An on-column fracture/end-column reaction interface for chemiluminescence detection in capillary electrophoresis

Chemiluminescence (CL) offers a sensitive detection method for capillary electrophoresis (CE), but the implementation of CE–CL is usually under compromised operating conditions for CE, such as the prerequisite of extreme pH buffer for optimal CL reaction at the capillary outlet. This has sometimes significantly deteriorated the separation of CE. In this study, the development of a new interface makes it possible to optimize the operating conditions for CE separation and CL detection independently. The interface consists of an on-column fracture being installed in a reservoir near the capillary end to create an electrical connection and also serve as reagent addition entrance. The capillary terminal is inserted into an end-column reservoir for CL reaction and detection. In this arrangement, the applied electric field has been decoupled from the CL detection, which is proved to effectively improve CE's performance by allowing the use of optimal CE buffers. At the same time, it enables the optimization of CL detection independently. The applicability of this interface was evaluated by using acridinium ester (AE) and luminol systems. For AE system, the interfering products of CL reagent (⁻OH, HO₂⁻) have been prevented, and the pH range of CE buffer can be independent to the optimal pH value of AE CL reaction, which is usually below 3. The AE was detected using running buffer at pH 8.7, giving a detection limit of 0.1 nM (S/N = 3), and the theoretical plate numbers is as high as 56 000. The on-column fracture based configuration is simple, sensitive and easy to implement.     

Separation and determination of amino acids by micellar electrokinetic chromatography coupling with novel multiphoton excited fluorescence detection

In this article, it was demonstrated that separation and determination of 20 amino acids were accomplished by micellar electrokinetic chromatography (MEKC) coupling with novel multiphoton excited fluorescence (MPEF) detection method. Different from MPEF achieved by expensive fs laser, continuous wave (CW) diode laser of ultra-low cost was uniquely employed in our MPEF system. Amino acids were fluorescently labeled with fluorescein isothiocyanate (FITC), and were subjected to sodium dodecyl sulfate (SDS)-based MEKC separation and CW-based MPEF detection. The result was compared with that by single photon excited fluorescence (SPEF), which indicated that MPEF had the advantages of better mass detectability and higher separation selectivity over SPEF. Quantitative analysis was performed and revealed linear dynamic range of over 2 orders of magnitude, with mass detection limit down to ymole level. To evaluate the reliability, this method was successfully applied for analyzing a commercial nutrition supplement liquid.     

Continuous wave-based multiphoton excitation fluorescence for capillary electrophoresis

It was reported that a novel detection method, continuous wave (CW)-based multiphoton excitation (MPE) fluorescence detection with diode laser (DL), has been firstly proposed for capillary electrophoresis (CE). Special design of end-column detection configuration proved to be superior to on-column type, considering the detection sensitivity. Three different kinds of fluorescent tags that were widely used as molecular label in bio-analysis, such as small-molecule dye, fluorescent protein and nano particle or also referred to as quantum dot (QD), have been evaluated as samples for the constructed detection scheme. Quantitative analyses were also performed using rhodamine species as tests, which revealed dynamic linear range over two orders of magnitude, with detection limit down to zeptomole-level. Simultaneous detection of fluorescent dyestuffs with divergent excitation and emission wavelengths in a broad range showed advantage of this scheme over conventional laser-induced fluorescence (LIF) detection. Further investigations on CW-MPE fluorescence detection with diode laser for capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations of fluorescein isothiocyanate (FITC) labeled amino acids indicated good prospect of this detection approach in various micro or nano-column liquid phase separation technologies.     

