Fast and simple sample introduction for capillary electrophoresis microsystems

A newly designed capillary electrophoresis (CE) microchip with a simple and efficient sample introduction interface is described. The sample introduction is carried out directly on the separation channel through a sharp inlet tip placed in the sample vial, without an injection cross, complex microchannel layouts or hardware modification. Alternate placement of the inlet tip in vials containing the sample and buffer solutions permits a volume defined electrokinetic sample introduction. Such fast and simple sample introduction leads to highly reproducible signals with no observable carry over between different analyte concentrations. The performance of the system was demonstrated in flow-injection and CE measurements of nitroaromatic explosives and for on-chip enzymatic assays of glucose in the presence of ascorbic acid. Employing an 8 cm long separation channel and a separation voltage of 4000 V it offers high-throughput flow-injection assays of 100 samples h(-1) with a relative standard deviation of 3.7% for TNT (n= 100). Factors influencing the analytical performance of the new microchip have been characterized and optimized. Such ability to continuously introduce discrete samples into micrometer channels indicates great promise for high-speed microchip analysis.     

On-line electrophoretic sample clean-up for sensitive and reproducible μCE immunoassay

We report a novel on-line electrophoretic sample clean-up approach for highly sensitive and reproducible microchip electrophoretic (μCE) immunoassay of low-abundance proteins in human serum. The method takes advantage of the differential effect of field-amplified sample stacking on molecules with different electrophoretic mobility. Large interfering proteins are removed from the loading channel by simple voltage control, resulting in selective concentration and injection of smaller target analytes to the separation channel. As a proof of concept, an antibody-free injection mode was developed for direct μCE immunoassay of human insulin-like growth factor-I (IGF-I) in serum samples without any additional purification steps. Clear and sharp peaks were obtained for IGF-I with low background and excellent reproducibility. Besides, the assay sensitivity was further increased by addition of ethanol to the sample buffer at a concentration of 50% right before performing the μCE detection. The lower limit of detection of IGF-I achieved 0.68 ng mL(-1), with an overall signal enhancement factor of 2750. The established on-line electrophoretic sample clean-up approach may find wide applications in the development of other microchip-based high-throughput analytical platforms for clinical and biological use.     

Hydrodynamic injection with pneumatic valving for microchip electrophoresis with total analyte utilization

A novel hydrodynamic injector that is directly controlled by a pneumatic valve has been developed for reproducible microchip CE separations. The PDMS devices used for the evaluation comprise a separation channel, a side channel for sample introduction, and a pneumatic valve aligned at the intersection of the channels. A low pressure (≤ 3?psi) applied to the sample reservoir is sufficient to drive sample into the separation channel. The rapidly actuated pneumatic valve enables injection of discrete sample plugs as small as ~ 100?pL for CE separation. The injection volume can be easily controlled by adjusting the intersection geometry, the solution back pressure, and the valve actuation time. Sample injection could be reliably operated at different frequencies (< 0.1?Hz to > 2?Hz) with good reproducibility (peak height relative standard deviation ≤ 3.6%) and no sampling biases associated with the conventional electrokinetic injections. The separation channel was dynamically coated with a cationic polymer, and FITC-labeled amino acids were employed to evaluate the CE separation. Highly efficient (≥ 7.0 × 103 theoretical plates for the ~2.4-cm-long channel) and reproducible CE separations were obtained. The demonstrated method has numerous advantages compared with the conventional techniques, including repeatable and unbiased injections, little sample waste, high duty cycle, controllable injected sample volume, and fewer electrodes with no need for voltage switching. The prospects of implementing this injection method for coupling multidimensional separations for multiplexing CE separations and for sample-limited bioanalyses are discussed.     

