as a concentration strategy to interface the two dimensions, which proved to be beneficial for the detection of metabolites. An artificial sample containing 118 standards, some of which lack chromophores or have weak UV absorbance, was used to optimize the 2-D separation system. Under the optimum conditions, 63 components in the artificial sample having absorbance at 254 nm could be well resolved and detected. The utility of the system was demonstrated by comprehensive analysis of E. coli metabolites. Comparing with the previous 2-D separation system we published in Anal. Chem. 2004, 76, 1419-1428, using a longer monolithic column in the first dimension improved the separation efficiency and offered the possibility of increasing the injection volume without compromising the separation efficiency. In the second dimension, field-enhanced stacking was used to improve the concentration sensitivity of the metabolites, and more metabolites in E. coli cell extract were detected and identified using the developed 2-D separation system. In addition, preliminary investigation for future CE-mass spectrometry coupling was also made in the study by using volatile buffers in the capillary LC and CE techniques.     

Interface‐free two‐dimensional heart‐cutting capillary electrophoresis for the separation and stacking of anionic and neutral analytes

Interface‐free two‐dimensional heart‐cutting capillary electrophoresis for two different classes of analytes (anionic and neutral) in a single capillary is presented. Simultaneous sample stacking and orthogonal separation were demonstrated. The anionic species were first analyzed by capillary zone electrophoresis in the first dimension. Then, the neutral compounds were separated in the second dimension by micellar electrokinetic chromatography using the common anionic surfactant sodium dodecyl sulfate. The first and second dimensions occurred automatically without changing the electrolyte and without polarity switching. Artificial mixtures (five anions and four neutral compounds) were successfully analyzed with sensitivity enhancement factors from 7 to 28. The orthogonal separation was complete within 8 min. Some analytical features and application to a spiked real river water sample were also studied.     

Multi-dimensional mapping of pyridylamine-labeled N-linked oligosaccharides by capillary electrophoresis

A simple, sensitive and reproducible multi-dimensional capillary electrophoresis (CE) oligosaccharide mapping method is reported. The structures of 20 identified N-linked oligosaccharides have been assigned mapping positions from which co-migrating unknown oligosaccharides can be characterized. The separation protocols developed have been demonstrated to separate both charged and neutral oligosaccharides. One dimension involves electroendosmotic flow-assisted CE in a sodium acetate buffer, pH 4.0. A second dimension involves separation based on borate complexation electrophoresis in a polyethylene glycol-containing buffer. A third dimension developed specifically for neutral oligosaccharides, using a sodium phosphate buffer, pH 2.5, has been shown to resolve neutral species not able to be separated by the other two dimensions. Thus, a three-dimensional map was generated to facilitate structural characterization of these oligosaccharides.     

Novel three-dimensional capillary electrophoresis system for complex and trace analysis

A novel triple column capillary electrophoresis system is described. Design specifications facilitate method development and analyses by providing on-line, selective, pre-concentration and clean-up of both high (ml) and low (microl) volumes of specific analytes in two dimensions and separation via an additional third dimension. The system described additionally provides four distinct detection capabilities via both contactless conductivity and UV. The addition of a third dimension to the previously reported "coupled-column" systems, and further modifications made, has allowed for optimal identification, separation, and quantitation of micro-components in complex mixtures. The ability to perform both capillary zone electrophoretic and isotachophoretic separations on-line and in any combination enhances the scope for rapid analytical method development and analysis of complex or trace sample components.     

毛细管等电聚焦-毛细管区带电泳二维蛋白质分离平台的构建及其性能

通常采用二维聚丙烯酰胺凝胶电泳 ( 2 D- PAGE)分析组织或细胞的全蛋白质 [1] ,但难与质谱 ( MS)直接联用 .用高效液相色谱 ( HPLC)和毛细管电泳 ( CE)分离分析蛋白质和多肽的一维分离模式的分辨率和峰容量有限 .多维柱联用技术比一维分离有更高的分辨率和峰容量 [2 ] ,便于和 MS直接联用 [3,4 ] ,易于实现自动化 .目前 ,有关 2 D- CE的报道相对较少 [5～ 7] ,我们初步实现了将 2 D- PAGE由平板转移到毛细管中[6 ,7] ,但凝胶柱的制作烦琐 ,存在交叉污染 ,不能与 MS直接联用 .本文用微透析中空纤维膜为接口构建了毛细管等电聚焦 ( …     

Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

无死体积蚀刻多孔膜接口的制作及其在二维毛细管电泳分离系统中的应用

介绍了一种在毛细管柱上原位腐蚀而成的多孔膜接口的制作方法,并用该接口构建了一类毛细管电泳二维分离技术平台。柱上原位腐蚀刻成的多孔膜接口具有零死体积、制作过程简易、成本低廉、耐用、柱间切换便捷等优点,特别适合作为基于毛细管柱的二维及多维电泳联用中的接口,是目前二维及多维毛细管柱联用中一类较为新型、实用、理想的接口。以鹿茸冻干粉可溶物样品为例,验证了该接口在二维毛细管电泳联用系统中的可行性和分离效能。实验结果表明：鹿茸冻干粉可溶物整个二维分离分析的时间在1 h内完成,二维分离系统的分辨率和总峰容量都比一维的高。