毛细管电泳-安培检测法分离分析手性药物索他洛尔

采用毛细管电泳-柱端喷壁式安培检测技术,建立了痕量手性药物索他洛尔的分离检测新方法。以1.5%(w/V)羧甲基-β-环糊精为手性选择试剂,借助于环糊精-客体包合物的拆分原理,索他洛尔对映异构体在优化的分离条件:50mmol/LTris-H3PO4缓冲液(pH5.5),分离电压21kV,进样条件18kV/10s,工作电位1150mV(vs.Ag/AgCl),可实现基线分离,线性范围为5～500μg/L;异构体Ⅰ和Ⅱ的检出限(S/N=3)分别为2.0和1.9μg/L。本方法用于模拟血清样品分析,结果令人满意。     

Off-Column Amperometric Detection for Pressurized Capillary Electrochromatography

A system enabling coupling of pressurized capillary electrochromatography (pCEC) with off-column amperometric detection (AD) is reported in which conduction of the current in pCEC was achieved through a cellulose acetate-coated porous polymer joint, and the effect of the high-voltage field applied to pCEC for AD was also eliminated. Effects of supplementary pressure on the porous polymer joint and the effects on AD of capillary columns of different i.d. were investigated. The performance of the pCEC–AD system with the porous polymer joint was evaluated with phenol and hydroquinone using sulfonated stearyl methacrylate monolithic columns. The separation efficiency of the column in pCEC–AD, using the proposed off-column detection with the cellulose acetate membrane joint, was comparable with that of pCEC–UV using on-column detection. Compared with end-column detection using a 50 μm i.d. capillary column without a joint, a higher signal-to-noise ratio was achieved, even using a 100 μm i.d. capillary column with a joint. Successful separation and detection of dopamine and epinephrine were also achieved by use of this system.     

Cyclic chronopotentiometric determination of sugars at Au and Pt microelectrodes in flowing solutions

The main advantage of the application of cyclic chronopotentiometry (CCP) in end-column CE detection arises from the fact that the detection parameters and the magnitude of the analytical signal are (in contrast with other electrochemical detection methods) independent of the ohmic polarization of the solution caused by the separation current at the detection end of the capillary. CCP was used to determine sugars on platinum and gold microelectrodes after separation by CE. The results obtained with a gold microelectrode were better. Subsequently this detection method was used for quantitative determination of sugars in honeys and for their authentication.     

Capillary electrophoresis and capillary flow injection analysis with electrochemical detection

Measurements by capillary flow injection analysis (CFIA) and capillary electrophoresis (CE) in conjunction with electrochemical detection are described. The detection is based on an end-column electrode arrangement. Several novel electrodes, such as a spherical gold electrode and a dual-microdisk electrode, are presented and characterized regarding their analytical utility. In order to improve the selectivity of CFIA, dual-electrode and multiple-pulse detection are studied using couples of cyanometallates or metallocenes. Capillary electrophoretic experiments with amperometric detection are performed using 50 m i.d. capillaries without any electrical-field decoupler. The practicality and analytical characteristics of this detection strategy are illustrated for the separation of serotonin and some biological precursors and metabolites of neurotransmitter substances.     

Combination of amperometric detector & UV detector for capillary electrophoresis

A new electrochemical cell assembly with the combination of UV and amperometric detector (AD) based on their complementarity was described. A Nafion tubing junction was used to decouple the high voltage from the separation capillary in the rear of on-column UV detector. In this mode, the electroactive and inert compounds could be detected by UV and AD at the same time. Aromatic amines were determined with the UV and the end-column AD detection to evaluate the performances of the cell assembly. Such an improved electrochemical detector could match the capillary with different diameters. By simple adjustment of the screws, the positioning of the working electrode and the detection capillary was easily gained without microscope. It is also very easy to assemble and disassemble the working electrode when needed.     

