Two-dimensional electrochromatography/capillary electrophoresis on a microchip

A two-dimensional separation system on a microfabricated device was demonstrated using open-channel electrochromatography as the first dimension and capillary electrophoresis as the second dimension. The first dimension was operated under isocratic conditions, and the effluent from the first dimension was repetitively injected into the second dimension every few seconds. A 25-cm separation channel with spiral geometry for open-channel electrochromatography was chemically modified with octadecylsilane and coupled to a 1.2-cm straight separation channel for capillary electrophoresis. Fluorescently labeled products from tryptic digests of beta-casein were analyzed in 13 min with this system.     

Frequency-doubled microchip lasers

Microchip lasers with internal second harmonic generation allow the efficient generation of visible light from particularly compact and readily manufactured devices. This paper reviews the underlying physics of these devices, and points to some of the reasons for their success.     

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.     

Flow separation control in open-channel bends

The investigation of fluid flow in sharp open-channel bends is key to controlling undesired sedimentation in natural river reaches. The difficulties are associated with controlling the flow separation in meanderings. Flow separation decreases the width of the flow, and consequently, the conveyance capacity while increasing erosion and mixing. This study proposes a novel approach to reduce the flow separation at the inner banks of sharp open-channel bends. Three-dimensional numerical experiments were conducted. To find the most reliable procedure, five turbulence models were examined. The employed numerical approach is formulated within the framework of the finite volume method and the volume of fluid (VOF) technique to solve the Navier–Stokes equations. Water levels and velocity profiles are obtained in different sections of the channel and are compared to experimental studies of a 90° sharp open-channel bend. A close agreement is observed using the RSM (Reynolds stress model) turbulence model. Moreover, the evaluation of acquired velocity profiles demonstrates that in a regular bend, the lowest velocity occurs near the inner bank, where it has a flow separation tendency. The same numerical procedure is employed to simulate water flow through a sharp converging open-channel bend. The measurements of velocity profiles and velocity vectors in the curved sections support the idea that decreasing the channel width considerably reduces the overall velocity variations in cross-sectional areas of the test case and is effective to control flow separation.     

Alternating voltage capillary electrochromatography

A phenomenon was found whereby the application of an alternating voltage to a capillary column can vary the capacity factor of a sample solute. The alternating voltage-induced variation in the capacity factor was studied using an anion exchange mini-bed (a short capillary column 12 mm long). The capacity factor varied according to both the amplitude and frequency of an applied alternating voltage. The variation greatly depended on the kinds of sample solutes and packing materials. A new separation mode for capillary electrochromatography using an alternating voltage, that is, alternating voltage capillary electrochromatography (AV-CEC), was proposed as an application of this phenomenon to control the retention of a sample solute. The chromatographic behavior of three organic acids (benzoic acid, phthalic acid, and salicylic acid) was studied in AV-CEC using an anion exchange column.     

Capillary electrochromatography of cannabinoids

The applicability of capillary electrochromatography (CEC) with photodiode array UV detection for the analysis of cannabinoids is presented. Baseline separation of seven cannabinoids (cannabigerol, cannabidiol, cannabinol, delta-9-tetrahydrocannabinol, delta-8-tetrahydrocannabinol, cannabichromene, delta-9-tetrahydrocannabinolic acid) is obtained using a 3-micron CEC Hypersil C18 capillary with an acetonitrile/phosphate (pH 2.57) mobile phase. The effects of acetonitrile concentration, buffer concentration, voltage, temperature, stationary phase, and column length on the separation of the cannabinoids were investigated. Good short- and long-term precision in retention times are observed, with significant improvement obtained using relative retention times with cannabinol as reference compound. Although short- and long-term peak area precisions are poor, satisfactory reproducibility is obtained using relative peak areas with cannabinol as reference compound. The applicability of the CEC methodology to drug seizures was demonstrated on marijuana and hashish. Using a high-sensitivity UV flow cell with an extended path length of 1.2 mm, concentration sensitivities approaching HPLC were obtained.     

Gradient elution in capillary electrochromatography

In analogy to pressure-driven gradient techniques in high-performance liquid chromatography, a system has been developed for delivering electroosmotically driven solvent gradients for capillary electrochromatography (CEC). Dynamic gradients with submicroliter per minute flow rates are generated by merging two electroosmotic flows that are regulated by computer-controlled voltages. These flows are delivered by two fused-silica capillary arms attached to a T-connector, where they mix and then flow into a capillary column that has been electrokinetically packed with 3-μm reversed-phase particles. The inlet of one capillary arm is placed in a solution reservoir containing one mobile phase, and the inlet of the other is placed in a second reservoir containing a second mobile phase. Two independent computer-controlled, programmable, high-voltage power supplies (0-50 kV) [Formula: see text] one providing an increasing ramp and the other providing a decreasing ramp [Formula: see text] are used to apply variable high-voltage potentials to the mobile phase reservoirs to regulate the electroosmotic flow in each arm. The ratio of the electroosmotic flow rates between the two arms is changed with time according to the computer-controlled voltages to deliver the required gradient profile to the separation column. Experiments were performed to confirm the composition of the mobile phase during a gradient run and to determine the change of the composition in response to the programmed voltage profile. To demonstrate the performance of electroosmotically driven gradient elution in CEC, a mixture of 16 polycyclic aromatic hydrocarbons was separated in less than 90 min. This gradient technique is expected to be well-suited for generating not only solvent gradients in CEC but also other types of gradients, such as pH and ionic strength gradients, in capillary electrokinetic separations and analyses.     

