Sensitive determination of carbohydrates labelled with p-nitroaniline by capillary electrophoresis with photometric detection using a 406 nm light-emitting diode

p-Nitroaniline was explored as a derivatising reagent for UV absorbance detection of carbohydrates after separation by CE. This derivatising agent has three advantages: first, it has excellent water solubility; second, it has high molar absorptivity; and third, it is possible to obtain sensitive detection using a UV or blue light-emitting diode (LED) as the light source. The labelling reaction took less than 30 min to complete with high reaction yield. The separation process was modelled and optimised using an artificial neural network. Nine carbohydrates were separated by a CE system within 16 min using a 0.17 M boric acid buffer at pH 9.7. On-column LED detection at 406 nm allowed the detection of carbohydrates with good detection limits (<1.1 microM or 8.8 fmol) and reproducible quantification in the concentration range of 2.6-200 microM. This method was applied successfully to the determination of component carbohydrates in some food samples.     

Multisyringe flow injection analysis coupled to capillary electrophoresis (MSFIA-CE) as a novel analytical tool applied to the pre-concentration, separation and determination of nitrophenols

For the first time, a multisyringe flow injection analysis capillary electrophoresis system is described. The potential of the hyphenation for sample treatment including analyte pre-concentration is demonstrated by its successful application to the determination of mono-nitrophenols (NPs) in different water samples. The analytical system was used to automate in-line sample acidification, analyte pre-concentration, elution, hydrodynamic injection, electrophoretic separation, and detection as well as the maintenance and re-conditioning of the solid-phase extraction (SPE) column and the separation capillary. A pre-concentration factor of better than 115 and detection down to 0.11 micromol L(-1) were achieved. Detection was carried out at visible wavelength using a blue LED as a low baseline-noise light source. High repeatability was obtained each for migration times and for peak heights with relative standard deviations typically below 2.5 and 6% including the pre-concentration procedure, respectively. Three injections per hour were achieved by running in parallel the pre-concentrating and the electrophoretic separation procedures. Instrumental control and data registration and evaluation were carried out with the software package AutoAnalysis, allowing autonomous operation of the analytical system.     

微流控芯片上同工酶的孵育及活性检测

建立了一种在微流控芯片上进行同工酶孵育及活性检测的方法. 该方法在集成温控装置的微流控芯片上实现对同工酶与辅酶反应进程的控制, 完成同工酶的进样、孵育反应、电泳分离和活性检测的实验步骤. 建立了基于微流控芯片的同工酶荧光检测系统, 使用360 nm光源激发辅酶产生荧光, 在460 nm处选择性采集荧光信号. 在微流控芯片上实现了同工酶样品的快速活性检测, 酶活性检测限达到0.5 U/L.     

Spatio-temporally resolved detection on a microfluidic chip for monitoring the dynamic processes of molecular events

Detection is an essential aspect in analytical approaches. In liquid phase separations, many attempts have been focused on the capability to detect a partial or an entire column. However, detection in both spatial and temporal resolutions has not gained much attention yet. Here we present the concept of spatio-temporally resolved detection (STRD) and a proof-of-the-concept microchip electrophoresis (MCE)-STRD system. The MCE-STRD system was mainly composed of a microchip and an STRD unit, which were designed completely based on the requirements for spatial and temporal resolutions. In the STRD unit, a linear light beam expanded from a UV LED light source was employed to illuminate the whole separation channel of the microchip while a linear CCD sensor that has an identical effective length as the separation channel and more pixels per unit length was used to detect the absorbance signals through the separation channel. As each pixel of the CCD sensor can detect a corresponding channel space in real time, the CCD provides both spatial and temporal resolutions. A significant advantage of STRD over conventional detection schemes is its capability for monitoring the dynamic processes of molecular events occurring in the separation channel. This was demonstrated through the monitoring of the dynamic processes of protein-DNA and protein-drug interactions in chip isoelectric focusing (chip IEF). The MCE-STRD system provided not only whole pictures of the entire dynamic processes at-a-glance but also quantitative kinetic information (dissociation rate constants) of the dynamic processes. With further development, we anticipate that STRD could be a promising tool for the characterization of biomolecular interactions and the observation of migration behaviours of analytes.     