Instrumentation for capillary electrochromatography

One of the reasons for the immense interest in capillary electrochromatography (CEC) is its feature to combine chromatographic selectivity with the high efficiency and the miniaturization potential of capillary electrophoresis (CE). The capability of commercial CE instruments to run CEC has enforced the readiness of users and researchers to work on this separation technique. Nevertheless, to fully exploit the potential of CEC, a routine CE device can certainly not fulfill all requirements. Two different approaches have been made to overcome this problem. The first was to modify commercial CE instruments for various demands. Pressurization of the packed capillary to prevent "air" bubble formation, gradient elution capabilities and thermostating devices allowing a greater flexibility in column designs have been implemented in CE instruments of several manufacturers. A completely different approach is the development of modular laboratory-made instrumentation dedicated to special CEC requirements. In order to increase mobile phase velocity and thus the speed of analysis the availability of voltages higher than 30 kV was accomplished in some of these devices. Gradient elution was achieved by either coupling of gradient LC systems or an electroosmotic generation of the changing eluent composition. When a pressure gradient is applied between both column ends in addition to the voltage gradient, a hybrid between capillary HPLC and CEC results. This chromatographic mode is named pressure-assisted electrochromatography (PEC). Either CE instruments equipped with additional HPLC pumps or modular laboratory-made devices are suitable for PEC. In CEC, sensitivity for UV detection is rather poor due to the short optical path length for on-column detection in capillary separation techniques. A special cell design with enhanced light path is presented and further principles like, e.g., fluorescence detection and coupling to mass spectrometry are discussed.     

Improving signal to background ratio for on-the-fly fluorescence lifetime detection in capillary electrophoresis

On-the-fly fluorescence lifetime detection (OFLD) in capillary electrophoresis (CE) was previously demonstrated using a commercial multiharmonic Fourier transform (MHF) spectrofluorometer interfaced to a commercial CE system. This paper discusses optimization of the interface design for minimization of background fluorescence and scattered light, thereby maximizing the signal-to-background ratio (S/B) of the dynamic measurement. Strategies included using various combinations of optical filters including a holographic filter and longpass or bandpass filters, tilting the capillary relative to the incident laser beam, employing a confocal design and adding an iris to remove out-of-focus light, using a microscope objective in the emission beam to increase the collection efficiency, and using square instead of ciruclar capillary columns. Significant improvements in S/B for on-column, on-the-fly detection of fluorescein in CE were achieved with most modifications.     

21世纪毛细管电泳技术及应用发展趋势

在21世纪，毛细管电泳技术面临着新的挑战和机遇，在其检测手段，仪器的小型化和集成化，以及分离模式上都存在着极大的发展空间，文中针对这三方面的发展趋势和毛细管电泳的应用进行了讨论。     

Wall-jet conductivity detector for microchip capillary electrophoresis

A new end-column ‘hybrid’ contactless conductivity detector for microchip capillary electrophoresis (CE) was developed. It is based on a “hybrid” arrangement where the receiving electrode is insulated by a thin layer of insulator and placed in the bulk solution of the detection reservoir of the chip, whereas the emitting electrode is in contact with the solution eluted from the channel outlet in a wall-jet arrangement. The favorable features of the new detector including the high sensitivity and low noise, can be attributed to both the direct contact of the ‘emitting’ electrode with the analyte solution as well as to the insulation of the detection electrode from the high DC currents in the electrophoretic circuit. Such arrangement provides a 10-fold sensitivity enhancement compared to currently used on-column contactless conductivity CE microchip detector as well as low values of noise and easy operation. The new design of the wall-jet conductivity detector was tested for separation of explosive-related methylammonium, ammonium, and sodium cations. The new detector design reconsiders the wall-jet arrangement for microchip conductivity detection in scope of improved peak symmetry, simplified study of inter-electrode distance, isolation of the electrodes, position of the wall-jet electrode to the separation channel, baseline stability and low limits of detection.     

Analysis of glyphosate and glufosinate by capillary electrophoresis-mass spectrometry utilising a sheathless microelectrospray interface

The potential of capillary electrophoresis combined with mass spectrometry for the simultaneous determination of two herbicides (glyphosate and glufosinate) and their metabolites (aminomethylphosphonic acid and methylphosphinicopropionic acid) as the native species is demonstrated utilising a simple microelectrospray interface. The interface uses the voltage applied to the CE capillary to drive separation and generate the electrospray, avoiding sample dilution associated with the use of a sheath liquid interface. The chemistry of the internal walls of the capillary has a marked influence on peak shape, and appropriate choice is essential to successful operation of the interface. A linear polyacrylamide coated capillary, which has no electroosmotic flow, gave best reproducibility, with precision of migration time and peak area in the range 1-2 and 7-12% RSD, respectively, for the four analytes. Limits of detection, low-pg on-column, are substantially better than for previous methods and calibration curves over the range 1-100 microM have R2 values greater than 0.97. The observed concentration limit of detection for glyphosate in water is 1 microM and for a water-acetone extract of wheat is 2.5 microM, allowing the underivatised herbicide to be detected at 10% of the maximum residue limit in wheat.     

