High-throughput screening of genetic mutations/polymorphisms by capillary electrophoresis

Genetic mutations/polymorphisms analyses play a great role in genetic and medical research, and clinical diagnosis. Most conventional methods for genetic assay are based on slab gel electrophoresis that is both labor-intensive and time-consuming. Recently, capillary electrophoresis (CE) has been used for genetic analysis instead of conventional slab gel electrophoresis. This technique can be automated and is characterized by short analysis time, small sample and reagents requirements, and high separation efficiency. CE has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes and disease-causing genes, and has shown a great potential for genetic mutation/polymorphism screening of large numbers of clinical samples. In this article, an overview of the fundamental aspects of mutation/polymorphism assay methods in combination with CE is given and some key applications are summarized.     

Recent advances in the study of biomolecular interactions by capillary electrophoresis

Capillary electrophoresis (CE) has been abundantly used in the study of molecular interactions owing to such advantages as short analysis time, low sample size requirement, high separation efficiency, and flexible applications. The focus of this paper is to review recent studies and advances (mainly from 1998 to now) in biomolecular interactions using CE. Five CE modes: zone migration CE, affinity CE, frontal analysis (FA), Hummel-Dreyer (HD) and vacancy peak (VP) are cited and compared. Quantitative aspects of the thermodynamics and kinetics of biomolecular interaction are reviewed. Several biomolecular binding systems, including protein-protein (polypeptide), protein-DNA (RNA), protein(polypeptide)-carbohydrate, protein-small molecule, DNA-small molecule, small molecule-small molecule, have been well characterized by CE. CE is shown to be a powerful tool for the determination of the binding parameters of various bioaffinity interactions.     

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

This review is in support of the development of selective, precise, fast, and validated capillary electrophoresis (CE) methods. It follows up a similar article from 1998, Wätzig H, Degenhardt M, Kunkel A. “Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications,” pointing out which fundamentals are still valid and at the same time showing the enormous achievements in the last 25 years. The structures of both reviews are widely similar, in order to facilitate their simultaneous use. Focusing on pharmaceutical and biological applications, the successful use of CE is now demonstrated by more than 600 carefully selected references. Many of those are recent reviews; therefore, a significant overview about the field is provided. There are extra sections about sample pretreatment related to CE and microchip CE, and a completely revised section about method development for protein analytes and biomolecules in general. The general strategies for method development are summed up with regard to selectivity, efficiency, precision, analysis time, limit of detection, sample pretreatment requirements, and validation.     

Quantitative determination of sulfur-containing anions in complex matrices with capillary electrophoresis and conductivity detection.

The analysis of sulfur species in complex matrices, like environmentally related samples, requires selective and sensitive as well as robust determination methods. As many as possible different anions need to be quantified in a reasonable analysis time. Besides ion chromatography, capillary electrophoresis has proven to be a very efficient technique for the separation and determination of ionic compounds. With the advantages of less sample and solvent consumption compared to conventional LC, short separation time, inexpensive and robust capillaries, CE was used to separate the anions sulfate, sulfite, thiosulfate, thiocyanate and sulfide. Detection and injection modes and the composition of the separation buffers have been varied to find the most suitable conditions. Conductivity detection after electrokinetic sample injection and improvement of calibration linearity allowed the determination of sulfur containing anions with low limits of detection (8 to 50 micrograms/l). The developed CE method was applied to the analysis of water from an open-pit mining lake.     

Analysis of amino acids in latent fingerprint residue by capillary electrophoresis‐mass spectrometry

The analysis of the chemical composition of fingerprints is important for the development and improvement of existing fingerprint enhancement techniques. This study demonstrates the first analysis of a latent fingerprint sample, using an optimized CE‐MS method. In total 12 amino acids were detected in the fingerprint sample. MS/MS fragmentation was used to provide additional identity confirmation, for which eight of the twelve detected amino acids generated confirmatory product ions. Nine amino acids were quantified and their relative abundances were consistent with previous studies with serine and glycine being the most abundant. The successful detection of amino acids from latent fingerprints demonstrates that CE‐MS is a potential future technique for further study of such compounds in fingerprint samples.     

