Nanoinjector for capillary electrophoresis and capillary electrochromatography

A rotary valve nanoinjector was devised for use in capillary electrophoresis (CE) and capillary electrochromatography (CEC). A fused-silica capillary tip was inserted in a small through-hole in the rotor. The narrow and short capillary tip, with an inner volume of 6-24 nL, was embedded in the hole using epoxy resin. The injection volume was confirmed chromatographically by comparing the peak areas obtained with the nanoinjector to those of a conventional injector. In addition, both the rotor and stator of the injector were made of a nonconducting material, polyimide resin, to be utilized for CE and CEC. The application of the nanoinjector for CE was demonstrated.     

Gold nanoparticle-enhanced microchip capillary electrophoresis

We describe here the use of gold nanoparticles in conjunction with chip-based capillary electrophoresis to improve the selectivities between solutes and to increase the efficiency of the separation. We coated the microchannel wall of a microfluidic device with a layer of poly(diallyldimethylammonium chloride) (PDADMAC) and then collected on it citrate-stabilized gold nanoparticles. The resolutions and the plate numbers of the solutes were doubled in the presence of the gold nanoparticles. Such selectivity improvements reflect changes in the observed mobility accrued from interactions of solutes with the particle surface. The electrochemical detection and the quantitation of the solutes were not effected by the PDADMAC and the gold nanoparticles.     

Dual conductivity/amperometric detection system for microchip capillary electrophoresis

The performance characteristics and advantages of a new dual electrochemical microchip detection system based on simultaneous conductivity and amperometric measurements are described. The system relies on the combination of a contactless conductivity detector with an end-column thick-film amperometric detector. Such coupling of the conductivity and amperometric detection modes in a single separation channel greatly enhances the sample characterization to offer simultaneous measurements of both ionic and electroactive species, improved reproducibility, and confirmation of peak identity. The simultaneous measurement of nitroaromatic and ionic explosives is used for demonstrating the ability to detect both electroactive and ionic species. Major improvements are also observed for analytes responding at both detectors. For example, the generation of dual response ratios can be used to improve the reproducibility and confirm the peak identity/integrity. Such dual response ratios reflect the distinct redox and conductivity properties of the individual analytes. The independence of the two detectors is reflected in the absence of "cross-talk" effects. The behavior of the dual detector is comparable with those of the individual detectors. Such a dual electrochemical detection system is easy to implement and requires inherently portable low-cost instrumentation.     

Movable contactless-conductivity detector for microchip capillary electrophoresis

A new movable contactless-conductivity detection system for microchip capillary electrophoresis is introduced. Such a versatile system relies on positioning the detector at different points along the separation channel via "sliding" the electrode holder. The new movable microchip detection system offers distinct improvements compared to common fixed-location conductivity detectors. For example, placing the detector at different locations along the microchannel offers useful insights into the separation process. Three-dimensional plots of resolution/channel length/separation voltage can be used for optimizing the separation process and selecting the analysis time. The system enables rapid switching between "total" (unresolved) and "individual" (resolved/fingerprint) signals on the basis of placing the detector at the beginning and end of the separation channel, respectively. By moving the detector to a shorter effective separation length, after eluting fast-migrating ions, shorter analysis times can be achieved (through faster detection of late-eluting analytes). These and other improvements in the analytical performance and insights into the separation process are illustrated in connection with the detection of low-energy ionic explosives and nerve agent degradation products.     

Contactless conductivity detector for microchip capillary electrophoresis

A microfabricated electrophoresis chip with an integrated contactless conductivity detection system is described. The new contactless conductivity microchip detector is based on placing two planar sensing aluminum film electrodes on the outer side of a poly(methyl methacrylate) (PMMA) microchip (without contacting the solution) and measuring the impedance of the solution in the separation channel. The contactless route obviates problems (e.g., fouling, unwanted reactions) associated with the electrode-solution contact, offers isolation of the detection system from high separation fields, does not compromise the separation efficiency, and greatly simplifies the detector fabrication. Relevant experimental variables, such as the frequency and amplitude of the applied ac voltage or the separation voltage, were examined and optimized. The detector performance was illustrated by the separation of potassium, sodium, barium, and lithium cations and the chloride, sulfate, fluoride, acetate, and phosphate anions. The response was linear (over the 20 microM-7 mM range) and reproducible (RSD = 3.4-4.9%; n = 10), with detection limits of 2.8 and 6.4 microM (for potassium and chloride, respectively). The advantages associated with the contactless conductivity detection, along with the low cost of the integrated PMMA chip/detection system, should enhance the power and scope of microfluidic analytical devices.     