Deviceless decoupled electrochemical detection of catecholamines in capillary electrophoresis using gold microband array electrodes

Samples containing microM concentrations of dopamine, (+/-)-isoproterenol, para-aminophenol and chlorogenic acid have been separated by capillary electrophoresis (CE) and detected using end-column amperometric detection based on a novel decoupling method. The present decoupling approach involves the use of an electrochemical detector chip containing an array of microband electrodes where the working and reference electrodes are positioned only 10 microm from each other. The short distance between the working and reference electrodes ensures that both electrodes are very similarly affected by the presence of the CE electric field. With this method, no shift in the detection potential was seen when the CE high voltage was applied. This eliminated the need for a reoptimization of the detection potential to compensate for the influence of the separation voltage on the detection. It is also demonstrated that catecholamines can be detected using gold microband electrodes by careful adjustment of the detection potential to avoid the formation of gold oxide. Such careful adjustments of the detection potential are straightforward using the present decoupling method.     

Capillary electrophoresis with end-capillary potentiometric detection using a copper electrode.

Potentiometric end-capillary detection in capillary electrophoresis has the advantage of relatively easy miniaturisation without having to compromise the concentration sensitivity. Potentiometric end-capillary detection using a copper electrode is also attractive because of the sensitive detection of many inorganic and organic UV-transparent ions and the ability to work in both direct and indirect mode. In this work, detection of a number of common anions in a tartrate electrolyte at pH 3 was studied. The influence of the end-capillary detection geometry on the detection performance was investigated. An end-capillary detection cell allowing the separation capillary to be changed without the need to realign the detection electrode was constructed and fitted into a commercial CE apparatus. Under the optimal configuration, which was a 25 microm diameter copper electrode aligned coaxially with a 25 microm capillary and positioned at a distance of about 25 microm from the capillary end, excellent peak shapes were achieved and comparison with simultaneous on-capillary photometric detection showed no additional peak broadening. Good sensitivity was obtained, resulting in concentration limits of detection (LODs) in the low microM range and mass LODs in the low amol range. Examples of separations of inorganic and organic anions are presented and the analytical potential of the detection method is assessed.     

Separation of miRNA and its methylation products by capillary electrophoresis

Methylation is a crucial step in plant microRNA biogenesis. To improve our understanding of the methylation process and its regulation, a rapid and convenient high-throughput method should be sought to help with the study of reaction kinetics and assist the search for chemical inhibitors of the methyltransferase, HEN1. In this short communication, we report a pioneering work that used capillary electrophoresis (CE) to separate the miRNA and its methylation product. Capillary zone electrophoresis (CZE) with UV-absorption detection and a reduced running temperature achieved good separation of miR173/miR173* and miR173-m/miR173*-m with a detection limit of around 1 microM. To enhance detection sensitivity, capillary gel electrophoresis (CGE) coupled with laser-induced fluorescence (LIF) detection was also tested, and base-line separation of nanomolar duplex RNA samples was achieved using 4% polyvinylpyrrolidone (PVP) as the sieving matrix and SYBR Green II RNA gel stain for on-column labeling. Although further study is needed to investigate if the separation is sequence dependent, our study demonstrated, for the first time, that CE could be an effective and rapid method in monitoring the miRNA methylation process.     