Fast and simultaneous detection of heavy metals using a simple and reliable microchip-electrochemistry route: An alternative approach to food analysis

This paper reports, for the first, the fast and simultaneous detection of prominent heavy metals, including: lead, cadmium and copper using microchip CE with electrochemical detection. The direct amperometric detection mode for microchip CE was successfully applied to these heavy metal ions. The influences of separation voltage, detection potential, as well as the concentration and pH value of the running buffer on the response of the detector were carefully assayed and optimized. The results clearly show that reliable analysis for lead, cadmium, and copper by the degree of electrophoretic separation occurs in less than 3min using a MES buffer (pH 7.0, 25mM) and l-histidine, with 1.2kV separation voltage and -0.8V detection potential. The detection limits for Pb(2+), Cd(2+), and Cu(2+) were 1.74, 0.73 and 0.13microM (S/N=3). The %R.S.D. of each peak current was <6% and migration times <2% for prolonged operation. To demonstrate the potential and future role of microchip CE, analytical possibilities and a new route in the raw sample analysis were presented. The results obtained allow the proposed microchip CE-ED acts as an alternative approach for metal analysis in foods.     

Top-down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry

A system of microchip capillary electrophoresis/electrospray ionization mass spectrometry (microchip-CE/ESI-MS) for rapid characterization of proteins has been developed. Capillary electrophoresis (CE) enables rapid analysis of a sample present in very small quantity, such as at femtomole levels, at high resolution. Faster CE/MS analysis is expected by downsizing the normal capillary to the microchip (microchip) capillary. Although rapidity and high resolution are advantages of CE separation, electroosmotic flow (EOF) instability caused by the interaction between proteins and the microchannel surface results in low reproducibility in the analysis of basic proteins under neutral pH conditions. By coating the microchannel surface with a basic polymer, polyE-323, basic proteins, which have pI values of over 7.5, could be separated and detected by microchip-CE/MS on quadrupole (Q) and time-of-flight (TOF) hybrid instruments. By increasing the cone and collision voltages during the analysis by microchip-CE/ESI-MS of a small protein, some product ions, which contain the sequence information, could also be obtained, i.e., 'top-down' analysis of the protein could be accomplished with this microchip-CE/MS system. To our knowledge, this is the first report of 'top-down' analysis of a protein by microchip-CE/MS. Since it requires a much shorter time and a smaller sample amount for analysis than the conventional liquid chromatography (LC)/ESI-MS method, microchip-CE/MS promises to be suitable for the high-throughput characterization of proteins.     

Flow manipulation for sweeping with a cationic surfactant in microchip capillary electrophoresis

Flow manipulation in sweeping microchip capillary electrophoresis (CE) is complicated by the free liquid communication between channels at the intersection, especially when the electroosmotic flows are mismatched in the main channel. Sweeping in traditional CE with cationic micelles is an effective way to concentrate anionic analytes. However, it is a challenge to transfer this method onto microchip CE because the dynamic coating process on capillary walls by cationic surfactants is interrupted when the sample solution free of surfactants is introduced into the microchip channels. This situation presents a difficulty in the sample loading, injection and dispensing processes. By adding surfactant at a concentration around the critical micelle concentration and by properly designing the voltage configuration, the flows in a microchip were effectively manipulated and this sweeping method was successfully moved to microchip CE using tetradecyltrimethylammonium bromide (TTAB). The sweeping effect of cationic surfactant in the sample solution was discussed theoretically and studied experimentally in traditional CE. The flows in a microchip were monitored with fluorescence imaging, and the injection and sweeping processes were studied by locating the detection point along the separation channel. A detection enhancement of up to 500-fold was achieved for 5-carboxyfluorescein.     

Field amplified sample stacking coupled with chip-based capillary electrophoresis using negative pressure sample injection technique

A multi-T microchip for integrated field amplified sample stacking (FASS) with CE separation to increase the chip-based capillary electrophoresis (chip-based CE) sensitivity was developed. Volumetrically defined large sample plug was formed in one step within 5s by the negative pressure in headspace of the two sealed sample waste reservoirs produced using a syringe pump equipped with a 3-way valve. Stacking and separation can proceed only by switching the 3-way valve to release the vacuum in headspace of the two sample waste reservoirs. This approach considerably simplified the operations and the equipments for FASS in chip-based CE systems. Migration time precisions of 3.3% and 1.3% RSD for rhodamine123 (Rh123) and fluorescien sodium salt (Flu) in the separation of a mixture of Flu and Rh123 were obtained for nine consecutive determinations with peak height precisions of 4.8% and 3.4% RSD, respectively. Compared with the chip-based CE on the cross microchip, the sensitivity for analysis of FlTC, FITC-labeled valine (Val) and Alanine (Ala) increased 55-, 41- and 43-fold, respectively.     