In-column fiber-optic laser-induced fluorescence detection for CE

A highly sensitive in-column fiber-optic LIF detector for CE has been constructed and evaluated. In this detection system, a 457-nm diode-pumped solid-state blue laser was used as the excitation light source and an optical fiber (40 mum od) was used to transmit the excitation light. One end of the optical fiber was inserted into the separation capillary and was in situ positioned at the detection window. The other end of the fiber was protruded from the capillary to capture the excitation light beam from the blue laser. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a yellow color filter before reaching the photomultiplier tube. The present CE-fluorescence detection is a simple and compact optical system. It reduces the laser scattering effect from the capillary and fiber as compared to the conventional LIF detection for CE. Its utility was successfully demonstrated by the separation and determination of D-penicillamine labeled with naphthalene-2,3-dicarboxaldehyde. The detection limit was 0.8 nM (S/N = 3). The present detection scheme has been proven to be attractive for sensitive fluorescence detection for CE.     

An investigation of peak-broadening effects arising when combining CE with MS

In this study, peak-broadening effects caused by nebulizing gas flow and lack of temperature control have been investigated for separation capillaries with three different inner diameters. The study was performed with serial UV/ESI-MS detection in an effort to distinguish between peak broadening arising in the separation and peak broadening arising in the ion source. The nebulizing gas was found to significantly affect both migration time and separation efficiency when using capillaries with 50 and 75 microm id. If the nebulizing gas is on during injection, the injection volume increases to such an extent that significant peak broadening is induced. Reducing the id to 25 microm minimizes the parabolic flow induced by the nebulizing gas. Results indicate that the nebulizing gas pressure can be optimized to minimize peak broadening in the ion source. A decrease in detection sensitivity, possibly related to the orthogonal design of the interface, was observed when the nebulizing gas pressure was increased. A tapered capillary tip was found to provide superior separation efficiency as well as sensitivity.     

A simple and compact fluorescence detection system for capillary electrophoresis and its application to food analysis

A novel fluorescence detection system for CE was described and evaluated. Two miniature laser pointers were used as the excitation source. A Y‐style optical fiber was used to transmit the excitation light and a four‐branch optical fiber was used to collect the fluorescence. The optical fiber and optical filter were imported into a photomultiplier tube without any extra fixing device. A simplified PDMS detection cell was designed with guide channels through which the optical fibers were easily aligned to the detection window of separation capillary. According to different requirements, laser pointers and different filters were selected by simple switching and replacement. The fluorescence from four different directions was collected at the same detecting point. Thus, the sensitivity was enhanced without peak broadening. The fluorescence detection system was simple, compact, low‐cost, and highly sensitive, with its functionality demonstrated by the separation and determination of red dyes and fluorescent whitening agents. The detection limit of rhodamine 6G was 7.7 nM (S/N = 3). The system was further applied to determine illegal food dyes. The CE system is potentially eligible for food safety analysis.     

Capillary isoelectric focusing-mass spectrometry: analysis of protein mixtures from human body fluids

Isoelectric focusing within a fused silica capillary (cIEF) has proved to be a powerful and practical method for high-resolution separation of analytes from complex biological mixtures. This technique overcomes many of the problems of isoelectric focusing within slab gel media. However current cIEF systems commonly utilize UV detection which limits the detail of analyte structural information that is obtained during analysis. The use of mass spectrometry (MS) as the detection system provides much greater structural information about the detected analytes allowing accurate relative molecular mass (M(r)) determination for proteins and polypeptides. We have constructed a cIEF-MS interface and compared the separation of standard proteins analyzed by cIEF-UV with cIEF-MS. This allowed rapid optimization of the cIEF-MS system performance. Further we have demonstrated the use of MS as a detection system provides accurate M(r) information and can provide analyte modification details. These factors increase the likelihood of absolute identification for physiological proteins within complex in vivo-derived mixtures. To demonstrate the value of cIEF-MS in such analyses we have undertaken an examination of cerebrospinal fluid (CSF), and tentatively identified a number of constituent proteins. We have also analyzed whole blood from control and diabetic patients. We show that glycated alpha- and beta- chains of hemoglobin are found in almost equal abundance in diabetic patient blood. From these results we suggest cIEF-MS is an efficient and useful tool for the separation and examination of in vivo-derived analytes within physiological fluids.     

