On‐line sample preconcentration by polarity switching in floating electrode‐integrated microchannel

In this study, we found that the polarity switching was effective to enrich and separate fluorescent analytes which have weakly‐dissociated groups in a floating platinum electrode (width, 50 µm; thickness, 2.5 µm)‐integrated straight‐channel in microchip electrophoresis (MCE). In the straight channel filled with an Alexa Flour 488 (AF488) solution, a sharp peak was observed after the polarity inversion with a 530‐fold enhancement of the sensitivity relative to the conventional MCE analysis. By using a fluorescent pH indicator, we verified that a sharp high‐pH zone was generated nearby the floating electrode and moved toward the anode with maintaining the high pH, which induced the sample enrichment like a dynamic pH junction mechanism. In the floating electrode‐embedded channel, the mixture of AF488‐labeled proteins was also well concentrated and separated within 100 s.     

Acupuncture injection for field amplified sample stacking and glass microchip‐based capillary gel electrophoresis

Acupuncture sample injection is a simple method to deliver well‐defined nanoliter‐scale sample plugs in PDMS microfluidic channels. This acupuncture injection method in microchip CE has several advantages, including minimization of sample consumption, the capability of serial injections of different sample solutions into the same microchannel, and the capability of injecting sample plugs into any desired position of a microchannel. Herein, we demonstrate that the simple and cost‐effective acupuncture sample injection method can be used for PDMS microchip‐based field amplified sample stacking in the most simplified straight channel by applying a single potential. We achieved the increase in electropherogram signals for the case of sample stacking. Furthermore, we present that microchip CGE of ΦX174 DNA‐HaeⅢ digest can be performed with the acupuncture injection method on a glass microchip while minimizing sample loss and voltage control hardware.     

Application of capillary electrophoresis with different sample stacking strategies for the determination of a group of nonsteroidal anti-inflammatory drugs in the low microg x L(-1) concentration range

Several on-column sample preconcentration modes--large-volume sample stacking using the EOF pump (LVSEP), LVSEP with anion-selective exhaustive injection (LVSEP-ASEI) and field-amplified sample injection with sample matrix removal using the electroosmotic flow (EOF) pump (FAEP)--were used to analyze some nonsteroidal anti-inflammatory drugs (NSAIDs) by capillary electrophoresis, and then compared. Methanol was the background electrolyte solvent to suppress the EOF. The effect of the type and length of the solvent plug, and the sample injection time were investigated in FAEP to determine the conditions that provided the best response. LVSEP, LVSEP-ASEI, and FAEP improved the sensitivity of the peak area by 100-, 1200-, and 1800-fold, respectively. The methodology developed, in combination with solid-phase extraction (SPE), was applied to the analysis of water samples.     

Sensitivity enhancing injection from a sample reservoir and channel interface in microchip electrophoresis

The stacking of a cationic analyte (i.e., rhodamine B) at the interface between a sample reservoir and channel in a microchip electrophoresis device is described for the first time. Stacking at negative polarity was by micelle to solvent stacking where the dye was prepared in a micellar solution (5 mM sodium dodecyl sulfate in 25 mM phosphoric acid, pH 2.5) and the channel was filled with high methanol content background solution (70% methanol in 50 mM phosphoric acid, pH 2.5). The injection of the stacked dye into the channel was by simple reversal of the voltage polarity with the sample solution and background solution at the anodic and cathodic reservoirs of the straight channel, respectively. The enrichment of rhodamine B at the interface and injection of the stacked dye into the channel was clearly visualized using an inverted fluorescence microscope. A notable sensitivity enhancement factor of up to 150 was achieved after 2 min at 1 kV of micelle to solvent stacking. The proposed technique will be useful as a concentration step for analyte mixtures in simple and classical cross‐channel microchip electrophoresis devices or for the controlled delivery of enriched reagents or analytes as narrow plugs in advanced microchip electrophoresis devices.     

Large volume sample stacking in capillary electrophoresis of weakly acidic compounds using coated capillaries at high pH

Large volume stacking using the electroosmotic flow (EOF) pump (LVSEP) in capillary electrophoresis under a reverse potential is a convenient and straightforward approach for on-line concentration of dilute anionic sample solutions. LVSEP achieves automatic sample matrix removal and subsequent separation without intermediate polarity switching nor complicated instrumental setup. Since anionic analytes should move against the EOF in LVSEP, EOF needs to be suppressed. We extended the range of LVSEP up to pH 11 using various EOF suppression methods, such as dynamic coating by polymer pretreatment and permanent coating. Weakly acidic organic compounds (pK_a<5.2), chlorinated phenols (pK_a=7-9), and aromatic amino acids (pK_a2∼9.3) were concentrated and separated. By hydrodynamically filling the whole capillary of 27 cm long with the sample solution, fast and reliable injection was achieved and sensitivity enhancement factors as large as 170 were readily obtained in less than 8 min.     