A triple-injection method for microchip electrophoresis

We report here a novel triple injection method for microchip electrophoresis (micro-CE) that results in a higher intensity of DNA peaks. This new method includes a triple-repeated process of a combination of a sample loading voltage and a separation voltage in each interval, namely (loading time) + (separation time) + (loading time) + (separation time) + (loading time), prior to electrophoretic separation. All these injections were electrokinetically controlled by a software. Although the usual sample injection, which included the process of one 60 s electrokinetically application, was limited by the amount of sample, peaks of 40% higher intensity were obtained using the new method within half of the conventional injection time compared to the conventional method. Maximum peak intensity was successfully achieved with integration of the intensities of the triple-repeated peaks by adjusting the application period of the separation voltage. Repetition of the sample loading voltage for an adjusted period with a further adjusted period of separation voltage in each interval may be an effective method for injection of samples that results in peaks with higher intensity.     

Recent developments in electrochemical detection for microchip capillary electrophoresis

Significant progress in the development of miniaturized microfluidic systems has occurred since their inception over a decade ago. This is primarily due to the numerous advantages of microchip analysis, including the ability to analyze minute samples, speed of analysis, reduced cost and waste, and portability. This review focuses on recent developments in integrating electrochemical (EC) detection with microchip capillary electrophoresis (CE). These detection modes include amperometry, conductimetry, and potentiometry. EC detection is ideal for use with microchip CE systems because it can be easily miniaturized with no diminution in analytical performance. Advances in microchip format, electrode material and design, decoupling of the detector from the separation field, and integration of sample preparation, separation, and detection on-chip are discussed. Microchip CEEC applications for enzyme/immunoassays, clinical and environmental assays, as well as the detection of neurotransmitters are also described.     

Continuous monitoring with microfabricated capillary electrophoresis chip devices

We report on the direct coupling of hydrodynamically flowing stream to a microchip capillary electrophoresis (CE) for continuous assays of liquid samples. The new interface relies on mounting the sample tubing onto a sharp inlet tip and allows rapid, convenient and reproducible electrokinetic loading from a continuously flowing stream directly into the narrow separation microchannel. The sharp inlet interface is characterized by its efficiency, stability and simplicity. The effect of the sample flow rate, applied voltages and other relevant variables, is described. It was found that the peak intensity is independent of the flow rate. The performance of the new interface is illustrated for on-line CE-electrochemical monitoring of phenolic and explosive compounds. Conditions simulating continuous long-term monitoring, led to a highly stable response for a 15 ppm 1,3,5-trinitrobenzene solution (RSD = 3.7%, n= 40). Such ability to continuously introduce flowing samples into micrometer channels makes 'lab-on-a-chip' devices highly compatible with real-life monitoring applications.     

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.     

Electrochemical Detection for Capillary Electrophoresis Microchips: A Review

Electrochemistry detection offers considerable promise for capillary‐electrophoresis (CE) microchips, with features that include remarkable sensitivity, portability, independence of optical path length or sample turbidity, low cost and power requirements, and high compatibility with modern micromachining technologies. This article highlights key strategies in controlled‐potential electrochemical detectors for CE microchip systems, along with recent advances and directions. Subjects covered include the design of the electrochemical detection system, its requirements and operational principles, common electrode materials, isolation from the separation voltage, derivatization reactions, typical applications, and future prospects. It is expected that electrochemical detection will become a powerful tool for CE microchip systems and will lead to the creation of truly portable (and possibly disposable) devices.     

