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-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.     

Light-emitting-diode-induced fluorescence detection of fluorescent dyes for capillary electrophoresis microchip with cross-polarization method

A cross-polarization scheme is presented to filter out the excitation light from the emission spectrum of fluorescent dyes using green light emitting diodes as a light source and a linear charge coupled device as an intensity detector. The excitation light was linearly polarized and was then used to illuminate the fluorescent dyes in the microchannels of a capillary electrophoresis microchip. The detector was shielded by the second polarizer, oriented perpendicular to the excitation light. The fluorescent signals from Rhodamine B dyes were measured in a dilution series with resulting emission signals and four different concentrations of fluorescent dyes were detected simultaneously with the same excitation source and detector. A limit-of-detection of 1 μM was demonstrated for Rhodamine B dye under the optimal conditions.     

Adsorption-based electrochemical detection of nonelectrochemically active analytes for capillary electrophoresis

A sensitive electrochemical detection method (ECD) for capillary electrophoresis has been developed that is applicable to a much wider range of analytes than more conventional ECD methods. Using a modified Osteryoung square-wave voltammetry method, the adsorption of what are normally considered nonelectrochemically active analytes onto a platinum electrode was found to produce a concentration-proportional response. Although the mechanisms that cause this response may be complex, it appears that it is due to changes in the electrode/solution interface that accompany adsorption of the analyte onto the electrode rather than a simple redox process. Analytes that possess pi-electron density appeared to chemisorb rather than only physically adsorb onto the electrode and gave the best response with detection limits of < 10(-8) M while maintaining good linearity. Because this detection method requires only that the analyte adsorb onto the electrode, it has the advantage of much wider applicability than previously reported electrochemical detection methods. The applicability of this detection method was investigated for a variety of analytes and background electrolyte conditions (varied pH, ionic strength, buffer additives). Comparisons of the sensitivity of this method to UV detection showed that, even for analytes that have good UV chromophores, sensitivities greater than 1 order of magnitude were obtained using adsorption-based electrochemical detection.     

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.     

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.     

Dual fluorescence and electrochemical detection on an electrophoresis microchip

Simultaneous amperometric and fluorescence detection in a microfabricated electrophoresis chip is reported. Detection limits of 448 nM and 1.52, 16, and 28 microM have been achieved for dopamine, catechol, NBD-arginine, and NBD-phenylalanine, respectively. These two orthogonal detection schemes allow analysis of a wider spectrum of compounds per separation, leading to higher throughput and enabling resolution of two neutral analytes, NBD-arginine and catechol. In addition, insight into the detection and separation mechanisms is realized. Differences in migration time and peak widths between the two detectors are compared, providing a better understanding of detector alignment. A common problem encountered in electrophoresis is run-to-run migration time irreproducibility for certain samples. This novel microchip dual detection system has been utilized to reduce this irreproducibility. An unknown sample is monitored with one detector while a standard (i.e., ladder) is added to the sample and monitored simultaneously with the other detector. This dual detection method is demonstrated to normalize unknown peak mobilities in a cerebral spinal fluid sample.     

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.     

Nonaqueous electrophoresis microchip separations: conductivity detection in UV-absorbing solvents

The use of organic solvents in microfabricated capillary electrophoresis (CE) devices is demonstrated in connection with the separation of aliphatic amines in pure dimethylformamide, dimethylacetamide, dimethyl sulfoxide, or propylene carbonate media. Contactless conductivity detection is employed for monitoring the separated solutes in these UV-absorbing solvents. The effect of the physicochemical properties of the organic solvent upon the migration behavior is investigated. The apparent mobility increases nearly linearly with the reciprocal of the solvent viscosity, while the electroosmotic mobility increases in a linear fashion with the dielectric constant/ viscosity ratio. Some deviation from theoretical predictions is observed using propylene carbonate. The nonaqueous CE microchip offers high separation efficiency, reflected in plate numbers ranging from 93,680 to 127,680, using a separation voltage of +3,000 V, a dimethylformamide medium, and a contactless conductivity detection. Experimental parameters affecting the analytical performance of the nonaqueous CE/conductivity microchip are examined. Calibration and precision experiments indicate a linear and reproducible response. Such use of organic solvents can benefit microchip separations through extended scope (toward nonpolar solutes) and tunable selectivity.     

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.     

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.     

Deep UV laser-induced fluorescence detection of unlabeled drugs and proteins in microchip electrophoresis

Deep UV fluorescence detection at 266-nm excitation wavelength has been realized for sensitive detection in microchip electrophoresis. For this purpose, an epifluorescence setup was developed enabling the coupling of a deep UV laser into a commercial fluorescence microscope. Deep UV laser excitation utilizing a frequency quadrupled pulsed laser operating at 266 nm shows an impressive performance for native fluorescence detection of various compounds in fused-silica microfluidic devices. Aromatic low molecular weight compounds such as serotonin, propranolol, a diol, and tryptophan could be detected at low-micromolar concentrations. Deep UV fluorescence detection was also successfully employed for the detection of unlabeled basic proteins. For this purpose, fused-silica chips dynamically coated with hydroxypropylmethyl cellulose were employed to suppress analyte adsorption. Utilizing fused-silica chips permanently coated with poly(vinyl alcohol), it was also possible to separate and detect egg white chicken proteins. These data show that deep UV fluorescence detection significantly widens the application range of fluorescence detection in chip-based analysis techniques.     