On-column electrochemical detection for microchip capillary electrophoresis

The development of a cellulose acetate decoupler for on-column electrochemical detection in microchip capillary electrophoresis is presented. The capillary based laser-etched decoupler is translated to the planar format to isolate the detector circuit from the separation circuit. The decoupler is constructed by aligning a series of 20 30-microm holes through the coverplate of the microchip with the separation channel and casting a thin film of cellulose acetate within the holes. The decoupler shows excellent isolation of the detection circuit for separation currents up to 60 microA, with noise levels at or below 1 pA at a carbon fiber electrode. Detection limits of 25 nM were achieved for dopamine. This decoupler design combines excellent mechanical stability, effective shunting of high separation currents, and ease of manufacture.     

Solvent-programmed microchip open-channel electrochromatography

Open-channel electrochromatography in combination with solvent programming is demonstrated using a microchip device. Channel walls were coated with octadecylsilanes at ambient temperatures, yielding stationary phases for chromatographic separations of neutral dyes. The electroosmotic flow after coating was sufficient to ensure transport of all species and on-chip mixing of isocratic and gradient elution conditions with acetonitrile-buffer mixtures. Chips having different channel depths between 10.2 and 2.9 μm were evaluated for performance, and van Deemter plots were established. Channel depths of about 5 μm were found to be a good compromise between efficiency and ease of operation. Isocratic and gradient elution conditions were easily established and manipulated by computer-controlled application of voltages to the terminals of the microchip. Linear gradients with different slopes, start times, duration times, and start percentages of organic modifier proved to be powerful tools to tune selectivity and analysis time for the separation of a test mixture. Even very steep gradients still produced excellent efficiencies. Together with fast reconditioning times, complete runs could be finished in under 60 s.     

Miniaturisation of a capillary electrophoresis microchip for the sensing of endocrine disruptors

Although capillary electrophoresis amperometric detector (CE-AD) involving double-T microchannel configuration is a powerful analytical tool in terms of sensitivity and selectivity, its long microchannel configuration hinders further miniaturisation. Therefore a twisted CE microchannel configuration was used in the present study to fabricate CE-AD devices for detection of endocrine disruptors. The analyte separation time varied slightly for the twisted microchannel structure, whereas the detector sensitivities were similar for the two configurations. The conventional indium tin oxide amperometric detector in the device with twisted microchannel configuration was later modified with Prussian blue to enhance the sensitivity of detection.     

High-voltage capacitively coupled contactless conductivity detection for microchip capillary electrophoresis

Contactless conductivity detection was carried out on a planar electrophoresis device by capacitive coupling using an ac excitation voltage of 500 V(p-p) and a frequency of 100 kHz. It was possible to carry out detection in this way through a cover plate of 1 mm thickness. Better sensitivity is obtained, however, by placing the electrodes into troughs that allow tighter coupling to the separation channel. The 3 x S/N detection limits are 0.49, 0.41, and 0.35 microM for the small inorganic ions K+, Na+, and Mg2+. The detection of heavy metals is demonstrated with the example of Mn2+, Zn2+, and Cr3+ with detection limits of 2.1, 2.8, and 6.8 microM, respectively. The universal nature of the method is further illustrated by the detection of citric and lactic acids, which are of interest in food and beverage analysis, and detection of three antiinflammatory nonsteroid drugs, 4-acetamidophenol, ibuprofen, and salicylic acid, as examples of species of pharmaceutical interest.     