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.     

毛细管电泳分析中手性化合物的定性检测

毛细管电泳(CE)作为一种新型分离分析技术,具有分离效率高、分析速度快、样品用量少、分离模式灵活多样等众多优势,在手性物质分离等领域应用广泛。在以往的工作中,手性化合物的CE分离模式、手性拆分剂选择及提高分离度等研究已作了详尽报道,而成功分离后的手性物质定性、对映体出峰顺序确认等问题也至关重要。该文以CE手性化合物分离分析中是否依赖标准品分类,及其定性检测方法进行了总结。利用CE分离分析手性样品,若待测物有手性对映体标准品时,其定性通常通过比较标准品迁移时间或标准加入法完成。基于检测器的不同,依赖标准品的CE分析主要分为光学、质谱和电化学3类检测模式。其中光学检测又包含紫外-可见(UV)、激光诱导荧光(LIF)、化学发光(CL)等多种检测方式。不同的检测方式决定了样品前处理方式的差异,随之形成的谱图及手性化合物定性方式也大相径庭。当缺少手性对映体标准品时,可用的CE手性分离分析方法主要有酶消解和抗体添加法、计算法等。前两种方法主要依据对映体与特定消解酶或抗体间的相互作用,完成手性物质的选择定性,适用范围均较局限。相比较,借助理论计算,使CE结合圆二色光谱(CD)的计算法操作简单,定性准...     

Fast determination of paracetamol and its hydrolytic degradation product p-aminophenol by capillary and microchip electrophoresis with contactless conductivity detection

Paracetamol (PAC) is one of the most extensively used analgesics and antipyretic drugs to treat mild and moderate pain. P-aminophenol (PAP), the main hydrolytic degradation product of PAC, can be found in environmental water. Recently, CE has been developed for the detection of a wide variety of chemical substances. The purpose of this study is to develop a simple and fast method for the detection and separation of PAC and its main hydrolysis product PAP using CE and microchip electrophoresis with capacitively coupled contactless conductivity detection. The determination of these compounds using microchip electrophoresis with capacitively coupled contactless conductivity detection is being reported for the first time. The separation was run for all analytes using a BGE (20 mM β-alanine, pH 11) containing 14% (v/v) methanol. The RSDs obtained for migration time were less than 4%, and RSDs obtained for peak area were less than 7%. The detection limits (S/N = 3) that were achieved ranged from 0.3 to 0.6 mg/L without sample preconcentration. The presented method showed rapid analysis time (less than 1 min), high efficiency and precision, low cost, and a significant decrease in the consumption of reagents. The microchip system has proved to be an excellent analytical technique for fast and reliable environmental applications.     

Multiplexed DNA sizing by capillary electrophoresis using entangled polymer solutions and diode array detection

We developed a method for the analysis of multiplexed double-stranded DNA (dsDNA) samples complexed to various intercalating dyes using entangled polymer solution. A commercial single-column capillary electrophoresis (CE) instrument with diode array detection was used for multiplexed detection of DNA samples by addition of intercalating fluorescent molecules. A Phi X174HinfI and a pGEM DNA ladder (1 mg/mL) were used for the electrophoretic separation of dsDNA fragments ranging in size from 24 to 726 and 36 to 2645 bp, respectively. The results suggested that simultaneous electrophoretic separation of different DNA ladders multiplexed with different dyes could be performed in the same capillary yielding fast DNA sizing separations. CE analysis, which is often overpowered by slab gel in sample throughput, could now overcome this disadvantage by allowing multiplexed sample analysis in a fraction of the time needed for slab gel analysis. The separation efficiency of stained DNA molecules with both dyes were dramatically improved with buffers containing a large cation such as tetrapentylammonium ion (Npe(4) (+)) as the only cation in the buffer.     