Pressure-assisted field-amplified sample injection with reverse migrating micelles for analyzing trace steroids in MEKC

This paper describes a novel method that applies pressure-assisted field-amplified sample injection with reverse migrating micelles (PA-FASI-RMM) for the online concentration of neutral analytes in MEKC with a low-pH BGE. After injection of a plug of water into the separation capillary, negative voltage and positive pressure were simultaneously applied to initialize PA-FASI-RMM injection. The hydrodynamic flow generated by the positive pressure compensated the reverse EOF in the water plug and allowed the water plug to remain in the capillary during FASI with reverse migrating micelles (FASI-RMM) to obtain a much longer injection time than usual, which improved stacking efficiency greatly. Equations describing this injection mode were introduced and were supported by experimental results. For a 450-s online PA-FASI-RMM injection, three orders of magnitude sample enhancement in terms of peak area could be observed for the steroids and an achievement of detection limits was between 1 and 10 ng/mL.     

Impact of reservoir potentials on the analyte behavior in microchip electrophoresis: computer simulation and experimental validation for DNA fragments

Fundamental understanding of the impact of reservoir potentials on the analyte behavior on the microfluidic chips is an important issue in microchip electrophoresis (MCE) for suitable injection and separation of analytes, since the applied potentials may significantly affect the shape of sample plug, sample leakage from the injection channel to the separation channel, injected sample amount, and separation efficiency. This study addressed this issue for the case of a conventional cross-geometry microchip with four reservoirs using computer simulations, the results of which were verified by the analysis of DNA fragments. For the microchip with a definite structure and migration distance, the injected sample amount was shown to be the vital parameter for improving the limit of detection and resolution. During injection, the shape of the sample plug could be adjusted by varying the reservoir potentials. It was demonstrated that a "magnified injection" (applying high voltage on the three reservoirs to the sample reservoir) is useful to enhance the detection sensitivity depending on the analyte composition, although such injection was previously avoided because of introducing too large amounts of the analyte in comparison with two established modes, floating and pinched injection. Optimal magnified injection was proved to improve the sensitivity for about 4 times over that of pinched injection for the analysis of DNA step ladders using microchip gel electrophoresis (MCGE). Sample leakage of DNA fragments could be suppressed by applying a high positive voltage on injection channel during separation, but the voltage degraded the injected amount and resolution.     

Recent progress of on-line sample preconcentration techniques in microchip electrophoresis

This review highlights recent developments and applications of on-line sample preconcentration techniques to enhance the detection sensitivity in microchip electrophoresis (MCE); references are mainly from 2008 and later. Among various developed techniques, we focus on the sample preconcentration based on the changes in the migration velocity of analytes in two or three discontinuous solutions system, since they can provide the sensitivity enhancement with relatively easy experimental procedures and short analysis times. The characteristic features of the on-line sample preconcentration applied to microchip electrophoresis (MCE) are presented, categorized on the basis of "field strength-" or "chemically" induced changes in the migration velocity. The preconcentration techniques utilizing field strength-induced changes in the velocity include field-amplified sample stacking, isotachophoresis and transient-isotachophoresis, whereas those based on chemically induced changes in the velocity are sweeping, transient-trapping and dynamic pH junction.     

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。     

Single‐step CE for miniaturized and easy‐to‐use system

We developed a novel single‐step capillary electrophoresis (SSCE) scheme for miniaturized and easy to use system by using a microchannel chip, which was made from the hydrophilic material polymethyl methacrylate (PMMA), equipped with a capillary stop valve. Taking the surface tension property of liquids into consideration, the capillary effect was used to introduce liquids and control capillary stop valves in a partial barrier structure in the wall of the microchannel. Through the combined action of stop valves and air vents, both sample plug formation for electrophoresis and sample injection into a separation channel were successfully performed in a single step. To optimize SSCE, different stop valve structures were evaluated using actual microchannel chips and the finite element method with the level set method. A partial barrier structure at the bottom of the channel functioned efficiently as a stop valve. The stability of stop valve was confirmed by a shock test, which was performed by dropping the microchannel chip to a floor. Sample plug deformation could be reduced by minimizing the size of the side partial barrier. By dissolving hydroxyl ethyl cellulose and using it as the sample solution, the EOF and adsorption of the sample into the PMMA microchannel were successfully reduced. Using this method, a 100‐bp DNA ladder was concentrated; good separation was observed within 1 min. At a separation length of 5 mm, the signal was approximately 20‐fold higher than a signal of original sample solution by field‐amplified sample stacking effect. All operations, including liquid introduction and sample separation, can be completed within 2 min by using the SSCE scheme.     