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.     

Protein sizing on a microchip

We have developed a microfabricated analytical device on a glass chip that performs a protein sizing assay, by integrating the required separation, staining, virtual destaining, and detection steps. To obtain a universal noncovalent fluorescent labeling method, we have combined on-chip dye staining with a novel electrophoretic dilution step. Denatured protein-sodium dodecyl sulfate (SDS) complexes are loaded on a chip and bind a fluorescent dye as the separation begins. At the end of the separation channel, an intersection is used to dilute the SDS below its critical micelle concentration before the detection point. This strongly reduces the background due to dye molecules bound to SDS micelles and also increases the peak amplitude by 1 order of magnitude. Both the on-chip staining and SDS dilution steps occur in the 100-ms time scale and are approximately 10(4) times faster than their conventional counterparts in SDS-PAGE. This represents a much greater speed increase due to microfabrication than has been obtained in other assay steps such as electrophoretic separations. We have designed and tested a microchip capable of sequentially analyzing 11 different samples, with sizing accuracy better than 5% and high sensitivity (30 nM for carbonic anhydrase).     

Multiple open-channel electroosmotic pumping system for microfluidic sample handling

The development of a novel, fully integrated, miniaturized pumping system for generation of pressure-driven flow in microfluidic platforms is described. The micropump, based on electroosmotic pumping principles, has a multiple open-channel configuration consisting of hundreds of parallel, small-diameter microchannels. Specifically, pumps with microchannels of 1-6 microm in depth, 4-50 mm in length, and an overall area of a few square millimeters, were constructed. Flow rates of 10-400 nL/min were generated in electric-field-free regions in a stable, reproducible and controllable manner. In addition, eluent gradients were created by simultaneously using two pumps. Pressures up to 80 psi were produced with the present pump configurations. The pump can be easily interfaced with other operational elements of a micrototal analysis system (micro-TAS) device with multiplexing capabilities. A new microfluidic valving system was also briefly evaluated in conjunction with these pumps. The micropump was utilized to deliver peptide samples for electrospray ionization-mass spectrometric (ESI-MS) detection.     

Human low-density lipoprotein-coated capillaries in electrochromatography

Low-density lipoprotein (LDL) particles were immobilized on the inner wall of a fused-silica capillary and used in a study of the interactions between LDL and neutral drugs in electrochromatography. The effect of coating parameters (pH, ionic strength of the coating solution, duration of the coating procedure) on the properties and stability of the coating was examined. The stability of the coating was highest when the pH of the coating solution was under the pI value of the LDL particles. Interactions of unmodified LDL coatings with drugs were compared with those of acetylated LDL coatings. Acetylation of LDL neutralizes the positive charge on the lysine residues of the protein component of LDL particles, and acetylated LDL was used as a reference to examine the effect of the positively charged amino acids in the unmodified coating. Under similar coating conditions, acetylated LDL coating yielded stronger EOF evidently due to the decreased number of positive charges on LDL particles. The interactions of the unmodified and acetylated LDL coatings with steroids aldosterone, testosterone, and progesterone were comparable, which indicates that the density of immobilized LDL particles is not appreciably altered by acetylation. As expected, the strength of the interactions between steroids and the LDL coating increased with hydrophobicity of the drug.     

Macroporous photopolymer frits for capillary electrochromatography

Macroporous polymer frits have been fabricated in fused-silica capillaries by the UV photopolymerization of a solution of glycidyl methacrylate and trimethylopropane trimethacrylate. This in situ preparation is a simple, rapid, and reproducible process. The frit can be placed at any desired position along the column. Photopolymer frits can withstand the short exposure to a high pressure (over 6000 psi). Bubble formation is no observed to occur with these frits under our experimental conditions. By choice of porogens, it is possible to control the porous properties. The use of such frits in capillaries to retain particles of chromatographic packing has been demonstrated to be stable and robust with continuous operation over 3 days.     

Separation of explosives using capillary electrochromatography

The identification of explosives and their degradation products is important in forensic and environmental applications. Complete separation of these structurally similar compounds using reversed-phase liquid chromatography has proven to be a challenge. Here we present a demonstration of the use of capillary electrochromatography on the separation of a series of 14 nitroaromatic and nitramine explosive compounds. A separation with baseline resolution is achieved for all of the compounds in under 7 min, featuring efficiencies of over 500?000 theoretical plates/m. Using more aggressive running conditions, 13 of the 14 compounds are separated in under 2 min.     