Frontal analysis on a microchip

Conventional microchip applications involving capillary electrophoresis (CE) typically inject a sample along one channel and use an intersection of two channels to define the sample plug--the portion of sample to be analysed along a second channel. In contrast to this method of zone separation, frontal analysis proceeds by injecting sample continuously into a single channel or column. Frontal analysis is more common in macroscopic procedures but there are benefits in sensitivity and device density to its application to electrophoresis on microchips. This work compares conventional microchip zone analysis with frontal analysis in the separation of PCR products. Although we detect on the order of 5000 fluorophores with a compact instrument using the zone separation CE method, we found a several-fold increase in the effective signal-to-noise ratio by using a frontal analysis method. By removing the need for additional channels and reservoirs the frontal method would allow device densities to be significantly increased, potentially improving the cost-effectiveness of microchip analyses in applications such as medical diagnostics.     

Highly efficient approach for characterizing nanometer-sized gold particles by capillary electrophoresis

This paper demonstrates that capillary electrophoresis (CE) can be employed for characterizing the sizes of nanometer-scale gold particles. We characterized the gold nanoparticles by effecting CE separation using a buffer of SDS (70 mM) and 3-cyclohexylamino-1-propanesulfonic acid (CAPS; 10 mM) at pH 11.0 and an applied voltage of 18 kV and obtained a linear relationship (R² > 0.99) between electrophoretic mobilities and size for nanoparticles whose diameters fall in the regime from 5.0 ± 0.5 to 41.2 ± 3.3 nm; the relative standard deviations of these electrophoretic mobilities are <0.8%. We evaluated the feasibility of employing these separation conditions for the size characterization by of gold nanoparticle samples that were synthesized by a rapid microwave heating method. We confirmed that this CE method is a valid one for size characterization by comparing the results obtained by CE with those provided by scanning electron microscopy (SEM); a good correlation exists between these two techniques. Our results demonstrate that CE can be employed to accelerate the analysis of the sizes of nanomaterials.     

Improved hydrostatic pressure sample injection by tilting the microchip towards the disposable miniaturized CE device

A simple method of hydrostatic pressure sample injection towards a disposable microchip CE device was developed. The liquid level in the sample reservoir was higher than that in the sample waste reservoir (SWR) by tilting microchip and hydrostatic pressure was generated, the sample was driven to pass through injection channel into SWR. After sample loading, the microchip was levelled for separation under applied high separation voltage. Effects of tilted angle, initial liquid height and injection duration on electrophoresis were investigated. With enough injection duration, the injection result was little affected by tilted angle and initial liquid heights in the reservoirs. Injection duration for obtaining a stable sample plug was mainly dependent on the tilted angle rather than the initial height of liquid. Experimental results were consistent with theoretical prediction. Fluorescence observation and electrochemical detection of dopamine and catechol were employed to verify the feasibility of tilted microchip hydrostatic pressure injection. Good reproducibility of this injection method was obtained. Because the instrumentation was simplified and no additional hardware was needed in this technology, the proposed method would be potentially useful in disposable devices.     

Determination of small phosphorus-containing compounds by capillary electrophoresis

The review focuses on the analysis of small phosphorus-containing compounds by capillary electrophoresis (CE) with different detection modes including UV absorption, laser-induced fluorescence, conductometry, amperometry, atomic spectrometry, and mass spectrometry. Determinations of phosphates, organophosphate, and chemical warfare agents in environmental samples such as water, soil and grains are emphasized. To achieve better sensitivity, high-resolving power, and reproducibility, the optimum separation conditions for various analytes (samples) are different. We compare the merits and demerits of the different detection modes for the detection of the analytes. Although the present methods are successful in many cases, there is still a need to develop high-throughput CE techniques for screening numerous environmental samples and sample stacking techniques in CE for the analysis of trace analytes.     

Determination of haloacetic acids by the combination of non-aqueous capillary electrophoresis and mass spectrometry