On-line coupling of capillary electrochromatography, capillary electrophoresis, and capillary HPLC with nuclear magnetic resonance spectroscopy

A novel capillary NMR coupling configuration, which offers the possibility of combining capillary zone electrophoresis (CZE), capillary HPLC (CHPLC), and for the first time capillary electrochromatography (CEC) with nuclear magnetic resonance (NMR), has been developed. The hyphenated technique has a great potential for the analysis of chemical, pharmaceutical, biological, and environmental samples. The versatile system allows facile changes between these three different separation methods. A special NMR capillary containing an enlarged detection cell suitable for on-line NMR detection and measurements under high voltage has been designed. The acquisition of 1D and 2D NMR spectra in stopped-flow experiments is also possible. CHPLC NMR has been performed with samples of hop bitter acids. The identification and structure elucidation of humulones and isohumulones by on-line and stopped-flow spectra has been demonstrated. The suitability of the configuration for electrophoretic methods has been investigated by the application of CZE and CEC NMR to model systems.     

Electrokinetic injection for stacking neutral analytes in capillary and microchip electrophoresis

An on-column mechanism for electrokinetically injecting long sample plugs with simultaneous stacking of neutral analytes in capillary electrokinetic chromatography is presented. On-column stacking methods allow for the direct injection of long sample plugs into the capillary, with narrowing of the analyte peak width to allow for an increase in the detected signal. Low-pressure injections (approximately 50 mbar) are commonly used to introduce sample plugs containing neutral analytes. We demonstrate that injection can be accomplished by applying an electric field from the sample vial directly into the capillary, with neutral analytes injected by electroosmotic flow at up to 1 order of magnitude faster than the corresponding pressure injections. Since stacking occurs simultaneously with electrokinetic injection, stacking is initiated at the capillary inlet, resulting in an increased length of capillary remaining for separation. Reproducibility obtained for peak height and peak area with electroosmotic flow injection is comparable to that obtained with the pressure injection mode, while reproducibility of analysis time is markedly improved. Electrokinetic stacking of neutral analytes utilizing electroosmotic flow is demonstrated with discontinuous (high conductivity, high mobility) as well as continuous (equal conductivity, equal mobility) sample electrolytes. Injecting neutral analytes by electroosmotic flow affords a 10-fold or greater decrease in analysis times when capillaries of 50-microm i.d. or smaller are used. This stacking method should be exportable to dynamic pH junction stacking and electrokinetic chromatography with capillary arrays. Equations describing this electrokinetic injection mode are introduced and stacking of a neutral analyte on a microchip by electrokinetic injection using a simple cross-T channel configuration is demonstrated.     

Dual-channel method for interference-free in-channel amperometric detection in microchip capillary electrophoresis

A novel in-channel amperometric detection method for microchip capillary electrophoresis (CE) has been developed to avoid the interference from applied potential used in the CE separation. Instead of a single separation channel as in conventional CE microchips, we use a dual-channel configuration consisting of two different parallel separation and reference channels. A working electrode (WE) and a reference electrode (RE) are placed equally at a distance 200 microm from its outlet on each channel. Running buffer flows through the reference channel. Our dual-channel CE microchips consist of a poly(dimethylsiloxane) (PDMS) upper plate and a glass lower plate to form a PDMS/glass hybrid chip. Amperometric signals are measured without any potential shift and interference from the applied CE potential, and CE separation maintains its high resolution because this in-channel configuration does not allow additional band broadening that is notorious in end-channel and off-channel configurations. The high performance of this new in-channel electrochemical detection methodology for CE has been demonstrated by analyzing a mixture of electrochemically active biomolecules: dopamine (DA), norepinephrine, and catechol. We have achieved a 0.1 pA detectability from the analysis of DA, which corresponds to a 1.8 nM concentration.     

On-line sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis

Previous reports describing sample stacking on microchip capillary electrophoresis (microCE) have regarded the microchip channels as a closed system and treated the bulk flow as in traditional capillary electrophoresis. This work demonstrates that the flows arising from the intersection should be investigated as an open system. It is shown that the pressure-driven flows into or from the branch channels due to bulk velocity mismatch in the main channel should not be neglected but can be used for liquid transportation in the channels. On the basis of these concepts, a sample preconcentration scheme was developed in a commercially available single-cross glass chip for microCE. Similar to field-amplified stacking injection in traditional CE, a low conductivity sample buffer plug was introduced into the separation channel immediately before the negatively charged analyte molecules were injected. The detection sensitivity was improved by 94-, 108-, and 160-fold for fluorescein-5-isothiocyanate, fluorescein disodium, and 5-carboxyfluorescein, respectively, relative to a traditional pinched injection. The calibration curves for fluorescein and 5-carboxyfluorescein demonstrated good linearity in the concentration range (1-60 nM) investigated with acceptable reproducibility of migration time and peak height and area ratios (4-5% RSD). This preconcentration scheme will be of particular significance to the practical use of microCE in the emerging miniaturized analytical instrumentation.     