Photopolymerized sol-gel monoliths for capillary electrochromatography

A solution of methacryloxypropyltrimethoxysilane in the presence of an acid catalyst, water, toluene, and a photoinitiator was irradiated at 365 nm for 5 min in a 75-microm i.d. capillary to prepare a porous monolithic sol-gel column by a one-step, in situ, process. The photopolymerized sol-gel (PSG) column shows reversed-phase behavior. Using this column, a variety of low-molecular-weight neutral compounds, including polycyclic aromatic hydrocarbons, alkyl benzenes, alkyl phenyl ketones, and steroids are separated from mixtures. Various different operational parameters, such as buffer composition, field strength, and column temperature, were varied to assess their influence on column performance. Use of PSG as a stationary phase for a pressure-driven separation is also demonstrated.     

Electrochemistry-based real-time PCR on a microchip

The development of handheld instruments for point-of-care DNA analysis can potentially contribute to the medical diagnostics and environmental monitoring for decentralized applications. In this work, we demonstrate the implementation of a recently developed electrochemical real-time polymerase chain reaction (ERT-PCR) technique on a silicon-glass microchip for simultaneous DNA amplification and detection. This on-chip ERT-PCR process requires the extension of an oligonucleotide in both solution and at solid phases and intermittent electrochemical signal measurement in the presence of all the PCR reagents. Several important parameters, related to the surface passivation and electrochemical scanning of working electrodes, were investigated. It was found that the ERT-PCR's onset thermal cycle ( approximately 3-5), where the analytical signal begins to be distinguishable from the background, is much lower than that of the fluorescence-based counterparts for high template DNA situations (3 x 10(6) copies/microL). By carefully controlling the concentrations of the immobilized probe and the enzyme polymerase, improvements have been made in obtaining a meaningful electrochemical signal using a lower initial template concentration. This ERT-PCR technique on a microchip platform holds significant promise for rapid DNA detection for point-of-care testing applications.     

Adaptive nanowires for switchable microchip devices

This paper demonstrates for the first time the use of adaptive functional nickel nanowires for switching on-demand operation of microfluidic devices. Controlled reversible magnetic positioning and orientation of these nanowires at the microchannel outlet offers modulation of the detection and separation processes, respectively. The former facilitates switching between active and passive detection states to allow the microchip to be periodically activated to perform a measurement and reset it to the passive ("off") state between measurements. Fine magnetic tuning of the separation process (postchannel broadening of the analyte zone) is achieved by reversibly modulating the nanowire orientation (i.e., detector alignment) at the channel outlet. The concept can be extended to other microchip functions and stimuli-responsive materials and holds great promise for regulating the operation of microfluidic devices in reaction to specific needs or unforeseen scenarios.     

Magnetically immobilized beds for capillary electrochromatography

Fritless packed beds comprised of magnetically responsive octadecylsilane bonded silica particles have been constructed for reversed-phase electrochromatography. The magnetic particles were immobilized in the capillary by applying an external magnetic field transverse to the direction of electroosmotic flow. Being subjected to the interplay of fluid dragging and magnetic forces, the initial loosely packed particle assembly was compacted into a uniform packing structure. The magnetically immobilized beds obtained were used as stationary phases for separation of neutral compounds, with retention behavior and column efficiency similar to those of slurry-packed columns. The results suggest that the magnetic attraction approach to fritless column packing may be used for construction of advanced chip-based chromatography, especially in complex architectures comprising curved and intersecting channels.     

Single-particle fritting technology for capillary electrochromatography

Large perfusive silica beads (particle size 110 microm, through pore approximately 2 microm) held in place by the keystone effect were used as single-particle frits for the manufacture of particulate packed capillary columns. High-quality capillary electrochromatographic separations of a standard test mixture of alkylbenzenes were obtained over the full voltage range of 5-30 kV, with no requirement for pressurization. Excellent robustness was demonstrated by the reproducibility of migration times, peak efficiencies, and resolution during 100 consecutive runs at the highest voltage (30 kV) without thermostating and pressurization. Superior performance relative to traditional sinter-fritted columns is ascribed to the heat-free fritting process and short frit length of approximately 110 microm.     

Magnetic microchip traps and single-atom detection

Microchip traps provide a promising approach to quantum information processing and communication (QIPC) with neutral atoms: strong and complex potentials can be produced for acting on the qubit atoms, and the potentials can be scaled to higher qubit numbers by virtue of the microfabrication process. We describe experimental results that are relevant to use in QIPC, such as the transport of Bose-Einstein-condensed atomic ensembles along the chip surface with the help of a magnetic conveyor belt. The second part of the paper is devoted to single-atom detection on the chip.