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.     

Autonomous polymer loading and sample injection for microchip electrophoresis

We have developed an extremely simple method for microchip electrophoresis. Loading of a sieving polymer solution and injection of a sample solution are autonomously executed by a microchip fabricated in poly(dimethylsiloxane) (PDMS). In advance, the energy for the fluid pumping is stored in bulk PDMS by evacuating air dissolved in PDMS, and the information for the sample plug regulation is coded into the microchannel design. Besides the simplicity, our method brings about an advantageous effect: sample compaction due to the discontinuous electrophoretic mobility at the sample/polymer interface. The sample compaction effect was moderate in ordinary size-dependent separation for double-stranded DNA and was extreme in affinity electrophoresis for single-stranded DNA (ssDNA). In the latter separation mode, ssDNA components were sequence-specifically separated by difference in affinity to a probe oligonucleotide immobilized to the sieving polymer matrix. We separated up to 60-mer ssDNA mixtures based on single-base substitutions. The separation processes included typically 100-fold sample compaction and were completed within 15-30 s. This technology provides easy, simple, and sensitive method for detection of gene point mutations and typing of single-nucleotide polymorphisms.     

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.     

Fabrication of integrated microelectrodes for electrochemical detection on electrophoresis microchip by electroless deposition and micromolding in capillary technique

A new method for the fabrication of an integrated microelectrode for electrochemical detection (ECD) on an electrophoresis microchip is described. The pattern of the microelectrode was directly made on the surface of a microscope slide through an electroless deposition procedure. The surface of the slide was first selectively coated with a thin layer of sodium silicate through a micromolding in capillary technique provided by a poly(dimethylsiloxane) (PDMS) microchannel; this left a rough patterned area for the anchoring of catalytic particles. A metal layer was deposited on the pattern guided by these catalytic particles and was used as the working electrode. Factors influencing the fabrication procedure were discussed. The whole chip was built by reversibly sealing the slide to another PDMS layer with electrophoresis microchannels at room temperature. This approach eliminates the need of clean room facilities and expensive apparatus such as for vacuum deposition or sputtering and makes it possible to produce patterned electrodes suitable for ECD on microchip under ordinary chemistry laboratory conditions. Also once the micropattern is ready, it allows the researchers to rebuild the electrode in a short period of time when an electrode failure occurs. Copper and gold microelectrodes were fabricated by this technique. Glucose, dopamine, and catechol as model analytes were tested.     

Simple and sensitive electrode design for microchip electrophoresis/electrochemistry

A simple and sensitive electrode design for microchip capillary electrophoresis/electrochemistry (CE-EC) is presented. The system employs metal microwires as the working electrodes for electrochemical detection. Two general approaches for integration of electrodes in microchip CE-EC are commonly used, end-channel and microfabrication. The end-channel approach allows electrode cleaning and the use of chemically modified electrodes; however, the designs generally lack portability and the ability to incorporate multiple electrodes. Microfabrication allows the incorporation of multiple electrodes on-chip and is easily made portable; however, it requires the use of expensive metallization and clean room facilities, and integration of more than one electrode material is challenging. The reported approach aligns a solid metal microwire through the separation channel allowing integration of multiple electrodes and the use of different electrode materials without sacrificing the portability. A detection limit of 100 nM for dopamine was achieved without the use of a decoupler as a result of a higher collection efficiency with the new design.     

A universal concept for stacking neutral analytes in micellar capillary electrophoresis

Unlike recent studies that have depended on manipulation of separation buffer parameters to facilitate stacking of neutral analytes in micellar capillary electrophoresis (MCE) mode, we have developed a method of stacking based simply on manipulation of the sample matrix. Many solutions for sample stacking in MCE are based on strict control of pH, micelle type, electroosmotic flow (EOF) rate, and separation-mode polarity. However, a universal solution to sample stacking in MCE should allow for free manipulation of separation buffer parameters without substantially affecting separation of analytes. Analogous to sample stacking in capillary zone electrophoresis by invoking field amplification of charged analytes in a low-conductivity sample matrix, the proposed method utilizes a high-conductivity sample matrix to transfer field amplification from the sample zone to the separation buffer. This causes the micellar carrier in the separation buffer to stack before it enters the sample zone. Neutral analytes moving out of the sample zone with EOF are efficiently concentrated at the micelle front. Micelle stacking is induced by simply adding salt to the sample matrix to increase the conductivity 2-3-fold higher than the separation buffer. This solution allows free optimization of separation buffer parameters such as micelle concentration, organic modifiers, and pH, providing a method that may complement virtually any existing MCE protocol without restricting the separation method.