Enhanced sensitivity and resolution for the analysis of paralytic shellfish poisoning toxins in water using capillary electrophoresis with amperometric detection and field‐amplified sample injection

The profiling of the most lethal paralytic shellfish poisoning toxins (PSTs) in freshwater has increased the need to establish an alternative analytical method with high sensitivity and resolution. In this paper, a coupling technique of field‐amplified sample injection (FASI) and CE with end‐column amperometric detection (CE‐AD) was developed to improve the detection sensitivity and separation of PSTs by electrokinetically injecting a water plug of analytes to the capillary filled with a high‐conductivity BGE. Parameters affecting FASI and CE process were carefully adjusted to achieve the highest response and resolution. Separation selectivity for PSTs, especially for the analogues and epimers, was greatly enhanced by using 40 mM Britton–Robinson buffer (pH 9.5) as BGE, which altered the EOF and mobility of the analytes that interacted with polyborate ions. Satisfactory linear relationship between peak current and concentration of toxins were gained over a wide range of 1.95–254 μg/L. The detection limits (S/N = 3) for five PSTs ranged from 0.63 to 3.11 μg/L, which are below the health alert level in drinking water. In comparison with the up‐to‐date reporting chromatographic methods, the FASI‐CE‐AD method was simple, low‐cost, selective, and sensitive enough for direct quantification of PSTs at very low levels, implying a potential for screening and monitoring of PSTs in surface waters.     

On‐line synergistic stacking in capillary zone electrophoresis featuring field‐amplified sample stacking and micelle to cyclodextrin stacking in the determination of two alkaloids in complicated matrix samples

A novel on‐line synergistic proconcentration strategy coupling field‐amplified sample stacking and micelle to cyclodextrin stacking for cationic analytes in capillary zone electrophoresis has been proposed and applied for the separation and determination of two alkaloids, matrine, and oxymatrine in complicated matrix samples. The approach was performed by the long injection of sample in a low‐conductivity sodium dodecyl benzene sulfonate solution followed by the injection of hydroxypropyl‐β‐cyclodextrin solution in higher conductivity. The stacking mechanism of this method has been expounded and parameters affecting stacking effect have been optimized in our study. Under the optimum experimental conditions, 169‐ and 218‐fold sensitivity improvements were achieved for matrine and oxymatrine when compared with normal injection. Analytical indicators including linearity, limits of detection, and reproducibility (intra‐ and inter‐day relative standard deviations) were evaluated. Moreover, sample matrix effect was studied using compound flavescent sophora and salicylic acid powder and spiked urine samples. The developed method is an attempt for the combination of micelle to cyclodextrin stacking with other stacking methods. It could be a good alternative choice for the determination of alkaloids in a complex sample matrix.     

Simultaneous determination of aripiprazole and its active metabolite,dehydroaripiprazole, in plasma by capillary electrophoresis combining on‐column field‐amplified sample injection and application in schizophrenia

A sensitive high‐performance CZE combining on‐column field‐amplified sample injection (FASI) has been developed for simultaneous determination of aripiprazole and its active metabolite, dehydroaripiprazole, in human plasma. A sample pretreatment by means of liquid–liquid extraction (LLE) (diethyl ether) with subsequent quantitation by FASI‐CZE was used. The separation of aripiprazole and dehydroaripiprazole was performed using a BGE containing 150 mM phosphate buffer (pH 3.5) with 40% methanol and 0.02% PVA as a dynamic coating to reduce interaction of analytes with the capillary wall. Before sample loading, a methanol plug (0.3 psi, 6 s) was injected to permit FASI for stacking. The samples were injected electrokinetically (10 kV, 30 s) to introduce sample cations and the applied voltage was 20 kV with on‐column detection at 214 nm. Several parameters affecting the separation and sensitivity of the drug and its active metabolite were studied, including reconstitution solvent, organic modifier, pH and concentration of phosphate buffer. The linear ranges of the method for test drug and its active metabolite, in plasma using amlodipine as an internal standard, were over the range 5.0–100.0 ng/mL. One female volunteer (25 years old) was orally administered a single dose of 10 mg aripiprazole (Abilify^®, Otsuka) and blood samples were drawn over a 60 h period for pharmacokinetic study. The method was also applied to monitor the concentration of aripiprazole and dehydroaripiprazole in plasma collected after oral administration of 20 or 30 mg aripiprazole (Abilify^®, Otsuka) daily at steady state in one schizophrenic patient.     