Optical fiber light-emitting diode-induced fluorescence detection for capillary electrophoresis

A highly sensitive optical fiber light-emitting diode (LED)-induced fluorescence detector for CE has been constructed and evaluated. In this detector, a violet or blue LED was used as the excitation source and an optical fiber with 40 microm OD was used to transmit the excitation light. The upper end of the fiber was inserted into the separation capillary and was situated right at the detection window. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a cutoff filter before reaching the photomultiplier tube. Output signals were recorded and processed with a computer using in-house written software. The present CE/fluorescence detector deploys a simple and inexpensive optical system that requires only an LED as the light source. Its utility was successfully demonstrated by the separation and determination of amino acids (AAs) labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and FITC. Low detection limits were obtained ranging from 17 to 23 nM for NDA-tagged AAs and 8 to 12 nM for FITC-labeled AAs (S/N=3). By virtue of such valuable features as low cost, convenience, and miniaturization, the presented detection scheme was proven to be attractive for sensitive fluorescence detection in CE.     

Determination of phenolic acids and flavonoids of apple and pear by high-performance liquid chromatography

A new HPLC stationary phase has been applied to the analysis of phenolic acids and flavonoids with diode array and mass spectrometric detection. The separation of 26 standard compounds was achieved within 1 h. The stationary phase displayed excellent resolution especially of flavonol glycosides. The analytical system has been used for the determination of phenolic compounds in apple pomace and apple juice, and in extracts of pear fruits of different cultivars. Apple pomace was found to be a promising source of phenolics. However, yields are affected by the drying conditions applied. Furthermore, the applicability of the analytical system for the authenticity control of apple and pear juice was demonstrated by determination of characteristic quercetin and isorhamnetin glycosides, and dihydrochalcones, respectively. Since isorhamnetin-3-glucoside was present in all pear cultivars investigated, the usefulness of arbutin as a specific marker of pear products appears to be doubtful.     

Detailed characterization of cationic hydroxyethylcellulose derivatives using aqueous size-exclusion chromatography with on-line triple detection

A more complete understanding of polymeric, cationic cellulose derivatives, including polyquaterium-10 (Polymer JR), has become increasingly important in the eye care industry as thorough characterization of raw materials helps promote product quality and process control. Often such detailed information requires utilization of a combination of analytical techniques. In this work three Polymer JR samples with different viscosities were characterized using aqueous size exclusion chromatography (SEC) with a light scattering detector, a differential viscometer, and a differential refractometer (triple detection). Detailed molecular information such as absolute molecular weights, molecular weight distributions, intrinsic viscosities, and molecular conformations were obtained. One major challenge of analyzing cationic polymers is abnormal size exclusion separation, which could be caused by the ionic interaction between sample molecules and the column packing material. A selection of mobile phases varying in pH, buffer, organic solvent content, and molar concentration of salts was employed to evaluate the correlation of obtained molecular weight values and mobile phase composition. Universal calibration concept was used to examine the abnormal size exclusion separation phenomenon of Polymer JR samples when using different mobile phases. It was observed that the abnormal size exclusion was dependent on both the separation conditions and molecular weights of the samples. Despite the changes in separation parameters and uncharacteristic polymeric structure compared to conventional SEC samples, the use of aqueous SEC with triple detection provided reproducible and valuable molecular information of Polymer JR samples with low to medium molecular weights. By using a combination of high buffer content and adding organic solvent, the abnormal exclusion separation of high molecular weigh Polymer JR could be considerably reduced.     

A pico-HPLC-LIF system for the amplification-free determination of multiple miRNAs in cells

MicroRNAs are a class of important biomarkers,and the simultaneous detection of multiple miRNAs can provide valuable information about many diseases and biological processes.Amplification-free determination has been developed for the analysis of multiple miRNAs because of its characteristic low cost and high fidelity.Herein,a method for the amplification-free analysis and simultaneous detection of multiple miRNAs based on a so-called pico-HPLC-LIF system is described.In this process,a bare open capilla ry with an inner diameter of 680 nm is used as a sepa ration column for a sample volume of several hundreds of femtoliters(300 fL),followed by separation and detection.The technique has a zeptomolar limit of detection.The method was applied to detect cellular miRNA from adenocarcinomic human alveolar basal epithelial(A549) cell extracts,and the simultaneous detection of the mir-182,miR-155,and let-7 a was achieved.The results showed that the expression of mir-182 and miR-155 was up-regulated and that of let-7 a was down-regulated in A549 cells.This method for multiple miRNAs detection is expected to have broad applications in miRNA-based disease diagnosis,prognosis,treatment,and monitoring.     