The applicability of capillary electrophoresis (CE) in combination with atmospheric pressure ionization mass spectrometry (API-MS) is demonstrated for the determination of organic acids and in particular for haloacetic acids. CE-conditions, sheath flow and MS-parameters were optimized with respect to the separation of the analytes and mass spectrometric sensitivity. CE/MS turned out to be an attractive alternative for the determination of haloacetic acids to existing methods based on GC-ECD. Employing CE/MS derivatization is not necessary which saves time and avoids possible sources of errors. In the present work the sample pre-treatment is performed by liquid-liquid extraction using methyl tert.-butyl ether as the extraction solvent. The organic phase is brought to dryness in a stream of nitrogen gas and the residue is dissolved in methanol and analyzed by CE/MS using a mixture of 2-propanol/water 80?:?20 containing triethylamine as the sheath liquid in the interface. Best results for the separation of all nine possible bromo- and chloroacetic acids together with two internal standards are obtained with a carrier electrolyte consisting of ammonium acetate/acetic acid in methanol; to resolve the strongly acidic trihaloacetic acids as well as the less acidic monohaloacetic acids, a careful optimization of the acetic acid content is necessary. The method was applied to the determination of haloacetic acids in real water samples. With optimized CE and MS conditions detection limits between 0.3 and 7.6 μg/L in the original water samples were achieved, employing a sample volume of 30 mL.     

Fast and simultaneous detection of prominent natural antioxidants using analytical microsystems for capillary electrophoresis with a glassy carbon electrode: a new gateway to food environments

This paper examines for the first time the analytical possibilities of fast and simultaneous detection of prominent natural antioxidants including examples of flavonoids and vitamins using a CE microchip with electrochemical detection (ED). Unpinched injection conditions, zone electrophoretic separation and amperometric detection were carefully assayed and optimised. Analysis involved the zone electrophoretic separation of arbutin, (+)-catechin and ascorbic acid in less than 4 min using a borate buffer (pH 9.0, 50 mM), employing 2 kV as the separation voltage and +1.0 V as the detection potential. In addition, the separation of different 'couples' of natural antioxidants of food significance including (+)-catechin and ascorbic acid, (+)-catechin and rutin, as well as arbutin and phlorizdin is proposed. To demonstrate the potential and future role of CE microsystems, analytical possibilities and a new route in the raw sample analysis are presented. The preliminary results obtained allow the proposal of CE-ED microchips as a real gateway to microanalysis in foods.     

Chemometrics on Microchips: Towards the Classification of Wines

The coupling of a capillary electrophoresis (CE) microchip with electrochemical detection with a chemometric approach was used for the classification of wines. Cabernet Sauvignon wine samples from 3 different geographical regions were used for verifying the discriminating power of the microchip electrophoretic technique using high separation voltage (3000 V) with analysis time of only 60 seconds. Electrophoretic data were transformed into initial 14 variables, concerning peak's area and peak's height. Principal component analysis was applied to optimize the information given by the electrophoretic data sets. Results of classification using LDA and CART were promising and the preliminary model was able to classify samples on the basis of their geographical origin. While the coupling of microchips with chemometrics approaches is illustrated for a former tentative of the classification of wines it could be readily extended to a wide range of other important applications.     

Elimination of suction effect in interfacing microchip electrophoresis with inductively coupled plasma mass spectrometry using porous monolithic plugs

A suction-free interfacing method was developed for microchip electrophoresis hyphenated with inductively coupled plasma mass spectrometry (MCE-ICP-MS). The hyphenated system was composed of a microchip, a demountable capillary microflow nebulizer (d-CMN) combined with a heated single pass spray chamber, a negative pressure sampling device, a high voltage power supply, a syringe pump and an ICP-MS. To eliminate the nebulizer suction generated by the pneumatic nebulizer and to ensure that the makeup solution flowed into the nebulizer, two porous polymer plugs were fabricated in the microchip. As a result, reasonably true electropherograms were obtained when compared to the CE separation performed in the traditional MCE-ICP-MS mode without porous polymer plugs. Electrophoretic separation of I(-) and IO(3)(-) was achieved within 25 s in a microchip with an effective separation length of only 15 mm at an electric field of 857 V cm(-1) using 10 mmol L(-1) borate (pH 9.2) as the running buffer. A resolution of 1.3 was obtained and the absolute detection limits for I(-) and IO(3)(-) were 0.12 and 0.13 fg, respectively. The precisions (RSD, n = 10) of the migration time and peak height for I(-) and IO(3)(-) were in the range of 1.1-1.6% and 2.5-2.8%, respectively. Two table salt samples were analyzed by an external calibration method. The iodate contents were in accordance with their labeled values. The recoveries of I(-) and IO(3)(-) in the table salt samples were in the range of 92-105%.