Improving sensitivity by large-volume sample stacking using the electroosmotic flow pump to analyze some nonsteroidal anti-inflammatory drugs by capillary electrophoresis in water samples

Large-volume sample stacking using the electroosmotic flow (EOF) pump (LVSEP) has been used to analyze some nonsteroidal anti-inflammatory drugs (NSAIDs) in water samples. With methanol as the run buffer solvent to suppress the EOF, sensitivity was enhanced by 80-100-fold. The sample for the analysis of real water sample was pretreated by solid-phase extraction (SPE). When the method was based on off-line SPE-LVSEP-CE, sensitivity improved by as much as 1000 times.     

Analysis of tetrabromobisphenol A and other phenolic compounds in water samples by non-aqueous capillary electrophoresis coupled to photodiode array ultraviolet detection

Non-aqueous capillary electrophoresis (NACE) with large-volume sample stacking injection using the electroosmotic flow pump (LVSEP) has been developed for the determination of tetrabromobisphenol A (TBBPA) and other phenolic compounds in environmental matrices. Methanol has been used as run buffer solvent to reduce the electroosmotic flow (EOF). Identification and quantification of the analytes was performed by photodiode array ultraviolet detection. LVSEP-NACE improved sensitivity of the peak height by 90-300-fold. The method developed was applied to the analysis of TBBPA in river water and wastewater samples, using solid-phase extraction (SPE) as sample pretreatment process. The average recoveries of the analytes were in the range of 96-106% and 73-103% for 1 L of river water and 0.5 L of wastewater samples, respectively. When the method was based on off line SPE-LVSEP-NACE, sensitivity was improved by 3300-4500-fold and 1600-2200-fold for river water and wastewater samples, respectively.     

Simultaneous stacking of cationic and anionic compounds in single run capillary zone electrophoresis by two-end field amplified sample injection

In order to extend the application of field amplified sample injection (FASI) in high throughput analysis, a convenient and simple procedure, namely two-end field amplified sample injection (TE-FASI), was developed for the simultaneous stacking of cationic and anionic compounds in a single run capillary zone electrophoresis (CZE). Following the capillary-filling with a buffer of high conductivity, water plug was loaded into each end of the capillary; and two high-field strength zones were generated at both heads of the column when high voltage was applied. Therefore, under suppressed EOF cations and anions can be selectively FASI stacked at anode and cathode head, respectively. After separation, the stacked anions and cations are detected by a common detector placed in the center of the capillary. Under the optimized conditions, the limits of detection for the model cationic (matrine and oxymatrine) and anionic (5-sulfosalicylic acid) compounds were determined as 0.2, 0.2 and 0.06 ng/mL, respectively. Compared with non-stacking conditions, the sensitivities of these compounds were enhanced 1003-, 1330- and 1380-fold, respectively. The results of reproducibility, linearity and real sample analysis show that the proposed procedure is promising to be applied for the simultaneous quantification detection of trace cationic and anionic analytes.     

Surfactant‐assisted dispersive liquid–liquid microextraction combined with field‐amplified sample stacking in capillary electrophoresis for the determination of mexiletine and lidocaine

A sensitive method for the determination of mexiletine and lidocaine using surfactant‐assisted dispersive liquid–liquid microextraction coupled with capillary electrophoresis was developed. Triton X‐100 and dichloromethane were used as the dispersive agent and extraction solvent, respectively. After the extraction, mexiletine and lidocaine were analyzed using capillary electrophoresis with ultraviolet detection. The detection sensitivity was further enhanced through the use of field‐amplified sample stacking. Under optimal extraction and stacking conditions, the calibration curves were linear over a concentration range of 0.05–1.00 μM for mexiletine and 0.03–1.00 μM for lidocaine. The limits of detection (signal‐to‐noise ratio of 3) were 0.01 and 0.01 μM for mexiletine and lidocaine, respectively. An approximately 1141‐ to 1250‐fold improvement in sensitivity was observed for the two analytes compared with the injection of a standard solution without the surfactant‐assisted dispersive liquid–liquid microextraction and field‐amplified sample stacking procedures. This developed method was successfully applied to the determination of mexiletine and lidocaine in human urine and serum samples. Both precision and accuracy for urine and serum samples were less than 8.7 and 6.7%, respectively. The recoveries of the two analytes from urine and serum samples were 54.7–64.9% and 16.1–56.5%, respectively.     