Enhancement of signal-to-noise level by synchronized dual wavelength modulation for light emitting diode fluorimetry in a liquid-core-waveguide microfluidic capillary electrophoresis system

The signal-to-noise level of light emitting diode (LED) fluorimetry using a liquid-core-waveguide (LCW)-based microfluidic capillary electrophoresis system was significantly enhanced using a synchronized dual wavelength modulation (SDWM) approach. A blue LED was used as excitation source and a red LED as reference source for background-noise compensation in a microfluidic capillary electrophoresis (CE) system. A Teflon AF-coated silica capillary served as both the separation channel and LCW for light transfer, and blue and red LEDs were used as excitation and reference sources, respectively, both radially illuminating the detection point of the separation channel. The two LEDs were synchronously modulated at the same frequency, but with 180°-phase shift, alternatingly driven by a same constant current source. The LCW transferred the fluorescence emission, as well as the excitation and reference lights that strayed through the optical system to a photomultiplier tube; a lock-in amplifier demodulated the combined signal, significantly reducing its noise level. To test the system, fluorescein isothiocyanate (FITC)-labeled amino acids were separated by capillary electrophoresis and detected by SDWM and single wavelength modulation, respectively. Five-fold improvement in S/N ratio was achieved by dual wavelength modulation, compared with single wavelength modulation; and over 100-fold improvement in S/N ratio was achieved compared with a similar LCW-CE system reported previously using non-modulated LED excitation. A detection limit (S/N = 3) of 10 nM FITC-labeled arginine was obtained in this work. The effects of modulation frequency on S/N level and on the rejection of noise caused by LED-driver current and detector were also studied.     

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。     

On-line simultaneous and rapid separation of anions and cations from a single sample using dual-capillary sequential injection-capillary electrophoresis

A novel capillary electrophoresis (CE) approach has been developed for the simultaneous rapid separation and identification of common environmental inorganic anions and cations from a single sample injection. The method utilised a sequential injection-capillary electrophoresis instrument (SI-CE) with capacitively-coupled contactless conductivity detection (C⁴D) constructed in-house from commercial-off-the-shelf components. Oppositely charged analytes from a single sample plug were simultaneously injected electrokinetically onto two separate capillaries for independent separation and detection. Injection was automated and may occur from a syringe or be directly coupled to an external source in a continuous manner. Software control enabled high sample throughput (17 runs per hour for the target analyte set) and the inclusion of an isolation valve allowed the separation capillaries to be flushed, increasing throughput by removing slow migrating species as well as improving repeatability. Various environmental and industrial samples (subjected only to filtering) were analysed in the laboratory with a 3 min analysis time which allowed the separation of 23 inorganic and small organic anions and cations. Finally, the system was applied to an extended automated analysis of Hobart Southern Water tap water for a period of 48 h. The overall repeatability of the migration times of a 14 analyte standard sample was less than 0.74% under laboratory conditions. LODs ranged from 5 to 61 μg L⁻¹. The combination of automation, high confidence of peak identification, and low limits of detection make this a useful system for the simultaneous identification of a range of common inorganic anions and cations for discrete or continuous monitoring applications.     

Design and performance of a microchip electrophoresis instrument with sensitive variable-wavelength fluorescence detection

A modular instrument for high-speed microchip electrophoresis (MCE) equipped with a sensitive variable-wavelength fluorescence detection system was developed and evaluated. The experimental setup consists mainly of a lamp-based epifluorescence microscope for variable-wavelength fluorescence detection and imaging and a programmable four-channel bipolar high-voltage source capable of delivering up to +/- 10 kV per channel. The optical unit was equipped with a high-sensitivity photomultiplier tube and an adjustable aperture. The system was applied to MCE separations of flurescein isothiocyanate (FITC)-labelled amines utilizing blue light (450-480 nm) for excitation as well as for the separation of rhodamines utilizing excitation light in the green spectral region (531-560 nm). At optimized conditions baseline separation of four FITC-labelled amines could be obtained in less than 50 s at a detection limit of 460 ppt (1 nM) with a signal-to-noise ratio of 3:1. Three rhodamines could be baseline-separated in less than 6 s at a detection limit of 240 ppt (500 pM). The relative standard deviations of absolute migration times determined in repetitive MCE separations of FITC-labelled amines were below 2.5% (n= 25). By the application of cyclodextrin-modified electrolytes, chiral separation of FITC-labelled amines could be performed in seconds demonstrating the potential of microchip electrophoresis for chiral high-throughput screening.     