Chemiluminescence detection for a microchip capillary electrophoresis system fabricated in poly(dimethylsiloxane)

Chemiluminescence (CL) detection integrated with a microchip capillary electrophoresis (MCE) system that was fabricated in poly(dimethylsiloxane) was demonstrated for chemical and biochemical analyses. Two model CL systems were involved here: metal ion-catalyzed luminol-peroxide reaction and dansyl species conjugated peroxalate-peroxide reaction. Different strategies based on three chip patterns (cross, cross combining with Y, and cross combining with V) to perform on-line CL detection for MCE were evaluated and compared in terms of sensitivity, reproducibility, and peak symmetry. The chip pattern of cross combining with Y proved to be promising for the luminol-peroxide CL system, while the chip pattern of cross combining with V was preferred for the peroxalate-peroxide system where CL reagent could not be effectively transported by electroosmotic flow. A detection limit down to submicromolar concentrations (midattomole) was achieved with good reproducibility and symmetric peak shape. Successful separation of three metal cations such as Cr(III), Co(II), and Cu(II) and chiral recognition of dansyl phenylalanine enantiomers within 1 min revealed distinct advantages of combining MCE with CL detection for rapid and sensitive analyses.     

Analysis of neuroactive amines in fermented beverages using a portable microchip capillary electrophoresis system

A portable microfabricated capillary electrophoresis (CE) instrument is used for the determination of neurologically active biogenic amines, especially tyramine and histamine, in fermented beverages. The target molecules are labeled on their primary amino groups with fluorescamine in a 10-min reaction, and the samples analyzed directly, producing a detailed electropherogram in only 120 s on a microfabricated glass CE device containing 21.4-cm-long separation channels. Tyramine was found mainly in red wines at <1-3.4 mg/L, while the histamine content of these samples ranged from 1.8 to 19 mg/L. The highest levels of histamine (20-40 mg/L) were found in sake. The analysis of samples drawn from grape crush through malolactic fermentation in four varieties of zinfandel red wines revealed that histamine and tyramine are produced during yeast and malolactic fermentation, respectively. Following malolactic fermentation, the histamine content in these samples ranged from 3.3 to 30 mg/L, and the tyramine content ranged from 1.0 to 3.0 mg/L. This highly sensitive and rapid lab-on-a-chip analysis method establishes the feasibility of monitoring neurologically active amine content and potentially other chemically and allergenically important molecules in our food supply.     

Semipreparative capillary electrochromatography

Capillaries with inner diameters of 550 microm have successfully been packed with 1.5-microm octadecyl silica particles using frits made of macroporous polymers by the UV photopolymerization of a solution of glycidyl methacrylate and trimethylolpropane trimethacrylate. This type of frit is found superior to one made of low-melting point poly(styrene-co-divinylbenzene) beads. Bubble formation is not observed to occur within these capillary columns under our experimental conditions. Separations can be achieved with sample injection volumes as high as 1 microL. To demonstrate its semipreparative use, a mixture of 500 nL of taxol (20 mM) and its precursor, baccatin III (30 mM), is separated using such a column with a Tris buffer.     

In-channel electrochemical detection for microchip capillary electrophoresis using an electrically isolated potentiostat

A new electrode configuration for microchip capillary electrophoresis (CE) with electrochemical (EC) detection is described. This approach makes it possible to place the working electrode directly in the separation channel. The "in-channel" EC detection was accomplished without the use of a decoupler through the utilization of a specially designed, electrically isolated potentiostat. The effect of the working electrode position on the separation performance (in terms of plate height and peak skew) of poly(dimethylsiloxane)-based microchip CEEC devices was evaluated by comparing the more commonly used end-channel configuration with this new in-channel approach. Using catechol as the test analyte, it was found that in-channel EC detection decreased the total plate height by a factor of 4.6 and lowered the peak skew by a factor of 1.3. A similar trend was observed for the small, inorganic ion nitrite. Furthermore, a fluorescent and electrochemically active amino acid derivative was used to directly compare the separation performance of in-channel EC detection to that of a widely used laser-induced fluorescence (LIF) detection scheme. In this case, it was found that the plate height and peak skew for both detection schemes were essentially equal, and the separation performance of in-channel EC detection is comparable to LIF detection.     