Evaluation of different nucleic acid stains for sensitive double-stranded DNA analysis with capillary electrophoretic separation

This paper outlines the first use of SYTOX Orange, SYTO 82 and SYTO 25 nucleic acid stains for on-column staining of double-stranded DNA (dsDNA) fragments separated by capillary electrophoresis (CE). Low-viscosity, replaceable poly(vinylpyrrolidone) (PVP) polymer solution was used as the sieving matrix on an uncoated fused-silica capillary. The effects of PVP concentration, electric field strength, and incorporated nucleic acid stain concentrations on separation efficiency were examined for a wide range of DNA fragment sizes. Our study was focused on using nucleic acid stains efficiently excitable at a wavelength of 532 nm. Among the five tested nucleic acid stains, SYTOX Orange stain was shown to have the best sensitivity for dsDNA detection by CE. About a 500-fold lower detection limit was obtained compared to commonly used ethidium bromide and propidium iodide. SYTOX Orange stain also provided a wide linear dynamic range for direct DNA quantitation with on-line CE detection. Use of SYTOX Orange stain can greatly improve the measurement of DNA fragments by CE, which will enable an expanded set of applications in genomics and diagnostics.     

Application of a Square‐Wave Potential Program for Time‐Dependent Amperometric Detection in Capillary Electrophoresis

Use of a square‐wave potential program for time‐dependent amperometric detection of analyte zones in capillary electrophoresis (CE) is described. Electrochemical detection for CE requires that the separation field be isolated from that of the electrochemical detection. This is generally done by physically separating the CE separation field from that of the detection. By applying a time variant potential program to the detection electrode, the detector current has a time dependence that can be used to help isolate the electrochemical detection current from that of the separation. When using a 20 μm inner‐diameter capillary, we find that a square‐wave potential program decreases the RMS baseline current from 4.5×10^?10 A, found with a constant potential amperometric detection, to 1.1×10^?10 A when using a square‐wave potential program. With a 75 μm inner‐diameter capillary, the improvement is even more dramatic, from 2.3×10^?9 A with amperometric detection to 2.06×10^?10 A when using a 1 Hz square‐wave potential program. When not using the time‐dependent detection with the 75 μm capillary, the analyte zones were beneath the S/N for the system and not detected. With the square‐wave potential program and time‐dependent detection, however, the analyte zones for an electrokinetic injection of 200 μM solution of 2,3‐dihydroxybenzoic acid were observed with the 75 μm inner‐diameter capillary. The improvement in the ability to discriminate the analytical signal from the background found experimentally is consistent with modeling studies.     

Niacin Determination in Legumes by Capillary Electrophoresis (CE). Comparison with High Performance Liquid Chromatography (HPLC)

Available and total niacin content in lentils and faba beans have been analyzed by capillary electrophoresis (CE), and the results compared with those obtained by high performance liquid chromatography (HPLC). Acidic and enzymatic hydrolysis have been carried out for available niacin determination, and an alkaline extraction performed for total niacin. The extracts were subsequently purified using a strong anion exchanger resin. Precise conditions for purification had to be worked out for each one of the two analytical methods (HPLC and CE). The HPLC analysis for available and total niacin was carried out in an ion-pair reverse phase column with UV detection at 261 nm. For the CE separation, the following conditions were employed: a 20 mM sodium tetraborate; 15 mM sodium dodecyl sulfate and 20% isopropyl alcohol solution as separation buffer; 30 kV and 25 or 30°C. Separation was carried out in a 70 cm effective length × 75 μm i.d. fused-silica capillary using on-column UV detection at 254 nm. The results obtained by CE for lentils and faba beans were similar to those obtained by HPLC.     

A handy detection cell for end-column electrochemical detection in capillary electrophoresis.

A handy and simple detection cell was constructed using a mixing joint for end-column electrochemical detection in capillary electrophoresis (CE). The cell allows for positioning of the working electrode at the end of the separation capillary without the aid of micropositioners. The design facilitates the exchange of electrodes and capillaries without the need to refabricate the entire capillary-electrode setup. The cell can be assembled in a short period of time. Alignment with the joint screw proved to be reproducible for working electrodes of copper and gold. The advantages of reduced time and low cost make the device very attractive for the routine analysis of electroactive species, such as carbohydrates and their derivatives, purine bases and nucleosides, amino acids, and catecholamines etc. by CE with electrochemical detection.