Capillary electrophoresis time-of-flight mass spectrometry of an opium powder

Summary Intensive work has been invested in recent years to evaluate the performance of capillary electrophoresis (CE) in forensic analysis. Tremendous progress has also been achieved in interfacing CE to sensitive and specific detection systems such as the mass spectrometer (MS). We have recently developed an electrospray time-of-flight mass spectrometer (ESI-TOFMS) for use as a detector for fast and efficient liquid phase separations. In the present paper we investigated ESI-TOFMS for the analysis of an opium powder. Both continuous infusion and CE were studied for direct sample introduction into the TOFMS and mixture separation, respectively. CEMS analysis of the opium was performed in a citrate buffer, using aqueous or mixed aqueous/organic eluents. Low fmol detection was achieved.     

Molecularly imprinted dispersive solid-phase microextraction for determination of sulfamethazine by capillary electrophoresis

We have developed a rapid, selective and efficient method for dispersive solid-phase microextraction (DSPME) using microbeads of a molecularly imprinted polymer (MIP). It enables the pre-concentration of sulfamethazine and sample clean-up prior to capillary electrophoresis with UV detection. The microbeads were synthesized via precipitation polymerization using sulfamethazine, methacrylic acid and ethylene glycol dimethacrylate (EGDMA) as the template molecule, the functional monomer and the cross-linking monomer, respectively. Characterization by SEM displayed the high uniformity and dispersibility of the MIP microbeads. The adsorption and desorption of sulfamethazine and the parameters for CE were optimized to result in a limit of detection of 1.1?μg?L^?1, which is 373-fold lower than that of direct CE detection. The equilibration time of extraction was reduced to 5?min, and the selectivity of the microbeads was significantly improved compared to the non-imprinted polymer. The method was successfully applied to the determination of trace sulfamethazine in several milk samples, with recoveries in the range of 89?% to 110?%.

Determination of enantiomeric excess by capillary electrophoresis

Capillary electrophoresis (CE) is becoming an established method for the determination of chiral trace impurities. This paper provides an overview of the state of the art of CE for such determinations. Detection limits of 0.1% impurity is widely accepted as a minimum requirement for chiral trace impurity determinations. This can be relatively easily achieved with CE. However, determination of lower concentrations requires careful optimization of the separation system. Four factors that are of particular significance for trace enantiomeric determinations: resolution, limit of detection, linear range and type of detection, are discussed. Further, the advantages and disadvantages of derivatization in this context are treated as well as the separation approach, ie., direct chiral separation or separation after the formation of diastereomers. It is concluded that the limit of impurity detection can be about 0.05% when UV detection is employed. Using laser-induced fluorescence detection, a quantitative determination at the 0.005% level is often possible.     

Determination of Anionic Surfactant Turkey Red Oil by Capillary Electrophoresis with Direct UV Detection

A method using capillary electrophoresis with direct UV detection has been developed and validated for the determination of Turkey Red Oil (sulfonated castor oil). The highest performance with respect to separation efficiency and analysis time was achieved with 30 mM Tris (pH 8.0) buffer containing 7.5 mM HP-β-CD. The feasibility of the proposed CE method for the analysis of Turkey Red Oil surfactant in industrial water samples is demonstrated. Spiking of real samples gave recoveries between 90 and 106%. The CE results were compared with that obtained by GC-MS. It was concluded that CE can be a good alternative for fast determination of Turkey Red Oil component distribution in industrial process waters with no sample preparation other than dilution. However, the method sensitivity is not satisfactory for monitoring surfactant level in a waste effluent stream.     