High-throughput genotyping of factor V Leiden mutation by ultrathin-layer agarose gel electrophoresis.

Ultrathin-layer agarose gel electrophoresis is a novel combination of the established methodologies of slab gel electrophoresis and capillary gel electrophoresis. This new format provides a multilane separation platform with rapid analysis time and excellent sensitivity by using laser-induced fluorescence scanning detection system. Sample injection onto the ultrathin-layer separation platform is easily accomplished by membrane mediated loading technology. In this paper, we demonstrate the sensitivity and high-throughput fashion of this novel separation and detection system for rapid genotyping of the coagulation factor V Leiden mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. The PCR amplified fragment from exon 10 of the factor V gene was digested by the Mnl I restriction enzyme, followed by automated ultrathin-layer agarose gel electrophoresis analysis with "in migratio" fluorescent labeling during the separation process. Due to its speed and automation, this method should be considered for large scale screening of factor V Leiden mutation.     

微流控芯片测定单细胞内化学组分的进展

细胞是生命的基本单元。由于细胞的个体差异,传统分析群体细胞的方法难以得到单细胞的重要信息。准确可靠地测定单细胞内化学组分的含量能大大提高从正常细胞中辨别不正常细胞的能力,为进一步研究和发展生物化学、医学和临床检验等领域奠定基础。近年来,用微流控芯片进行单细胞分析已引起广泛的兴趣。微流控芯片可以集成单细胞进样、溶膜、电泳分离胞内化学组分和高灵敏度测定等一系列操作步骤,为分析单细胞内的化学组分提供了新的技术平台。本文主要综述了近年来微流控芯片测定单细胞内化学组分的进展。重点在于利用电渗流、压力结合电渗流和激光镊子等技术操控单细胞在微流控芯片上完成单细胞进样、溶膜、细胞内化学组分的电泳分离和高灵敏度测定等一系列操作步骤。对在微流控芯片上的衍生技术也做了较为详细的阐述。     

Detection method for microchip separations

The features of analytical systems utilizing microfluidic devices, especially detection methods, are described. Electrochemical detection (EC), laser-induced fluorescence (LIF), mass spectrometry (MS), and chemical luminescence (CL) methods are covered. EC enables detection without labeling and has been used in recent years because of its low cost and sensitivity. LIF is the most generally used detection method in microchip separations. Use of LED as an excitation source for fluorescence measurement was also developed for the purpose of miniaturization of the entire system, including detection and separation. Although MS enables highly sensitive analysis, the interface between MS and micro channels is still under examination. This review with fifty-two references introduces interesting detection methods for microchip separations. Related separation methods using microfluidic devices are also discussed.     

Analysis of pharmaceutical impurities using multi‐heartcutting 2D LC coupled with UV‐charged aerosol MS detection

To overcome challenges in HPLC impurity analysis of pharmaceuticals, we developed an automated online multi‐heartcutting 2D HPLC system with hyphenated UV‐charged aerosol MS detection. The first dimension has a primary column and the second dimension has six orthogonal columns to enhance flexibility and selectivity. The two dimensions were interfaced by a pair of switching valves equipped with six trapping loops that allow multi‐heartcutting of peaks of interest in the first dimension and also allow “peak parking.” The hyphenated UV‐charged aerosol MS detection provides comprehensive detection for compounds with and without UV chromophores, organics, and inorganics. It also provides structural information for impurity identification. A hidden degradation product that co‐eluted with the drug main peak was revealed by RP × RP separation and thus enabled the stability‐indicating method development. A poorly retained polar component with no UV chromophores was analyzed by RP × hydrophilic interaction liquid chromatography separation with charged aerosol detection. Furthermore, using this system, the structures of low‐level impurities separated by a method using nonvolatile phosphate buffer were identified and tracked by MS in the second dimension.