Portable high-voltage power supply and electrochemical detection circuits for microchip capillary electrophoresis

Miniaturized, battery-powered, high-voltage power supply, electrochemical (EC) detection, and interface circuits designed for microchip capillary electrophoresis (CE) are described. The dual source CE power supply provides +/- 1 kVDC at 380 microA and can operate continuously for 15 h without recharging. The amperometric EC detection circuit provides electrode potentials of +/-2 VDC and gains of 1, 10, and 100 nA/V. The CE power supply power is connected to the microchip through an interface circuit consisting of two miniature relays, diodes, and resistors. The microchip has equal length buffer and separation channels. This geometry allows the microchip to be controlled from only two reservoirs using fixed dc sources while providing a consistent and stable sample injection volume. The interface circuit also maintains the detection reservoir at ground potential and allows channel currents to be measured likewise. Data are recorded, and the circuits are controlled by a National Instruments signal interface card and software installed in a notebook computer. The combined size (4 in. x 6 in. x 1 in.) and weight (0.35 kg) of the circuits make them ideal for lab-on-a-chip applications. The circuits were tested electrically, by performing separations of dopamine and catechol EC and by laser-induced fluorescence visualization.     

Development of a microfabricated palladium decoupler/electrochemical detector for microchip capillary electrophoresis using a hybrid glass/poly(dimethylsiloxane) device

The fabrication and evaluation of a palladium decoupler and working electrode for microchip capillary electrophoresis (CE) with electrochemical detection is described. The use of the Pd decoupler allows the working electrode to be placed directly in the separation channel and eliminates the band-broadening characteristic of the end-channel configuration. The method used for fabrication of the decoupler and working electrode was based on thin-layer deposition of titanium followed by palladium onto a glass substrate. When employed as the cathode in CE, palladium absorbs the hydrogen gas that is generated by the hydrolysis of water. The effect of the decoupler size on the ability to remove hydrogen was evaluated with regard to reproducibility and longevity. Using boric acid and TES buffer systems, 500 microm was determined to be the optimum decoupler size, with effective voltage isolation lasting for approximately 6 h at a constant field strength of 600 V/cm. The effect of distance between the decoupler and working electrode on noise and resolution for the separation of dopamine and epinephrine was also investigated. It was found that 250 microm was the optimum spacing between the decoupler and working electrode. At this spacing, laser-induced fluorescence detection at various points around the decoupler established that the band broadening due to pressure-induced flow that occurs after the decoupler did not significantly affect the separation efficiency of fluorescein. Limits of detection, sensitivity, and linearity for dopamine (500 nM, 3.5 pA/microM, r(2) = 0.9996) and epinephrine (2.1 microM, 2.6 pA/microM, r(2) = 0.9996) were obtained using the palladium decoupler in combination with a Pd working electrode.     

Three-electrode electrochemical detector and platinum film decoupler integrated with a capillary electrophoresis microchip for amperometric detection

This article demonstrates that a three-electrode electrochemical (EC) detector and an electric decoupler could be fabricated in the same glass chip and integrated with an O2-plasma-treated PDMS layer using microfabrication techniques to form the capillary electrophoresis (CE) microchip. The platinized decoupler could mostly decouple the electrochemical detection circuit from the interference of an separation electric field in 10 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 6.5) solution. The baseline offset of background current recorded from the working electrode with and without application of a separation electric field was maintained at less than 0.05 pA in 10 mM MES. In addition, the platinized pseudoreference electrode was demonstrated to offer a stable potential in electrochemical detection. As a consequence, the limit of detection of dopamine was 0.125 microM at a S/N = 4. The responses for dopamine to different concentrations were found to be linear between 0.25 and 50 microM with a correlation coefficient of 0.9974 and a sensitivity of 11.76 pA/microM. The totally integrated CE-EC microchip should be able to fulfill the ideal of miniaturization and commercialization.