Poly（dimethylsiloxane） Microchips with Two Sharpened Stretching Tips and Its Application to Protein Separation Using Dynamic Coating

An integrated poly（dirnethylsiloxane） （PDMS） microchip with two sharpened stretching tips for convenient sample injecting, running buffer refreshing and channel cleaning has been presented. The sample was directly introduced into the separation channel through the stretching inlet tip without complicated power switching supplies and injection cross channel. The operation of running buffer refreshing or channel cleaning was simplified by vacuuming one end of the tip and placing the other tip into the solution vial. Therefore, this fabrication method can be easily applied to most analytical laboratories economically without soft lithography and plasma bonding equipments. The attractive performance of the novel PDMS microchips has been demonstrated by using laser-induced fluorescence detection for separation of proteins. The addition of 0.04% Brij 35 in 0.04 mol/L phosphate buffer （pH 7.0） can reduce the adhesion of proteins in multienzyme tablet and make separation more easily. The electroosmotic flow （EOF） exhibits pH-independence in the range of 3-1 1 in dynamic modified microchannel.     

In‐line preconcentration capillary zone electrophoresis for the analysis of haloacetic acids in water

Two in‐line enrichment procedures (large volume sample stacking (LVSS) and field amplified sample injection (FASI)) have been evaluated for the CZE analysis of haloacetic acids (HAAs) in drinking water. For LVSS, separation on normal polarity using 20 mM acetic acid–ammonium acetate (pH 5.5) containing 20% ACN as BGE was required. For FASI, the optimum conditions were 25 s hydrodynamic injection (3.5 kPa) of a water plug followed by 25 s electrokinetic injection (?10 kV) of the sample, and 200 mM formic acid–ammonium formate buffer at pH 3.0 as BGE. For both FASI and LVSS methods, linear calibration curves (r²>0.992), limit of detection on standards prepared in Milli‐Q water (49.1–200 μg/L for LVSS and 4.2–48 μg/L for FASI), and both run‐to‐run and day‐to‐day precisions (RSD values up to 15.8% for concentration) were established. Due to the higher sensitive enhancement (up to 310‐fold) achieved with FASI‐CZE, this method was selected for the analysis of HAAs in drinking water. However, for an optimal FASI application sample salinity was removed by SPE using Oasis WAX cartridges. With SPE‐FASI‐CZE, method detection limits in the range 0.05–0.8 μg/L were obtained, with recoveries, in general, higher than 90% (around 65% for monochloroacetic and monobromoacetic acids). The applicability of the SPE‐FASI‐CZE method was evaluated by analyzing drinking tap water from Barcelona where seven HAAs were found at concentration levels between 3 and 13 μg/L.     

A polymeric microchip with integrated tips and in situ polymerized monolith for electrospray mass spectrometry

We describe the integration of a cyclo-olefin polymer based microchip with a sheathless capillary tip for electrospray ionization-mass spectrometry (ESI-MS). The microchip was fabricated by hot embossing and thermal bonding. Its design includes a side channel for adjusting the composition of the electrospray solution so that analytes in 100% water can be analyzed. The fused silica capillaries, used for sample introduction, and the electrospray tips for MS coupling were directly inserted into the microchannel before thermal bonding of the device. A microfabricated on-chip gold microelectrode was used to apply the electrospray voltage. Annealing the device after thermal bonding increased the pressure resistance of the microchip. The cross section of the microchannel was imaged by scanning electron microscopy to estimate the effects of the annealing step. The relationship between the applied electrospray voltages and MS signal was measured at different flow rates by coupling the device to an ion trap mass spectrometer. The performance of the microchip was evaluated by MS analysis of imipramine in ammonium acetate buffer solution by direct infusion. An alkylacrylate based monolith polymer bed for on-chip sample pretreatment and separation was polymerized in the microchannel and tested for ESI-MS applications.