Evaluation of Liquid Chromatography and Capillary Electrophoresis for the Elucidation of the Artificial Colorants Brilliant Blue and Azorubine in Red Wines

Summary Liquid chromatography (LC) and capillary electrophoresis (CE) have been compared for the analysis of the dyes brilliant blue and azorubine in red wines. A liquid-liquid extraction procedure followed by an ion-pair LC method was developed to separate the dyes from the wine polyphenols allowing reliable UV-spectral identification of the target dyes with limits of detection of 10 and 20 ppb for azorubine and brilliant blue, respectively. Because adulteration of wine with dyes is usually in the ppm level, CE proved to be a good alternative for the LC method. CE could be applied after a simple sample clean-up step by SPE eliminating interference from the bulk of the polyphenols. Although LC proved to be more sensitive compared to CE, the latter is more effective in reducing interferences from other wine components and showed the typical advantages of CE such as low solvent consumption and speed of analysis.     

Integrated circuit microchip system with multiplex capillary electrophoresis module for DNA analysis

In this paper, we describe the use of an integrated circuit (IC) microchip system as a detector in multiplex capillary electrophoresis (CE). This combination of multiplex capillary gel electrophoresis and the IC microchip technology represents a novel approach to DNA analysis on the microchip platform. Separation of DNA ladders using a multiplex CE microsystem of four capillaries was monitored simultaneously using the IC microchip system. The IC microchip-CE system has advantages such as low cost, rapid analysis, compactness, and multiplex capability, and has great potential as an alternative system to conventional capillary array gel electrophoresis systems based on charge-coupled device (CCD) detection.     

Determination of Nonelectroactive Cations by Capillary Electrophoresis with Amperometric Detection

Capillary electrophoresis with amperometric detection(CE/AD) is a highly efficient and sensitive technique capable of separation and determination of charged analytes in ultrasmall-volume samples. However, the extension of CE/AD application was limited due to the requirement for the analytes inherently possessing electroactivity. Several approaches have been proposed to improve the applicability of the technique such as indirect detection, sample derivatization and utilization of a variety of chemically modified electrodes.     

毛细管电泳化学发光在线检测

评论了毛细管区带电泳化学发光检测联用技术这一新兴的研究领域。化学发光检测具有背景低、热力学范围宽、灵敏度高的优点，适于毛细管电泳柱后微量样品的在线检测。论述了该检测器与毛细管电泳联用的接口和应用状况。     

Capillary electrophoresis for the determination of norfloxacin and tinidazole in pharmaceuticals with multi-response optimization

Capillary electrophoresis (CE) with UV photo-diode array detection technique was utilized to adopt new method for the analysis of norfloxacin and tinidazole in pharmaceuticals. Many CE aspects including separation, rapidity, sensitivity, ruggedness as well as the repeatability of qualitative and quantitative analyses were considered simultaneously for the purpose of optimization. Experimental design approach including factorial design and response surface methods were applied to optimize electrolyte concentration and the pH while injection time, voltage and column temperature were optimized using the univariate method. Successful results were obtained using 32.5 mmol l^−l phosphate electrolyte at pH 2.5, injection time 8.0 s, voltage 25 kV and column temperature 25 °C with detection at wavelength 301 nm. The analytical characteristics including recovery, intermediate precision, linear dynamic ranges, linearity and selectivity as well as limits of detection and quantification were demonstrated and the applicability to pharmaceuticals was studied. The newly provided method enjoys the advantages of CE over HPLC with respect to rapidity, ruggedness, simplicity in reagents and sample preparation as well as saving in reagents and samples.     

Analysis of natural food pigments by capillary electrophoresis

Lac, cochineal, safflower, gardenia, Monascus and elderberry pigments are used as food color additives in Japan. These natural pigments can be analyzed by capillary electrophoresis (CE). CE has several advantages over thin layer chromatography, gas chromatography and high-performance liquid chromatography, such as low capillary cost, reduced operating costs, small sample amounts, low production of waste materials and short analysis time. CE is shown to be a useful technique for the analysis of these natural food pigments and the pigments extracted from commercial food samples by solid-phase extraction method.